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CORTICOBASAL SYNDROME: CLINICAL, NEUROPSYCHOLOGICAL, IMAGING,

GENETIC AND PATHOLOGICAL FEATURES

by

Mario Masellis

A thesis submitted in conformity with the requirements for the degree of Doctorate of

Philosophy in the Graduate Department of Institute of Medical Sciences, University of Toronto

© Copyright by Mario Masellis (2012)

ii

Corticobasal Syndrome: Clinical, Neuropsychological, Imaging, Genetic and Pathological

Features

Doctorate of Philosophy 2012

Mario Masellis

Graduate Department of Institute of Medical Sciences, University of Toronto

ABSTRACT

Corticobasal Syndrome (CBS) is a rare movement and cognitive disorder. There is significant

heterogeneity observed in it clinical presentation, neuroimaging, pathology and genetics.

Understanding this heterogeneity is a priority and may help to shed light on underlying

pathogenic mechanisms. We first demonstrated that truncating mutations in the progranulin gene

(PGRN) can cause familial CBS associated with frontotemporal lobar degeneration (FTLD)-

ubiquitin pathology. This study identified a mutation in PGRN (Intervening Sequence 7+1

guanine > adenine [IVS7+1G>A]) that segregated with CBS in a family. The mutation was

predicted to result in a shortened messenger RNA (mRNA) sequence and the absence of the

mutant PGRN allele was confirmed in the reverse transcriptase-polymerase chain reaction (RT-

PCR) product, which supported the model of haploinsufficiency for PGRN-linked disease. In a

second familial study, clinical, radiological, genetic, and pathological studies were performed to

contrast clinical features of the affected members. Sequencing PGRN revealed a novel,

heterozygous cytosine-adenine dinucleotide deletion in exon 11 (g.2988_2989delCA,

P439_R440fsX6). The proband`s clinical diagnosis was frontotemporal dementia and

parkinsonism (FTDP). The proband‟s brother with the same mutation presented initially as a

progressive non-fluent aphasia (PNFA), and later evolved into a CBS. Pathological analysis

iii

revealed Frontotemporal Lobar Degeneration-Ubiquitin (FTLD-U)/ TAR DNA-binding protein

43 (TDP43) positive pathology. The next studies shift away from pathogenic mechanisms to

focus on brain-behavioural correlations and phenotypic heterogeneity in a prospective sample of

31 CBS cases. We provide the first direct correlative analysis between the severity of ideomotor

apraxia, a common sign in CBS, and cerebral SPECT perfusion imaging. Reductions in perfusion

within the left inferior parietal lobule (t=5.7, p=0.03, Family-Wise Error [FWE] corrected),

including the left angular gyrus (t=5.7, p=0.02, FWE corrected), were associated with more

severe ideomotor apraxia. We stratified the sample into CBS presenting with early motor

features (CBS-M; n=9) or early dementia (CBS-D; n=22), which identified that CBS-M were

more likely to have cortical sensory loss than CBS-D (p=0.005). In contrast, the presence of

aphasia was found to be more common and severe in CBS-D compared to CBS-M (p=0.02).

CBS-M patients had significantly reduced perfusion in the right supplementary and premotor

areas compared to CBS-D (p<0.05).

iv

ACKNOWLEDGEMENTS

I would like to first and foremost thank my supervisor, Dr. Sandra Black, for her support and

mentorship over the many years that I have known her. I first met Dr. Black (a.k.a. Sandy) back

in 2001 when I began my residency program in psychiatry. I attended many of her cognitive

neurology clinics as an intern and it was this initial exposure to the field of neurology and

neurodegenerative disease that made me decide to transfer into the neurology residency program.

The commitment and passion that she displayed towards treating patients and their families

afflicted with these devastating diseases was truly an inspiration for me. She taught me that every

patient has something unique to offer not only in terms of developing my clinical skills, but also

importantly in terms of asking novel questions about the diseases and their heterogeneous

presentations that could be assessed using the scientific method and valuable data gathered from

clinical and neuroimaging studies. In 2004, I had an opportunity to do my fourth year project

course in her lab, which further stimulated me to pursue a career in research and enroll in the

Ph.D. program following completion of my residency. Needless to say, my experiences in her lab

have been outstanding. As a result of this training, I have learned a new research method, applied

neuroimaging, which I now can add to my repertoire of techniques to use in my clinical and

genetic studies. Sandy‟s enthusiasm for research is incredible and her passion to understand and

to investigate novel therapies to treat these devastating disorders has also stimulated me to

pursue a career as a clinician-scientist. I would also like to thank Sandy for her support of my

research ideas, which have led to several peer-reviewed funding projects during and beyond my

training. Thank you Sandy for your ongoing support and I look forward to collaborating with you

on many interesting projects to come!

v

I would also like to thank Dr. James Kennedy (a.k.a. Jim) for his mentorship over many years

and also for his contribution to and participation on my program advisory committee. Jim

stimulated my initial interest in scientific research in the mid-1990s when I completed a M.Sc. in

his laboratory. My thesis was on pharmacogenetics and I am pleased to say that I continue this

very interesting line of research using the combination of my genetic, clinical and neuroimaging

training. I would also like to thank Dr. Robert Chen for his commitment and contributions that he

has made as a member of my program advisory committee. I appreciate the efforts of Dr.

Antonio Strafella for reading my thesis under tight time lines and also for being present at my

final program advisory committee meeting to participate as an additional examiner.

I would like to thank my friends and colleagues: Dr. Brad MacIntosh, Dr. Kie Honjo, Dr. Galit

Kleiner-Fisman, Dr. Ekaterina Rogaeva, Dr. Anthony E. Lang, Dr. Eric Roy, Isabelle Guimont,

Philip Francis, and Gregory Szilagyi. Their technical and thought-stimulating advice and

suggestions have helped to bring me to this point today. I would also like to thank Kayla

Sherborn for her impeccable organizational skills in helping to assemble components of this

thesis.

I would like to thank my in-laws for their support over the last few years and also for their

assistance in making home life more manageable. I would like to thank my parents for providing

me with the opportunities to pursue higher level education and for providing the right

environment for me to succeed. I greatly appreciate their continued support and inspiration and I

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am indebted to them for their patience especially over the last few years during the preparation of

this thesis.

Last but not least, I would like to thank my beautiful wife, Paola Masellis, for her incredible

patience and support that she has provided since the first day that I met her and over the course

of the last few years during the completion of this thesis. We have been through a lot together,

especially in recent years, and I am indebted to her kindness, love, and caring attitude. With the

completion of this thesis, I look forward to many good times ahead and many more years of

positive and happy life experiences together with her. Thanks for all that you do!

vii

TABLE OF CONTENTS

TITLE PAGE i

ABSTRACT ii

ACKNOWLEDGEMENTS iv

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xiii

ABBREVIATIONS xvii

CONTRIBUTIONS xxv

1.0: GENERAL INTRODUCTION 1

1.1 Corticobasal Degeneration: Historical Perspective 2

1.2 Epidemiology of CBS 4

1.3 Illustrative case examples 4

1.4 Symptoms and signs of corticobasal syndrome 10

1.4.1 Clinical motor and sensory features 11

1.4.2 Clinical cognitive features 14

1.4.3 Apraxia 19

1.5 Neuroimaging in CBS 21

1.5.1 Structural neuroimaging studies 22

1.5.2 Functional neuroimaging studies: PET and SPECT 25

1.6 Pathological heterogeneity in CBS 31

1.7 Genetics of CBS and CBD 37

1.8 Synopsis and overall research objective 42

1.8.1 Specific objectives 43

1.9 Description of chapters 45

1.9.1 Chapter 2: Novel splicing mutation in the progranulin gene causing

familial corticobasal syndrome

45

1.9.2 Chapter 3: Intra-Familial Clinical Heterogeneity due to FTLD-U with

TDP43 Proteinopathy Caused by a Novel Deletion in Progranulin Gene

(PGRN)

46

1.9.3 Chapter 4: Ideomotor Apraxia in Corticobasal Syndrome: Brain

Perfusion and Neuropsychological Correlates

46

1.9.4 Chapter 5: Clinical, neuropsychological, MRI and SPECT

characterization of a prospective sample of patients with corticobasal

syndrome

47

2.0: NOVEL SPLICING MUTATION IN THE PROGRANULIN GENE CAUSING

FAMILIAL CORTICOBASAL SYNDROME

48

2.1 Summary 49

2.2 Introduction 50

2.3 Methods 53

2.3.1 Subjects 53

2.3.2 Neuropathology 54

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2.3.3 Genetic Analysis 54

2.4 Results 55

2.4.1 Clinical features and autopsy results 55

2.4.1.1 Case #4150 (Proband) 57

2.4.1.2 Case #4993 (sister of proband) 61

2.4.1.3 Neuropathology (Case #4993) 61

2.4.2 Genetic analysis 63

2.5 Discussion 64

2.6 Acknowledgements 67

2.7 Addendum 68

3.0: INTRA-FAMILIAL CLINICAL HETEROGENEITY DUE TO FTLD-U WITH

TDP43 PROTEINOPATHY CAUSED BY A NOVEL DELETION IN

PROGANULIN GENE (PGRN)

69

3.1 Abstract 70

3.2 Introduction 71

3.3 Materials and methods 72

3.3.1 Subjects 72


3.3.3 Neuropathological analysis 74

3.4 Results 74

3.4.1 Clinical, neuropsychological, and radiographic features 74

3.4.2 Neuropathology (III:2) 81

3.4.3 Family history 82


3.5 Discussion 86


4.0 IDEOMOTOR APRAXIA IN CORTICOBASAL SYNDROME: BRAIN

PERFUSION AND NEUROPSYCHOLOGICAL CORRELATES

91

4.1 Abstract 92

4.2 Introduction 93

4.3 Materials and methods 96

4.3.1 Subjects 96

4.3.2 Description of neuropsychological measures 97

4.3.3 Brain SPECT acquisition and processing 99

4.3.3.1 Regional perfusion ratios 99

4.3.4 Data analysis 100

4.3.4.1 Demographic, clinical and neuropsychological measures 100

4.3.4.2 Statistical Parametric Mapping (SPM) SPECT analysis 100

4.3.4.3 Region of interest (ROI) SPECT analysis 101

4.3.4.3.1 Comparison of CBS cases to controls 101

4.3.4.3.2 Confirmation of voxel-wise correlation with WAB praxis using

ROI method

102

4.3.4.4 Brain MRI acquisition and processing 102

4.3.4.4.1 Brain extraction and automated tissue segmentation 103

4.3.4.4.2 Post-hoc MRI analysis 103

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4.4 Results 104

4.4.1 CBS vs controls 104

4.4.1.1 Demographic data 104

4.4.1.2 Clinical features 105

4.4.1.3 SPM and ROI SPECT analysis 105

4.4.1.4 CBS sample with praxis scores available 107

4.4.1.5 Comparison of apraxic to borderline/non-apraxic CBS patients:

Neuropsychological and SPECT analysis

109

4.4.1.6 Perfusion versus ideomotor apraxia 113

4.4.1.7 Post-hoc atrophy analysis 117

4.5 Discussion 117


5.0 CLINICAL, NEUROPSYCHOLOGICAL, MRI, AND SPECT

CHARACTERIZATION OF A PROSPECTIVE SAMPLE OF PATIENTS WITH

CORTICOBASAL SYNDROME

132

5.1 Abstract 133

5.2 Introduction 134

5.3 Methods 137

5.3.1 Subjects 137

5.3.2 Neuropsychological, neuropsychiatric, and functional measures 139

5.3.3 Brain MRI 140

5.3.4 Brain SPECT 141

5.3.5 Regional perfusion ratios 142

5.3.6 Pathological analysis 142

5.3.7 Data analysis 143

5.3.7.1 Demographic, clinical and neuropsychological measures 143

5.3.7.2 Region of interest (ROI) SPECT analysis 143

5.3.7.3 Statistical Parametric Mapping SPECT analysis 144

5.4 Results 145

5.4.1 CBS cases versus controls 145

5.4.1.1 Neuropsychological, behavioural and functional assessment 145

5.4.1.2 MRI features 146

5.4.2 Early dementia vs. early motor presentations 149

5.4.2.1 Demographic and clinical characteristics 149

5.4.2.2 Neuropsychological, behavioural and functional evaluation 150

5.4.2.3 MRI features 154

5.4.2.4 SPM and ROI SPECT 155

5.4.3 Description of pathological series and relation to MRI findings 156

5.5 Discussion 158

5.5.1 CBS presenting with early dementia vs. early motor features 158

5.5.2 Pathology 164

5.5.3 MRI investigation 166

5.5.4 Limitations 168

5.5.5 Conclusions 169

6.0 SUMMARY AND GENERAL DISCUSSION 171

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6.1 Representative sample 172

6.1.1 Demographic features 172

6.1.2 Clinical and neuropsychological features 173

6.1.3 Neuropsychiatric features 176

6.2 Apraxia in CBS 178

6.3 Comment on the neuroimaging methods 180

6.4 Can CBS serve as a model of etiology for common sporadic disorders? 185

7.0 CONCLUSIONS AND FUTURE DIRECTIONS 188

8.0 REFERENCES 192

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LIST OF TABLES

Chapter 2

Table 1 Scores on neuropsychological and functional measures for

case #4150 compared to standardized scores calculated based

on normal population matched for age and years of education.

Page 58

Chapter 3

Table 1 Raw scores on neuropsychological and functional measures

for proband (III:1) and proband‟s brother (III:2).

Page 76

Chapter 4

Table 1 Demographics of patients with corticobasal syndrome and

control group.

Page 104

Table 2 Clinical characteristics of CBS sample. Page 105

Table 3 Demographic features of CBS presenting with apraxia vs.

those without significant apraxia.

Page 109

Table 4 Mean scores on neuropsychological, neuropsychiatric and

functional measures in CBS presenting with apraxia vs. those

without significant apraxia.

1

Table 5 Areas of hypoperfusion on SPECT in the CBS group that

correlate with WAB praxis scores in the regression analyses.

Page 114

Supplementary

Table 1

Areas of hypoperfusion on SPECT in all CBS patients, CBS

with left side of body most affected, and CBS with right side

of body most affected relative to controls.

Page 130

Chapter 5

Table 1 Case summaries of clinical, pathological, and MRI features of

CBS patients.

Page 147

Table 2 MRI atrophy patterns in CBS cases stratified according to Page 148

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body side most affected by motor symptoms.

Table 3 Demographic features of CBS groups presenting with early

dementia versus early motor features.

Page 149

Table 4 Mean scores on neuropsychological measures in CBS patients

presenting with early dementia vs. early motor symptoms.

Page 151

Table 5 Mean scores on behavioural and functional measures in the

CBS group.

Page 154

Table 6 MRI atrophy patterns in CBS cases stratified by the presence

or absence of aphasia as determined by the WAB.

Page 154

Table 7 Areas of relative hypoperfusion on SPECT in CBS patients

presenting with early dementia versus those presenting with

early motor features.

Page 156

Table 8 Areas of relative hypoperfusion on SPECT in CBS patients

presenting with early motor versus those presenting with early

dementia.

Page 156

Supplementary

Table 1

Mean scores on behavioural and functional measures in the

CBS group.

Page 170

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LIST OF FIGURES

Chapter 1

Figure 1 (A) Loss of neurons in the outer layers of the right parietal

cortex and disorganized cortical architecture in the deeper

layers; (B) Swollen, pale neurons with eccentric nuclei in the

left superior parietal region.

Page 2

Figure 2 (A) Brain SPECT showing right parieto-occipital

hypoperfusion, and (B) T1-weighted MRI showing right > left

biparietal atrophy and also a lesser degree of frontal atrophy.

Page 7

Figure 3 (A) Brain SPECT showing left > right bifrontal hypoperfusion,

and (B) T1-weighted MRI showing superior left superior

frontal > parietal atrophy.

Page 10

Figure 4 Macroscopic brain specimen showing left frontal > temporal

atrophy of Pick‟s disease.

Page 32

Figure 5 Microscopic Lewy body pathology showing Lewy bodies,

cytoplasmic stippling, neuropil grains and Lewy neurites

immunostained by antibodies to alpha-synuclein.

Page 33

Figure 6 Microscopic pathology of CBD stained with Gallyas

demonstrating (A) oligodendroglial coils, (B) neuronal pre-

tangles in the precentral region, (C) ballooned neurons, and

(D) astrocytic plaques in the basal ganglia.

Page 35

Figure 7 Microscopic agyrophilic grain disease pathology showing (A)

branched astrocytes in the amygdale, and (B) agyrophilic

grains and coiled bodies in the prosubiculum.

Page 36

Figure 8 Microscopic Alzheimer‟s pathology showing (A) astrocytic

plaques in frontal regions, and (B) neurofibrillary tangles in

the CA1 region of the hippocampus.

Page 36

Figure 9 Schematic representation of the MAPT genomic region and 3-

repeat and 4-repeat Tau transcripts.

Page 39

Figure 10 (A) H1 and H2 linkage disequilibrium blocks showing a 900

kb region of inversion, and (B) sub-structure of the MAPT

gene and associated H1 and H2 haplotypes.

Page 41

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Chapter 2

Figure 1 (A) The pedigree structure of the Canadian family showing the

inheritance of the disease (with age-at-onset). (B) Genomic

DNA (gDNA) and RT-PCR (cDNA) sequence fluorescent

chromatograms around the PGRN mutation (IVS7+1G>A)

observed in the patients and the sequence around common

synonymous variation rs25646; (C) An agarose gel photograph

of the PGRN product from RT-PCR, using RNA obtained from

white blood cells of the affected family member (#4150) and

normal control.

Page 56

Figure 2 Corresponding (A) T1-weighted Magnetic Resonance Imaging

(MRI) and (B) Technetium 99m-ethyl cysteinate dimer (99m

Tc-

ECD) Single Photon Emission Computed Tomography

(SPECT) scans of the brain of Case #4150.

Page 59

Chapter 3

Figure 1 T1-weighted brain MRI and corresponding 99m

Tc-ECD brain

SPECT images of proband‟s brother (III:2) in radiographic

axial orientation- Session 1

Page 79


Tc-ECD brain

SPECT images of proband‟s brother (III:2) in radiographic

axial orientation - Session 2

Page 79


Tc-HMPAO

(800MBq) brain SPECT images of proband (III:1) in standard

radiographic axial orientation.

Page 79

Figure 4 Micrographs demonstrating a large number of TDP43

inclusions found in the fascia dentata, substantia nigra, and

CA1 region.

Page 82

Figure 5 Detection of PGRN mutation P439_R440fsX6. A) Pedigree

showing family history of neurodegenerative condition. B)

Electropherogram showing start of deletion marked with an

arrow.

Page 84

Figure 6 Amplification from genomic DNA (gDNA; lane 1) using

primers specific for the mutant allele demonstrate the mutant

Page 85

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fragment of 153 bp as expected. Amplification from cDNA

(lane 2) shows an absence of the expected product supportive

of non-sense mediated decay.

Chapter 4

Figure 1 Statistical parametric maps (SPM) of bilateral frontal, parietal

and temporal surface regions of the brain showing decreased

perfusion in (A) all CBS cases compared to controls and (B)

CBS cases with predominant symptoms on their left side

(CBS-L) compared to controls overlaid on brain MRI

template.

Page 106

Figure 2 Frequency of different aphasia categories on the Western

Aphasia Battery (WAB) distributed according to the CBS

group with apraxia versus those with borderline/no apraxia.

Page 113

Figure 3 Statistical parametric map of surface regions of the brain

showing decreased perfusion in the left inferior parietal region,

including the angular gyrus, that correlate with WAB praxis

scores in the regression analyses.

Page 115

Supplementary

Figure 1A

Mean proportion of different MRI tissue classes underlying the

FWE-corrected SPM mask.

Page 128

Supplementary

Figure 1B

Mean proportion of different MRI tissue classes underlying the

FDR-corrected SPM mask.

Page 129

Chapter 5

Figure 1 Normalized (z-) scores of neuropsychological measures in

patients with CBS compared to control group.

Page 145

Figure 2 Frequency of (A) extrapyramidal and (B) cortical features of

CBS patients presenting with early dementia vs. early motor

symptoms.

0

Figure 3 Frequency of CBS patients with early dementia vs. early motor

presentation stratified according to category on the Western

Aphasia Battery (WAB).

Page 153

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Figure 4 Statistical parametric maps overlaid on multi-slice brain MRI

template showing decreased perfusion in left fusiform gyrus

(uncorrected p<0.001) in CBS cases presenting with early

dementia versus early motor features.

Page 155

xvii

ABBREVIATIONS

β-CIT 2-β-carbomethoxy-3-β-(4-iodophenyl)-tropane

C Degrees Celsius

μg Micrograms

18F-dopa

18F-6-fluorodopa

3R Three repeat

4R Four repeat

99mTc-ECD Technetium-99m ethyl cysteinate dimer

A Adenine

ABA-2 Apraxia Battery for Adults-2

ACTB Beta Actin

AD Alzheimer‟s disease

ADL Activities of Daily Living

AGD Agyrophilic Grain Disease

AIR Automated Image Registration package

ANCOVA Analysis of covariance

ANOVA Analysis of variance

ANT Anterior

AOO Age of onset

APOE Apolipoprotein E

APX Apraxia

AQ Aphasia quotient

AT Anterior temporal

BAs Brodmann Areas

BNT Boston naming test

xviii

BOLD Blood Oxygen Level Dependent

bp Base pair

bvFTD Behavioral variant of frontotemporal dementia

C Cytosine

CAA Cerebral Amyloid Angiopathy

CBD Corticobasal degeneration

CBS Corticobasal syndrome

CBS-D Corticobasal syndrome presenting with early dementia

CBS-L Corticobasal syndrome cases with left-sided symptoms

CBS-M Corticobasal syndrome presenting with early motor features

CBS-R Corticobasal syndrome cases with right-sided symptoms

cDNA Complimentary deoxyribonucleic acid

CDR Clinical Dementia Rating

CHMP2B Chromatin-modifying protein 2B

CJD Creutzfeldt-Jakob disease

cm Centimeters

Cog Cognitive Neurology Clinic

CSDD Cornell Scale for Depression in Dementia

CSF Cerebrospinal fluid

CT Computerized Tomography

CVLT California Verbal Learning Test

D Aspartic acid

D2 Dopamine D2 receptor

DAD Disability Assessment for Dementia

DAT Dopamine transporter

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DEM Dementia onset

D-KEFS Delis-Kaplan Executive Function System

DLB Dementia with Lewy Bodies

DNA Deoxyribonucleic acid

DRS Dementia Rating Scale

DSM-IV Diagnostic and Statistical Manual - IV

DTI Diffusion tensor imaging

EEG Electroencephalograph

F Female

FAS F-, A-, S-word phonemic fluency

FBI Frontal Behavioural Inventory

FDG Fluorodeoxyglucose

FDR False discovery rate

FLAIR Fluid Attenuated Inversion Recovery

fMRI Functional Magnetic Resonance Imaging

FOV Field of View

Fr Frontal

FTD Frontotemporal dementia

FTLD Frontotemporal Lobar Degeneration

FTDP Frontotemporal dementia and parkinsonism

FTDP-17 Frontotemporal dementia with parkinsonism linked to chromosome

17

FTLD-U Frontotemporal Lobar Degeneration-Ubiquitin

FWE Family-Wise Error

FWHM Full width at half maximum

xx

g Gram

G Guanine

gDNA Genomic deoxyribonucleic acid

Gen Generalized

GLM General linear model

GRN Granulin

GWAS Genome wide association studies

HMPAO Hexamethylpropyleneamine Oxime

iADL Instrumental Activities of Daily Living

IBZM 123

I-iodobenzamide

IF Inferior frontal

IP Inferior parietal

IMA Ideomotor apraxia

IMP N-isopropyl-p[123

I]iodoamphetamine

IVS7+1G>A Intervening Sequence 7+1 guanine > adenine

L Left

L-dopa Levodopa

LD Linkage disequilibrium

LFB Luxol fast blue

LKA Limb-kinetic apraxia

M Male

MANCOVA Multivariate analysis of covariance

MAPT Microtubule-Associated Protein Tau

mCi Millicurrie

Mb Megabases

xxi

mBq Megabecquerel

MD Movement Disorders Clinic

MDRS Mattis Dementia Rating Scale

Min Minutes

miRNA Micro ribonucleic acid

mL Milliliter

mm Millimeter

MMSE Mini Mental Status Examination

MND Motor neuron disease

MNI Montreal Neurological Institute

Motor Motor onset

MR Magnetic Resonance

mRNA Messenger ribonucleic acid

MRI Magnetic Resonance Imaging

ms Millisecond

MSA Multiple system atrophy

n Sample size

N/T Not testable

nAPX Those without significant apraxia

NART-R National Adult Reading Test-Revised

NCO Normal cut-off

NEX Number of excitations

NPI Neuropsychiatric Inventory

O Occipital

OMIM On-line Mendelian Inheritance in Man

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p Probability value

P Parietal

P301S Proline301Serine

PCR Polymerase Chain Reaction

PD Parkinson‟s disease

PET Positron Emission Tomography

PGRN Progranulin

PNFA Progressive non-fluent aphasia

POST Posterior

PPA Primary Progressive Aphasia

PSEN1 Presenilin 1

PSP Progressive Supranuclear Palsy

PT Posterior temporal

Q-Q Quantile-Quantile

R Right

rCBF Regional cerebral blood flow

RNA Ribonucleic acid

ROI Regions of interest

RT-PCR Reverse transcriptase-polymerase chain reaction

SD Standard deviation

Sec Second

SEM Standard Error of Mean

SF Superior frontal

SNCA Alpha-synuclein

SNPs Single Nucleotide Polymorphisms

xxiii

SP Superior parietal

SPECT Single-Photon Emission Computed Tomography

SPGR Spoiled gradient

SPM Statistical Parametric Mapping

SPM5 Statistical Parametric Mapping version 5

SPSS Statistical Package for the Social Sciences

SS Scaled Score

SYM Symmetrical

T Thymine

T2/PD T2/Proton density

TDP43 TAR DNA-binding protein 43

Te Temporal

TE Echo time

TMEM106B Transmembrane protein 106B

TMT-A Trail Making Test A

TMT-B Trail Making Test B

TOLA Test of Oral and Limb Apraxia

TR Repetition time

TRODAT [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-

yl]methyl](2mercaptoethyl)amino]ethyl]amino]ethanethiolato(3-)-

N2,N2‟,S2,S2‟]oxo-[1R-(exo-exo)]- [99m

Tc] technetium)

([99m

Tc]TRODAT-1)

VBM Voxel-based morphometry

WAB Western Aphasia Battery

WCST Wisconsin Card Sort Test

WMH White Matter Hyperintensities

xxiv

WMS-R Wechsler Memory Scale-Revised

ZS Z-score

xxv

CONTRIBUTIONS

Chapter 2.0 Novel splicing mutation in the progranulin gene causing familial corticobasal

syndrome

Mario Masellis,* Parastoo Momeni,

* Wendy Meschino, Reid Heffner Jr., Christine Sato, Yan

Liang, Peter St George-Hyslop, John Hardy, Juan Bilbao, Sandra Black, and Ekaterina Rogaeva

As published in: Brain (2006); 129: 3115-3123

Mario Masellis extracted the clinical information on all family members, interpreted and

integrated the clinical, neuropsychological, neuroimaging, genetic and pathological data, and was

responsible for writing the manuscript. Sequencing and genotyping was performed by Parastoo

Momeni and Ekaterina Rogaeva. Pathological analysis was done by Reid Heffner Jr. and Juan

Bilbao.

Chapter 3.0 Intra-Familial Clinical Heterogeneity due to FTLD-U with TDP43

Proteinopathy Caused by a Novel Deletion in Progranulin Gene (PGRN)

Tomasz Gabryelewicz*, Mario Masellis*, Mariusz Berdynski

*, Juan M. Bilbao, Ekaterina

Rogaeva, Peter St. George-Hyslop, Anna Barczak, Krzysztof Czyzewski, Maria Barcikowska,

Zbigniew Wszolek, Sandra E. Black and Cezary Zekanowski As published in: J Alzheimers Dis

(2010); 22: 1123-1133.

Mario Masellis extracted the clinical information on the brother of the proband, interpreted and


responsible for writing a significant proportion of the manuscript with contribution from Tomasz

xxvi

Gabryelewicz. Sequencing of the proband and genotyping of the controls was performed by

Mariusz Berdynski and Cezary Zekanowski. Ekaterina Rogaeva performed the genotyping in the

brother of the proband. Pathological analysis was done by Juan Bilbao.

Chapter 4.0 Ideomotor Apraxia in Corticobasal Syndrome: Brain Perfusion and

Neuropsychological Correlates

Mario Masellis, Philip L. Francis, Kie Honjo, Bradley J. MacIntosh, Isabelle Guimont, Gregory

M. Szilagyi, Wendy R. Galpern, Galit Kleiner-Fisman, James L. Kennedy, Robert Chen, Eric A.

Roy, Curtis B. Caldwell, Anthony E. Lang, Sandra E. Black. As submitted to: Cortex

Mario Masellis clinically assessed several of the patients included in this study, extracted the

clinical information, designed the study, performed the data analysis and wrote the manuscript.

Philip Francis assisted with the SPM analysis of SPECT data and Kie Honjo assisted with the

MRI segmentation procedure. Bradley J. MacIntosh assisted with the atrophy correction

procedure. Wendy R. Galpern, Galit Kleiner-Fisman and Anthony E. Lang assessed and

collected clinical data on patients ascertained from a movement disorders clinic.

Chapter 5.0 Clinical, neuropsychological, MRI and SPECT characterization of a

prospective sample of patients with corticobasal syndrome

Mario Masellis, Philip L. Francis, Isabelle Guimont, Wendy Galpern, Juan Bilbao, Kie Honjo,

Fuqiang Gao1, Gregory Szilagyi, Farrell Leibovitch, James L. Kennedy, Galit Kleiner-Fisman,

Lisa Ehrlich, Robert Chen, Anthony E. Lang, Sandra E. Black

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Philip Francis assisted with the SPM analysis of SPECT data and Kie Honjo assisted with an

independent visual read of the MRI data. Wendy R. Galpern, Galit Kleiner-Fisman and Anthony

E. Lang assessed and collected clinical data on patients ascertained from a movement disorders

clinic. Juan Bilbao performed the neuropathological analysis.

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1.0 General Introduction

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1.0 General Introduction

1.1 Corticobasal Degeneration: Historical Perspective

Rebeiz and colleagues described the first case of corticobasal degeneration (CBD) in 1967 and

subsequently characterized three cases from the clinical and pathological perspective in 1968 in

their seminal paper „Corticodentatonigral Degeneration with Neuronal Achromasia‟ in which

they coined the term based on pathological changes noted in the brain (figure 1) [Rebeiz et al.

1967;Rebeiz et al. 1968].

Figure 1. (A) Loss of neurons in the outer layers of the right parietal cortex and disorganized cortical

architecture in the deeper layers, and (B) swollen, pale neurons with eccentric nuclei in the left superior

parietal region. Adapted from Rebeiz et al. [Rebeiz et al. 1968]

A B

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Since then, many terms have been used to describe this enigmatic disorder of interest to

cognitive and movement disorder neurologists worldwide. These include: cortical degeneration

with swollen chromatolytic neurons, corticobasal ganglionic, cortical basal ganglionic, and the

most common designation, corticobasal degeneration (CBD) [Mahapatra et al. 2004].

Patients suffering from CBD pathology or from the typical clinical syndrome have an insidious

onset and gradual decline in function due to a combination of cortical and subcortical/

extrapyramidal clinical features not attributable to other etiologies such as stroke or tumour

[Boeve et al. 2003]. The cortical features may include focal or asymmetric ideomotor apraxia,

alien limb phenomenon, cortical sensory loss, visual or sensory hemi-neglect, constructional

apraxia, focal or asymmetric myoclonus, and apraxia of speech/nonfluent aphasia. The

extrapyramidal features may consist of appendicular rigidity lacking prominent and sustained L-

dopa response, and appendicular dystonia. Supportive criteria include cognitive dysfunction with

relative preservation of learning and memory on psychometric testing, asymmetric atrophy on

computed tomography or magnetic resonance imaging, typically maximal in frontoparietal

cortical regions, and asymmetric hypoperfusion or hypometabolism on single-photon emission

computed tomography (SPECT) and positron emission tomography (PET), respectively,

typically maximal in frontoparietal cortex ± basal ganglia ± thalamus. The clinical syndrome

produced by CBD pathology is most often markedly asymmetrical with either left or right

hemisphere signs noted in the early stages of the disease although symmetrical cases at onset

have been infrequently described [Hassan et al. 2010].

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1.2 Epidemiology of CBS

CBS and its most commonly associated underlying pathology, CBD, are extremely rare

syndromic/ pathologic entities and, as a result, it is difficult to estimate their true incidence and

prevalence. Corticobasal syndrome typically presents in the sixth to eighth decade of life and has

a mean age of onset of approximately 63 (standard deviation 7.7) years [Wenning et al. 1998]. It

is estimated that CBS accounts for four to six percent of all cases of parkinsonism and, based on

the incidence of Parkinson‟s disease, it is speculated that the incidence of CBS lies somewhere

between 0.62 to 0.92 per 100,000 per year [Mahapatra et al. 2004;Togasaki and Tanner

2000;Wenning et al. 1998]. Based on the average duration of survival of approximately 7.9

years, prevalence can be estimated at about 4.9 to 7.3 per 100,000 [Mahapatra et al.

2004;Togasaki and Tanner 2000;Wenning et al. 1998]. Despite its rarity, CBS is an extremely

interesting syndrome particularly pertaining to the enormous amount of heterogeneity that can be

seen on multiple levels including clinical, neuropsychological, neuroimaging, genetic and

pathological features. A selection of some of the more common symptoms and signs of CBD will

now be illustrated through a review of two representative cases whose clinical and research data

were included in the thesis experiments.

1.3 Illustrative case examples

Case 1: CBD with early motor presentation

This 65 year old right-handed woman with no relevant past medical history presented at age 62

with the insidious onset and progressive decline in the use of her left arm. Her presenting

complaint was that she could not knit because her “left hand was somewhat awkward.” It would

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not do what she “wanted it to do.” Shortly thereafter, she noted difficulty using her left hand to

cut steak and onions with a knife and fork, de-bone chicken, button up her jacket and fold

laundry. She also endorsed troubles with going down stairs. She also noted that she was

becoming more “impatient.” She saw a neurologist early on in the disease course at age 62 and

was noted on exam to have difficulties with fine motor coordination of her left hand and to a

lesser degree her left lower extremity. There was also mild pseudoathetosis of the left fingers.

Otherwise, her neurological exam, including “higher mental functions”, was intact. She was

diagnosed with “left upper extremity apraxia” and referred on to a movement disorders clinic

where a provisional diagnosis of corticobasal syndrome was made. This was based on history

and the emergence of left-sided rigidity and overflow dystonic posturing of the left arm while

walking – slight abduction at the shoulder, extended at the elbow and wrist with a clenched fist,

in addition to an action tremor, but none at rest on the left. Initial brain MRI and SPECT scans

were reported as normal. An EEG demonstrated “non-specific bitemporal slow waves.” A trial of

levodopa was initiated for several months with no response; she eventually discontinued it. Her

motor symptoms continued to slowly worsen.

Over time, she noted that her left hand and arm “has a mind of its own.” It moved “against” her

will and she used her right arm to keep her left in check. She also lost the ability to write with

even her right hand. Her husband also endorsed that she was not seeing things as easily in her

left visual world. Her medications at this time included amantadine 100 mg tid. Although there

were no cognitive issues endorsed by the patient or caregiver, cognitive screening revealed an

MMSE of 21/28 (total score reduced to 28 given that apraxia interfered with tasks involving

drawing and writing) with points lost predominantly on attention and delayed recall. A cognitive

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screening battery revealed difficulties with tasks involving sustained attention, working memory,

executive functions and praxis. Delayed verbal recall was impaired, but benefited from cueing.

Neurological exam revealed left greater than right-sided rigidity and paratonia. She had a classic

alien-limb phenomenon involving the left upper extremity. Proprioception was reduced on the

left and there was bilateral agraphesthesia. There was a left-sided grasp reflex. About two

months after this initial visit, she continued to decline with worsening left-sided dystonia and

apraxia creating an essentially useless left arm. A repeat brain SPECT revealed decreased

perfusion in the right parietal and lateral occipital region (Figure 2). Brain MRI revealed

generalized atrophy most prominent in the right posterior region (Figure 2). Neuropsychological

testing revealed a preservation of frontal lobe executive function. About seven months later (age

66), she was having increasing difficulties with ambulation requiring a cane and wheelchair for

distances. MMSE was 25/28. Shortly thereafter, she lost the ability to ambulate and became

wheel-chair bound. She had moderate dysarthria. Rigidity was present in all four limbs although

it remained worse on the left. Ideomotor apraxia was also becoming worse in the right hand.

There was also evidence for a mild orofacial and oculomotor apraxia. About eight months later

(age 67) she continued to decline with slower speech, increasing word-finding difficulties, and

occasional semantic paraphasia. There were no complaints of memory loss. Her MMSE was

18/28. She developed a classic alien limb phenomenon of her right arm, with constant

involuntary grabbing of the left arm and touching of faces. Her rigidity was severe with

superimposed spasticity and hyperreflexia. Over the next nine months (age 68), her speech

became severely dysarthric and eventually progressed to mutism. Her swallowing also became

impaired and she developed recurrent pneumonia presumably on the basis of aspiration. She

developed severe, generalized rigidity and it was uncomfortable to move her. Approximately,

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one year later (age 69) she passed away from respiratory complications related to her

neurodegenerative condition. Pathological diagnosis was CBD.

Figure 2. (A) Brain SPECT showing right parieto-occipital hypoperfusion, and (B) T1-weighted MRI showing

right > left biparietal atrophy and also a lesser degree of frontal atrophy.

Case 2: CBD with early dementia presentation

This 61 year old woman presented with the insidious onset and gradual decline in expressive

language and speech production. Her past medical history was significant for hypertension,

diabetes mellitus, and hypercholesterolemia. Her initial cognitive symptoms occurred at age 59

with word-finding difficulties and difficulties putting together sentences. As an example, she

occasionally left out verbs and prepositions when speaking, for example, “the dog - the

backyard.” There were no complaints of memory loss initially. Her initial MMSE was 28/30. She

lost one point on delayed recall and on figure copying. She also had difficulties with serial 7s.

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Her language was described as non-fluent. Naming, reading, writing, comprehension and

repetition were intact. On this initial assessment, her neurological exam was otherwise normal. A

CT head revealed generalized cerebral atrophy and a brain SPECT revealed mild hypoperfusion

in the posterior parietotemporal regions as well as bifrontally. Her initial diagnosis was primary

progressive non-fluent aphasia (PNFA).

Approximately one year later (age 62), her problems with fluency progressed. Occasionally, she

would substitute in the incorrect word while speaking. She would also repeat words that

someone else had said representing echolalia. Her word pronunciation declined and her speech

became more strained. Despite these difficulties, she was able to sing along to songs. Even

though the patient denied any short-term memory difficulties, her family noted that there was

some forgetfulness as she would often not recall things on a grocery list. Apathy and depressive

symptoms were present with the patient becoming more withdrawn from interaction with others

and also less interested in doing things that she enjoyed. She was frequently tearful. She began to

have postural instability with episodes of spontaneously falling backwards. Her MMSE had

declined to 18/30 with five points lost on orientation, four points lost on attention/calculation,

and one point lost on each of delayed recall, three-step command, and figure-copying. A

cognitive screening battery revealed prominent deficits on tasks involving sustained attention,

working memory, executive functions and ideomotor praxis. Language assessment revealed

strained, effortful speech with paraphasias and decreased spontaneous output. Naming for low

frequency words was impaired as was repetition. Comprehension remained relatively spared.

Short-term memory was also impaired, but benefited from cueing. Visuospatial function was

relatively preserved. Neurological exam revealed slow, hypometric saccades horizontally and

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difficulty with eliciting downward saccades. She had a positive grasp reflex bilaterally as well as

a snout/pout response. Tone was increased moreso on the right. Strength was within normal

limits as were reflexes and plantar responses. There was some evidence for mild bradykinesia on

rapid alternating movements. Gait revealed some slowing with decreased arm swing on the right.

On pull-test, there was an absence of the postural reflex; she fell straight backwards. The

diagnostic impression at this time was primary progressive non-fluent aphasia evolving into CBS

with some features of Progressive Supranuclear Palsy (PSP). Re-assessment three months later

revealed continual decline in terms of memory, language, falls, apathy and loss of instrumental

activities of daily living. Her exam revealed ongoing troubles with saccadic eye movements and

increasing rigidity and bradykinesia on the right greater than left side. A repeat SPECT scan

revealed moderate to severe hypoperfusion of the left frontal lobe extending to the left temporal

lobe, caudate, and less so to the thalamus (Figure 3). MRI revealed left greater than right-sided

atrophy involving the frontal, temporal and parietal regions (Figure 3). Neuropsychological

testing revealed deficits across all cognitive domains. Her WAB category was anomic aphasia.

Evaluation approximately eight months later (age 63) revealed worsening expressive language

function with preserved comprehension; she could say only one to two words at a time. She

continued to have frequent falls. She progressed to dependence on all activities of daily living.

She could only walk if assisted. Her MMSE score was 8/28. She was able to name 5/6 objects,

and followed some commands. She had no extraocular movements to command or pursuit, but

they were present on vestibular oculoreflex testing. There was increased axial tone with nuchal

hyperextension. There was marked rigidity of the right arm and leg with significantly less

rigidity on the left. There was a severe ideomotor apraxia on the right greater than left. Re-

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assessment nine months later (age 64) revealed worsening aphasia; she was now only able to say

single word sentences and had difficulties comprehending even simple instructions. Gait had

worsened and she required a two-person assist to transfer, and was wheel-chair bound. On exam,

the MMSE score was 2/28. The physical exam was unchanged except for worsening rigidity and

postural instability. She died at age 65 due to respiratory complications related to the

neurodegeneration. Pathological diagnosis was CBD.

Figure 3. (A) Brain SPECT showing left > right bifrontal hypoperfusion, and (B) T1-weighted MRI showing

superior left superior frontal > parietal atrophy.

1.4 Symptoms and signs of corticobasal syndrome

The two cases described in the preceding section provide an illustrative account of several

common symptoms and signs associated with CBS and also demonstrate the evolution of the

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clinical syndrome over time. Several classical papers detailing the frequency of clinical signs in

CBS will now be reviewed.

1.4.1 Clinical motor and sensory features

A large prospective study from a movement disorders clinic identified that 64% (23/36) of

patients presenting with CBS had “clumsiness of one hand or arm with loss of manual dexterity”

as the most common initial complaint [Rinne et al. 1994]. A disturbance of gait due to leg

stiffness, jerking, clumsiness, imbalance or combinations thereof, was the next most frequent

presenting complaint (28%; 10/26) [Rinne et al. 1994]. Rare initial presentations included

prominent sensory symptoms, isolated speech disorder with dysarthria, or a prominent

behavioural syndrome [Rinne et al. 1994]. Another early clinical study of 15 patients

demonstrated that postural-action tremor, apraxia, limb dystonia or cortical sensory loss were the

most frequent initial presenting symptoms [Riley et al. 1990]. Wenning and colleagues

[Wenning et al. 1998] found a similar distribution of the most common clinical signs mentioned

above. A retrospective chart review of 147 CBS cases from multiple centers found that rigidity

(92%), apraxia (82%), bradykinesia (80%) and gait disorder (80%) were the most common signs

observed in their sample [Kompoliti et al. 1998].

The disorder progressed over time to involve the ipsilateral limb, typically the leg, and then

eventually involved the contralateral side usually starting with the arm. With progression, other

cortical and extrapyramidal features of the syndrome emerge although many of these signs can

also be present early on. Of the extrapyramidal features, asymmetric rigidity and akinesia/

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bradykinesia that typically do not respond to levodopa were common findings and eventually

occurred in all patients [Kompoliti et al. 1998;Riley et al. 1990;Rinne et al. 1994]. Limb

dystonia was also a common finding and usually involved the most affected limb with adduction

at the shoulder, flexion posturing at the elbow and clawing of the fingers around the adducted

thumb into the palm, often with skin breakdown – the so-called “clenched fist” [Rinne et al.

1994;Vanek and Jankovic 2001]. Extension of one or more fingers has also been observed

[Rinne et al. 1994;Vanek and Jankovic 2001]. Limb dystonia has been associated with pain in

prior studies [Rinne et al. 1994;Vanek and Jankovic 2001] and may respond to local botulinum

toxin injectons into the affected muscles [Cordivari et al. 2001].

Cortical features that involve the limbs also typically present asymmetrically, but will also

progress to bilateral involvement over time. Apraxia is the most common cortical feature and

will invariably occur in all patients at some point during the course of the disease [Leiguarda et

al. 1994;Rinne et al. 1994;Stamenova et al. 2009]. Apraxia will be discussed in more detail in a

subsequent section. Cortical sensory loss, manifest as agraphesthesia, astereognosis, sensory

extinction, hemi-neglect, and/or loss of two-point discrimination and/or proprioception,

presented asymmetrically in several studies [Kompoliti et al. 1998;Mahapatra et al. 2004;Riley

and Lang 2000;Rinne et al. 1994]. Irregular jerking (myoclonus) in CBS was focal involving one

limb, typically occurred in distal regions, and was elicited with action and/ or evoked by a

stimulus [Riley et al. 1990;Thompson et al. 1994]. The myoclonus was cortical in origin as

determined by electrophysiological studies demonstrating evidence of enhanced cortical

excitability via cutaneous or mixed nerve stimulation [Thompson et al. 1994]. Alien limb

phenomenon is a particularly interesting cortical feature whereby the affected limb acts out on its

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own, sometimes without the patient being aware of its movement and behavior [Riley et al.

1990]. As in our first case, her left hand had a “mind of its own” and it moved “against her will”

representing what is now considered to be the true form of alien limb phenomenon [Boeve et al.

2003]. “Levitation” of a limb, originally described by Denny-Brown et al. [Denny-Brown et al.

1952], was thought to originate from lesions in the parietal lobe and should be distinguished

from the true alien limb phenomenon. Although both phenomena occur in CBS, levitation is

more common than alien limb phenomenon [Riley et al. 1990] and previous studies that have

grouped these signs together have likely artificially inflated the frequency of “alien limb” in this

syndrome [Kompoliti et al. 1998]. Similar to case 1, asymmetric pyramidal findings including

superimposed spasticity, hyperreflexia, and positive Babinski signs have also been observed

[Kompoliti et al. 1998;Mahapatra et al. 2004;Riley et al. 1990;Rinne et al. 1994;Wenning et al.

1998]. Frontal release signs were often present and can be more pronounced on the most affected

side of the body [Kompoliti et al. 1998;Mahapatra et al. 2004;Riley et al. 1990;Rinne et al.

1994;Wenning et al. 1998].

Eye movement abnormalities similar to that described in case 2, were observed in 72% (26/36)

of CBS cases and were considered supranuclear in nature manifesting as oculomotor apraxia,

saccadic (jerky) pursuit movements, and/or restriction in the range of saccadic and pursuit

movement vertically more than horizontally [Rinne et al. 1994]. In four cases, a frank limitation

of vertical downgaze was noted reminiscent of that observed in progressive supranuclear palsy

(PSP) [Rinne et al. 1994]. Similar findings were noted in other studies [Kompoliti et al.

1998;Mahapatra et al. 2004;Riley and Lang 2000]. The constellation of clinical exam features is

variable across individual patients. That is, not all patients manifest every sign that has been

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associated with CBS. In addition, the body side most affected and the timing in which the

different clinical signs present is also variable across patients although over time the signs are

present bilaterally.

1.4.2. Clinical cognitive features

The majority of the studies reviewed in the preceding section were conducted in specialist

movement disorder clinics. While several of the early studies acknowledged that a few of their

cases presented with an early dementia syndrome [Rinne et al. 1994;Wenning et al. 1998], a

general conclusion drawn was that early dementia was not a common initial presentation of the

CBS. This viewpoint changed when Grimes et al. [Grimes et al. 1999b] reported that dementia

was the most common initial presentation in a case series of patients selected based on having a

pathological diagnosis of CBD. In a retrospective review of clinical features of 13 patients with a

post-mortem diagnosis of CBD, only four patients had a diagnosis of CBS in life, while six

patients had a primary diagnosis of Alzheimer‟s disease and three were diagnosed with atypical

dementia (two with frontotemporal dementia or Pick‟s disease and one with dementia and

Parkinsonism) [Grimes et al. 1999b]. In longitudinal follow-up, three of the four cases who

presented initially with the classic perceptuomotor disorder went on to develop clinical evidence

for dementia [Grimes et al. 1999b]. In addition, 11 of 13 cases with dementia during the disease

course developed a motor disorder initially, concurrently or at a later time point and the majority

of these patients would have retrospectively met criteria for CBS underscoring the importance of

longitudinal follow-up [Grimes et al. 1999b]. The heterogeneity observed in the initial clinical

presentation and evolution over time of patients with CBD pathology likely results from the

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differences in the distribution and severity of the underlying histopathological lesions [Lang

2003]. A subsequent study also identified two patients who presented with a frontotemporal

dementia syndrome in life who subsequently were found to have a pathological diagnosis of

CBD demonstrating the overlap of these disorders [Mathuranath et al. 2000].

Several studies have attempted to clarify the nature of the underlying cognitive deficits

associated with CBS and CBD pathology. One of the earliest cognitive studies compared the

neuropsychological profile of 15 patients with a clinical diagnosis of CBS to that of 19 matched

normal controls, as well as to that of patients with PSP (n=15) or Alzheimer‟s disease (AD;

n=15) [Pillon et al. 1995]. CBS patients demonstrated a moderate degree of dementia based on

global measures of cognition used, such as the Mattis Dementia Rating Scale and Raven‟s

Progressive Matrices. They also demonstrated prominent troubles with executive dysfunction

similar to that seen in PSP, but more severe than that observed in Alzheimer‟s disease and this

was thought to be due to abnormal function of the frontal-subcortical circuit including damage to

the basal ganglia and connections with prefrontal cortical regions [Pillon et al. 1995]. Although

mild learning deficits on verbal episodic memory tasks were found in CBS and PSP, the deficits

significantly benefited from semantic cueing in contrast to that observed in AD cases, in which

both cued recognition and recall were impaired [Pillon et al. 1995]. This finding is also

consistent with impaired frontal-subcortical retrieval processes in CBS and PSP compared to

prominent hippocampal involvement of encoding and retrieval processes in AD. Similar to

patients with PSP, CBS patients demonstrated deficits in dynamic motor execution including

difficulties with control and inhibition as well as temporal organization and bimanual

coordination [Pillon et al. 1995]. These motor execution deficits were not observed in patients

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with AD. In contrast, asymmetric ideomotor apraxia was noted mainly in CBS patients reflecting

involvement of premotor and parietal regions and was not commonly observed in the PSP or AD

groups [Pillon et al. 1995].

Using the Delis-Kaplan Executive Function System, Huey et al. [Huey et al. 2009a] compared

51 patients with behavioural variant FTD and 50 patients with CBS on various standardized

measures of executive function and identified MRI correlates within each of the groups. Both

groups were more impaired on executive functions compared to their performance on an episodic

memory task – the Wechsler Memory Scale-third edition [Huey et al. 2009a]. A between group

comparison revealed that FTD patients were significantly more impaired on most executive

functions than the CBS group, except for those tasks weighted towards motor and/ or

visuospatial abilities, including the Trail Making Test and the two timed measures of the Tower

Test [Huey et al. 2009a]. Within the CBS group, atrophy on MRI in the dorsal frontal, parietal,

and temporal-parietal cortical regions in addition to the thalamus was correlated with

performance on executive tasks [Huey et al. 2009a]. This study confirms in the largest CBS

sample to date ascertained from a single site that executive dysfunction is a prominent feature

associated with CBS implicating significant frontal lobe dysfunction in this disorder. Graham et

al. [Graham et al. 2003b] reviewed the literature on cognitive dysfunction in CBS and

summarized that deficits on frontal lobe tasks such as the Wisconsin Card Sort Test, trail making

and initial letter and category fluency were invariably affected across most patients with CBS.

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Several studies of CBS have revealed that language impairment is a common cognitive feature of

this disorder. Frattali et al. [Frattali et al. 2000] studied 15 patients with a clinical diagnosis of

CBS and found that eight (53%) of these patients had a classifiable aphasia based on a

standardized language assessment using the Western Aphasia Battery. Six patients were

categorized as having an anomic aphasia, one patient had a Broca‟s aphasia, while one had a

transcortical motor aphasia. An additional patient demonstrated an apraxia of speech [Frattali et

al. 2000]. MRI scans were assessed visually and the patients with language dysfunction were

found to have more frontal, temporal and parietal cortical atrophy as well as subcortical white

matter and callosal changes [Frattali et al. 2000]. Another study followed 35 patients with CBS

longitudinally, 15 with a motor onset and 20 with a cognitive onset, and observed that 13 patients

(37%) in the cognitive onset group presented initially with a disorder of progressive aphasia

[Kertesz et al. 2000b]. Over longitudinal follow up, all but one patient in the motor onset group,

that is, 97% of the sample demonstrated a disorder of language [Kertesz et al. 2000b]. Formal

assessment of language using the Western Aphasia Battery was conducted in 21 CBS patients

and this demonstrated that patients with cognitive onset had significantly lower scores than the

motor-onset group [Kertesz et al. 2000b]. This indicated the presence of more severe forms of

aphasia in the cognitive onset group. Graham et al. [Graham et al. 2003a] also performed a

detailed assessment of language in a series of ten unselected patients with CBS and demonstrated

that eight patients (80%) had language impairment characterized by deficits in phonologic

processing and in spelling (orthographic processing). Only two of their patients demonstrated a

clinically evident non-fluent aphasia [Graham et al. 2003a]. These important early studies of

language function in CBS among others were reviewed and this has lead some authors to

conclude that presentation with a progressive apraxia of speech and/ or progressive non-fluent

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aphasia is strongly associated with the later development of a CBS and may also be predictive of

CBD pathology [Josephs and Duffy 2008].

There have been very few studies examining visuospatial functioning in CBS. Tang-Wai et al.

[Tang-Wai et al. 2003] reported two cases of patients with pathologically proven CBD, who

presented initially with a progressive focal visuospatial syndrome and then evolved into a full-

blown CBS. A clinical study of 88 patients with atypical parkinsonian syndromes using the

Visual Object and Space Perception battery, including 20 patients with multiple system atrophy,

43 with PSP, and 25 with CBS, demonstrated that only the CBS group had evidence for

significant visuospatial dysfunction that was independent of their performance on other cognitive

tasks [Bak et al. 2006]. They hypothesized that the observed visuospatial deficit reflects

dysfunction of the dorsal visual stream due to involvement of the parietal lobes by the

pathologies that can produce CBS [Bak et al. 2006].

The studies discussed in the preceding paragraphs described cognitive and neuropsychological

features of patients clinically diagnosed with CBS and only a small proportion of these patients

had pathologically confirmed CBD. We will now review the findings of a longitudinal clinical

and neuropsychological study of 15 patients with pathologically proven CBD [Murray et al.

2007]. Similar to prior studies, only six patients (40%) had a clinical diagnosis of CBS in life

whereas other primary or differential diagnoses included progressive non-fluent aphasia,

behavioural variant FTD, Alzheimer‟s disease, atypical dementia, atypical PSP, and dementia

with Lewy bodies [Murray et al. 2007]. Using a comprehensive neuropsychological battery, a

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specific cognitive profile of CBD was identified that included deficits in the performance of

gestural, language, visuospatial, executive, and social functioning with relative sparing of

episodic memory, even at the late stages of the disease. These neuropsychological deficits

correlated with burden of CBD Tau-related pathology in the frontal and parietal regions as well

as the basal ganglia with minimal involvement of the temporal lobes and hippocampi [Murray et

al. 2007].

1.4.3 Apraxia

In general terms, apraxia is “characterized by loss of the ability to execute or carry out skilled

movements and gestures, despite having the desire and the physical ability to perform them”

(http://www.ninds.nih.gov/disorders/apraxia/apraxia.htm). Apraxia is the main clinical feature

that distinguishes CBS from other parkinsonian disorders and it is observed in 100% of CBS

cases during longitudinal follow-up [Leiguarda et al. 1994;Rinne et al. 1994;Stamenova et al.

2009]. Different types of apraxia have been reported in CBS including subtypes of limb apraxia,

such as limb-kinetic apraxia, ideomotor apraxia (IMA), and ideational/conceptual apraxia.

Orofacial apraxia and apraxia of speech have also been observed. A full description of the types

of apraxia identified in CBS have been extensively reviewed elsewhere [Gross and Grossman

2008;Josephs and Duffy 2008;Leiguarda and Marsden 2000;Stamenova et al. 2009;Zadikoff and

Lang 2005]. Limb apraxia is the most common type observed in CBS and the remainder of this

section will focus on this topic.

http://www.ninds.nih.gov/disorders/apraxia/apraxia.htm

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Several models of limb apraxia have been described in the literature based on original case

studies and series, and the left parietal lobe has been implicated in most [Geschwind

1975;Goldenberg 2009;Heilman and Rothi 1993;Liepmann 1920;Roy 1996]. Although it is

beyond the scope of this thesis to describe these models in detail, highlights of several models

will be briefly reviewed. One of the earliest models postulated the existence of visual mental

images of the intended movement stored in posterior brain regions such as the parieto-occipito-

temporal junction in the dominant left hemisphere that are then transferred forward to central

sensorimotor regions for the task to be carried out [Liepmann 1920;Goldenberg 2009].

Geschwind alternatively proposed that comprehension of verbal commands to carry out a motor

task is achieved in Wernicke‟s area and then is carried forward to the sensorimotor cortex via the

arcuate fasciculus passing under the parietal lobes [Geschwind 1975;Goldenberg 2009].

Therefore, damage in the parietal region on the left can result in apraxia through disruption of

this circuit [Geschwind 1975]. A more recent neuroanatomical theory of praxis based on the

original Liepmann model suggests that „praxicons‟ or „movement formulae‟ are stored in the left

inferior parietal lobule, which then are transformed into „innervatory patterns‟ or „motor schema‟

in the premotor and supplementary motor areas, before being decoded by the primary motor

cortex to perform motor tasks both ipsilaterally and contralaterally [Heilman 1979;Heilman and

Rothi 1993;Ochipa and Gonzalez Rothi 2000]. An information-processing model of apraxia

proposes the existence of three systems: the sensory-perceptual, conceptual, and production

system [Roy 1996]. Depending on where damage occurs across these systems, specific praxis

deficits will be observed [Roy 1996]. The various pathologies that can produce the CBS localize

to the frontoparietal cortex and its subcortical connections and this is thought to be the reason

that limb apraxia is so commonly observed in CBS.

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As previously mentioned, ideomotor apraxia, limb-kinetic apraxia and, less often,

conceptual/ideational apraxia have been the main subtypes of limb apraxia studied in CBS in that

order [Gross and Grossman 2008;Stamenova et al. 2009;Zadikoff and Lang 2005]. Ideomotor

apraxia is best elicited through voluntary pantomime and/or imitation of hand gestures and tool

use and is characterized by disturbances of spatial organization, sequencing and timing of

gestural limb movements [Rothi et al. 1991]. Limb-kinetic apraxia (LKA) is defined as a loss of

hand and finger dexterity resulting in a breakdown and awkwardness of distal movements [Kleist

1907]. The definitions used for conceptual/ ideational apraxia have been more variable.

Conceptual/ideational apraxia was defined in this thesis as impairment in object/tool or action

knowledge [Stamenova et al. 2009]. However, some studies have distinguished between

conceptual and ideational apraxia with the latter being defined as a failure to sequence tasks

related to tool use correctly. This has resulted in phenomenological/ taxonomic confusion across

studies [Stamenova et al. 2009]. More research is required to better localize the regions of the

brain involved in limb apraxia associated with the CBS and to better understand the network

involved in this phenomenology.

1.5 Neuroimaging in CBS

The cognitive and physical symptoms and signs of the disorder correlate reasonably well with

the location of the maximally affected brain regions, which can often be identified in vivo using

structural neuroimaging (e.g., brain MRI) and functional neuroimaging (e.g., brain perfusion

SPECT or glucose metabolism PET).

22

1.5.1 Structural neuroimaging studies

Riley et al. [Riley et al. 1990] conducted one of the earliest clinical studies in a case series of 15

patients with CBS that examined brain computed tomography and MRI imaging. On visual

inspection of CT and/ or MRI of the brain, asymmetric atrophy worse contralateral to the most

affected side of the body was observed in eight patients, whereas six patients demonstrated

symmetric atrophy [Riley et al. 1990]. One patient did not have any notable atrophy on CT of the

brain [Riley et al. 1990]. Several years later Yamauchi and colleagues [Yamauchi et al. 1998b]

observed that, compared to controls, a group of eight CBS patients had atrophy on MRI of the

corpus callosum, which was most severe in the middle-posterior > middle-anterior > anterior >

posterior regions. The degree of callosal atrophy also was correlated with glucose metabolism as

measured by PET and the latter tended to be asymmetric [Yamauchi et al. 1998b]. Another MRI-

based study comparing 16 patients with CBS to 28 patients with PSP demonstrated that atrophy

on T1-weighted MRI images was most prominent in frontoparietal regions contralateral to the

most affected side of the body in approximately 14/16 (87.5%) of the CBS patients and was not

present in any patients with PSP, who demonstrated mainly midbrain atrophy [Soliveri et al.

1999]. This group also observed the presence of cortical and subcortical white matter signal

changes involving or underlying the atrophic region on proton density and T2-weighted images

in six (37.5%) CBS cases [Soliveri et al. 1999]. Similar findings were observed by a Japanese

group that demonstrated that the parietal, anterior middle, and inferior frontal lobes, and

paracentral regions were significantly more atrophic and tended to be asymmetric in CBS than in

PSP, whereas the brainstem was more atrophic in PSP using MRI-based hemisphere surface

display and volumetry [Taki et al. 2004]. Another study that compared 18 patients with CBS to

23

33 with PSP found that CBS cases as a group had reduced whole brain volumes, and more

selective atrophy involving the parietal lobes and the corpus callosum [Groschel et al. 2004]. The

most severe atrophy was observed in the white matter of the parietal lobes; however, in contrast

to prior studies, there was no tendency for the atrophy to be localized contralateral to the most

affected side of the body [Groschel et al. 2004]. Similar to Soliveri et al. [Soliveri et al. 1999],

midbrain atrophy also differentiated PSP from CBS [Groschel et al. 2004]. Finally, using a

discriminant function analysis in a subset of the sample with pathologically proven CBD or PSP

as well as controls, the combined volumes of the midbrain, brainstem, pons, frontal and parietal

white matter and temporal grey matter were found to differentiate the groups with high accuracy

[Groschel et al. 2004]. The first published voxel-based morphometry (VBM) study that

compared 14 patients with CBS to 15 with PSP identified that atrophy in CBS was more

prominent on the left than the right and involved bilateral premotor regions, superior parietal

lobes and the striatum whereas PSP patients had prominent atrophy involving the midline

subcortical structures including the midbrain, pons, thalamus and striatum as well as minimal

involvement of the frontal lobes [Boxer et al. 2006]. Using a voxel-wise discriminant function

analysis, they were able to correctly distinguish between CBS and PSP patients with 93%

accuracy by using the severity of atrophy in the dorsal pons, midbrain tegmentum and left frontal

eye field [Boxer et al. 2006].

There have been only a few published case series of pathologically proven CBD studied with

MRI. One study examined 17 patients with a clinical diagnosis of CBS of which six had a

pathologically confirmed diagnosis of CBD and 11 had other pathological diagnoses including

PSP, FTD, AD, and Creutzfeldt Jakob Disease [Josephs et al. 2004]. Using a semi-quantitative

24

visual assessment of pre-selected regions of interest bilaterally on MRI, they confirmed findings

of previous studies that demonstrated atrophy on T1-weighted imaging involving the posterior

frontal, superior parietal and middle corpus callosum in both groups and subcortical/

periventricular white matter changes on T2-weighted imaging [Josephs et al. 2004]. However,

there was no difference between the MRI findings in the CBD group vs. that with other

pathologies suggesting that it is the location and distribution of the pathology and not the specific

pathology itself that predicts the CBS [Josephs et al. 2004]. The same group later demonstrated

in a larger series of pathologically proven CBD patients (n=11) compared to controls that

atrophy predominated in the cortical regions bilaterally including the superior, middle and

posterior inferior frontal lobes, the posterior temporal and parietal lobes, and the superior

premotor cortex [Josephs et al. 2008]. The insular cortex and supplementary motor area also

demonstrated atrophy in CBD patients and subcortical grey matter atrophy was observed in the

globus pallidus, putamen and caudate head [Josephs et al. 2008]. There was also a small amount

of white matter atrophy identified in the posterior frontal lobes, the corpus callosum, the external

capsule and the right midbrain in the CBD group [Josephs et al. 2008].

Several recent studies have employed diffusion tensor imaging (DTI) to better characterize the

integrity of the white matter in CBS in order to follow up on prior studies that demonstrated T1-

weighted atrophy and T2-weighted hyperintensities of the white matter in this condition. Borroni

et al. [Borroni et al. 2008b] compared 20 patients with CBS to 21 normal controls using DTI

MRI and demonstrated reduced fractional anisotropy in the long frontoparietal connecting tracts,

the intraparietal associative fibers, and the corpus callosum. Reductions in fractional anisotropy

were also observed in the sensorimotor projections of the cortical hand areas [Borroni et al.

25

2008b]. Another study used tract-based statistics to study 10 patients with CBS and 10 normal

controls and found that CBS patients had higher average apparent diffusion coefficient values

and lower average fractional anisotropy values in the corticospinal tract in the most affected

hemisphere and also in the posterior trunk of the corpus callosum [Boelmans et al. 2009]. The

same group has more recently observed that higher mean diffusivity and lower fractional

anisotropy within the posterior trunk of the corpus callosum can distinguish CBS from

Parkinson‟s disease [Boelmans et al. 2010]. MRI studies to date demonstrate heterogeneity

across CBS patients in terms of the degree and localization of the cortical and subcortical grey

matter atrophy observed and also in the involvement of the white matter and this may be, in part,

responsible for the variability in clinical presentations.

1.5.2 Functional neuroimaging studies: PET and SPECT

Sawle et al. [Sawle et al. 1991] using PET to measure regional cerebral oxygen metabolism were

the first to demonstrate that patients with CBS have hypometabolism predominantly in the

posterior and superior temporal, inferior parietal, and occipital (association) cortices; frontal

association regions also demonstrated reduced metabolism although they did not achieve

statistical significance. This pattern of hypometabolism tended to be asymmetric being lower

contralateral to the most affected side of the body. Following this initial study, several other PET

studies using fluorodeoxyglucose (FDG) as the tracer demonstrated similar findings. One of the

first studies using FDG-PET also demonstrated asymmetric uptake of FDG in five patients with

CBS compared to PD patients and normal controls involving the thalamus, hippocampus and

inferior parietal lobule [Eidelberg et al. 1991]. Asymmetry of parietal lobe metabolic reduction

26

of 5% or more was found in the CBS group whereas the PD and normal control groups

manifested less than 5% reductions [Eidelberg et al. 1991]. Another study demonstrated

significant reductions in FDG uptake in frontal, temporal, sensorimotor and parietal association

cortices in five CBS patients compared to controls and additionally showed involvement of

subcortical structures including the caudate and lentiform nuclei and thalami; reductions were

noted predominantly contralateral to the most affected side of the body [Blin et al. 1992]. Similar

findings were observed in a Japanese study with asymmetric involvement of the parietal cortex,

including the primary sensorimotor and lateral parietal regions, the caudate, putamen and

thalamus contralateral to the most severely affected side in the CBS group [Nagasawa et al.

1996]. Another FDG-PET study compared nine patients with CBS to nine with PSP and

observed that CBS patients had significant metabolic reductions involving the inferior parietal,

lateral temporal, sensorimotor cortices as well as the striatum that were worse contralateral to the

most affected side of the body [Nagahama et al. 1997]. Symmetrical hypometabolism involving

the frontal and parietal lobes and thalami has also been demonstrated in some cases even though

asymmetry was present on the clinical exam suggesting the presence of heterogeneity in the

imaging findings [Taniwaki et al. 1998].

The previous FDG-PET studies described had sample sizes that were small, typically less than

10 CBS patients, and employed mainly region of interest approaches. Garraux et al. [Garraux et

al. 2000] conducted a voxel-based analysis of 22 patients with CBS, 21 PSP patients, and 46

healthy controls. They largely confirmed findings of earlier studies demonstrating asymmetric

metabolic reductions involving the thalamus, putamen, supplementary motor and lateral

premotor areas, the dorsolateral prefrontal cortex and the anterior part of the inferior parietal

27

lobule, which includes the intraparietal sulcus [Garraux et al. 2000]. PSP patients could be

differentiated from CBS patients based on metabolic reductions involving the midbrain, anterior

cingulate and orbitofrontal regions, whereas CBS patients had reductions in posterior frontal

regions including the supplementary motor area as well as the inferior parietal lobule in contrast

to PSP [Garraux et al. 2000]. These FDG-PET findings were also observed in several smaller

case series using voxel-based approaches [Hosaka et al. 2002;Juh et al. 2005;Klaffke et al.

2006]. Finally, an FDG-PET study using a visual assessment method as opposed to semi-

quantitative or quantitative techniques demonstrated the clinical utility of visual assessment in

detecting asymmetric hypometabolism involving the peri-rolandic area, striatum and thalamus

[Coulier et al. 2003].

Other PET tracers have been used to characterize patients with CBS. Sawle et al. [Sawle et al.

1991] were the first to report reductions in basal ganglia uptake of 18

F-6-fluorodopa (18

F-dopa) in

CBS. They found that uptake of 18

F-dopa was most reduced in the caudate contralateral to the

most affected side of the body in all patients [Sawle et al. 1991]. Putaminal reduction of 18

F-dopa

uptake was also most prominent contralateral to the most affected side of the body in all but one

patient who demonstrated bilateral reductions [Sawle et al. 1991]. This study provided the first in

vivo evidence of nigrostriatal dopaminergic denervation, that is, reduction in the number of

functioning nigrostriatal dopaminergic neurons, in CBS. Other studies have confirmed the

finding of reduced 8F-dopa uptake in CBS [Laureys et al. 1999;Nagasawa et al. 1996].

28

Perfusion SPECT, using a variety of tracers, has also been used to image series of patients with

CBS and largely show similar cortical and subcortical involvement as that observed with FDG-

PET metabolic studies. Markus et al. [Markus et al. 1995] were the first to demonstrate that, in

eight CBS patients compared to controls, markedly reduced perfusion on

Hexamethylpropyleneamine Oxime (HMPAO)-SPECT was present bilaterally, but worse

contralateral to the most affected side of the body in subcortical regions including the caudate,

putamen and thalamus, and in cortical regions including the posterior frontal cortex and in all

divisions of the parietal cortex (anterior, superior, posterior, and inferior). In comparison to PD

patients, perfusion was also reduced in the most affected hemisphere in the thalamus, posterior

frontal, as well as the anterior and inferior parietal cortices [Markus et al. 1995]. Using N-

isopropyl-p[123

I]iodoamphetamine (IMP) SPECT in nine patients with CBS, limb apraxia was

the most common clinical finding and hypoperfusion contralateral to the most affected limb was

most prominent in the sensorimotor cortex and posterior parietal cortex [Okuda et al. 1999]. In a

small IMP-SPECT study, asymmetric reductions in regional perfusion were observed in the

frontoparietal regions including the inferior prefrontal, posterior parietal and sensorimotor

cortices in CBS, but not in PSP [Okuda et al. 2000b]. Medial prefrontal perfusion reductions,

however, were seen in both disorders [Okuda et al. 2000b]. Another SPECT study using

Technetium-99m ethyl cysteinate dimer (99m

Tc-ECD SPECT) as a tracer compared nine patients

with CBS to nine with PSP and found that asymmetrical hypoperfusion in the frontal, parietal,

and temporal cortex and basal ganglia, as well as, to a lesser degree, the occipital cortex

differentiated CBS from PSP [Zhang et al. 2001]. The first ECD-SPECT study of CBS to

employ an unbiased, whole brain, voxel-wise analytical technique (statistical parametric

mapping; SPM) demonstrated more widespread brain hypoperfusion than previously observed by

29

region of interest studies, including the frontal, parietal and temporal cortices, as well as the

basal ganglia, thalamus and pontocerebellar regions [Hossain et al. 2003]. However, to our

knowledge, this study did not correct for multiple testing using more modern techniques such as

correcting for the family-wise error [Hossain et al. 2003]. Using HMPAO SPECT and a factor

discriminant analysis applied to regions of interest, Kreisler et al. [Kreisler et al. 2005] identified

seven variables, including the more affected temporoinsular region, the more affected medial

frontal region, the less affected and more affected lateral frontal regions, the less affected

temporoparietal region, and the lateral frontal and parietal asymmetry indices, that correctly

classified patients as having CBS or PD with 100% and 95% accuracy, respectively. A more

recent clinical study that performed both MRI and ECD-SPECT in 16 patients with CBS that

were read by two neuroradiologists blinded to the diagnosis and clinical information found that

SPECT was more sensitive than MRI in detecting asymmetries [Koyama et al. 2007].

Frisoni et al. [Frisoni et al. 1995] were the first to report reductions in uptake of the SPECT

tracer 123

I-iodobenzamide (IBZM; binds to post-synaptic dopamine D2 receptors) in the right

basal ganglia in a case of CBS with prominent left-sided motor involvement and proposed that

reduction in the number of D2 receptors may account for the lack of levodopa responsiveness

seen in CBS. This finding was largely refuted by two papers showing that IBZM uptake on

SPECT was mostly normal in most patients with CBS as combined results across the studies

demonstrated that only three of 17 patients had reductions in IBZM uptake in the basal ganglia

[Klaffke et al. 2006;Plotkin et al. 2005].

30

Finally, several studies have demonstrated the value of using dopamine transporter (DAT)-

SPECT imaging in CBS and other parkinsonian disorders. One of the earliest studies used the

DAT-SPECT tracer, 2-β-carbomethoxy-3-β-(4-iodophenyl)-tropane (β-CIT), to compare 18

patients with multiple system atrophy (MSA), eight with PSP, four with CBS and 48 with PD in

terms of their striatal binding [Pirker et al. 2000]. They found that all patient groups

demonstrated reduced β-CIT striatal binding compared to controls and this tended to be

asymmetric in the PD and CBS groups [Pirker et al. 2000]. Another study by the same group

followed longitudinal changes in striatal β-CIT striatal dopamine transporter binding over time in

36 patients with PD, 10 patients with atypical parkinsonian syndromes including three CBS

cases, and nine patients with essential tremor [Pirker et al. 2002]. They found that the uptake of

β-CIT was reduced in PD and the atypical parkinsonian syndromes, but not in essential tremor

compared to controls [Pirker et al. 2002]. They also observed that the β-CIT striatal uptake

declined more rapidly in those with atypical parkinsonian syndromes compared to PD [Pirker et

al. 2002]. These initial findings of reduced β-CIT striatal uptake in CBS were supported by other

studies [Klaffke et al. 2006;Plotkin et al. 2005]. A single case of pathologically proven CBD did

not demonstrate any reductions in β-CIT striatal uptake visually after four years from disease

onset refuting prior studies [O'Sullivan et al. 2008]. The largest and most recent study using β-

CIT SPECT in 36 patients with CBS, 37 patients with PD and 24 healthy controls demonstrated

that striatal binding reduction was variable across CBS cases and more uniformly reduced with

more hemispheric asymmetry than that observed in PD [Cilia et al. 2011]. There was also no

correlation between striatal β-CIT and clinical features of the disease including severity [Cilia et

al. 2011]. Four CBS patients had normal striatal uptake compared to controls, while four had

strictly unilateral uptake despite all showing bilateral extrapyramidal signs [Cilia et al. 2011].

31

Many of the neuroimaging studies described employed small sample sizes and only a few of the

studies provided a detailed clinical and neuropsychological characterization of their CBS

subjects. Therefore, further neuroimaging studies in a prospective sample that has been well-

characterized clinically are required in order to further understand the heterogeneity observed in

the presentation of CBS and how this correlates with neuroimaging features.

1.6 Pathological Heterogeneity in CBS

CBS is not only heterogeneous in its clinical presentation and in its neuroimaging as previously

described, but there is also substantial pathological heterogeneity that can produce the syndrome.

Ball and colleagues [Ball et al. 1993] described a case of CBS presenting with an alien left limb,

memory loss, cortical myoclonus and bilateral parietal dysfunction with a pathological diagnosis

of AD. Lang and colleagues [Lang et al. 1994] described a case of pathologically proven

“parietal” Pick‟s disease that presented as corticobasal syndrome. Since these two early studies,

there have been several case series published that have demonstrated similar pathological

heterogeneity underlying the corticobasal syndrome. This section reviews these case series and

provides images that demonstrate the variety of pathologies that have been associated with the

CBS.

32

Figure 4. Macroscopic brain specimen showing left frontal > temporal atrophy of Pick’s disease

A study of 11 cases with pathologically confirmed CBD identified overlapping pathology of one

or more of AD, PD, hippocampal sclerosis and PSP in six cases (54%) suggesting that mixed

pathology can be associated with CBD [Schneider et al. 1997]. The cases with mixed CBD and

other pathologies all presented with early memory loss in comparison to those with CBD alone

and the authors proposed that the mixed pathology may account for the variable clinical

presentations observed in CBS [Schneider et al. 1997]. Another study presented clinical

vignettes of several different pathologically confirmed cases of neurodegenerative disease,

including ten with CBD, to six neurologists who then had to make a clinical diagnosis based on

the information that was provided [Litvan et al. 1997]. The accuracy of the clinical diagnosis

33

was then determined and it was observed that the specificity was high at 99.6% meaning that less

than 1% of patients without CBD were diagnosed as having it [Litvan et al. 1997]. However, the

sensitivity was low at 48.3% meaning that only about 50% of patients were accurately diagnosed

with CBD in life [Litvan et al. 1997]. Boeve and colleagues [Boeve et al. 1999] identified 13

Figure 5. Microscopic Lewy body pathology showing Lewy bodies, cytoplasmic stippling, neuropil grains and

Lewy neurites immunostained by antibodies to alpha-synuclein

patients from the Mayo clinic records with a diagnosis of CBS who also had a neuropathological

examination at autopsy and demonstrated that seven cases had a pathological diagnosis of CBD

(53.8%) while six had other diagnoses (46.2%; two with AD, one with Creutzfeldt-Jakob disease

(CJD), one with PSP, one with Pick‟s disease and one with non-specific histopathology) [Boeve

et al. 1999]. Frontotemporal lobar degeneration (FTLD) with motor neuron disease-like

34

inclusions, today known as FTLD-Ubiquitin (U)/TAR DNA-binding protein 43 (TDP43) with

motor neuron disease (MND), has also been documented to produce the CBS [Grimes et al.

1999a]. CJD has also been observed to cause the CBS [Kleiner-Fisman et al. 2004], as has

agyrophilic grain inclusion disease [Rippon et al. 2005]. Another case report identified that

bilateral strokes involving the frontoparietotemporal and occipital regions, worse on the right,

due to ipsilateral occlusion of the distal internal carotid and middle cerebral arteries and severe

stenosis of the left middle cerebral artery was associated with a corticobasal syndrome [Kim et

al. 2009].

An important pathological study that screened all archival data from the Queen Square Brain

Bank over a 20 year period identified 19 pathologically confirmed cases of CBD and 21

clinically diagnosed cases of CBS [Ling et al. 2010]. Of the pathologically confirmed cases, only

five were accurately diagnosed as having CBD in life yielding a sensitivity of 26.3% [Ling et al.

2010]. Alternative clinical diagnoses were eight cases with PSP, two with PD, two with FTD,

one with spastic quadriparesis with myoclonus of unknown etiology, and one incidental case

with Tourette‟s syndrome who died before symptoms of CBS manifested [Ling et al. 2010].

From the clinical standpoint, of the 21 cases diagnosed as having CBS in life, only five had

confirmed CBD pathology, while the rest had alternative pathological diagnoses including six

with PSP, five with AD, two with PD, one with frontotemporal lobar degeneration-

Ubiquitin/TDP43 (FTLD-U/TDP43) with MND, one with FTLD-U/TDP43 subtype 2, and one

with dementia lacking distinctive histopathology resulting in a positive predictive value of 23.8%

[Ling et al. 2010]. Finally, a larger study of 18 cases with pathologically proven CBD and 40

35

cases of CBS due to other histopathologies will now be discussed [Lee et al. 2011]. The

pathologically confirmed cases of CBD presented with four distinct clinical syndromes including

Figure 6. Microscopic pathology of CBD stained with Gallyas demonstrating (A) oligodendroglial coils, (B)

neuronal pre-tangles in the precentral region, (C) ballooned neurons, and (D) astrocytic plaques in the basal

ganglia

executive-motor (n=7; 38.9%), progressive non-fluent aphasia (n=5; 27.8%), behavioural variant

FTD (n=5; 27.8%), and posterior cortical atrophy (n=1; 5.5%) [Lee et al. 2011]. Conversely,

those presenting with a CBS had various underlying pathologies including AD (n=9; 22.5%),

CBD (n=14; 35%), PSP (n=5; 12.5%), FTLD-U/TDP43 (n=5; 12.5%), mixed pathologies (n=5

comprised of two PSP+AD, one CBD+AD, and one FTLD-U/TDP43+AD; 12.5%), Pick‟s

disease (n=1; 2.5%), and one with multiple system tauopathy without agyrophilia (n=1; 2.5%)

[Lee et al. 2011]. As can be seen from this review of prior clinicopathological studies of CBS

36

Figure 7. Microscopic agyrophilic grain disease pathology showing (A) branched astrocytes in the amygdale,

and (B) agyrophilic grains and coiled bodies in the prosubiculum

and CBD, the rate at which CBD pathology is predicted based on having a CBS is highly

variable and in general is low. The variability is likely explained by the small samples sizes used

in even the larger studies. Future studies are required that follow patients longitudinally to death

and characterize them with multiple modalities including clinical examination,

neuropsychological and neuroimaging with subsequent pathological analyses as only this type of

study will improve our ability to predict the specific pathological diagnosis underlying the CBS

in life.

Figure 8. Microscopic Alzheimer’s pathology showing (A) astrocytic plaques in frontal regions, and (B)

neurofibrillary tangles in the CA1 region of the hippocampus

37

1.7 Genetics of CBS and CBD

Genetic analysis of complex syndromes, such as CBS, may be complicated by many factors such

as incomplete penetrance, multiple disease susceptibility loci, gene-environment interactions and

diagnostic uncertainties [Nothen et al. 1993]. The latter is particularly important given the

significant pathological heterogeneity underlying the CBS described in the preceding section.

Two main strategies have been utilized for the genetic study of complex illnesses, such as CBS:

a) linkage or candidate gene studies involving families or affected pairs of relatives, and b)

association studies using candidate genes or genome wide approaches in unrelated cases and

controls. Traditional family-linkage studies follow the segregation of marker alleles in pedigrees

that are multiply affected with the disease phenotype of interest. A model is then proposed to

explain the inheritance pattern of phenotypes and genotypes in the pedigree [Lander and Schork

1994]. Although this is the method of choice for simple Mendelian traits, linkage analysis of

complex traits has limited power in identifying disease susceptibility loci, that is, estimating the

large number of unknown parameters required to model complex traits is extremely difficult [Ott

1990]. A genetic association study design does not require specification of a genetic model and

therefore overcomes many of the limitations inherent in the linkage-based familial approaches

[Crowe 1993;Kidd 1993]. Risch & Merikangas [Risch and Merikangas 1996] have suggested

that association analyses have far greater power than linkage analyses to identify genes involved

in complex genetic diseases. Both approaches have been applied to elucidate genetic factors

contributing to the etiology of CBD.

38

From a genetic epidemiologic perspective, CBD is mainly a sporadic disorder with very few

reported familial cases [Mahapatra et al. 2004]. In sporadic cases, genetic association studies

have identified a particular haplotype that spans the MAPT gene among several other loci, as

being associated with CBD and PSP pathology. Please refer to Caffrey and Wade-Martins

[Caffrey and Wade-Martins 2007] for a comprehensive review. The MAPT gene is localized to

chromosome 17q21 and encodes for the Microtubule-Associated Protein Tau (MAPT)

[Andreadis et al. 1992]. Tau is highly expressed within both central and peripheral nervous

system neurons where it is involved in the assembly and stabilization of microtubules, signal

transduction and maintaining neuronal polarity [Shahani and Brandt 2002]. Hyperphosphorylated

Tau can aggregate in neurons producing pathological Tau inclusions called neurofibrillary

tangles, which are present in several neurodegenerative diseases including AD, PSP, CBD,

agyrophilic grains, FTD Parkinsonism-17 (FTDP-17), and Pick‟s disease [Caffrey and Wade-

Martins 2007].

MAPT is comprised of 16 exons and alternative splicing of exons 2, 3 and 10 yields six mRNA

transcripts that are translated into unique protein isoforms [Goedert et al. 1988;Goedert et al.

1989]. Exons 9 through 12 of MAPT encode for imperfect repeat sequences that code for

microtubule-binding domains and thus play an important role in the main function of the protein

[Caffrey and Wade-Martins 2007]. When exon 10 is spliced out, three repeat (3R) sequences are

generated, whereas the presence of exon 10 results in the generation of four repeat (4R)

sequences [Goedert et al. 1988;Goedert et al. 1989]. The major tangle isoform observed in CBD

is comprised of 4R Tau [Caffrey and Wade-Martins 2007]. MAPT is located within the largest

known block of linkage disequilibrium in the human genome that spans approximately 1.8

39

megabases (Mb) [Caffrey and Wade-Martins 2007]. Two major haplotypes, H1 and H2, have

been defined based on tagging with eight single nucleotide polymorphisms (SNPs; inherited with

H1 haplotype) and a 238 base pair (bp) deletion (inherited with rarer H2 haplotype). Two early

studies demonstrated that the H1 haplotype is over-represented in sporadic CBD cases compared

to controls [Di Maria E. et al. 2000;Houlden et al. 2001].

Figure 9. Schematic representation of the MAPT genomic region and 3-repeat and 4-repeat Tau transcripts.

Adapted from Caffrey and Wade-Martins [Caffrey and Wade-Martins 2007].

In the rare event that CBS and/or CBD pathology are observed to segregate in a family, other

members are typically affected with FTD and/or PSP demonstrating overlap in these conditions

[Boeve et al. 2002;Brown et al. 1996;Brown et al. 1998;Bugiani et al. 1999;Gallien et al.

1998;Tuite et al. 2005;Casseron et al. 2005;Uchihara and Nakayama 2006]. Brown et al. [Brown

et al. 1996] described two families in which a progressive dementia segregated in 15 affected

individuals. Ten individuals were clinically studied and the main presenting features were that of

40

personality change or memory loss with invariable progression into a frontal dementia [Brown et

al. 1996]. Additional features observed were aphasia, limb clumsiness, parkinsonism and gait

imbalance [Brown et al. 1996]. Pathological examination in two individuals revealed swollen

achromatic cortical neurons and corticobasal inclusion bodies in the basal ganglia [Brown et al.

1996]. Patients had features of frontotemporal dementia and/or CBS and the pathology most

closely resembled CBD [Brown et al. 1996]. A similar family was described by the same group

with one member clinically having CBS and a sibling having FTD, while the mother presented

with an early onset dementia with features of a movement disorder [Brown et al. 1998]. The

pathological diagnosis of the individual with CBS was dementia lacking distinctive

histopathological features confirming heterogeneity even in familial cases of the syndrome

[Brown et al. 1998]. Pathological findings of non-distinctive histopathology has also been found

in another study of a kindred that included a patient with CBS and that also segregated FTD in

other individuals [Boeve et al. 2002]. An Italian group identified a family with two afflicted

members, the father presenting as FTD and the son presenting as CBS, with the etiological cause

being a Proline301Serine (P301S) mutation in exon 10 of MAPT that lead to extensive

filamentous hyperphosphorylated Tau pathology [Bugiani et al. 1999]. A Japanese group

identified three siblings, all of whom presented with parkinsonism and frontal dementia, with

typical CBD pathology [Uchihara and Nakayama 2006]. Tuite et al. [Tuite et al. 2005] identified

a consanguinous family with members having clinical diagnoses of PSP and CBS. In two

members who presented as CBS, one had confirmed CBD pathology while the other

demonstrated PSP pathology demonstrating the overlap at both the clinical and pathological level

[Tuite et al. 2005]. Interestingly, no MAPT mutations were identified and only the H1/H1

haplotype was found in the four affected individuals studied [Tuite et al. 2005]. CBS has also

41

been associated with a leucine-rich repeat kinase 2 mutation [Chen-Plotkin et al. 2008]. Given

the pathologic and genetic heterogeneity observed in CBS, future genetic studies of families with

this syndrome and association studies of unrelated individuals are required to identify other

causative genes and/ or genetic risk factors that predispose to this syndrome.

42

Figure 10. (A) H1 and H2 linkage disequilibrium blocks showing a 900 kb region of inversion, and (B) sub-

structure of the MAPT gene and associated H1 and H2 haplotypes. Adapted from Caffrey and Wade-Martins

[Caffrey and Wade-Martins 2007].

1.8 Synopsis and Overall Research Objective

Despite the rarity of CBS compared to other neurodegenerative disorders such as AD and PD, it

represents an important neurodegenerative syndrome to study given the substantial heterogeneity

that is observed in its initial presentation and evolution over time. Understanding this

heterogeneity in CBS may facilitate our understanding of heterogeneity in the more common

neurodegenerations. From a clinical perspective, it is unclear why some CBS patients present

with an early motor syndrome while others present initially with mainly symptoms of dementia.

Comparing these different presentations of CBS in terms of clinical, neuropsychological and

neuroimaging features may help to shed light on the brain regions involved in determining the

type of symptom onset in CBS and this may help to determine which patients presenting with an

early dementia would be at risk of evolving into a CBS. Additionally, there have been few CBS

samples that have been extensively characterized to allow for brain-behaviour correlations using

structural and functional neuroimaging, and this remains an important line of investigation to

understand the localization of some of the observed phenomenology in the brain. Novel genetic

studies are required in order to elucidate additional genes that can cause or increase risk for the

CBS. Finally, more work needs to be done in understanding how clinical and neuroimaging

features map on to the various pathologies that can underlie the CBS, as this will provide insight

into clinicopathological correlations, which may help in the prediction of underlying in vivo

pathological state. Therefore, the overall objective of this thesis is to characterize a prospective

sample of CBS patients in terms of the heterogeneity observed across clinical,

neuropsychological, and neuroimaging features and, in a subset of the sample, to describe

43

genetic and pathological features and how these relate to the clinical phenotype and

neuroimaging findings.

1.8.1 Specific Objectives

The specific objectives of this thesis and related hypotheses are as follows:

A) Objective 1: To characterize the genetic and pathological heterogeneity observed in a

family segregating corticobasal syndrome.

Hypothesis 1: Affected patients will harbor a mutation in one of the genes known to

cause diseases occurring along the spectrum of frontotemporal dementia, including CBS,

and will have associated pathological features that are typical of the identified genetic

mutation.

B) Objective 2: To characterize members of a family that segregate a novel mutation in the

progranulin gene (PGRN) associated with FTD spectrum disorders, including CBS, and

to contrast the heterogeneity observed in their clinical presentation, neuropsychological

testing, and neuroimaging findings.

Hypothesis 2: There will be significant heterogeneity in clinical, neuropsychological, and

neuroimaging features among patients with the same PGRN mutation and this will be

dependent on the hemisphere and lobar region most prominently affected in the early

stages of the disease.

C) Objective 3: To identify brain SPECT perfusion and neuropsychological correlates of

severity of ideomotor apraxia in CBS.

44

Hypothesis 3A: Compared to controls, CBS patients will demonstrate reduced perfusion

on SPECT in an asymmetrical fashion in frontoparietotemporal cortical and subcortical

regions.

Hypothesis 3B: Hypoperfusion within the left frontoparietal network will correlate with

severity of ideomotor apraxia in CBS.

Hypothesis 3C: Patients with more severe apraxia will demonstrate more impairment on

language-based measures.

D) Objective 4: To describe the initial neuropsychological and neuropsychiatric, MRI, and

pathological features of a prospective sample of CBS patients.

Hypothesis 4A: Compared to controls, CBS patients will demonstrate reduced

performance globally on neuropsychological testing with worse performance on

measures assessing executive, visuospatial, language and praxis functions.

Hypothesis 4B: Compared to controls, asymmetric atrophy on MRI contralateral to the

most affected side of the body will be observed.

Hypothesis 4C: In a subset of the CBS sample that came to autopsy, underlying

neuropathological diagnoses will be heterogeneous.

Hypothesis 4D: Atrophy and white matter hyperintensities on MRI in vivo will be

associated with the underlying neuropathology.

E) Objective 5: To compare the clinical, neuropsychological, MRI, and SPECT features of

CBS presenting with early dementia versus those presenting with early motor features.

45

Hypothesis 5A: The CBS group with early dementia will be more likely to have their

right side of the body affected by motor signs, have more profound language deficits, and

have hemispheric atrophy and reduced perfusion in left frontotemporal regions.

Hypothesis 5B: The CBS group with early motor features will be more likely to have

their left side of the body affected by motor signs, and have hemispheric atrophy and

hypoperfusion on SPECT that is more pronounced on the right.

1.9 Description of Chapters

1.9.1 Chapter 2: Novel splicing mutation in the progranulin gene causing familial

corticobasal syndrome

This study was the first to report that mutations in the PGRN gene, discovered in 2006 as a major

cause of frontotemporal dementia, can also cause familial corticobasal syndrome. It provides a

detailed account of two family members afflicted with corticobasal syndrome and characterizes

one of the family members from the clinical, neuropsychological, neuropsychiatric and

neuroimaging perspective. The other sibling is characterized from the pathological standpoint as

having underlying FTLD-U pathology, which, shortly after this publication was accepted, was

found to be a marker of TDP43 pathology [Neumann et al. 2006]. This study extends the

literature on genetic and phenotypic heterogeneity associated with FTD and set the stage for

several follow-up papers confirming our initial findings.

46

1.9.2 Chapter 3: Intra-Familial Clinical Heterogeneity due to FTLD-U with TDP43

Proteinopathy Caused by a Novel Deletion in Progranulin Gene (PGRN)

This paper identifies a novel mutation in the PGRN gene that caused neurodegenerative

presentations in a kindred originally from Poland and characterizes two affected brothers from

the clinical, neuropsychological, and neuroimaging perspective comparing important differences

in presentation and how these correlate with heterogeneous neuroimaging findings between

them. One of the brothers, who initially presented with symptoms of progressive non-fluent

aphasia (PNFA) and then evolved into CBS, is studied from the pathological perspective

demonstrating the expected FTLD-U/ TDP43 pathology. This study extends on the literature

demonstrating allelic and phenotypic heterogeneity in FTD and proposes molecular mechanisms,

which likely underly some of this heterogeneity.

1.9.3 Chapter 4: Ideomotor Apraxia in Corticobasal Syndrome: Brain Perfusion and

Neuropsychological Correlates

This paper replicates findings from previous studies showing that perfusion reductions on

SPECT occur in frontoparietotemporal regions in CBS compared to controls and is the first to

identify that severity of ideomotor apraxia in CBS correlates strongly with reduced perfusion in

the left inferior parietal lobule in patients afflicted with this syndrome. It is the largest SPECT

study of CBS that attempts to understand the neuroanatomical correlates of ideomotor apraxia

and also identifies that several other posterior cognitive functions are more impaired in the CBS

group with significant apraxia compared to those without this feature. The study is one of the

first to provide a comprehensive discussion of limitations in the field of apraxia research and

47

identifies that many of the limitations originate from variable definitions that are currently

applied to the different types of apraxia.

1.9.4 Chapter 5: Clinical, neuropsychological, MRI and SPECT characterization of a

prospective sample of patients with corticobasal syndrome

This paper provides a comprehensive and multi-modal assessment of a prospective sample of

CBS patients using clinical and neuropsychological assessments, MRI and brain SPECT

neuroimaging. It then compares a subgroup of CBS patients presenting with early dementia to

one presenting with early motor features identifying a tendency for the early dementia group to

have symptoms involving the right side of the body and to have more severe language

disturbances whereas the early motor group has symptoms prominently involving the left side of

their body. A subset of the patients came to autopsy and heterogeneity in pathological diagnoses

was observed. The burden and location of the pathology mostly correlated with neuroimaging

features irrespective of the specific underlying pathological diagnoses.

48

2.0 Novel splicing mutation in the progranulin gene causing

familial corticobasal syndrome

Mario Masellis,* Parastoo Momeni,

* Wendy Meschino, Reid Heffner Jr., Christine Sato, Yan

Liang, Peter St George-Hyslop, John Hardy, Juan Bilbao, Sandra Black, and Ekaterina Rogaeva

As published in: Brain (2006); 129: 3115-3123

Mario Masellis extracted the clinical information on all family members, interpreted and


responsible for writing the manuscript. Sequencing and genotyping was performed by Parastoo

Momeni and Ekaterina Rogaeva. Pathological analysis was done by Reid Heffner Jr. and Juan

Bilbao.

* These authors contributed equally to the work as co-first authors

49

2.1 SUMMARY

Corticobasal Syndrome (CBS) is a rare cognitive and movement disorder characterized by

asymmetric rigidity, apraxia, alien-limb phenomenon, cortical sensory loss, myoclonus, focal

dystonia, and dementia. It occurs along the clinical spectrum of Frontotemporal Lobar

Degeneration (FTLD), which has recently been shown to segregate with truncating mutations in

progranulin (PGRN), a multifunctional growth factor thought to promote neuronal survival. This

study identifies a novel splice donor site mutation in the PGRN gene (IVS7+1G>A) that

segregates with CBS in a Canadian family of Chinese origin. We confirmed the absence of the

mutant PGRN allele in the RT-PCR product which supports the model of haploinsufficiency for

PGRN-linked disease. This report of mutation in the PGRN gene in CBS extends the evidence

for genetic and phenotypic heterogeneity in FTLD spectrum disorders.

Keywords: Corticobasal Syndrome; Frontotemporal Lobar Degeneration; progranulin; gene;

mutation

Abbreviations: CBS = Corticobasal Syndrome; CBD = Corticobasal Degeneration; FTD =

Frontotemporal Dementia; MAPT = microtubule-associated protein tau; FTDP-17 = FTD with

parkinsonism linked to chromosome 17; PSP = Progressive Supranuclear Palsy; MMSE = Mini-

Mental Status Examination; PGRN = progranulin; MND = Motor Neuron Disease; CHMP2B =

Chromatin-modifying protein 2B; MRI = Magnetic Resonance Imaging; SPECT = Single Photon

Emission Computed Tomography; FTLD = Frontotemporal Lobar Degeneration; LFB = luxol

fast blue

50

2.2 INTRODUCTION

In 1967, Rebeiz and colleagues [Rebeiz et al. 1967] described three cases of a progressive,

perceptuo-motor disorder characterized by an asymmetric akinetic-rigid syndrome and apraxia.

They termed the disorder “corticodentatonigral degeneration with neuronal achromasia” based

on identified pathological features. Since then, a variety of terms have been applied to this

enigmatic disorder of interest to cognitive and movement disorder neurologists worldwide

including corticonigral degeneration with neuronal achromasia, cortical degeneration with

swollen chromatolytic neurons, cortical basal ganglionic, corticobasal ganglionic, and the most

common designation, corticobasal degeneration (CBD) [Mahapatra et al. 2004]. This

terminology has caused considerable nosological confusion over the years presumably because

some terms refer to the underlying pathological changes, while others refer to the neural

substrates causing the recognized clinical syndrome.

To add to this nosological uncertainty, extensive research has demonstrated significant clinical

and pathological heterogeneity in CBD [Boeve et al. 2003;Lang 2003]. Specifically, cases

presenting with the “classical” clinical syndrome of CBD often have alternative pathologies (i.e.,

not CBD) underlying the clinical manifestations such as Progressive Supranuclear Palsy (PSP),

Frontotemporal Dementia (FTD), Alzheimer‟s Disease (AD), Dementia with Lewy Bodies

(DLB), and Creutzfeldt-Jacob Disease (CJD). Conversely, pathologically-confirmed cases of

CBD [Dickson et al. 2002] may present with a variety of clinical phenotypes in addition to

“classical” CBD including Primary Progressive Aphasia (PPA) and Frontotemporal Dementia

(FTD). As a result, it has been suggested that the term Corticobasal Syndrome (CBS) be applied

51

to clinically-diagnosed cases presenting with the “classical” features of asymmetric rigidity,

apraxia, alien-limb phenomenon, cortical sensory loss, myoclonus, and focal dystonia [Boeve et

al. 2003;Kertesz et al. 2000a;Lang 2003;Litvan et al. 2003]. Herein, we use the term

Corticobasal Syndrome (CBS) to refer to clinically diagnosed cases without proof of typical

CBD pathology conforming to the clinical diagnostic criteria [Boeve et al. 2003]. Included in

this syndromic definition are patients presenting with early dementia, for which there is evidence

suggesting this to be the most common initial presentation [Bergeron et al. 1998;Grimes et al.

1999b;Mathuranath et al. 2000]. The cognitive symptoms and underlying pathologies of CBS

have many overlapping features with those of FTD prompting current nosological classification

to include CBS as part of the spectrum of FTD [Josephs et al. 2006;Kertesz et al. 2000b;Neary et

al. 1998]. Similar to CBS, several terms have been applied to describe this heterogeneous

disorder including FTD/Pick Complex [Kertesz 2003], Frontotemporal Lobar Degeneration

[Neary et al. 1998], Pick‟s Disease [Pick 1892], and FTD [The Lund and Manchester Groups

1994]. We have adopted the term Frontotemporal Lobar Degeneration (FTLD) in this paper.

FTLD encompasses a wide spectrum of clinical entities ranging from FTD, Primary Progressive

Aphasia, Semantic Dementia, CBS, PSP, FTD-Motor Neuron Disease (FTD-MND), and FTD

with Parkinsonism linked to chromosome 17 (FTDP-17) [Kertesz 2003;Kertesz 2005]. It

represents a group of primary degenerative dementias with predominant frontal and/or temporal

lobe symptoms (e.g. decline in social and personal behavior, apraxia, stereotyped behavior,

hyperorality and aphasia) [Kertesz 2005] and consensus diagnostic and neuropathological criteria

have been proposed [McKhann et al. 2001;Neary et al. 1998]. The neuropathological

characteristics of FTLD include variable frontal, temporal, and basal ganglia atrophy with

52

neuronal loss and gliosis (with tau or ubiquitinated inclusions). The deposition and abnormal

processing of tau encoded by the gene named microtubule-associated protein tau (MAPT) play an

important role in the development of several forms of FTLD, including CBS [Goedert et al.

2000;Hutton 2001;McKhann et al. 2001]. However, up to 60% of FTLD cases lack tau-positive

neuronal inclusions, primarily displaying a microvacuolization of the superficial neuropil in the

cortex (often with ubiquitin-positive inclusions in cortical neurons) [Ince and Morris 2006;Ince

and Morris 2006;Kertesz et al. 2000a].

FTLD is a genetically complex disorder with at least three known causal genes. The aberrant

splicing mutation in Chromatin-modifying protein 2B (CHMP2B) is responsible for autosomal

dominant FTLD in a large Danish family [Skibinski et al. 2005]. However, the CHMP2B is not a

common cause of FTLD since several large series of FTLD patients failed to detect any

CHMP2B mutations [Cannon et al. 2006;Momeni et al. 2006]. Many of the autosomal dominant

FTDP-17 families are explained by mutations in the MAPT gene [Hutton et al. 1998;Poorkaj et

al. 1998;Spillantini et al. 1998]. However, in several FTLD families linked to chromosome

17q21, MAPT mutations were excluded. Recently the disease in many of these families was

explained by truncating mutations in the progranulin gene (PGRN) which was mapped ~1.7 Mb

centromeric of the MAPT locus [Baker et al. 2006;Cruts et al. 2006]. The PGRN gene encodes a

secreted multifunctional growth factor involved in development, wound repair and inflammation.

Patients with PGRN mutations do not have tau-pathology. Instead there are ubiquitin-

immunoreactive neuronal cytoplasmic and intranuclear inclusions, the protein identity of which

remains unknown [Baker et al. 2006;Cruts et al. 2006;Mackenzie et al. 2006].

53

Neurodegeneration in mutation carriers is caused by PGRN haploinsufficiency due to nonsense-

mediated decay since transcript analysis demonstrated the absence of the mutant allele.

Herein, we describe the clinical, neuropathological and genetic findings of a CBS-like disease

which is segregating a novel PGRN mutation in a Canadian family of Chinese origin. This

finding extends knowledge on the clinical, pathologic and genetic heterogeneity of CBS and

FTLD.

2.3 METHODS

2.3.1 Subjects

The proband (Case 4150) was recruited through the Linda C. Campbell Cognitive Neurology

Research Unit at Sunnybrook Health Sciences Centre in Toronto as part of the Sunnybrook

Dementia Study. This is a prospective, longitudinal study of dementia and aging with well over

800 subjects enrolled to date approved by the local Research Ethics Boards. Patients or their

substitute decision makers provide written, informed consent to participate in accordance with

the Declaration of Helsinki. The proband underwent a detailed clinical assessment including:

history and physical examination, and standardized behavioural neurology assessment. Routine

biochemical screening was done to exclude any other causes for their presentation. The patient

was seen every six months for routine clinical follow-up and had yearly prospective longitudinal

assessments which included: detailed neuropsychological battery (measures of general

intelligence and cognition, language, praxis, visuospatial ability, attention and working memory,

and executive functions), measures of neuropsychiatric symptoms and of functional status.

54

Structural and functional neuroimaging of the brain with Magnetic Resonance Imaging (MRI)

and Single Photon Emission Computed Tomography (SPECT), respectively, were performed.

The sister of the proband (Case #4993) was identified through clinical history from the proband.

Information pertaining to this case is limited to that ascertained through a telephone interview

with her caregiver and through an autopsy report as this patient was residing out of country. The

normal control group consisted of 200 unrelated subjects of North American origin (mean age at

time of examination of 72.7 8.4 years).

2.3.2 Neuropathology

Neuropathological examination was carried out by two of the authors (R.H.; J.B.). Paraffin-

embedded sections were stained with Hematoxylin and Eosin, luxol fast blue (LFB),

Bielschowski and Gallyas. Immunostains using commercial antibodies for tau (Dako, #A0024)

and ubiquitin (Vector Labs, #ZPU576) were performed.

2.3.3 Genetic Analysis

Genomic DNA and RNA were extracted from whole blood using Qiagen kits. Two affected

members of the family (Case 4150 and Case 4993) were tested for mutations in exons 1 and 9-13

of the MAPT gene by direct sequencing as previously described [Kertesz et al. 2000a]. The entire

open reading frame with the exon-intron boundaries of the CHMP2B and PGRN genes were

sequenced in both affected individuals as previously described [Baker et al. 2006;Skibinski et al.

55

2005]. RT-PCR primers were designed for PGRN exon 3 (5‟- GCCACTCCTGCATCTTTACC-

3‟) and exon 8 (5‟-TTCTCCTTGGAGAGGCACTT-3‟). The RT-PCR conditions were 94C for

5 min, followed by 40 cycles of 94C for 30 sec, 58C for 30 sec, 72C for 30 sec, and 7 min at

72C. Mutations were detected by direct inspection of the fluorescent chromatographs and by

analysis using the SeqScape software version 1.0 (Applied Biosystems, Foster City, CA).

2.4 RESULTS

2.4.1 Clinical features and autopsy results

This family of Chinese origin presented with inheritance of a progressive neurodegenerative

disorder characterized by dementia and motor decline, including rigidity, dystonia, apraxia,

cortical sensory loss, visuospatial dysfunction and behavioural changes (Figure 1A). Family

records indicate that two out of 12 siblings have been affected with Corticobasal Syndrome. A

third family member has developed early parkinsonism. Two patients were available for the

genetic and clinical study.

56

Figure 1.

(A) The pedigree structure of the Canadian family showing the inheritance of the disease (with age-at-onset).

Affected individuals are shown as filled symbols and the arrow points to the proband. The gender of the

individuals has been masked to protect family confidentiality;

(B) Genomic DNA (gDNA) and RT-PCR (cDNA) sequence fluorescent chromatograms around the PGRN

mutation (IVS7+1G>A) observed in the patients and the sequence around common synonymous variation

rs25646;

(C) An agarose gel photograph of the PGRN product from RT-PCR, using RNA obtained from white blood

cells of the affected family member (#4150) and normal control (the 586bp band corresponds to the PGRN

fragment containing exons 3-8 confirmed by sequencing analysis).

57

2.4.1.1 Case #4150 (Proband)

This 71 year old right-handed woman with a previous history of hyperthyroidism treated with

radioablation and requiring thyroid replacement presented at age 62 with the insidious onset of

behavioural changes including increased irritability, depression, social withdrawal, and

suspiciousness. Subsequently, she began to experience difficulties with short-term memory,

planning, attention, word-finding difficulties, and getting lost in familiar environments.

Abnormalities on her initial examination (age 65) were a left visual field defect which was

thought to be, in part, secondary to profound left hemi-neglect, left cortical sensory loss

(specifically, sensory extinction and agraphesthesia), left-hand ideomotor apraxia, and a dressing

apraxia. These exam features are consistent with right parieto-occipital dysfunction. She scored

20/30 on the Mini-Mental Status Exam (MMSE) putting her in the moderate range of dementia

severity. Cognitive testing confirmed severe visual perceptual dysfunction and also revealed

short-term memory deficits, impaired executive functions, anomic aphasia and apraxia. The

results of the neuropsychological battery and standardized scores are summarized in Table 1. An

MRI of the brain revealed right greater than left hemispheric cortical atrophy and ventricular

dilatation, slightly more prominent in the posterior regions; there were also some periventricular

white matter changes (Figure 2A). A brain SPECT scan demonstrated a large right

58

Demographics, Neuropsychological Battery and Functional Measures (Test name /maximum raw score)

Raw Scores for Case #4150

Standardized Score

Category

Age of Onset 62 - - Age at this testing 65 - - Duration of disease at testing 3 - - Years of education 12 - - General cognition

Folstein’s Mini-Mental Status Examination /30 20 ≥ 28 (NCO) Impaired Mattis Dementia Rating Scale /144 92 2 (SS) Impaired

Memory California Verbal Learning Test – Long Delay Free

Recall /16 4 -2 (ZS) Impaired

Delayed Visual Reproduction /41 0 1st percentile Impaired Language

Western Aphasia Battery – total /100 83 -2 (ZS) Impaired Western Aphasia Battery – comprehension /10 8 -2 (ZS) Impaired Boston Naming /30 19 2 (SS) Impaired Semantic Fluency /20 6 < 10th percentile Borderline-Impaired

Praxis Western Aphasia Battery – praxis /60 48 -2 (ZS) Impaired

Attention & working memory Digit span – forward /12 6 30th percentile Normal Digit span – backward /12 2 5th percentile Borderline

Visuospatial abilities Rey Osterieth Complex Figure – copy /36 0 < 1st percentile Impaired Benton Line Orientation /30 N/A ≤ 4 (SS) Impaired Executive functions

Phonemic fluency (F-, A-, S-words) 16 3 (SS) Impaired Wisconsin Card Sort Test – categories /6 0 > 1 (NCO) Impaired Wisconsin Card Sort Test – perseverative errors 20 0 (NCO) Impaired

Activities of daily living Disability Assessment for Dementia (%) 53 100 (NCO) Impaired

Neuropsychiatric symptoms Neuropsychiatric Inventory – total /144 24 0 (NCO) Abnormal Neuropsychiatric Inventory – apathy /12 8 0 (NCO) Abnormal Neuropsychiatric Inventory – depression /12 8 0 (NCO) Abnormal Neuropsychiatric Inventory – disinhibition /12 0 0 (NCO) Normal Cornell Depression Scale (%) 53 < 25 (NCO) Depressed

Table 1. Scores on neuropsychological and functional measures for case #4150 compared to standardized scores calculated based on normal population matched for age and years of education. Abbreviations: NCO = Normal cut-off; SS = Scaled score (Mean = 10; 1 standard deviation = 3); ZS = Z-score; N/A = Not assessable

parieto-occipital perfusion deficit extending into the temporal and frontal regions with a milder

decrease in perfusion in the left parietal lobe (Figure 2B). The neuropsychological data was

59

collected within a one month time period of the MRI and SPECT images. The provisional

diagnosis was thought to be posterior cortical atrophy, a possible variant of Alzheimer‟s disease.

She was initiated on a cholinesterase inhibitor with no major change in symptoms apart from

some improvement in attention.

Figure 2.

Corresponding (A) T1-weighted Magnetic Resonance Imaging (MRI) and (B) Technetium 99m-ethyl cysteinate

dimer (99mTc-ECD) Single Photon Emission Computed Tomography (SPECT) scans of the brain of Case #4150.

Areas of relative atrophy on MRI and decreased cerebral perfusion on SPECT in the right inferior frontal (IF),

inferior parietal (IP), superior frontal (SF), superior parietal (SP), and occipital (O) regions are demonstrated.

There is a clear asymmetry in cortical atrophy and regional perfusion with the right being more affected than the

left. For the SPECT images, orange-yellow colours represent areas of high perfusion while blue-purple colours

represent areas of low perfusion.

A year after her initial assessment (age 66), the patient‟s cognitive performance continued to

decline (MMSE = 12) and she required assistance in all activities of daily living. She also was

60

developing an asymmetric akinetic-rigid syndrome including prominent rigidity of the left upper

and lower extremities, bradykinesia and a stooped posture with a shuffling gait. The provisional

diagnosis was changed to CBS based on the emergence of an asymmetric akinetic-rigid

syndrome and severe left-sided ideomotor apraxia. She met clinical criteria for CBS [Boeve et al.

2003]. Over the next three months, the patient became verbally and physically aggressive

towards her day-time caregiver. Cognitively, her dementia had progressed into the severe range

and she was completely dependent for self care. She had a positive glabellar tap and bilateral

grasp reflexes consistent with frontal release phenomena. At this point, she was observed to be

constantly biting her finger nails, likely representing repetitive, stereotyped behavior. Clinically,

her akinetic-rigid syndrome had progressed and she now developed dystonic posturing of her left

hand, and worsening left-sided apraxia, the combination of which produced a useless left arm.

Approximately three years after her initial assessment (age 68), she lost the ability to ambulate

and developed corticospinal tract findings on the left side of her body (i.e., left hyperreflexia and

extensor plantar response). Her verbal output declined and she would often repeat phrases such

as “you‟re killing me”. She continued to decline and four years after the initial assessment (age

69), her speech output diminished to the point where she was only able to grunt to indicate her

needs, with relative preservation of verbal comprehension. Eventually, she became mute and lost

the ability to comprehend and interact with others. Recently, she developed dysphagia to liquids

and is able to eat only pureed foods. Currently (age 71), she is bed-bound with end-stage CBS

about nine years into the course of her illness.

61

2.4.1.2 Case #4993 (sister of proband)

This deceased 61 year old woman had a history of dementia and motor decline since age 57

consisting of axial and extremity rigidity and aphasia. She had significant contractures and

flexion posturing of her upper extremities and right lower extremity. She required complete

personal care and gastrostomy tube feeds for nutrition towards the end of her disease course. Her

clinical diagnosis by a neurologist was CBS. She passed away at age 61 from medical

complications related to her neurodegenerative disorder. Disease duration in this patient was

about four years.

2.4.1.3 Neuropathology (Case #4993).

Gross: The whole brain weighed 940 grams unfixed. Examination of the right half of the fixed

brain demonstrated mild to moderate sulcal widening in the frontotemporal regions. Coronal

sections showed a well-defined and regular cortical ribbon without focal defects. Significant

widening of the circular sulcus and Sylvian fissure was noted. The caudate nucleus and putamen

were atrophic. The hippocampus was normal in size. The substantia nigra was normally

pigmented. There were no gross abnormalities of the cerebellum, pons, medulla, or cervical

spinal cord.

Microscopic: Severe pancortical micro-vacuolation associated with neuronal loss and gliosis was

seen in the frontal cortex. Similar changes were seen in the insular and temporal cortices and in

the basal ganglia. The vacuoles varied in size and were more numerous in the superficial layers

of the cortex. Vacuoles were not encountered in the thalamus, brainstem, cerebellum and spinal

cord. The vacuoles were located within neuronal cytoplasm and the neuropil. Patchy myelin

62

pallor was demonstrated in the white matter underlying the atrophic cortical areas. This finding

was best seen in LFB stains. The hippocampus was well-preserved. There was some neuronal

loss in the substantia nigra with an absence of Lewy bodies in the brainstem or cerebral cortex.

Bunina bodies were not seen in the motor nuclei of the cranial nerves. Bielschowski stains

demonstrated no neocortical senile plaques but rare, probably age-related, plaques were

identified in the hippocampus. No astrocytic plaques were observed in Gallyas stains. There

were no axonal spheroids. Immunostains for tau protein were performed and showed no

reactivity in neurons or other cells. Immunostains for ubiquitin demonstrated ubiquitin-reactive

neuronal cytoplasmic inclusions. Ubiquitin-reactive neuronal intranuclear inclusions were not

seen. Scattered neurites in the frontotemporal cortex were also ubiquitin positive. These findings

are compatible with the diagnosis of FTD with ubiquitin-only positive inclusions also referred to

as FTD-MND-type inclusion or FTD-U pathology [Lipton et al. 2004;Mackenzie and Feldman

2005;Mann et al. 2000;Taniguchi et al. 2004].

The third affected family member (brother of proband), after retiring at age 65, experienced

“dizzy” spells and did not feel well. He saw a number of doctors and he was told that he had

early Parkinsonism. Although he was never diagnosed with a dementing illness, he has been

unable to drive or prepare meals for himself. Information pertaining to this brother was limited to

history from a family member. There was no history of dementia or Parkinsonism in either

parent. The father died in his sixties from tuberculosis. The mother died at age 65 from

“pulmonary edema”. The other siblings are unaffected.

63

2.4.2 Genetic Analysis

Due to the clinical course and strong family history of disease, we performed mutation analysis

of all three known FTLD genes (MAPT, CHMP2B and PGRN) for patients #4150 and #4993. We

did not observe any sequence variations in the MAPT and CHMP2B genes. However, in the

PGRN gene we identified a novel heterozygous single nucleotide G-to-A mutation in the

invariant “GT” splice donor site 3‟of exon 7 (genomic position 5680; Accession Number

AC003043) (Figure 1B). The exon numbering was according to the National Center for

Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and our exon 7 corresponds to exon 6

in the published report [Baker et al. 2006]. The IVS7+1G>A mutation segregates with the

disease in the two affected family members (#4150 and #4993) tested and was not found in 200

normal controls.

The mutation is predicted to have dramatic consequences on the maturation process of PGRN

mRNA leading to the removal of exon 7 which would create a frame shift and truncate the

protein to half its normal length (amino acid position 236). Likely such a transcript will be

destroyed by nonsense mediated decay. In agreement with this, the result of the RT-PCR, using

RNA isolated from the white blood cells of patient #4150, revealed only the wildtype product on

agarose gel (Figure 1C). The specificity of this RT-PCR product, containing exons 3-8 of the

PGRN gene, was confirmed by direct sequencing analysis. Importantly, this patient, who is

heterozygous (T>C) for a common synonymous polymorphism in exon 5 (D128D; rs25646)

using genomic DNA, showed only the “C” allele in the RT-PCR product (Figure 2B). Hence, the

RT-PCR result demonstrates the absence of the mutant PGRN transcript.

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=Nucleotide&dopt=GenBank&val=2828776&from=35486&to=43465&strand=2

http://www/

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2.5 DISCUSSION

In the family reported here, the novel splice donor site mutation in the PGRN gene

(IVS7+1G>A) affects the sequence that is important in the recognition of the intron/exon

boundary and removal of the intron [Berget 1995]. There are no doubts about the pathological

nature of this mutation. It segregates with the disease in two affected family members and was

absent in 200 unrelated normal controls. The predicted consequence of the splicing mutation is

either the expression of the truncated protein or the haploinsufficiency of PGRN due to nonsense

mediated decay. According to the published reports the second possibility is more likely [Baker

et al. 2006;Cruts et al. 2006]. Indeed, our attempt to evaluate the pathological consequences of

the IVS7+1G>A mutation by RT-PCR using RNA from the blood cells of patient #4150 did not

identify aberrant PGRN transcripts (Figure 1C). Instead we confirmed the absence of the mutant

PGRN allele in the RT-PCR product (Figure 1B). Hence, the progression from normal function

to the disease state would result from the reduction of the PGRN level, further supporting the

model of haploinsufficiency for PGRN-linked FTLD. Previously, a different splicing mutation

(named IVS8+1G>A) was reported in one family; however, a source of RNA was not available

to confirm the haploinsufficiency mechanism [Baker et al. 2006].

The cases described in this family met clinical criteria for CBS [Boeve et al. 2003]. Pathology in

one affected individual demonstrated ubiquitin-positive, tau-negative cytoplasmic inclusions

consistent with the pathology reported in the original FTLD families in which PGRN mutations

co-segregate with disease [Baker et al. 2006;Cruts et al. 2006]. To our knowledge, this is the first

report of mutation in PGRN causing familial CBS with underlying FTD-MND-type inclusion

pathology. This type of pathology has been demonstrated previously in sporadic cases of CBS

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[Grimes et al. 1999a;Kertesz et al. 2005]. One could surmise that these previously reported

“sporadic” cases may come from families with PGRN mutations that were non-penetrant.

Previous familial studies have demonstrated that CBS coexists with PSP, and/or FTLD [Boeve et

al. 2002;Brown et al. 1996;Brown et al. 1998;Bugiani et al. 1999;Gallien et al. 1998;Tuite et al.

2005;Uchihara and Nakayama 2006]. Only two of these studies had more than one affected

individual with CBS making this a relatively uncommon presentation in FTLD families [Tuite et

al. 2005;Uchihara and Nakayama 2006]. Our study extends this literature in that two of the

affected family members have CBS, while one has early parkinsonism which may be evolving

into a dementing condition based on history. Perhaps, the novel splice donor site mutation in

PGRN identified in this family predicts the phenotypic expression of CBS as opposed to FTLD

or PSP. However, this would be unlikely given the current haploinsufficiency model proposed

for PGRN mutation. Another possibility may be that the FTLD phenotype may be differentially

expressed in Asians such that CBS is more likely to occur. Reasons for this might include

epigenetic factors, modifier genes, and/or environmental influences that “tip the balance” in

favour of one particular manifestation of FTLD over another.

The proband in our study presented initially with behavioural symptoms consisting of increased

irritability, depression, social withdrawal, and suspiciousness. Prominent visuospatial

dysfunction was present early on in the clinical course. Subsequently, she had difficulties with

short-term memory, executive functions, and expressive language. MRI and SPECT imaging of

the brain (Figures 2A and 2B) demonstrated cortical atrophy and reduced perfusion, respectively,

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in the right parieto-occipital greater than right frontotemporal regions which was clearly

asymmetrical when compared to the left hemisphere. This suggested an initial diagnosis of

posterior cortical atrophy although clinically there were also deficits of anterior cerebral

dysfunction. Once the extrapyramidal features evolved, the diagnosis of CBS became clear.

We have previously reported a case of a patient with sporadic CBS who presented initially with

prominent visuospatial dysfunction and a hemi-neglect syndrome similar to the proband in the

current study [Kleiner-Fisman et al. 2003]. Interestingly, final pathological diagnosis in this

patient confirmed ubiquitin positive, tau negative inclusions consistent with FTD-MND-type

inclusion pathology (unpublished data) similar to the pathology observed in the current study.

Visuospatial dysfunction in CBS has also been observed rarely [Mendez 2000;Okuda et al.

2000a] with one study demonstrating underlying typical CBD pathology [Tang-Wai et al. 2003].

Therefore, CBS presenting with prominent visuospatial dysfunction does not necessarily predict

the specific underlying pathological diagnosis.

Although both cases described in this family were diagnosed with CBS, there were significant

differences in their clinical course. The proband presented at age 62 with behavioural symptoms

and posterior cerebral dysfunction and evolved over a few years into CBS and is still living nine

years after disease onset, although nearing end-stage disease. The sister of the proband presented

at a younger age (57 years old) and had early and prominent motor features which eventually

lead to death at age 61, four years after symptom onset. Unknown environmental or genetic

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factors or stochastic events must contribute to this variability in age of onset and disease severity

within families and will require further investigation.

Clinical diagnosis along the FTLD spectrum is challenging and frequently longitudinal follow-up

of patients is required to ascertain the most likely provisional diagnoses. Take, for example, the

prospective, clinic-based cohort of FTLD patients of Kertesz et al. [Kertesz et al. 2005] that was

followed longitudinally to autopsy. In this cohort, the authors describe patients presenting with

initial syndromes ranging from behavioural variants of FTLD, CBS, PSP, to primary progressive

aphasia. The majority of these patients then went on to develop second and/or third syndromes

with significant clinical overlap along the FTLD spectrum. Added to this complexity is the fact

that there were a variety of pathologies underlying each of the clinical phenotypes ranging from

tau positive to tau negative types. For the most part, the clinical syndrome of FTLD observed is

dependent more on “the distribution of the underlying pathological state rather than on its

nature” [Lang 2003]. It is hoped that as we learn more about the underlying molecular

pathogenic mechanisms of FTLD spectrum disorders, diagnostic accuracy in life will improve

and this will also lead to potential therapies to prevent or cure these debilitating disorders.

2.6 ACKNOWLEDGEMENTS

This work was supported by grants from the Japan-Canada and Canadian Institutes of Health

Research Joint Health Research Program, Parkinson Society of Canada (ER), Canadian Institutes

of Health Research (PSGH; SB – MT13129), Howard Hughes Medical Institute, Canada

Foundation for Innovation (PH). MM is supported by a Medical Scientist Training Fellowship

68

from the McLaughlin Centre for Molecular Medicine, University of Toronto. JH was supported

by the NIA/NIH Intramural Program. The authors would like to thank Edward Huey, MD for

constructive criticisms; Mr. Shahryar Rafi-Tari for his assistance in preparing the neuroimaging

figure; Ms. Isabel Lam for her assistance in preparing the table of neuropsychological data.

2.7 ADDENDUM

Since this original paper was published, we have subsequently confirmed that the proband

described in this study had a pathological diagnosis of FTLD-U/ TDP43 consistent with that

observed in association with PGRN mutation.

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3.0 Intra-Familial Clinical Heterogeneity due to FTLD-U

with TDP43 Proteinopathy Caused by a Novel Deletion in

Progranulin Gene (PGRN)

Tomasz Gabryelewicz*, Mario Masellis*, Mariusz Berdynski

*, Juan M. Bilbao, Ekaterina

Rogaeva, Peter St. George-Hyslop, Anna Barczak, Krzysztof Czyzewski, Maria Barcikowska,

Zbigniew Wszolek, Sandra E. Black and Cezary Zekanowski

As published in: J Alzheimers Dis (2010); 22: 1123-1133.

Mario Masellis extracted the clinical information on the brother of the proband, interpreted and


responsible for writing a significant proportion of the manuscript with contribution from Tomasz

Gabryelewicz. Sequencing of the proband and genotyping of the controls was performed by

Mariusz Berdynski and Cezary Zekanowski. Ekaterina Rogaeva performed the genotyping in the

brother of the proband. Pathological analysis was done by Juan Bilbao.

* These authors contributed equally to the work as co-first authors

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3.1 ABSTRACT

Frontotemporal dementia (FTD) is one of the commonest forms of early-onset dementia,

accounting for up to 20% of all dementia patients. Recently, it has been shown that mutations in

progranulin gene (PGRN) cause many familial cases of FTD. Members of a family affected by

FTD spectrum disorders were ascertained in Poland and Canada. Clinical, radiological,

molecular, genetic, and pathological studies were performed. A sequencing analysis of PGRN

exons 1-13 was performed in the proband. Genotyping of the identified PGRN mutation and

pathological analysis was carried out in the proband‟s brother. The onset of symptoms of FTD in

the proband included bradykinesia, apathy, and somnolence followed by changes in personality,

cognitive deficits, and psychotic features. The proband‟s clinical diagnosis was FTD and

parkinsonism (FTDP). DNA sequence analysis of PGRN revealed a novel, heterozygous

mutation in exon 11 (g.2988_2989delCA, P439_R440fsX6). The mutation introduced a

premature stop codon at position 444. The proband‟s brother with the same mutation had a

different course first presenting as progressive non-fluent aphasia, and later evolving symptoms

of behavioral variant of FTD. He also developed parkinsonism late in the disease course

evolving into corticobasal syndrome. Pathological analysis in the brother revealed

Frontotemporal Lobar Degeneration-Ubiquitin (FTLD-U)/TDP43 positive pathology. The novel

PGRN mutation is a disease-causing mutation and is associated with substantial intra-familial

clinical heterogeneity. Although presenting features were different, rapid and substantial

deterioration in the disease course was observed in both family members.

Keywords: corticobasal syndrome, frontotemporal dementia, haploinsufficiency, parkinsonism,

progranulin mutation, progressive non-fluent aphasia

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3.2 INTRODUCTION

Frontotemporal dementia (FTD) is a clinically, genetically, and neuropathologically

heterogeneous disorder, accounting for 20% of early-onset dementia [Neary et al.

1998;Neumann et al. 2009]. FTD is characterized by behavioral and language dysfunction,

without amnesia, and consensus clinical and pathological diagnostic criteria have been proposed

[McKhann et al. 2001;Neary et al. 1998;The Lund and Manchester Groups 1994;Cairns et al.

2007].

Progranulin gene (PGRN, GRN [OMIM 138945]) mutations were shown to be common in

sporadic and familial FTD [Baker et al. 2006;Cruts et al. 2006;Gass et al. 2006]. PGRN is a 593

amino acid glycoprotein, composed of 7.5 evolutionary conserved tandem repeats, which are

cleaved, forming a family of granulin peptides. It is a growth factor important in neural

development [Ahmed et al. 2007]. A haploinsufficiency mechanism was identified to be the

etiology underlying PGRN-associated neurodegeneration, which causes frontotemporal lobar

degeneration with ubiquitin-positive, tau-negative inclusions (FTLD-U) [Baker et al. 2006;Cruts

et al. 2006]. TDP43 was found to be the major pathological protein underlying FTLD-U

pathology [Neumann et al. 2006].

From a clinical perspective, there is much to learn about how specific symptoms of FTD map

onto FTLD pathological subtypes. PGRN mutations have been associated with substantial

phenotypic heterogeneity in clinical presentation with a variety of diagnoses being observed:

behavioral variant FTD (bvFTD), progressive non-fluent aphasia (PNFA), corticobasal syndrome

(CBS), Alzheimer's disease, parkinsonism, and FTD with Parkinson‟s (FTDP) [Benussi et al.

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2008;Kelley et al. 2009;Masellis et al. 2006;Rademakers et al. 2007;Rohrer et al. 2009;Yu et al.

2010]. This clinical heterogeneity results from the same PGRN mutation causing pathology in

different hemispheres and lobar regions [Rademakers et al. 2007]. The molecular mechanism

underlying this clinical variability in different family members is unknown.

In this report, we describe the clinical, neuropsychological, and radiographic features at onset

and longitudinally in two brothers from the first Polish kindred identified to have a novel PGRN

mutation. Pathological characterization was performed in the index case‟s brother.

3.3 MATERIALS AND METHODS

3.3.1 Subjects

Genealogical data was ascertained in Poland. The proband was living in Warszawa, Poland. His

brother was living in Toronto, Canada. They underwent assessment in specialized dementia

clinics. Clinical evaluation included history, physical examination, and cognitive screening.

Routine biochemical screening was done. Brain MRI and SPECT were performed.

Neuropsychological, neuropsychiatric, and functional measures were completed. Baseline and

ten month follow-up data are presented for the brother.

The case-control group for genetic analysis consisted of 90 patients with familial or sporadic

FTD (age-matched) and 200 elderly, neurologically healthy controls from the Polish population.

All participants or their relatives provided written, informed consent in accordance with the

Helsinki Declaration and the study was approved by the appropriate ethics committees.

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3.3.2 Genetic analysis

DNA was isolated from peripheral leukocytes using standard procedures [Zekanowski et al.

2003b]. Intronic primers were used to amplify and sequence all (1-13) PGRN exons [Baker et al.

2006;Cruts et al. 2006]. Additionally, all exons of MAPT and PSEN1 were amplified and

sequenced to exclude mutations or rare polymorphisms [Zekanowski et al. 2003b;Zekanowski et

al. 2003a]. Amplification products were purified with ExoSAP IT (USB) and sequencing was

carried out using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and

the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). RNA was extracted from the

proband‟s leukocytes using TRIzol reagent (Ambion) according to manufacturer's instructions.

cDNA prepared from 5 μg of RNA using Superscript II (Invitrogen) was used as a template for

quantitative PCR with Power SYBR Green PCR Master Mix on an ABI PRISM 7500 instrument

(Applied Biosystems) according to manufacturer‟s` protocols. Relative 2-ΔΔCt

method with ACTB

as a reference gene was used to estimate levels of PGRN mRNA. Primers were designed for

PGRN cDNA: forward 5'-ATCCAGAGTAAGTGCCTCTCCAA-3', reverse 5'-

CTCACCTCCATGTCACATTTCAC-3', and for ACTB: 5'-CCGCAAAGACCTGTACGCCA-3'

and 5'-TGGACTTGGGAGAGGACTGG-3'.

Absence of mutated mRNA was confirmed using the PCR method with reverse primer specific

for the frameshifted region (5‟-GTCTGCTGCTCGGACCAC-3‟ and 5‟-

GTCACAGCCGATGTCTCG-3‟). Absence of the mutation in the case-control groups was

confirmed using restriction fragment length polymorphism analysis with AvaI (Fermentas) or

direct sequencing.

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3.3.3 Neuropathological analysis

Paraffin-embedded sections were stained with haematoxylin and eosin, Luxol fast blue,

Bielschowski and Gallyas. Immunostains using commercial antibodies for tau (Dako, A0024),

ubiquitin (Vector Labs, ZPU576), α-synuclein (Vector Labs), and subsequently with TDP43

(ProteinTech Group, Inc.) were performed.

3.4 RESULTS

3.4.1 Clinical, neuropsychological, and radiographic features

Proband (III:1). The proband was a 65-year-old right-handed male with no medical history. He

had 16 years of education and worked as a managing director of a company. At age 62, the first

symptoms were slowness, apathy and somnolence. The patient became withdrawn, less talkative,

gave up hobbies and had trouble handling familiar objects. After several months, his social

judgment deteriorated with a breakdown in formalities. He became disinhibited and significant

personality changes were observed. He developed cognitive symptoms thereafter including

aphasia and memory impairment.

Two years later (age 64), he stopped working and driving. Urinary incontinence occurred. The

patient underwent neurologic assessment and had evidence for dementia and parkinsonism.

Mini-Mental State Exam (MMSE) was 20. The patient deteriorated rapidly over the next few

months with insomnia and psychotic symptoms. He had significant irritability when opposed.

Motor re-examination showed moderately impaired monotone, slurred speech; minimal

hypomimia; resting tremor of upper extremities, moderate in amplitude; moderate rigidity;

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severe motor slowness and multi-step turning with postural instability. The symptoms progressed

throughout the ensuing observation period.

Neuropsychological assessment (age 64) showed impairment of executive functions, speech,

attention, and visuospatial functions (Table 1, III:1). He had spared autobiographical memory.

Word-finding difficulties were pronounced both in spontaneous speech and in verbal fluency

tasks with perseverations. He had problems switching between categories. The proband‟s verbal

learning was impaired, with a flat, plateau-like curve, and with intact delayed memory. Working

memory was severely disturbed. Copy of the Rey-Osterrieth Complex Figure was disorganized

with visuospatial and perseverative errors; most details were omitted on its delayed reproduction.

Naming and visual gnosis was intact. The patient had problems with gesture and spatial praxis

because of difficulties in motor switching. Sequencing of motor learning was severely impaired

with perseverations. This was also manifest as disturbed reciprocal coordination with a strong

tendency to repeat only one motor action without inhibition. The patient required help in

dressing and showering, and falls occurred daily. The patient manifested loss of initiative and a

lack of interest in daily routine activities. He had difficulties with speech and his handwriting

became illegible. Psychiatric examination showed psychotic features, including visual

hallucinations (faces on windows), bizarre delusions, and misidentifications.

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Table 1. Raw scores on neuropsychological and functional measures for proband (III:1) and proband’s brother (III:2).

III:2 Session1

III:2 Session 2

III:1

Demographics

Age of Onset (years) 55 - 62

Age at testing (years) 57 58 64

Duration of disease at testing (years) 2 3 2

Years of education 18 - 16

Neuropsychological Battery and Functional Measures (Test name/Maximum raw score)

General cognition

Folstein’s Mini-Mental Status Examination /30 19** 9** 22**

Mattis Dementia Rating Scale /144 96** N/T N/A

Blessed Information, Memory and Concentration Scale /37

N/A N/A 32

Memory

California Verbal Learning Test - Long Delay Free Recall /16

1** N/T N/A

Delayed Visual Reproduction /41 0** N/T N/A

Auditory verbal learning of 10 words list /First attempt/last attempt/delayed reproduction

N/A N/A 4/6/5

Rey Osterieth Complex Figure - reproduction /36 N/A N/A 6**

Address item from BIMC/5 N/A N/A 2

Language

Western Aphasia Battery - total /100 67.8** 40.4** N/A

Western Aphasia Battery – Aphasia Category Anomic Broca’s N/A

Western Aphasia Battery – Spontaneous Speech Content

7** 2** N/A

Western Aphasia Battery – Spontaneous Speech Fluency 5** 1** N/A

Western Aphasia Battery - comprehension /10 7.9** 5.8** N/A

Western Aphasia Battery – Repetition /10 8.4** 6.9** N/A

Western Aphasia Battery – Naming /10 5.6** 4.5** N/A

Boston Naming /30 13** N/T N/A

Boston Naming /20 N/A N/A 20

Semantic Fluency 6* 1** 10*

Praxis

Western Aphasia Battery - praxis /60 52** 34** N/A

Praxis of gesture /5 N/A N/A 4*

Reciprocal coordination I/10 II/10

N/A N/A 5 10*

Motor sequences learning I/5 N/A N/A 3

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II/5 2

Attention & working memory

Digit span - forward /12 6 N/T 4*

Digit span - backward /12 3* N/T 3*

Serial 7’s test /14 N/A N/A 1**

Visuospatial abilities

Rey Osterieth Complex Figure - copy /36 36 26.5 28

Benton Line Orientation /30 28 22 N/A

Visual gnosis /17 N/A N/A 14

Executive functions

Phonemic fluency (F-, A-, S-words) 3** N/T N/A

Phonemic fluency (K-words) N/A N/A 2**

Wisconsin Card Sort Test - categories /6 1** N/T N/A

Wisconsin Card Sort Test - perseverative errors 40** N/T N/A

Activities of daily living

Disability Assessment for Dementia (%) 96 24** N/A

Neuropsychiatric symptoms

Neuropsychiatric Inventory - total /144 4** 14** 23**

Neuropsychiatric Inventory- delusions/12 0 0 2**

Neuropsychiatric Inventory- hallucinations/12 0 0 6**

Neuropsychiatric Inventory - euphoria /12 2** 4** 0

Neuropsychiatric Inventory- anxiety/12 0 0 2**

Neuropsychiatric Inventory - apathy /12 2** 4** 6**

Neuropsychiatric Inventory – depression /12 0 0 1**

Neuropsychiatric Inventory - disinhibition /12 0 2** 2**

Neuropsychiatric Inventory – Irritability /12 0 0 4**

Neuropsychiatric Inventory - appetite /12 0 4** 0

Cornell Depression Scale (%) 8 3 11 Session 2 scores were obtained 10 months after session 1 scores for III:2. Unmarked scores are normal based on

comparison to healthy population matched for age and years of education. *Borderline-Impaired; **Impaired; N/T =

Not testable; N/A = Not available

He was diagnosed with FTDP based on neurologic, psychiatric, physical, and

neuropsychological examinations. Brain MRI and SPECT results supported this diagnosis and

correlated with his symptoms and findings (Figure 3). Specifically, there was atrophy in the right

anterior temporal region and bifrontally, more prominent on the right. There was reduced

perfusion bifrontally, more prominent on the right and extending into the right superior parietal

region.

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Proband’s brother (III:2). The proband‟s brother was a right-handed male with no relevant

medical history. He was assessed at age 57. He spoke Polish and English fluently. He had 18

years of education. Two years prior, he first presented with the insidious onset and gradual

decline in speech fluency; he had frequent word-finding difficulties that interrupted verbal

output. He often reverted to his native tongue. Comprehension was intact. However, he

continued to work as an engineer.

MMSE was 22/30. Spontaneous speech revealed word-finding difficulties with no paraphasic

errors. Comprehension, repetition, naming, and reading were intact. A written description of the

Cookie Theft Picture revealed use of simplified sentences with a sparse, but accurate description.

There was mild impairment in working memory and executive functions. His neurological exam

was normal except for mild increase in tone in the right arm with contralateral limb activation.

The initial diagnosis was PNFA.

Four months later, neuropsychological testing revealed moderate impairments in most domains

with relative sparing of visuospatial and visuoconstructive tasks (Table 1, session 1, III:2). On

the Western Aphasia Battery (WAB), his category was anomic. The aphasia over-estimated his

deficits. He, however, remained independent functionally with only minor troubles having a

phone conversation and taking messages. Initial MRI revealed bilateral frontal > anterior

temporal atrophy, which was prominent on the left (Figure 1A, B, C). Corresponding brain

SPECT revealed left > right bifrontal hypoperfusion extending into the left parietal region

(Figure 1B, C).

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Figures 1 and 2. T1-weighted brain MRI and corresponding 99mTc-ECD brain SPECT images of proband‟s brother

(III:2) in radiographic axial orientation. Asymmetric atrophy on MRI is seen affecting the left frontal > parietal

regions with ventricular enlargement (III:2 – Session 1) which progresses as seen in Figure 2 (III:2 – Session 2).

Perfusion deficits in the left > right frontoparietal regions in Figure 1 (III:2 – Session 1) also progress to more

bilateral involvement along with left temporal involvement seen in figure 2 (III:2 – Session 2).

Figure 3. T1-weighted brain MRI and corresponding 99mTc-HMPAO (800MBq) brain SPECT images of proband

(III:1) in standard radiographic axial orientation. Bilateral frontal and temporal regions demonstrate significant

atrophy with ventricular enlargement seen on axial slices of T1 weighted images in MRI. Corresponding axial

images of functional SPECT showing perfusion defect in frontal and temporal regions, bilaterally. There was a

predilection for the right hemisphere both in terms of atrophy and perfusion deficits. Orange-yellow color represents

areas of normal perfusion on SPECT, while blue-purple color represents relative decreases in perfusion.

AT=anterior temporal; PT=posterior temporal; O=occipital; IF=inferior frontal; IP=inferior parietal; SF=superior

frontal; SP=superior parietal.

Clinical assessment seven months later (age 58) revealed deterioration in multiple spheres of

cognition, behavior and function. He perseverated and had difficulties shifting sets. He giggled

excessively. He ate quickly cramming food into his mouth and pocketing it in his cheeks. He

developed a craving for chocolate. He became disinhibited and impulsive. He stopped

maintaining oral hygiene and had trouble eating with utensils. He had difficulties arising from a

chair and climbing stairs. His gait was slow with decreased arm swing on the right. Formal

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testing of praxis revealed both conceptual and ideomotor deficits. His score on the Frontal

Behavioral Inventory was 29, above the cut-off indicating FTD. The diagnosis remained PNFA,

but his syndrome evolved to include bvFTD.

Prospective re-evaluation on neuropsychological testing ten months after his first session

revealed significant deterioration (Table 1, session 2, III:2). MMSE was 9/30. WAB category

indicated a Broca‟s aphasia. He remained within normal limits on visuospatial tasks. From the

neuropsychiatric perspective, there was evidence for euphoria, disinhibition, apathy, and appetite

dysregulation. A repeat brain MRI demonstrated worsening atrophy of left > right

frontotemporoparietal regions (Figure 2A-C).

He became incontinent. He spoke with one word at a time. He was unable to follow instructions.

He required constant supervision. Physical exam revealed worsening parkinsonism with

hypomimia, right > left rigidity, difficulties arising from a chair, decreased right arm swing,

stooped posture and festinating gait. Re-evaluation on SPECT revealed progressive global

perfusion deficits with occipital sparing (Figure 2A-C). With the emergence of an asymmetric

akinetic-rigid syndrome associated with apraxia, his final diagnosis evolved to include CBS.

Eventually, he progressed to full mutism. At age 60, he was bed-ridden. He developed

progressive dysphagia. He passed away six years after disease onset (age 61) from complications

due to his neurodegeneration.

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3.4.2 Neuropathology (III:2)

The brain weighed 1,230 grams. Macroscopic examination disclosed atrophy of the frontal lobes,

worse on the left. Temporal and left parietal involvement was present. There was atrophy of the

caudate head.

Microscopic examination revealed severe pan-cortical atrophy, worse in anterior frontal regions

with microvacuolization. There was cell loss, gliosis, and pallor of the subcortical white matter.

Ubiquitin-positive threads co-localized with the microvacuolar changes. Many neurons displayed

“comma”-shaped perinuclear inclusions. Rare ubiquinated intranuclear inclusions were

demonstrable. Ubiquinated inclusions were abundant in the cingulum, mesiofrontal lobe,

precentral gyrus, temporal and parietal lobes, but less so in the latter with segmentally spared

areas. A dramatic decrease in ubiquitin pathology was noted in transition from the precentral to

postcentral gyrus. Primary visual cortex was spared. Silver stain and immunostaining for tau and

α-synuclein was negative.

Subcortical grey matter revealed neuronal ubiquinated granular and filamentous inclusions in

caudate, putamen, thalamus, posterior hypothalamus and nucleus accumbens. Globus pallidus

and nucleus basalis of Meynert were spared. In limbic regions, the cornu ammonis of CA1 was

severely gliotic and shrunken. Microvacuolar changes involving the parahippocampal gyrus were

noted, with sparing of the perirhinal cortex. Ubiquitin positive inclusions were observed in

neurons of the fascia dentata.

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The anterior 1/3 of the cerebral peduncles bilaterally was degenerated. The midbrain was small.

Estimated cell loss in the substantia nigra was 60% with severe gliosis and macrophages present.

There was no immunostaining for tau or α-synuclein. The pons was small with pallor of the

descending tracts. In the medulla, there was no α-synuclein staining. Rare neurons in the inferior

olive contained filamentous ubiquinated inclusions. Medullary motor nuclei were intact with no

ubiquinated inclusions observed. The cerebellum was unremarkable. Motor neurons in the spinal

cord were not affected.

Autopsy sections were re-examined with immunostains for TDP43 (Figure 4). TDP43 positive

neuropil threads, neuronal cytoplasmic stippled staining, neuronal cytoplasmic filamentous

inclusions, glial [oligo] cytoplasmic and neuronal intranuclear inclusions were found in the

frontal cortex, anterior striatum, fascia dentata, substantia nigra, and CA1 region. Final

pathological diagnosis was FTLD-U/TDP43 proteinopathy.

Figure 4. Micrographs demonstrating a large number of TDP43 inclusions (neuropil threads, neuronal cytoplasmic

stippled staining, neuronal cytoplasmic filamentous inclusions, glial [oligo] cytoplasmic and neuronal intranuclear

inclusions) found in the fascia dentata, substantia nigra, and CA1 region.

3.4.3 Family history

There was a strong family history of early-onset dementia and parkinsonism, suggesting

autosomal dominant inheritance (Figure 5). The proband‟s mother (II:2) died at age 64, with a

surmised progressive aphasia. Age of onset was 60. The maternal aunt had parkinsonism and

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dementia and died ca. 65 years (II:3). The proband‟s father (II:1) was neurologically intact and

died at age 62 of lung cancer.

3.4.4 Genetic analysis

A novel PGRN dinucleotide deletion in exon 11 (g.2988_2989delCA, c.1536_1537delCA,

P439_R440fsX6) was identified in the proband and his affected brother (Figure 5). Both the

sense and the complementary DNA strand were sequenced. The mutation causes a frameshift at

codon 441, and introduces a stop codon at position 444. The mutation was absent in a group of

90 Polish patients with FTD (mean age=59.7 ± 13 years) and 200 ethnically matched

neurologically healthy controls (mean age=72.7 ± 7 years; MMSE≥28).

RT-PCR analysis of PGRN mRNA levels in peripheral leukocytes from the proband revealed a

two-fold decrease of the cDNA transcript as compared to controls without the mutation. PCR

using a primer specific to the mutant cDNA resulted in absence of amplification product (Figure

6).

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Figure 5. Detection of PGRN mutation P439_R440fsX6. A) Pedigree showing family history of neurodegenerative

condition. Black symbols: patients affected with FTD and neurodegeneration; white symbols: unaffected individuals

or individuals with no clinical diagnosis available. B) Electropherogram showing start of deletion marked with an

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arrow. The resulting PGRN mutation P439_R440fsX6 is shown at the bottom of the chromatogram of the proband

and his affected brother.

Figure 6. Amplification from genomic DNA (gDNA; lane 1) using primers specific for the mutant allele

demonstrate the mutant fragment of 153 bp as expected. Amplification from cDNA (lane 2) shows an absence of the

expected product supportive of non-sense mediated decay. Ladder: GeneRuler 1kb DNA Ladder (Fermentas), the

lowest band is 250 bp (lane 3); positive control: cDNA amplified 84 bp fragment of β-actin gene (lane 4).

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3.5 DISCUSSION

We describe a novel PGRN mutation causing a frameshift introducing a premature stop codon.

RT-PCR analysis of PGRN mRNA levels confirmed the PGRN transcript decrease in the

proband as compared to normal. Additionally, no amplification products of the mutant allele

were detected suggesting that mRNA with the premature stop codon is rapidly degraded as a

result of non-sense mediated decay [Baker and Parker 2004]. There are no doubts about the

pathological nature of this mutation. It segregates with the disease in two affected family

members, it is absent in 200 normal controls, and immunohistochemistry confirms FTLD-

U/TDP43 pathology associated with mutant PGRN.

Patients affected with different PGRN mutations showed a broad range of age of onset (AOO;

48-83 years), with a mean of 597 years, often resulting in no family history recorded [Brouwers

et al. 2008;Gass et al. 2006]. Another study also showed a highly variable AOO ranging from 49

to 88 years, with variable disease duration ranging from one to 14 years [Kelley et al. 2009].

This novel PGRN deletion is associated with a rapid disease course and clear inheritance pattern.

Consistent with other studies, AOO was variable with the proband‟s brother developing

symptoms seven years earlier.

The clinical course of FTD in the two siblings was different, particularly at illness onset (Table

1). The proband‟s clinical features suggested early medial and dorsolateral prefrontal

involvement with slowing, lack of motivation, and apathy. Shortly thereafter, social impairment

and disinhibition were present, suggesting progression to orbitofrontal and right anterior

temporal structures. Parkinsonism was also present. The behavioral disturbance correlated well

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with bifrontal and anterior temporal atrophy and hypoperfusion, worse on the right (Figure 3).

Language problems were observed later than behavioral impairment. The proband also had

psychotic features including hallucinations, which is atypical in FTD.

In contrast, language disturbances came first in the proband‟s brother. These were expressive

with an early anomia progressing to a Broca‟s aphasia and then to mutism. The initial symptoms

of PNFA correlate with atrophy and hypoperfusion predominantly in the left frontal region

(Figure 1A-C). Later on, behavioral disturbance developed, which suggested progression to

orbitofrontal and right anterior temporal regions, with social impropriety and disinhibition,

culminating in apathy and cognitive decline. As the disease progressed from PNFA to include

bvFTD so did the atrophy and perfusion deficits involving frontotemporal regions bilaterally

(Figure 2A-C). Apraxia was likely accounted for by the left frontoparietal involvement (Figures

1C and 2C) and these findings supported the third diagnosis of CBS. Visuospatial function was

relatively preserved, correlating well with intact perfusion and absent pathology in the occipital

regions, bilaterally.

The most common clinical presentation of PGRN mutation includes behavioral symptoms, with

apathy as the dominant feature [Beck et al. 2008], similar to the proband. However, as is the case

with his brother, clinical presentations of PNFA due to PGRN mutation are also frequent

[Snowden et al. 2006]. Several studies have confirmed this strong association between PNFA

and PGRN mutations with the typical FTLD-U/TDP43 pathology [Beck et al. 2008;Moreno et

al. 2009;Pickering-Brown et al. 2008;Skoglund et al. 2009]. In particular, similar to the brother‟s

pathological findings, FTLD-U, type 3 pathology, was found to be most commonly associated

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with the clinical phenotype of PNFA [Snowden et al. 2007]. In one series, semantic dementia

cases were associated with MAPT mutations whereas PNFA with associated apraxia predicted

PGRN mutations [Pickering-Brown et al. 2008]. These particular case series were enriched with

familial forms of FTD or were selected for based on a priori identification of PGRN mutation.

Studies of predominantly sporadic cases of primary progressive aphasia selected for based on

availability of pathological material demonstrated the opposite trend. Specifically, non-fluent

presentations were associated with Tau pathology [Josephs et al. 2006;Knibb et al. 2006], while

fluent cases were associated with ubiquitin pathology [Knibb et al. 2006]. Longitudinal studies

of familial and sporadic aphasic variants of FTD followed clinically until death with subsequent

pathological characterization are warranted to clarify these apparent discrepant findings.

In the current study, both the proband and his brother developed parkinsonism. Indeed, FTD and

parkinsonism due to PGRN mutation is common [Josephs et al. 2007;Wong et al. 2009] and is

more variable than that due to FTDP-17 with MAPT mutations [Boeve and Hutton 2008]. In the

former, there are often posterior features, such as limb apraxia and visuospatial dysfunction,

which results in a wider clinical spectrum of diagnoses including dementia with Lewy bodies or

CBS [Boeve and Hutton 2008].

In general, the clinical heterogeneity and course of the affected siblings with this novel

P439_R440fsX6 dinucleotide deletion resembles the course of other FTD patients with short-

segment nucleotide PGRN deletions [Benussi et al. 2008;Borroni et al. 2008a;Llado et al.

2007;Skoglund et al. 2009]. The particular type of mutation does not predict the clinical

syndrome, but rather it is the location of the pathology which is most significant.

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To date, significant progress has been made in understanding the allelic heterogeneity of PGRN

mutation in FTD. This paper extends the literature on the allelic and phenotypic heterogeneity of

FTD. However, progress in terms of understanding the variable clinical presentation of FTD, i.e.,

specific diagnoses, age of onset, hemispheric and specific lobar involvement and duration of

disease remain to be explained. Studies examining polymorphism within PGRN miRNA binding

sites and peripheral expression levels of PGRN may help to shed light on this phenotypic

heterogeneity [Finch et al. 2009;Rademakers et al. 2008]. Using the approach of early

identification of those at risk of developing FTD by imaging and CSF biomarkers coupled with a

better understanding of genetic, epigenetic, and environmental modulators of disease will

facilitate future development of preventative treatments and/or disease-modifying therapies for

these devastating FTD syndromes.

3.6 ACKNOWLEDGMENTS

The authors would like to thank Dr Jaroslaw B.Cwikla from Dep. of Radiology and Diagnostic

Imaging, Medical Centre for Postgraduate Education and CSK, MSWiA in Warsaw for

comments and creating figure 1. The authors would also like to thank Mr. Mike Misch, Gregory

Szilagyi, and Mark Gravely for creating Figures 1, 2, and 3 and Ms. Isabel Lam for creating

Table 1. MM is supported by a Canadian Health Institutes of Research (CIHR) Clinician

Scientist Award and the Department of Medicine, Sunnybrook Health Sciences Centre. This

research is supported by operating grants from the CIHR (SEB, MT13129; PSGH & ER,

MT417763) and the Ontario Research Fund (PSGH). ZW is supported by NIH/NINDS

1P50NS072187-01, 1RC2NS070276-01, 1R01NS057567-01A2; Carl Edward Bolch, Jr. and

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Susan Bass Bolch Gift, and Mayo Clinic Florida Research Committee. CZ and MB are supported

by grant PBZ-MEiN-0/2/20/17.

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4.0 Ideomotor Apraxia in Corticobasal Syndrome: Brain

Perfusion and Neuropsychological Correlates

Mario Masellis, Philip L. Francis, Kie Honjo, Bradley J. MacIntosh, Isabelle Guimont, Gregory

M. Szilagyi, Wendy R. Galpern, Galit Kleiner-Fisman, James L. Kennedy, Robert Chen, Eric A.

Roy, Curtis B. Caldwell, Anthony E. Lang, Sandra E. Black

As submitted to: Cortex



Philip Francis assisted with the SPM analysis of SPECT data and Kie Honjo assisted with the

MRI segmentation procedure. Brad J. MacIntosh assisted with the atrophy correction procedure.

Wendy R. Galpern, Galit Kleiner-Fisman and Anthony E. Lang assessed and collected clinical

data on patients ascertained from a movement disorders clinic.

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4.1 Abstract

Ideomotor apraxia is one of the most common clinical features of corticobasal syndrome and is

associated with disability and reduced quality of life. Previous electrophysiological and

neuroimaging studies of apraxia implicated a role of the left frontoparietal network. However,

the specific nodes within this network have yet to be fully elucidated. The current study provides

the first direct correlative analysis between the severity of ideomotor apraxia in corticobasal

syndrome and cerebral perfusion imaging using brain SPECT. Reductions in perfusion within the

left inferior parietal lobule (t=5.7, p=0.03, Family-Wise Error [FWE] corrected), including the

left angular gyrus (t=5.7, p=0.02, FWE corrected), were associated with more severe ideomotor

apraxia as measured by the Western Aphasia Battery praxis scale. Results remained significant

even after controlling for the most affected side of the body. After categorizing the patients into

those with or without apraxia, language, visuospatial and visual memory functions were more

impaired in those with apraxia suggesting the involvement of overlapping networks, specifically,

bilateral occipitoparietal and left peri-Sylvian, subserving these related higher cognitive

processes. This study provides further evidence for the importance of the left inferior parietal

lobule in the dominant hemisphere frontoparietal praxis network and provides new insights into

associated cognitive dysfunction.

Keywords: apraxia; SPECT; perfusion; neuropsychology; corticobasal syndrome

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4.2 Introduction

Corticobasal Syndrome (CBS) is a rare and debilitating neurodegenerative syndrome

characterized by asymmetric rigidity, apraxia, dystonia, myoclonus, alien-limb phenomenon,

cortical sensory loss, frontosubcortical dementia, behavioral disturbances, and speech and

language abnormalities including apraxia of speech and progressive non-fluent aphasia (PNFA).

There is significant pathological heterogeneity that can produce the syndrome including

corticobasal degeneration, progressive supranuclear palsy (PSP), frontotemporal lobar

degeneration (FTLD)-Tau (Pick‟s disease) and FTLD-Ubiquitin/TDP43, and Alzheimer‟s

disease [Kertesz et al. 2005;Lee et al. 2011;McMonagle et al. 2006;Wadia and Lang 2007].

Apraxia is the hallmark that distinguishes CBS from other parkinsonian disorders in the early

stages of disease and it is the most common clinical feature occurring cross-sectionally in 70%

and longitudinally in 100% of cases [Leiguarda et al. 1994;Rinne et al. 1994;Stamenova et al.

2009]. Apraxia is defined as a higher-order “neurological disorder characterized by loss of the

ability to execute or carry out skilled movements and gestures, despite having the desire and the

physical ability to perform them” (http://www.ninds.nih.gov/disorders/apraxia/apraxia.htm).

There are many types of apraxia observed in CBS including apraxia of speech, limb-kinetic

apraxia, ideomotor apraxia (IMA), and ideational/conceptual apraxia, which have been

extensively described elsewhere [Gross and Grossman 2008;Josephs and Duffy 2008;Leiguarda

and Marsden 2000;Stamenova et al. 2009;Zadikoff and Lang 2005].

Ideomotor apraxia, best elicited by asking a patient to pantomime and/or imitate hand gestures

and tool use, is characterized by disturbances of timing, sequencing and spatial organization of

http://www.ninds.nih.gov/disorders/apraxia/apraxia.htm

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gestural movement of the limbs [Rothi et al. 1991]. It has been the most extensively studied

apraxia type in CBS [Zadikoff and Lang 2005], although there have been only a paucity of

studies that have directly correlated specific measures of praxis with brain imaging findings in

this disorder. Peigneux et al. [Peigneux et al. 2001] were the first to examine the association of

upper limb apraxia with fluorodeoxyglucose positron emission tomography (FDG-PET) imaging

in a case series of 18 patients with CBS. Their sample was stratified into CBS with and without

apraxia based on a standardized praxis measure. Using a global praxis performance score, the

bilateral anterior cingulate gyri demonstrated mild reductions in metabolism in the apraxic group

(uncorrected p < 0.001) [Peigneux et al. 2001]. Alternatively, stratification using a praxis

correction score resulted in hypometabolism contralateral to the most affected body side in the

superior parietal lobule, medial frontal gyrus and supplementary motor area, as well as the

middle frontal gyrus in the apraxic group (uncorrected p < 0.001) [Peigneux et al. 2001]. This

study, however, did not correlate PET images of metabolism with praxis measures, nor did their

imaging analysis correct for multiple comparisons on a voxel-by-voxel basis. Other small PET

and single photon emission computed tomography (SPECT) studies of apraxia have been

conducted in CBS samples, but these did not specifically look at the relationship between praxis

measure and functional imaging; rather they were a comparison of CBS versus controls and only

indirect associations with apraxia were made [Zadikoff and Lang 2005].

Functional neuroimaging studies with FDG-PET and SPECT have shown reduced metabolism

and perfusion, respectively, in frontoparietotemporal regions in CBS patients compared to

controls [Eidelberg et al. 1991;Garraux et al. 2000;Markus et al. 1995;Okuda et al. 1999]. The

hypoperfusion tends to be contralateral to the most affected side of the body. Similarly,

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asymmetrical atrophy on MRI can also be seen in CBS and the atrophy usually is more

prominent contralateral to the most affected side of the body [Riley et al. 1990;Savoiardo et al.

2000;Soliveri et al. 1999]. Perfusion SPECT was used in the current study given that prior

research of focal cortical atrophy syndromes, such as FTLD, have shown that perfusion

reductions on SPECT are more extensive than atrophy detected on MRI in the early stages of

disease and in longitudinal follow-up, indicating increased sensitivity of this modality as a

potential biomarker [Gregory et al. 1999;Gabryelewicz et al. 2010;Mendez et al. 2007].

The primary objectives of the current study were 1) to identify regions of reduced perfusion

using brain SPECT in a prospectively recruited sample of CBS cases compared to controls, 2) to

determine which of these regions directly correlate with performance on a standardized global

measure of ideomotor praxis using the Western Aphasia Battery (WAB) [Kertesz and Poole

1974;Kertesz 2007] accounting for effects of lateralization of motor symptoms and underlying

atrophy on MRI, and 3) to compare the demographic, clinical, neuropsychological and SPECT

characteristics of CBS patients with significant apraxia to those without this feature defined

based on performance on the WAB praxis subscale. The secondary objective of this study was to

explore the different subcomponents of the WAB praxis scale and their association with brain

SPECT perfusion.

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4.3 Materials and Methods

4.3.1 Subjects:

Thirty-one patients with a clinical diagnosis of CBS according to proposed diagnostic criteria

[Boeve et al. 2003] were recruited through two academic clinics: the Linda C. Campbell

Cognitive Neurology Research Unit at Sunnybrook Health Sciences Centre and the Movement

Disorders Centre at the Toronto Western Hospital, University Health Network. Patients provided

informed consent to participate according to the Declaration of Helsinki and were followed as

part of a prospective, longitudinal study of dementia and ageing approved by the local Research

Ethics Board. The patients underwent a detailed neurological exam, including a screening

assessment for apraxia in both upper limbs comprised of asking them to pantomime five gestures

(two intransitive and three transitive ones). Patient handedness was determined using a

standardized questionnaire [Bryden 1977]. The side of greatest rigidity and/or apraxia on clinical

examination by a cognitive and/or movement disorders neurologist with expertise in the clinical

assessment and diagnosis of CBS defined the motor-onset of symptoms. Although one side of

the body was more prominently affected than the other side in all patients initially, motor signs,

including apraxia, were indeed present bilaterally and became more evident as the disease

progressed. Diagnostic consensus was achieved through review by at least two neurologists

(AEL, MM and/or SEB). All patients were followed longitudinally to ensure diagnostic

accuracy. This is important because in advanced disease, the proportion of patients fulfilling

three of the most commonly applied diagnostic criteria for CBS was similar at approximately

90%, indicating that all criteria could be applied equally well in late stage disease [Mathew et al.

2011].

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4.3.2 Description of neuropsychological measures:

Neuropsychological tests assessing general cognitive functions included Folstein‟s Mini-Mental

State Examination (MMSE) [Folstein et al. 1975], and Mattis Dementia Rating Scale (DRS)

[Mattis 1976]. Measures of language function and naming included: the Western Aphasia

Battery (WAB), which calculates an aphasia quotient based on combined subscores of fluency,

content, comprehension, repetition and naming, with a maximum score of 100 and lower scores

representing more severe impairment [Kertesz and Poole 1974;Kertesz 2007]; the Boston

Naming Test (BNT) [Williams et al. 1989]; and semantic/categorical fluency [Gladsjo et al.

1999]. The visual reproduction subtest of the Wechsler Memory Scale-Revised (WMS-R) was

used to assess visual memory [Lezak 1983]. Visuospatial function was assessed using the Rey-

Osterrieth Complex Figure Test [Lezak 1983;Osterrieth 1944;Rey 1941], and the Benton Line

Orientation task, which is motor-free and assesses visuospatial orientation and attention [Lezak

1983]. Additional standardized neuropsychological, neuropsychiatric and functional measures

were performed as previously described [Masellis et al. 2006].

Praxis was assessed using the WAB praxis scale [Kertesz and Poole 1974;Kertesz 2007]. The

WAB is a valid and reliable measure of language and other higher cortical functions [Kertesz

2007] and the WAB praxis scale has been used to correlate stroke lesion localization and size

with severity of ideomotor apraxia [Kertesz and Ferro 1984]. Briefly, patients were asked to

pantomime gestures using their bucco-facial musculature and their less affected limb. Since CBS

is strikingly asymmetric in presentation and since the apraxia most often co-exists in the same

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limb where the extrapyramidal and cortical sensory features reside, the less affected limb was

selected for praxis assessment scoring to avoid contamination with the sensory and

extrapyramidal signs. Gestures fall into four categories: upper limb intransitive or transitive,

bucco-facial, or complex. Examples include waving goodbye (intransitive gesture), using a

toothbrush (transitive gesture), blowing out a match (bucco-facial gesture), and pretending to

drive a car (complex gesture). There were five different gestures asked in each of the four

categories. For all of the gestures, if the patient was unsuccessful at pantomime, they were asked

to imitate the gestures. For transitive gestures, if they were unsuccessful at both pantomime and

imitation, they were then handed the tool and asked to demonstrate how to use it. Three points

were given if the gesture was performed correctly on pantomime; two points were given if there

was approximate performance on pantomime or good performance on imitation only; one was

given if there was approximate performance on imitation or if performed correctly with the

actual tool or object; and no points were given if the patient was unable to perform the task, the

gesture was unrecognizable or unrelated, and for erroneous use of the actual object. Approximate

performance on gestural tasks was defined by the occurrence of the following types of errors:

inaccurate positioning of the hand or limb in space, improper finger configuration, a breakdown

in the core characteristics of the movement, and/or deficits in the sequence of an action, such as

omission or addition of movement elements, as well as a change in the order in which an action

should be carried out. Apraxia was scored out of 60 with lower scores indicating more severe

IMA [Kertesz and Poole 1974;Kertesz 2007]. Trained psychometrists with a Bachelor‟s or

Master‟s degree in Psychology administered all the tests, including the WAB, and were

completely blind to all neuroimaging measures.

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4.3.3 Brain SPECT acquisition and processing:

SPECT imaging employed a triple-head gamma camera (Prism 3000XP; Phillips Medical

Systems Inc., Cleveland, Ohio) and was performed a minimum of 30 minutes and a maximum of

120 minutes after injection of 20 mCi (740 MBq) of Technetium-99m ethyl cysteinate dimer

(99m

Tc-ECD SPECT). Patients were asked to rest quietly during the acquisition phase. 120 views

were acquired uniformly over 360 degrees using all three detectors fitted with ultra-high

resolution fan-beam collimators. Each view consisted of a 128 × 128 pixel image. Total imaging

time was 19 minutes. Reconstruction was performed using a ramp-filtered back-projection

algorithm followed by a 3-dimensional restoration post-filter (Wiener filter, multiplier 1.0).

Reconstructed image resolution was typically 9.7 mm full width at half maximum (FWHM).

Ellipses were fit to the approximate location of the outline of the head in each transaxial image,

and a calculated attenuation correction applied [Matsuda et al. 1995]. Voxel dimensions were

2.18 × 2.18 × 3.56 mm.

4.3.3.1 Regional perfusion ratios:

Reconstructed SPECT images were co-registered to a template that was an average of 14

healthy, elderly control scans. A T1-weighted MRI with dimensions similar to the SPECT

template was the source of 79 bilateral regions of interest (ROI) as previously described

[Lobaugh et al. 2000]. To obtain ROI intensity values, we used a common transformation to

move from the SPECT template space to MRI space. The cerebellum is frequently used to

normalize SPECT counts in studies of dementia [Stamatakis et al. 2001]. However, crossed

cerebellar diaschisis may lead to relative differences in perfusion between the left and right

cerebellar hemispheres, and, if whole cerebellum is used as the reference region in these cases,

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regional cerebral blood flow (rCBF) may be overestimated in a particular ROI. We, therefore,

applied the following rule: if there was more than a 5% difference in counts between left and

right cerebellar hemispheres, we use the hemisphere that was more perfused as the reference

region. If there was no difference then the whole cerebellum was used as the reference region. In

this way, semiquantitative perfusion ratios can be derived and regional Z scores calculated

[Lobaugh et al. 2000].

4.3.4 Data analysis:

Statistical analysis of demographic, clinical, neuropsychological and ROI SPECT variables was

performed using the Statistical Package for the Social Sciences (SPSS), version 16.

4.3.4.1 Demographic, clinical and neuropsychological measures:

Categorical demographic and clinical data were analyzed using chi-square or Fisher exact tests.

Normality of continuous demographic and neuropsychological data was assessed based on

examination of Q-Q probability plots. Normally distributed data were analyzed using

independent sample t-tests or ANOVA, otherwise, Mann Whitney U tests were performed.

4.3.4.2 Statistical Parametric Mapping (SPM) SPECT analysis:

SPECT scans were converted to Analyze 7.5 format. Statistical Parametric Mapping version 5

(SPM5, Wellcome Department of Imaging Neuroscience, University College London) was used

for all imaging processing. Images were spatially normalized to a standard SPECT template in

Montreal Neurological Institute (MNI) space [Stamatakis et al. 2001] with re-sampling of voxel

dimensions of 2 × 2 × 2 mm. Images were then smoothed using an isotropic Gaussian kernel (12

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mm FWHM). A thresholded mean voxel value was chosen for global calculation, and global

normalization was achieved by proportional scaling to an arbitrarily chosen constant value set at

50 mL/100 g/min. Voxel-by-voxel regression analysis was performed between perfusion and

praxis measures. Alternatively, voxel-by-voxel analyses were performed using unpaired t-tests to

compare 1) CBS patients to controls, and 2) apraxic to borderline/non-apraxic patients.

Covariates were incorporated if they were significantly different between groups. We reported

significance using a voxel-wise p-value threshold (p < 0.05) corrected for multiple comparisons

and an extent threshold of at least 20 contiguous voxels. Our correction methodologies included

either controlling the family-wise error (FWE) rate [Worsley et al. 1996] or controlling the false

discovery rate (FDR) [Genovese et al. 2002]. Controlling the FWE rate is more conservative but

is known to be associated with type II errors. A whole brain mask was used to exclude

extracranial voxels from the analysis. The maximal peak coordinates of the perfusion

differences were converted to Talairach space using the Yale Non-linear MNI to Talairach

Converter [Lacadie et al. 2008] (http://www.bioimagesuite.org/Mni2Tal/index.html). These

converted coordinates were translated into anatomical brain regions and Brodmann Areas (BAs)

using Talairach Daemon Client [Lancaster et al. 2000] (http://www.talairach.org/client.html).

4.3.4.3 Region of interest (ROI) SPECT analysis:

4.3.4.3.1 Comparison of CBS cases to controls

Normality of ROI SPECT data was confirmed as described above. Independent sample t-tests

between CBS and control groups were conducted to compare mean perfusion ratios of individual

ROIs within frontal, parietal, temporal, basal ganglia, and thalamic regions previously shown to

be affected in CBS [Markus et al. 1995;Okuda et al. 1999]. ROIs that were statistically

http://www.bioimagesuite.org/Mni2Tal/index.html

http://www.talairach.org/client.html

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significant on the t-test analyses were included in a multivariate analysis of covariance

(MANCOVA). „Years of education‟ was included as a covariate since it was the only

demographic variable that differed significantly between the cases and controls.

4.3.4.3.2 Confirmation of voxel-wise correlation with WAB praxis using ROI method

Regions of hypoperfusion identified on the voxel-by-voxel regression analysis allowed us to pre-

select the most relevant ROI to perform a Pearson correlation and linear regression analysis with

WAB praxis scores. This analysis provided for an independent confirmation of the voxel-wise

findings using the bottom up approach for data reduction given the sample size.

Since there is left hemisphere specialization for praxis control, and since CBS is typically

asymmetric in presentation, the potential confounding effect of symptom lateralization was

controlled for in the SPM and ROI regression analyses by incorporation of right- versus left-

sided motor presentation as a covariate.

4.3.4.4 Brain MRI acquisition and processing:

Structural MRI was obtained in 29 of the 31 patients using a standard protocol. Images were

acquired on a 1.5T Signa MR imager (GE Medical Systems, Milwaukee, Wis.) and consisted of

the following acquisitions: 1) T1-weighted (axial 3D spoiled gradient [SPGR] echo, with echo

time [TE] 5 ms, repetition time [TR] 35 ms, flip angle 35°, number of excitations [NEX] 1, field

of view [FOV] 22 × 16.5 cm, in-plane resolution 0.859 × 0.859 mm and slice thickness 1.2–1.4

mm), 2) proton-density and 3) T2-weighted images (interleaved axial spin echo, with TEs 30 and

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80 ms, TR 3 s, NEX 0.5, FOV 20 × 20 cm, in-plane resolution 0.781 × 0.781 mm and slice

thickness 3 mm).

4.3.4.4.1 Brain Extraction and Automated Tissue Segmentation:

Twenty-one of the 29 MRI scans were of sufficient quality to undergo semi-automated image

analysis. Poor image quality was primarily due to head motion artifacts. Brain extraction and

automated tissue segmentation were based on previously described methods [Kovacevic et al.

2002]. Images were co-registered to the T1-weighted image using the Automated Image

Registration package (AIR, v.5.2.3). T2/PD images were used collectively to extract brain and

subdural/ventricular CSF, then the masked T1 was segmented using a T1-based protocol

whereby local intensity histograms are fitted to four Gaussian curves to derive cut-offs for

classifying each voxel as white matter, grey matter, or cerebrospinal fluid (CSF) [Kovacevic et

al. 2002]. This is important for calculating the Total Intracranial Volume in correcting for head

size, especially in focal atrophy syndromes like CBS. The methods of Kovacevic et al. have been

updated and more details of the MRI image processing pipeline have been described [Ramirez et

al. 2011].

4.3.4.4.2 Post-hoc MRI analysis:

A post-hoc analysis was performed on the FWE- and FDR-corrected group statistical maps.

These two maps were outputted as masks and transformed into each participant‟s MRI T1-

weighted image space. Registering of the group SPECT SPM masked results into participant

coordinate space allowed for the characterization of tissue types that were defined by the group

SPM result. The proportion of grey matter, white matter, and CSF underlying the SPM masks

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was calculated for each patient. Mean and standard deviation values for each tissue class were

calculated across all patients to estimate the degree of atrophy underlying the SPM masks.

4.4 Results

4.4.1 CBS vs. controls

4.4.1.1 Demographic data

Demographic feature CBS

(n=31)

Controls

(n=31)

Gender 19 (61.3%) F

12 (38.7%) M

19F

12M

Handedness 29 (93.5%) R

2 (6.5%) L

29R

2L

Age of Onset (mean SEM years) 65.2 1.7 N/A

Age at Investigation (mean SEM years) 68.5 1.7 70.0 1.2

Duration of symptoms (mean SEM years) 3.3 0.4 N/A

Years of Education (mean SEM years)* 12.4 0.6 14.5 0.5

Initial body side most affected 16 (51.6%) R

15 (48.4%) L

N/A

Table 1. Demographics of patients with corticobasal syndrome (CBS) and control group.

F=female; M=male; R=right; L=left; SEM = standard error of mean; N/A = not applicable. * t(60) = -2.7, p = 0.008;

initial body side most affected was defined based on where most prominent motor symptoms were observed.

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4.4.1.2 Clinical features

Clinical characteristics Frequency (%) at

time of investigation

(N=31)

Frequency (%) at

follow-up

(N=31)

Extrapyramidal features

Rigidity (asymmetric) 28 (90.3%) 31 (100%)

Dystonia (asymmetric) 16 (51.6%) 18 (58.1%)

levodopa trial with poor

response*

13 (41.9%) 13 (41.9%)

Tremor – postural/action 8 (25.8%) 11 (35.6%)

Cortical features

Apraxia (asymmetric) 28 (90.3%) 31 (100%)

Cortical sensory loss 19 (61.3%) 19 (61.3%)

Alien-limb phenomenon 1 (3.2%) 3 (9.7%)

Limb levitation 7 (22.6%) 10 (32.3%)

Myoclonus 9 (29.0%) 13 (41.9%)

Early dementia 22 (71.0%) 22 (71.0%)

Language disturbance 24 (77.4%) 24 (77.4%) Table 2. Clinical characteristics of CBS sample.

* 13 patients had a trial of levodopa and all responded poorly based on clinical assessment. Average time for

emergence of additional signs on follow-up was 1.0 ± 0.3 years. All findings described above are based on clinical

examination.

Rigidity and IMA were asymmetric in the early stages of the disease and eventually occurred in

all patients. Early dementia was defined clinically according to DSM-IV criteria. Cortical

sensory loss in this study was defined by the presence of one or more of the following

abnormalities: extinction to double simultaneous tactile stimuli and/or astereognosis and/or

agraphesthesia. Limb levitation was distinguished from true alien limb phenomenon.

4.4.1.3 SPM and ROI SPECT analyses

Two methods, SPM and ROI analyses, were used to compare perfusion differences between all

CBS cases and controls, CBS cases with left-sided symptoms (CBS-L) vs. controls, and CBS

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cases with right-sided symptoms (CBS-R) vs. controls. See figure 1 and supplementary table 1,

which provides anatomical locations of the reduced perfusion and the statistical results.

Figure 1. Statistical parametric maps (SPM) of bilateral frontal, parietal and temporal surface regions of the

brain showing decreased perfusion in (A) all CBS cases compared to controls and (B) CBS cases with

predominant symptoms on their left side (CBS-L) compared to controls overlaid on brain MRI template.

N.B. Refer to Supplementary Table 1 for details of analysis and results. Green areas are corrected for multiple

testing using Family-Wise Error methods, while red areas are corrected using False Discovery Rate methods.

Areas of significantly reduced perfusion among CBS individuals compared to controls using ROI

and voxel-wise approaches were: bilateral dorsolateral prefrontal association cortices, bilateral

primary sensorimotor cortices, bilateral anterior cingulate regions, right superior and inferior

parietal lobules, left superior parietal lobule, right superior and middle temporal gyri, right

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fusiform gyrus and right insula. The left supramarginal gyrus ROI showed reduced perfusion in

CBS vs. controls in the individual t-test analysis, but when incorporated into the GLM

multivariate analysis, it showed a trend for significance (p=0.06). Three of these regions

remained significant after correcting the FWE: right middle frontal gyrus, left superior frontal

gyrus and left superior parietal lobule (Figure 1 and Supplementary Table 1). CBS patients with

predominant left-sided symptoms as compared to controls demonstrated reduced perfusion in the

same cortical regions as in the entire patient sample using the FDR correction except that these

regions lateralized mainly to the right hemisphere. Areas of reduced perfusion in CBS-L versus

controls were: superior and middle frontal gyri and post-central gyrus all lateralized to the right

hemisphere (FWE corrected; Supplementary Table 1 and Figure 1). An area of reduced perfusion

in CBS-R versus controls was the dorsal aspect of the left inferior frontal gyrus (ROI method).

When the stringency of the SPM analysis was reduced (uncorrected p-value <0.001), left

frontoparietal regions including the left inferior parietal lobule demonstrated reduced perfusion

in CBS-R vs. controls (data not shown). No significant areas of relative hyperperfusion were

observed in the CBS group.

4.4.1.4 CBS sample with praxis scores available

WAB praxis data were available on 87.1% (27/31) of the CBS patients. Severe dementia was the

reason for three CBS patients being unable to complete the WAB praxis task; MMSE scores

were 10 or less in these patients and they were unable to complete any other neuropsychological

tests as a result. One patient with an MMSE score of 24/30 and a DRS score of 99/144 was

unable to complete the WAB and most other tests due to poor effort secondary to severe apathy

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(apathy score on the Neuropsychiatric Inventory = 8) [Cummings 1997]. The body side most

affected by CBS symptoms and signs was right in 51.9% (14/27) of CBS patients and left in

48.1% (13/27; Table 3). Mean MMSE and total DRS scores were 23.2 ± 1.0 and 114.1 ± 1.2

(DRS cut-off for dementia in this age group = 123/144), respectively, suggesting that the CBS

patients were on average only mildly demented.

The mean WAB praxis scale total score of the CBS sample was 53.2 ± 1.6. There were no

statistically significant differences in mean WAB praxis scale total scores between those

presenting with their right side of the body most affected compared to those with the left side

most affected. Based on a normal control group matched for age and education obtained through

our longitudinal study, scores of greater than 57.1 are considered in the normal range (between 0

and -1.5 standard deviations [SD]), whereas scores of between 57.1 and 56.1 are considered

borderline apraxic (between -1.5 to -2 SD). Scores of less than or equal to 56.1 are considered in

the apraxic range; that is, -2 SD and below. Based on these cut-offs, 29.6% (8/27) of CBS

patients had no apraxia at the time of their initial investigation; 18.5% (5/27) had borderline

apraxia, while more than half (51.9%; 14/27) had clear IMA of varying severity. Importantly, all

patients eventually developed apraxia as their disease progressed over the longitudinal

observation period.

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Demographic variable CBS-APX (n=14) CBS-nAPX (n=13) Summary (n=27)

Gender 11 (78.6%) F

3 (21.4%) M

5 (38.5%) F

8 (61.5%) M

16 (59.3%) F

11 (40.7%) M


1 (7.1%)L

12 (92.3%) R

1 (7.7%) L

25 (92.6%) R

2 (7.4%) L

Site of recruitment 9 (64.3%) Cog

5 (35.7%) MD

8 (61.5%) Cog

5 (38.5%) MD

17 (63.0%) Cog

10 (37.0%) MD

Dementia vs. motor

onset

10 (71.4%) Dem

4 (28.6%) Motor

8 (61.5%) Dem

5 (38.5%) Motor

18 (66.7%) Dem

9 (33.3%) Motor

Age of Onset

(mean SEM years)

68.4 2.3

64.7 2.2

66.6 ± 1.6

Age at Investigation

(mean SEM years)

71.7 2.3

68.1 2.2

70 ± 1.6

Duration of symptoms

(mean SEM years)

3.4 0.6

3.4 0.5

3.4 ± 0.4

Years of Education

(mean SEM years)

12.1 0.5

12.8 0.1

12.4 ± 0.6

Body side most affected 7 (50.0%) R

7 (50.0%) L

7 (53.8%) R

6 (46.2%) L

14 (51.9%) R

13 (48.1%) L

Table 3. Demographic features of CBS presenting with apraxia (CBS-APX) vs. those

without significant apraxia (CBS-nAPX).

F = Female; M = Male; Cog = Cognitive Neurology Clinic; MD = Movement Disorders Clinic;

R = Right; L = Left; Dem = Dementia onset; Motor = Motor onset

4.4.1.5 Comparison of apraxic to borderline/non-apraxic CBS patients: Neuropsychological and

SPECT analysis

The CBS group was stratified into 1) those with apraxia and 2) those with borderline/no apraxia.

There were no significant differences in demographic features between CBS patients with

apraxia and those with borderline or no apraxia (Table 3). There were also no significant

differences in any of the clinical features between the two groups (data not shown). Table 4

compares neuropsychological, neuropsychiatric and functional measures between the two

groups. Mean MMSE scores were slightly lower in the apraxic vs. borderline/non-apraxic group

(21.2 ± 1.6 vs. 25.3 ± 1.1, respectively; t(25) = -2.1, p = 0.04). However, no significant

differences were observed on the mean DRS scores indicating that the groups did not differ

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significantly in terms of dementia severity. Mean scores on the delayed visual reproduction were

also worse in the apraxic vs. borderline/non-apraxic group (5.3 ± 1.9 vs. 14.4 ± 3.4, respectively;

t(15) = -2.4, p = 0.03 adjusted for unequal variances). A similar finding was observed for the

mean scores on the immediate visual reproduction task (apraxic: 12.9 ± 3.0 vs. non-apraxic: 22.6

± 3.0; t(17) = -2.2, p = 0.04). There was a good correlation noted between WAB praxis and

immediate visual reproduction scores (Pearson r=0.50, p=0.03) suggesting that the degree of

apraxia may account for some of the variance in this relationship. However, there was no

correlation observed between scores on the delayed visual reproduction and the WAB praxis

scale (Pearson r=0.32, p=0.17) suggesting that this association was mostly independent of degree

of motor impairment. Another significant difference between the apraxic and non-apraxic groups

was in the Benton Judgement of Line Orientation test (7.3 ± 2.9 vs. 18.6 ± 3.2, respectively;

t(19) = -2.6, p = 0.02) suggesting more right parieto-occipital involvement in the group with

apraxia.

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Psychometric Measures CBS-APX (n) CBS-nAPX (n)

General cognition

MMSE /30 [n=27]* 21.2 ± 1.6 (14) 25.3 ± 1.1 (13)

Clock total /10 [n=8] 6.3 ± 1.5 (4) 7.5 ± 1.3 (4)

MDRS /144 [n=25] 105.9 ± 6.0 (12) 121.6 ± 5.2 (13)

NART /127.8 [n=19] 108.4 ± 2.6 (8) 106.8 ± 2.9 (11)

Raven‟s Progressive Matrices [n=22] 19.6 ± 1.3 (10) 23.8 ± 2.6 (12)

Memory

CVLT Long Delay Free Recall /16 [n=21] 6.2 ± 0.8 (9) 6.8 ± 0.9 (12)

Delayed Visual Reproduction /41 [n=19]* 5.3 ± 1.9 (8) 14.4 ± 3.4 (11)

Language

WAB total /100 [n=23]* 81.1 ± 3.8 (13) 91.8 ± 1.9 (10)

WAB content /10 7.7 ± 0.5 8.8 ± 0.3

WAB fluency /10 7.8 ± 0.6 9.1 ± 0.2

WAB comprehension /10* 8.7 ± 0.3 9.8 ± 0.1

WAB repetition /10 8.4 ± 0.4 9.4 ± 0.2

WAB naming /10* 8.0 ± 0.3 8.9 ± 0.3

Boston Naming /30 [n=22] 23.8 ± 1.6 (10) 24.2 ± 1.4 (12)

Semantic Fluency /20 [n=26]* 7.4 ± 1.0 (14) 12.6 ± 2.2 (12)

Praxis

WAB praxis /60 [n=27] ¥ 48.7 ± 2.7 (14) 58.0 ± 0.3 (13)


Digit span - forward /12 [n=23] 7.1 ± 0.9 (11) 7.0 ± 0.8 (12)

Digit span - backward /12 [n=23] 4.6 ± 1.0 (11) 4.5 ± 0.8 (12)


Rey Osterieth Complex Figure – Copy /36 [n=20] 12.6 ± 4.1 (9) 20.5 ± 4.1 (11)

Benton Line Orientation /30 [n=21]* 7.3 ± 2.9 (10) 18.6 ± 3.2 (11)

Executive functions

Phonemic fluency (FAS) [n=21] 14.2 ± 2.3 (10) 23.6 ± 4.4 (11)

Trail Making Test A (time in seconds) [n=19] 133.4 ± 27.2 (8) 90.8 ± 14.7 (11)

Trail Making Test B (time in seconds) [n=13] 206.0 ± 43.4 (4) 218.2 ± 55.6 (9)

WCST categories /6 [n=22] 1.6 ± 0.4 (9) 2.0 ± 0.4 (13)

WCST perseverative errors [n=22] 16.9 ± 6.3 (9) 8.2 ± 1.9 (13)

Neuropsychiatric features

Neuropsychiatric Inventory – Total /144 [n=25] 11.8 ± 3.2 (12) 7.9 ± 2.3 (13)

Cornell Depression Scale (%) [n=26] 25.6 ± 4.7 (13) 19.0 ± 2.9 (13)

Functional measures

Disability Assessment for Dementia (DAD; %) [n=26] 70.4 ± 7.7 (13) 79.4 ± 6.8 (13)

DAD-Activities of Daily Living (%) 81.1 ± 8.0 86.8 ± 6.3

DAD-Instrumental Activities of Daily Living (%) 65.2 ± 9.3 73.9 ± 8.1 Table 4. Mean scores (± SEM) on neuropsychological, neuropsychiatric and functional measures in CBS

presenting with apraxia (CBS-APX) vs. those without significant apraxia (CBS-nAPX).

The number of patients (n) tested is listed next to individual measures. Missing data is secondary to the inability of

the patient/caregiver to complete the test. MMSE = Folstein‟s Mini-Mental State Exam; NART = National Adult

Reading Test; MDRS = Mattis Dementia Rating Scale; CVLT = California Verbal Learning Test; WAB = Western

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Aphasia Battery; FAS = F-, A-, and S-phonemic fluency; WCST = Wisconsin Card Sort Test. Independent samples

t-tests were used to compare MMSE, NART, Clock, MDRS, Boston naming, semantic fluency, visual reproduction,

forward and backward digit span, CVLT, Benton, Trails A and B mean scores between groups. Mann Whitney U

test was used to compare scores on FAS, WAB, Rey and WCST between groups. *p≤0.05; ≦≤0.005

The Western Aphasia Battery (WAB) praxis scores were correlated with WAB total scores

(Spearman‟s rho = 0.52, p<0.01). Furthermore, WAB total scores were significantly lower in the

apraxic compared to the non-apraxic group (81.1 ± 3.8 vs. 91.8 ± 1.9, respectively; Mann-

Whitney U test, p = 0.05; Table 4). This may account for the reduced MMSE scores in the

apraxic group since MMSE is heavily weighted towards language function. In support of this, a

strong correlation was observed between the WAB total and MMSE scores (Spearman‟s rho =

0.77, p<0.0005). Although all WAB subscores tended to be lower in the apraxic group, the

comprehension and naming subscores were significantly worse (Table 4). Figure 2 demonstrates

that CBS patients with apraxia tended to have more severe aphasic disturbances than those

without apraxia consistent with the mean WAB total score differences between the groups. Mean

semantic fluency scores were also lower in the apraxic vs. non-apraxic group (7.4 ± 1.0 vs. 12.6

± 2.2, respectively; t(16) = -2.2, p = 0.05; Table 4). With respect to the SPECT perfusion data, no

significant differences were observed between the apraxic and non-apraxic groups, after

correcting for multiple comparisons.

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Figure 2. Frequency of different aphasia categories on the Western Aphasia Battery (WAB) distributed

according to the CBS group with apraxia versus those with borderline/no apraxia.

4.4.1.6 Perfusion versus ideomotor apraxia

The SPECT scans were, on average, acquired within 3.9 ± 1.4 weeks of the neuropsychological

assessment including the WAB praxis measurement. Severity of IMA was positively correlated

with perfusion in the left inferior parietal lobule, including the left angular gyrus (i.e., WAB

praxis scores decrease as perfusion decreases). This was seen on the FWE- and FDR-corrected

maps shown in Figure 3 (see Table 5 for details). There were no negative correlations or areas of

relative hyperperfusion observed in association with the praxis measure. The use of the „body

side most affected‟ covariate did not significantly change the results.

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Anatomical locus

(Brodmann area)

Talairach

Coordinates

No. of

voxels

SPM t-score

(p-value)

ROI

x y z

Parietal region – SPM (FWE-corr)

Left angular gyrus (39) -42 -70 31 46 5.7 (p=0.02) Yes

Left inferior parietal lobule (40) -50 -52 47 59 5.7 (p=0.03) Yes

Parietal region – SPM (FDR-corr)

Left inferior parietal lobule (39) -44 -64 38 632 5.3 (p=0.01) Yes Table 5. Areas of hypoperfusion on SPECT in the CBS group that correlate with WAB praxis scores in the

regression analyses.

„Body side most affected‟ was included as a covariate in both Statistical Parametric Mapping (SPM) and Region of

Interest (ROI) analyses. All p-values were corrected for multiple testing in SPM analysis using Family Wise Error

(FWE-corr) and False Discovery Rate (FDR-corr), and in ROI analysis within a general linear model. Column

denoted ROI refers to overlapping regions of decreased perfusion between the SPM and ROI analyses.

Figure 3. Statistical parametric map of surface regions of the brain showing decreased perfusion in the left inferior parietal region, including the

angular gyrus, that correlate with WAB praxis scores in the regression analyses.

These have been overlaid on the „Collin‟ brain MRI template. Red areas are corrected for multiple testing using the False Discovery Rate, while green areas are

corrected using the more conservative Family Wise Error method. Refer to text and Table 5 for details of analysis and results

ROI analysis was also performed to serve as an independent confirmation of the SPM result.

Pearson correlation analysis revealed a significant positive correlation between perfusion in the

left inferior parietal ROI (comprised of left angular and supramarginal gyri) and WAB praxis

scores (r = 0.64, p < 0.001). To gauge the independent contribution of perfusion within the left

inferior parietal region in predicting WAB praxis scores and to control for the potential bias of

symptom lateralization in CBS, we conducted a hierarchical regression analysis with stepwise

variable entry. In this model, perfusion in the left inferior parietal region and „most affected side

of body‟ served as the independent predictors. WAB praxis scores represented the dependent

variable. These results were similar to the SPM analysis. Specifically, reduced perfusion in the

left inferior parietal region significantly predicted reduced performance on the WAB praxis scale

accounting for 42% of the variance in the relationship (F[1, 25] = 17.7, R2 = 0.42, p < 0.001).

„Body side most affected‟ did not enter into the overall model as significant.

To further explore the relationship between perfusion and praxis performance, separate SPM

regression analyses were conducted using individual subscores on the WAB praxis scale. No

individual WAB praxis subscores, including intransitive, transitive, bucco-facial, or complex

gestures, correlated with hypoperfusion or hyperperfusion after correction for multiple testing. In

an exploratory analysis set out to better delineate neural components of the praxis network, the

stringency of the SPM analyses was subsequently reduced by setting the threshold voxel level p-

value to <0.001, uncorrected. Intransitive gesture scores demonstrated correlation with reduced

perfusion in more posterior regions including the left inferior occipital, fusiform, and lingual

gyri, as well as the left and right superior parietal lobules. Transitive gesture scores were

associated with reduced perfusion in angular and supramarginal gyri, the superior parietal lobule,

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the precentral and postcentral gyri, and the inferior occipital gyrus all located only within the left

hemisphere. Complex gesture scores were correlated with the same left posterior regions as in

the transitive gesture regression; however, more left anterior regions demonstrated reduced

perfusion including the superior and middle frontal gyri. Finally, bucco-facial gestures were

correlated with hypoperfusion in the left inferior and middle frontal gyri as well as the left

precentral gyrus.

4.4.1.7 Post-hoc atrophy analysis

The average tissue type in the FWE-corrected SPM mask was found to be: 50% in white matter,

37% in grey matter, and 13% in CSF. Similar results were obtained with the FDR-corrected SPM

mask (white matter: 51%; grey matter: 35%; and CSF: 14%). Please refer to Supplementary

Figure 1.

4.5 Discussion

To our knowledge, this is the first brain SPECT study demonstrating that perfusion in the left

inferior parietal lobule is significantly correlated with severity of IMA in CBS. This result was

identified using a whole brain voxel-by-voxel SPM regression analysis that accounted for

multiple comparisons and was corroborated by a region of interest linear regression analysis.

Left inferior parietal atrophy, that is, the effect of partial volume averaging, is unlikely to be a

major contributor to this result based on the estimate that approximately 85% of the tissue

underlying the hypoperfused region was classified as brain parenchyma.

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Several models of apraxia have emerged in the literature based on original case studies and

series, and the majority of these implicate a role of the left parietal lobe [Geschwind

1975;Goldenberg 2009;Heilman and Rothi 1993;Liepmann 1920;Roy 1996]. Several animal and

human studies have attempted to identify the underlying neural substrates of IMA.

Neuroanatomical and electrophysiological studies in monkeys demonstrate the importance of the

parietofrontal circuit in transforming visual and tactile sensory information into knowledge for

limb movements [Leiguarda and Marsden 2000]. A functional MRI study of pantomiming use of

tools in healthy adults implicated the dominant, left intraparietal and dorsolateral frontal cortices

suggesting that these regions may be important in determining ideomotor praxis [Moll et al.

2000]. Lesional studies also confirm the role of the left hemisphere in apraxia, in particular the

inferior parietal and premotor/supplementary motor areas [Gross and Grossman 2008;Leiguarda

and Marsden 2000;Stamenova et al. 2009]. The majority of these studies have included patients

with strokes and CBS [Buxbaum et al. 2007;Goldenberg and Spatt 2009;Jacobs et al.

1999;Kertesz and Ferro 1984].

The main types of limb apraxia identified in CBS are IMA, limb-kinetic apraxia and, less often,

conceptual/ideational apraxia [Gross and Grossman 2008;Stamenova et al. 2009;Zadikoff and

Lang 2005]. Limb-kinetic apraxia (LKA; loss of hand and finger dexterity resulting in a

breakdown and awkwardness of distal movements) [Kleist 1907] is thought to reflect sensory-

motor control dysfunction [Liepmann 1920]. In CBS and in one study of pathologically-proven

corticobasal degeneration (CBD), it has been associated with involvement of the ventral

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premotor cortex bilaterally, although worse on the side contralateral to the LKA deficit

[Leiguarda et al. 2003;Tsuchiya et al. 1997;Zadikoff and Lang 2005]. Conceptual/ideational

apraxia, defined in this paper as impairment in object/tool or action knowledge, has been less

well studied in CBS [Stamenova et al. 2009]. We summarized data across nine studies that

looked for both ideomotor and conceptual/ideational apraxia in CBS, and approximately 27%

(30/112) of CBS patients demonstrated a conceptual deficit [Chainay and Humphreys

2003;Graham et al. 1999;Jacobs et al. 1999;Kertesz et al. 2000b;Leiguarda et al. 1994;Pillon et

al. 1995;Soliveri et al. 2005;Spatt et al. 2002;Stamenova et al. 2011]. There was a high degree of

variability in the occurrence of conceptual/ideational apraxia with several studies demonstrating

no conceptual deficit [Chainay and Humphreys 2003;Graham et al. 1999;Jacobs et al.

1999;Pillon et al. 1995;Soliveri et al. 2005;Stamenova et al. 2011], while three studies

demonstrated frequencies ranging from 30% to 60% [Kertesz et al. 2000b;Leiguarda et al.

1994;Spatt et al. 2002]. It is likely that these discrepancies across the studies occurred as a result

of differences in definition of this apraxia type, diagnostic heterogeneity, and/or methodological

differences in the assessments utilized. In terms of anatomical localization, conceptual

knowledge of tool use and action has been suggested to reside in the left inferior parietal lobule

[Heilman et al. 1982], and this was later shown to be restricted to mechanical knowledge

[Ochipa et al. 1992]. In contrast, semantic knowledge on the prototypical use of tools has been

shown to localize to the left temporal lobe [Hodges et al. 1999].

More recently, two studies have directly correlated structural changes on MRI to standardized

measures of ideomotor praxis in CBS [Borroni et al. 2008b;Huey et al. 2009b]. In 20 patients

with CBS, the first of these studies demonstrated a significant positive correlation between total

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score on the de Renzi test of praxis and grey matter density in the bilateral parietal operculum

[Borroni et al. 2008b]. Using a pre-specified hypothesis, they also found that total scores

positively correlated with fractional anisotropy in the left dorsolateral parietofrontal associative

fibers on diffusion tensor imaging [Borroni et al. 2008b]. A smaller study of 16 patients with

progressive non-fluent aphasia including three with CBS found that limb apraxia as assessed by

the Apraxia Battery for Adults-2 (ABA-2) correlated with loss of gray matter volume in the left

inferior parietal lobe [Rohrer et al. 2010b]. Notwithstanding important differences between these

studies and our current one (e.g., imaging modalities, praxis assessment tools, and diagnostic

heterogeneity), consistent findings are that the left hemisphere is invariably involved in IMA and

that the majority of studies identify the dominant inferior parietal lobule as an important

neuroanatomical correlate. From our structural MRI analysis, approximately 50% of the tissue

underlying the hypoperfused left inferior parietal region was white matter while approximately

35% was grey matter. These findings support those of Borroni et al. [Borroni et al. 2008b]

suggesting the importance of underlying white matter disease (either perfusion abnormalities or

loss of white matter tract integrity) as a potential contributor to IMA in CBS. The current finding

is also consistent with contemporary theories of apraxia previously described as well as a

correlational study using the subtraction method of lesion overlap in stroke, in which the critical

area of overlap in apraxic compared to non-apraxic patients was in the centrum semiovale deep

to the parietal cortex including the long association tracts, such as the superior longitudinal and

frontal occipital fasciculi [Roy et al. 1998].

Given that a deficit in tool or action knowledge (i.e., ideational/conceptual apraxia) is an

uncommon finding in CBS, why do our results demonstrate such a strong association between

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hypoperfusion in the left inferior parietal lobule where the purported „praxicons‟ are thought to

reside, and IMA as assessed by the WAB praxis scale? The reason, in part, may be the result of

what the WAB praxis assessment tool is actually evaluating. Although the total score on the

WAB praxis scale best represents severity of IMA, the score in patients with more severe

impairment is partly accounted for by failure of actual tool use, which reflects a conceptual

deficit. The definition of conceptual/ideational apraxia has also been variable from study to study

resulting in some degree of phenomenological/taxonomic confusion. Some studies distinguish

between ideational apraxia defined as a failure to sequence tasks related to tool use correctly and

“conceptual apraxia” defined (as in this paper) as a loss of knowledge relating to tool and action

use. Given the common feature of tool use across different assessments of apraxia (ideomotor,

conceptual and ideational), it is likely that there will be some degree of overlap in the brain

regions most correlated with deficits across the studies.

An alternative way of putting our finding into context is to explain the association of left inferior

parietal lobule hypoperfusion with IMA in CBS as being related to the dysfunction of a larger

circuit or network that is involved in determining both simple and more complex gestural

movements. Indeed, when we reduce the stringency of our analysis and examine different

subcomponents of the WAB praxis assessment, a larger network emerges. For example,

performance of transitive gestures correlate with hypoperfusion predominantly in the entire left

parietal lobe (inferior and superior divisions) as well as within the left sensorimotor cortex. In

examining complex gestures, the same regions are implicated; however, hypoperfusion extends

into the left premotor and supplementary motor areas as well. These results suggest that

performance of transitive and more complex gestures is more strongly linked to left hemispheric

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function [Kroliczak and Frey 2009]. Based on our hypoperfusion results in this CBS sample, it is

plausible that as gestures performed by healthy individuals increase in complexity more of the

left parietofrontal network is recruited to carry out the task. Although the reduced stringency of

this SPECT-WAB praxis subscore regression analysis increases the chances that the more

extensive area shown is a false positive result, prior studies have shown that it is acceptable to

use an uncorrected p-value of <0.005 in correlational analyses with SPM in a sample of this size

[Desgranges et al. 1998;Kas et al. 2011].

A large voxel-based morphometry study of 48 patients with CBS demonstrated that gray matter

volume loss in the left middle frontal and precentral gyri as well as the left caudate nucleus

correlated with reduced performance on the Test of Oral and Limb Apraxia (TOLA) [Huey et al.

2009b]. To some extent, these data are at odds with the results of the current study and we

propose as one possible explanation for this discrepancy that atrophy on MRI in the left frontal-

subcortical grey matter and hypoperfusion in the left inferior parietal lobule may each account

for unique variance associated with IMA severity in CBS. We did not look at volumetric

correlations and Huey et al. [Huey et al. 2009b] did not examine perfusion/metabolism so further

studies will be necessary to confirm this hypothesis. Another possible explanation for these

discrepant findings is diagnostic heterogeneity between the two studies. A prior study has shown

that patients with posterior lesions or fluent aphasia have a more severe form of apraxia –

including both ideomotor and conceptual/ideational types – than patients presenting with more

anterior lesions or non-fluent aphasia [Heilman et al. 1982]. Since about 67% of our sample

presented with early cognitive problems with the apraxic group having lower scores on the WAB

and more severe forms of aphasia compared to the borderline/non-apraxic group, then this might

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be one possible explanation for the discrepant findings. Although Huey et al. [Huey et al. 2009b]

did not specify the proportion of their sample presenting with early cognitive symptoms or

aphasia, scores on the Mattis DRS were comparable (Huey et al.: 116/144 vs. current: 114/144)

indicating a similar degree of global cognitive impairment in the two patient samples. Huey et al.

[Huey et al. 2009b] did not include any specific assessments of aphasia so we were not able to

compare the samples in this regard. The most affected side of the body was also similar between

samples as well (Huey et al.: 46% right-sided vs. current: 52% right-sided).

We further subdivided our sample into those with and without significant apraxia based on a -2

SD cut-off from controls (i.e. WAB praxis score ≤ 56.1). Our WAB praxis cut-off score was

higher than that of ≤ 49.7 used in one of the earliest studies of IMA in stroke patients [Kertesz

and Ferro 1984]. The control group in this original study was derived from non-brain injured

hospitalized patients, whereas our control group were healthy, elderly volunteers who were

living in the community. It is conceivable that the „non-brain‟ medical conditions or drug

therapies of the control group of Kertesz & Ferro [Kertesz and Ferro 1984] might have affected

their overall cognitive performance on the WAB praxis task.

In our stratified analysis, several interesting observations were made. As expected, given that

both language and praxis are most often lateralized to the dominant left hemisphere and are

represented in overlapping neuroanatomical networks, apraxic patients demonstrated

significantly lower WAB total scores and had more severe forms of aphasia than the

borderline/non-apraxic group. Comprehension difficulties and anomia were significantly more

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pronounced in the apraxic group in addition to an observed reduction in semantic fluency. This

suggests more left temporal lobe involvement in the apraxic group. Visuospatial orientation and

attention were also significantly worse in the apraxic group as evidenced by lower Benton

judgement of line orientation scores indicating more prominent right parieto-occipital

dysfunction. We have previously shown that performance on the Benton line orientation task

correlated with reduced perfusion of the right parietal lobe in Alzheimer‟s disease of varying

severity [Tippett and Black 2008]. The parietal lobes are prominently affected by the underlying

pathology in CBS [Wadia and Lang 2007] and although the neurodegenerative process usually

starts asymmetrically, it progresses relentlessly to involve bilateral structures. Indeed in the

current study, there was a significant correlation between performance on the WAB praxis and

Benton line orientation task with even stronger correlations observed between perfusion of left

and right parietal regions (data not shown) supporting our finding.

An unexpected finding was that the apraxic group demonstrated lower scores on the Wechsler

Memory Scale-Revised (WMS-R) delayed visual reproduction task than the non-apraxic group.

This finding could not be accounted for by the severity of the apraxia alone. In humans and in

monkeys, two pathways have been identified for the processing of visual information: the

occipitotemporal pathway or ventral stream and the occipitoparietal pathway or dorsal stream

[Ungerleider et al. 1998]. The ventral visual stream is important for object vision including

characteristics such as pattern, shape and colour, while the dorsal visual stream is important for

spatial perception (e.g., judging distance and orientation of objects relative to each other) and

also is involved in visually guided reaching [Goodale and Milner 1992;Ungerleider et al. 1998].

The visual reproduction task asks subjects to examine four drawings of several geometric figures

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oriented in space in relation to each other, each for ten seconds. After a ten second (immediate

visual reproduction) and 30 minute delay (delayed visual reproduction) they are asked to draw

the four pictures from memory. Scoring is based on the ability to accurately recall the shapes and

patterns as well as the distance and orientation in relation to each other thereby taxing both the

ventral and dorsal visual streams, respectively [Lezak 1983;Wechsler 1987]. A BOLD fMRI

study in healthy volunteers demonstrated that activity in the posterior parietal cortex bilaterally is

strongly correlated with the capacity limit to store visual information (i.e., visual short-term

memory) [Todd and Marois 2004]. We hypothesize that the poor performance of the apraxic

group on delayed visual reproduction may be due to involvement of the posterior parietal cortex

within the dorsal visual stream, a network which also overlaps with the frontoparietal praxis

system.

In the SPECT analyses of all CBS cases versus controls, there was reduced perfusion noted in

bilateral dorsolateral and medial frontal/prefrontal regions, as well as bilateral parietal regions in

the CBS group. Additionally, reduced perfusion was also evident to a lesser degree in right

temporal regions and insula. Our results confirm in a larger sample, previous SPECT studies of

CBS demonstrating reduced perfusion in frontoparietotemporal regions [Hossain et al.

2003;Koyama et al. 2007;Markus et al. 1995;Okuda et al. 1999;Okuda et al. 2000b;Zhang et al.

2001]. We further investigated whether perfusion reductions tended to be lateralized opposite to

the most affected side of the body by comparing CBS-L and CBS-R to their respective control

groups. In CBS-L, the regions of hypoperfusion localized to the right hemisphere including

dorsolateral prefrontal cortex, primary somatosensory cortex, superior parietal lobe and some

temporal regions. In contrast, the CBS-R group demonstrated reduced perfusion only in the left

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inferior frontal gyrus in the ROI analysis. However, when the stringency of the SPM analysis

was lowered, hypoperfusion in the left frontoparietal region was seen in the CBS-R group.

Overall, then, our study confirms a lateralization of perfusion defects contralateral to the most

affected side of the body in CBS.

Strengths of the present study include ascertainment of CBS cases from both cognitive and

movement disorders clinic, use of standardized neuropsychological assessments including a

language battery, use of brain SPECT perfusion that attempted to account for effects of

underlying atrophy on MRI, and the combined approach of an unbiased, whole brain voxel-by-

voxel analysis followed by confirmation using a more robust region of interest method. Although

the CBS sample size was relatively large considering the rarity of this diagnosis, from a

statistical perspective it is indeed a small sample. Although we did not have pathological

confirmation of CBD diagnosis on the entire sample, 25% of the sample came to autopsy with

pathological confirmation of CBD in 63% of cases (5/8 cases; unpublished data); this rate of

diagnostic accuracy is similar to prior studies [Wadia and Lang 2007]. Other pathologies

included PSP (12.5%; 1/8 cases), FTLD-U/TDP43 proteinopathy (12.5%; 1/8 cases), and

combined dementia with agyrophilic grains, CBD and cerebral amyloid angiopathy

(AGD/CBD/CAA; 12.5%; 1/8 cases). Other limitations include a cross-sectional design and

assessment of predominantly IMA. Furthermore, we only had volumetric MRI data on 21 of the

27 patients who had WAB and SPECT completed. Therefore, we were unable to estimate the

degree of atrophy underlying the SPM mask in these six patients. However, qualitative visual

examination of their MRI data did not reveal any tendency for the apraxic subgroup to have more

left parietal atrophy than the non-apraxic subgroup. Another limitation is that the WAB praxis

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measure does not provide a full picture of the nature of the disruption to limb praxis since it

confounds pantomime, imitation and tool use. The same limitation also applies to the ABA-2 and

de Renzi apraxia tests used in the other studies [Borroni et al. 2008b;Rohrer et al. 2010b].

This study suggests that severity of left inferior parietal lobule hypoperfusion corresponds to

IMA as it becomes more severely affected in CBS supporting a central role for this structure in

the dominant hemisphere frontoparietal praxis network. Dysfunction in language, visuospatial

and visual memory performance is more frequent in CBS patients with apraxia due to

involvement of overlapping brain networks that subserve these related higher cognitive

processes. Future work will involve use of a comprehensive assessment of apraxia using a

conceptual model [Stamenova et al. 2011] together with SPECT and MRI imaging modalities in

order to better identify the neuroanatomical correlates of the different apraxia types.

4.6 Acknowledgements

This work was supported by an operating grant from the Canadian Institutes of Health Research

[MT13129 to S.E.B.] and a New Investigator Award from the Parkinson Society Canada [2011-

19 to M.M.]. M.M. was supported by a Canadian Institutes of Health Research Clinician

Scientist Award. We thank the patients and their families for the time and effort that they

committed to participate in this study.

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Supplementary Figure 1A. Mean proportion of different MRI tissue classes underlying the FWE-corrected

SPM mask.

Error bars denote standard deviation.

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Supplementary Figure 1B. Mean proportion of different MRI tissue classes underlying the FDR-corrected

SPM mask.

Error bars denote standard deviation.

Group Anatomical locus

(Brodmann area)

Talairach

Coordinates

No. of

voxels

SPM t-score

(p-value) or

x Y z ROI F-score

(p-value)

CBS-all Frontal regions – SPM

vs. Right middle frontal gyrus (6) 48 8 44 10972 5.7 (p=0.003)

controls Right superior frontal gyrus (10) 30 56 23 184 4.2 (p=0.005)

Right inferior frontal gyrus (47) 44 17 -3 225 4.0 (p=0.006)

Left superior frontal gyrus (6) -18 9 68 10972 5.3 (p=0.003)


Left precentral gyrus (44) -52 12 3 100 3.7 (p=0.007)

Frontal regions – ROI

Left middle frontal gyrus - dorsal - - - 1740 3.5 (p=0.04)

Left inferior frontal gyrus - dorsal - - - 1105 8.0 (p=0.001)

Left anterior cingulate - middle - - - 623 4.8 (p=0.01)

Right precentral gyrus - - - 2723 3.4 (p=0.04)

Right inferior frontal gyrus - dorsal - - - 1128 5.8 (p=0.005)

Parietal regions – SPM

Left superior parietal lobule (7) -32 -55 60 10972 5.0 (p=0.003)

Right postcentral gyrus (2) 50 -25 42 1246 4.6 (p=0.004)

Right inferior parietal lobule (40) 65 -24 29 1246 4.0 (p=0.006)

Right angular gyrus (39) 50 -74 33 57 3.8 (p=0.007)

Parietal regions – ROI

Left postcentral gyrus - - - 2675 3.4 (p=0.04)

Right superior parietal lobule - - - 2132 3.3 (p=0.05)

Right supramarginal gyrus - - - 1295 3.3 (p=0.05)

Limbic regions – SPM

Right cingulate (24) 4 -8 41 107 3.9 (p=0.006)

8 8 35 107 3.4 (p=0.01)

Left cingulate (24) 0 -12 41 52 3.7 (p=0.007)

Temporal regions – SPM

Right middle temporal gyrus (21) 71 -45 2 138 3.8 (p=0.006)

Right fusiform gyrus (37) 42 -44 -15 34 3.7 (p=0.007)

Temporal regions – ROI

Right superior temporal gyrus lateral - - - 1473 3.5 (p=0.04)

Other regions – ROI

Right insula - - - 1973 4.9 (p=0.01)

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CBS-L Frontal regions – SPM

vs. Right superior frontal gyrus (6) 28 -3 67 2331 5.4 (p=0.008)

controls Right middle frontal gyrus (6) 48 8 44 2331 5.7 (p=0.008)

Right inferior frontal gyrus (47) 46 17 -1 1300 5.3 (p=0.008)


Frontal regions - ROI

Right superior frontal gyrus - dorsal - - - 1984 3.4 (p=0.05)

Right precentral gyrus - - - 2723 7.3 (p=0.003)

Left anterior cingulate - middle - - - 623 4.0 (p=0.03)

Parietal regions - SPM



Right superior parietal lobule (7) 24 -71 57 192 4.7 (p=0.008)

Temporal regions - SPM

Right transverse temporal gyrus (41) 50 -23 12 1300 4.8 (p=0.008)

Right superior temporal gyrus (22) 59 -6 4 1300 4.6 (p=0.008)

Temporal regions - ROI

Right middle temporal gyrus lateral - - - 1962 4.5 (p=0.02)

Limbic – ROI

Right insula - - - 1973 6.0 (p=0.007)

CBS-R Frontal regions – ROI

vs. Left inferior frontal gyrus - dorsal - - - 1105 6.2 (p=0.006)

controls Supplementary Table 1. Areas of hypoperfusion on SPECT in all CBS patients, CBS with left side of body

most affected, and CBS with right side of body most affected relative to controls.

CBS-all; n=31 cases and 31 matched, normal controls; refer to Figure 2A

CBS-L; n=15 cases and 15 matched, normal controls; refer to Figure 2B

CBS-R; n=16 cases and 16 matched, normal controls

„Years of education‟ was included as a covariate in both Statistical Parametric Mapping (SPM) and Region of

Interest (ROI) analyses. All p-values were corrected for multiple testing in SPM analysis using False Discovery Rate

(FDR), or included in ROI analysis within a general linear model multivariate ANCOVA.

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5.0 Clinical, neuropsychological, MRI and SPECT

characterization of a prospective sample of patients with

corticobasal syndrome

Mario Masellis, Philip L. Francis, Isabelle Guimont, Wendy Galpern, Juan Bilbao, Kie Honjo,

Fuqiang Gao1, Gregory Szilagyi, Farrell Leibovitch, James L. Kennedy, Galit Kleiner-Fisman,

Lisa Ehrlich, Robert Chen, Anthony E. Lang, Sandra E. Black



Philip Francis assisted with the SPM analysis of SPECT data and Kie Honjo assisted with an

independent visual read of the MRI data. Wendy R. Galpern, Galit Kleiner-Fisman and Anthony

E. Lang assessed and collected clinical data on patients ascertained from a movement disorders

clinic. Juan Bilbao performed the neuropathological analysis.

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5.1 Abstract

Corticobasal syndrome (CBS) is a rare and debilitating syndrome characterized by the unique

combination of lateralized cortical and extrapyramidal features that occurs due to a variety of

underlying neurodegenerative pathologies. In this paper, we describe the initial

neuropsychological, MRI and SPECT imaging profile of a prospective series of 31 consecutive

CBS patients ascertained from a movement disorders and a cognitive neurology clinic. The

sample was stratified into CBS presenting with early dementia (CBS-D; n=22) vs. early motor

features (CBS-M; n=9), which identified that CBS-M had a higher occurrence of cortical sensory

loss than CBS-D (100% vs. 45.5%, respectively; p=0.005). Conversely, the presence of aphasia,

as determined by the Western Aphasia Battery, was found to be more common and severe in

CBS-D compared to CBS-M (88.2% vs. 33.3%, respectively; p=0.02). These findings are

associated with lateralization of the motor signs to the right side in CBS-D. CBS-D also

demonstrated more difficulties with simple attention span and visuospatial orientation/attention

on neuropsychological testing. Atrophy patterns on MRI did not distinguish between CBS-D and

CBS-M. However, CBS-M patients had significantly reduced perfusion in the right

supplementary and premotor areas compared to CBS-D (p<0.05). A subset of eight patients was

followed to autopsy with 7 patients having a tauopathy and 1 patient exhibiting non-tau

pathology, specifically, frontotemporal lobar degeneration-ubiquitin/TDP43 proteinopathy

(FTLD-U/TDP43). Atrophy and white matter changes on MRI correlated with the burden of

underlying brain pathology. This study emphasizes the importance of performing detailed

clinical and multimodal phenotyping to characterize heterogeneity in CBS. It also provides new

insights into the neuropsychological and neuroimaging correlates of lateralized brain dysfunction

in the syndrome.

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5.2 Introduction

The first description of corticobasal syndrome (CBS) was in 1967 by Rebeiz and colleagues who

later characterized three cases of this syndrome from the clinical and pathological perspective

[Rebeiz et al. 1967;Rebeiz et al. 1968]. The scientific literature on this topic was sparse until the

late 1980s and early 1990s during which there were several case series published characterizing

the unique clinical features of CBS [Gibb et al. 1989;Mahapatra et al. 2004;Riley et al.

1990;Rinne et al. 1994]. Since then, much has been learned about the clinical, neuroimaging,

genetic and pathological heterogeneity of this enigmatic disorder.

The clinical diagnosis of CBS is made based on an insidious onset and progressive neurological

decline including at least one cortical (e.g., apraxia, non-fluent aphasia/apraxia of speech,

cortical sensory loss, myoclonus, alien limb phenomenon) and one extrapyramidal feature (e.g.,

rigidity, dystonia), which is not attributable to any other identifiable cause of brain dysfunction

[Boeve et al. 2003]. However, there have been no formally accepted, consensus clinical

diagnostic criteria [Mahapatra et al. 2004]. There are two main early clinical presentations of

CBS. The first is the “classical” perceptuo-motor disorder without early dementia, which often

presents to movement disorders clinics. The second subtype presents with an early dementia

occurring along the spectrum of frontotemporal lobar degeneration (FTLD), most commonly the

behavioural variant of frontotemporal dementia (bvFTD) or progressive non-fluent aphasia

(PNFA). This subtype is most likely to present first to dementia clinics. There is evidence

suggesting that early dementia is the more frequent initial presentation of CBS [Bergeron et al.

1998;Grimes et al. 1999b;Mathuranath et al. 2000] yet, because the initial symptoms may be

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non-specific, the movement disorder presentation is easier to recognize. This may have created a

referral bias in CBS research particularly in many of the early studies, which ascertained patients

predominantly from movement disorders clinics. Several studies have overcome this bias by

examining patients with both types of presentations [Josephs et al. 2008;Kertesz et al.

2000b;Kertesz et al. 2005;McMonagle et al. 2006;Murray et al. 2007;Riley et al. 1990]. Few

studies, however, have directly compared CBS patients presenting with early motor vs. early

dementia features [Josephs et al. 2008;Kertesz et al. 2000b;McMonagle et al. 2006], and, to our

knowledge, no studies have investigated whether perfusion SPECT can help differentiate

between these two subtypes of CBS.

Both structural and functional neuroimaging studies may support a diagnosis of CBS. Early MRI

studies have demonstrated asymmetrical cortical atrophy in frontoparietal regions and,

frequently, subcortical white matter T2/FLAIR hyperintensities contralateral to the most affected

side of the body [Riley et al. 1990;Savoiardo et al. 2000;Soliveri et al. 1999;Tokumaru et al.

1996;Winkelmann et al. 1999]. These initial findings have been confirmed by more recent MRI

studies in larger patient cohorts [Boxer et al. 2006;Groschel et al. 2004;Grossman et al.

2004;Josephs et al. 2008;Koyama et al. 2007;Taki et al. 2004;Yekhlef et al. 2003]. Sawle et al.

[Sawle et al. 1991] using PET to measure regional cerebral oxygen metabolism were the first to

demonstrate that patients with CBS have hypometabolism predominantly in the posterior and

superior temporal, inferior parietal, and occipital (association) cortices; frontal association

regions also demonstrated reduced metabolism although they did not achieve statistical

significance. This pattern of hypometabolism tended to be asymmetric, being more prominent

contralateral to the most affected side of the body. This frontoparietotemporal pattern of reduced

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activity has been confirmed by other studies employing 18-fluoro-deoxyglucose (18-FDG)-PET

[Blin et al. 1992;Coulier et al. 2003;Eidelberg et al. 1991;Garraux et al. 2000;Hosaka et al.

2002;Juh et al. 2005;Klaffke et al. 2006;Laureys et al. 1999;Nagahama et al. 1997;Nagasawa et

al. 1996;Taniwaki et al. 1998;Yamauchi et al. 1998a] and perfusion tracers (HMPAO, ECD and

IMP) using SPECT [Hossain et al. 2003;Koyama et al. 2007;Markus et al. 1995;Okuda et al.

1999;Okuda et al. 2000b;Zhang et al. 2001]. Several of these studies also demonstrated reduced

asymmetric activity in the basal ganglia and thalamus contralateral to the most affected side of

the body. Reduced dopamine transporter binding of TRODAT [Lai et al. 2004] and β-CIT

[Pirker et al. 2000;Plotkin et al. 2005] in the basal ganglia has also been demonstrated in CBS.

It is critically important to follow patients longitudinally to ensure that clinical criteria for CBS

have been met, as the neurological features of the full syndrome may not be present at onset, but

may develop over time. This was eloquently shown in a longitudinal, prospective cohort of

patients with initial diagnoses ranging from bvFTD, CBS, Progressive Supranuclear Palsy (PSP)

to PNFA, the majority of whom then went on to develop second and/or third syndromes with

significant clinical overlap along the FTLD spectrum [Kertesz et al. 2005;McMonagle et al.

2006]. In addition to the clinical heterogeneity in presentation and evolution of CBS, there is also

significant pathological heterogeneity [Lee et al. 2011] leading some to propose the term „Pick

Complex‟ to encompass the varying pathologies occurring along this disease spectrum [Kertesz

et al. 2000b]. Given this pathological heterogeneity, prospective, longitudinal studies that follow

patients with CBS to autopsy are required in order to obtain a more accurate estimate of the

neuropathological substrates of this rare syndrome.

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Our prior study provided the initial characterization of a prospectively recruited sample of CBS

cases vs. controls in terms of demographics, clinical, and SPECT imaging features and identified

that perfusion within the left inferior parietal lobule correlated with a measure of ideomotor

apraxia (chapter 4). The purpose of the current study using this sample was threefold: 1) to

describe the initial standardized neuropsychological and neuropsychiatric, and MRI

(qualitatively) profile of a prospective cohort of 31 CBS patients ascertained from either a

movement disorders or a cognitive neurology clinic; 2) to compare the clinical,

neuropsychological/neuropsychiatric, MRI, and, in particular, SPECT imaging features of CBS

patients presenting with early dementia vs. early motor symptoms; and 3) to identify the

underlying neuropathological substrates in a subset of this sample who came to autopsy. Novel

aspects of this study include the comparison of SPECT perfusion measures in the early motor vs.

early dementia subgroups and also the integration of clinical, neuropsychological, MRI, SPECT,

and pathological data, whenever possible. This study is also unique in that it used two different

techniques to analyze the SPECT data, namely, region of interest analysis and statistical

parametric mapping (SPM).

5.3 Methods

5.3.1 Subjects:

31 subjects with a clinical diagnosis of CBS according to proposed diagnostic criteria [Boeve et

al. 2003] were recruited consecutively through two academic clinics as previously described

(chapter 4). They were matched to 31 normal healthy controls as closely as possible with respect

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to age, sex, and years of education. CBS subjects and controls were ascertained and followed as

part of the Sunnybrook Dementia Study, a prospective, longitudinal study of dementia and

ageing, approved by the local Research Ethics Board. Participants or their substitute decision

makers provide written, informed consent to participate in accordance with the Declaration of

Helsinki. All subjects underwent detailed clinical evaluations including: history, general and

neurological physical exam, routine laboratory investigations, and standardized behavioural

neurology assessment [Darvesh et al. 2005]. The side of greatest rigidity and/or apraxia defined

the motor-onset of symptoms. Patients were seen every 6 months for routine clinical follow-up

and had yearly prospective, longitudinal assessments which included: standardized measures of

neuropsychological performance, neuropsychiatric symptoms and functional status. Structural

and functional neuroimaging of the brain with MRI and SPECT were also performed annually.

Additional inclusion criteria were: age between 40 and 90 years, have a knowledgeable

caregiver, minimum educational attainment of grade 6 and fluent in English. Their SPECT and

neuropsychological evaluations needed to be completed within three months of each other.

Exclusion criteria were: presence of secondary/reversible causes of dementia that were untreated,

concomitant neurological or psychiatric illness/substance use and abuse, including clinically

relevant depression, history of significant head trauma, early vertical gaze palsy, rest tremor,

autonomic disturbances, sustained responsiveness to levodopa, and lesions on neuroimaging

suggesting another pathological condition.

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5.3.2 Neuropsychological, neuropsychiatric and functional measures:

Neuropsychological tests assessing general intelligence and cognition included Folstein‟s Mini-

Mental State Examination (MMSE) [Folstein et al. 1975]; Mattis Dementia Rating Scale (DRS),

which ranges from 0 to 144, with lower scores representing more impairment [Mattis 1976];

Clock Drawing Test, which ranges from 0 to 10, with lower scores representing more

impairment [Rouleau et al. 1992]; the National Adult Reading Test-Revised (NART-R), which

provides a measure of premorbid verbal intelligence [Blair and Spreen 1989], and Raven‟s

Progressive Matrices, which provides a measure of premorbid non-verbal intelligence [Raven

1947]. Tests assessing learning and episodic memory included the California Verbal Learning

Test (CVLT), which assesses verbal memory [Delis et al. 1987], while the visual reproduction

subtest of the Wechsler Memory Scale-Revised (WMS-R) assesses visual memory [Lezak 1983].

Measures of language function and naming included: the Boston Naming Test (BNT), which is

scored out of 30 with lower scores representing more impairment [Williams et al. 1989];

semantic/categorical fluency [Gladsjo et al. 1999]; and the comprehension subscale of the

Western Aphasia Battery (WAB) [Kertesz and Poole 1974]. Initially, the full WAB was given to

all patients, but in the last few years it has only been administered if there is anomia detected on

the BNT. The WAB calculates an aphasia quotient based on combined subscores of fluency,

content, comprehension, repetition and naming, with a maximum score of 100 and lower scores

represent more severe impairment [Kertesz and Poole 1974]. Ideomotor praxis was assessed

using the WAB praxis subscale, which is scored out of 60 with lower scores indicating more

severe apraxia [Kertesz and Poole 1974]. Attention and working memory was assessed using the

Forward and Backward Digit Span tests from the WMS-R [Lezak 1983;Wechsler 1987]. Several

assessments of executive function were employed including: phonemic (F-, A-, and S-word)

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fluency [Gladsjo et al. 1999;Lezak 1983]; the Trail Making Test A and B (TMT-A and -B) that

measure speed of psychomotor processing and mental flexibility [Lezak 1983]; and the

Wisconsin Card Sort Test (WCST) [Heaton 1981]. Visuospatial function was assessed using the

Rey-Osterrieth Complex Figure Test scored out of 36 with lower scores indicating worse

visuospatial function [Lezak 1983;Osterrieth 1944;Rey 1941]; and the Benton Line Orientation

task, which is motor-free and assesses visuospatial orientation and attention [Lezak 1983].

Behavioural function was investigated using the Neuropsychiatric Inventory (NPI-12), a

caregiver-based interview assessing 12 common neuropsychiatric features of dementia;

maximum score is out of 144 with lower scores indicating lesser degrees of psychopathology

[Cummings 1997]. Severity of depressive symptoms was assessed using the Cornell Scale for

Depression in Dementia (CSDD); higher scores indicate more severe depressive symptoms

[Alexopoulos et al. 1988]. Functional assessment was performed using the Disability

Assessment for Dementia (DAD), which assesses both basic and instrumental activities of daily

living including subcomponents of initiation, planning and performance [Gelinas et al. 1999].

5.3.3 Brain MRI:

Structural MRI was obtained in 29 of the 31 patients using a standard protocol. Images were

acquired on a 1.5T Signa MR imager (GE Medical Systems, Milwaukee, Wis.) and consisted of

the following acquisitions: 1) T1-weighted (axial 3D spoiled gradient [SPGR] echo, with echo

time [TE] 5 ms, repetition time [TR] 35 ms, flip angle 35°, number of excitations [NEX] 1, field

of view [FOV] 22 × 16.5 cm, in-plane resolution 0.859 × 0.859 mm and slice thickness 1.2–1.4

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mm), 2) proton-density and 3) T2-weighted images (interleaved axial spin echo, with TEs 30 and

80 ms, TR 3 s, NEX 0.5, FOV 20 × 20 cm, in-plane resolution 0.781 × 0.781 mm and slice

thickness 3 mm). MRI images were qualitatively interpreted by a neurologist (KH) blinded to all

clinical, neuropsychological, neuropsychiatric, SPECT, and pathological data. Asymmetry and

lobar localization of the maximal atrophy was described. Localization of T2/PD white matter

changes was also noted.

5.3.4 Brain SPECT:

SPECT imaging was acquired with a triple-head gamma camera (Prism 3000XP; Phillips

Medical Systems Inc., Cleveland, Ohio) while the patient was resting comfortably and was

performed a minimum of 30 minutes and a maximum of 120 minutes after injection of 20 mCi

(740 MBq) of Technetium-99m ethyl cysteinate dimer (99m

Tc-ECD SPECT). Each view

consisted of a 128 × 128 pixel image with a typical reconstructed image resolution of 9.7 mm

full width at half maximum. The total imaging time was 19 minutes. Reconstruction was

performed by using a ramp-filtered back-projection algorithm followed by a 3-dimensional

restoration post-filter (Wiener filter, multiplier 1.0). Ellipses were fit to the approximate location

of the outline of the head in each transaxial image, and a calculated attenuation correction

applied [Matsuda et al. 1995]. Voxel dimensions were 2.18 × 2.18 × 3.56 mm.

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5.3.5 Regional perfusion ratios:

Uptake of 99m

Tc-ECD is approximately proportional to regional cerebral blood flow (rCBF)

[Matsuda et al. 1995] such that brain SPECT can be used to provide semi-quantitative measures

of regional brain perfusion. Reconstructed images were co-registered to a SPECT template that

was an average of 14 healthy, elderly control scans. A T1-weighted MRI with dimensions similar

to the SPECT template was the source of 79 bilateral regions of interest (ROI) as previously

described [Lobaugh et al. 2000]. In order to obtain ROI intensity values, we used a common

transformation to move from the SPECT template space to MRI space. The cerebellum is

frequently used to normalize SPECT counts in studies of dementia [Stamatakis et al. 2001].

However, crossed cerebellar diaschisis may lead to relative differences in perfusion between the

left and right cerebellar hemispheres, and, if whole cerebellum is used as the reference region

then this may overestimate rCBF in a particular ROI. We, therefore, applied the following rule: if

there was more than a 5% difference in counts between left and right cerebellar hemispheres, we

use the hemisphere that is more perfused as the reference region. If there is no difference then

we use the whole cerebellum as the reference region. In this way, semi-quantitative perfusion

ratios are derived and regional Z scores are calculated [Lobaugh et al. 2000].

5.3.6 Pathological analysis:

Neuropathological examination was carried out by one of the authors (J.B.). Paraffin-embedded

sections were stained with haematoxylin and eosin, Luxol fast blue (LFB), Bielschowski and

Gallyas. Immunostains using commercial antibodies for tau (Dako, A0024), ubiquitin (Vector

Labs, ZPU576), and α-synuclein (Vector Labs) were performed. Immunostaining with

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commercial antibodies for TDP43 (ProteinTech Group, Inc.) was performed, when

Frontotemporal Lobar Degeneration-Ubiquitin-positive, Tau-negative pathology (FTLD-U) was

demonstrated.

5.3.7 Data analysis:

Statistical analysis of demographic, clinical, neuropsychological, MRI and ROI SPECT variables

was performed using the Statistical Package for the Social Sciences (SPSS), version 16.

5.3.7.1 Demographic, clinical and neuropsychological measures:

Categorical demographic and clinical data were analyzed using chi-square or Fisher exact tests.

In comparing neuropsychological test results to the control sample, normalized z-scores were

calculated. Normality of continuous demographic and neuropsychological data was determined

by examining Q-Q probability plots. Parametric methods (e.g., independent samples t-test) were

used if the data fit a normal distribution, otherwise non-parametric tests (e.g., Mann Whitney U

test) were performed.

5.3.7.2 Region of interest (ROI) SPECT analysis:

Normality of ROI SPECT data was confirmed as described above. Independent sample t-tests

between CBS and control groups were conducted to compare mean perfusion ratios of individual

ROIs within frontal, parietal, temporal, basal ganglia, and thalamic regions, areas previously

shown to be affected in CBS. ROIs that were statistically significant on the t-test analysis were

included in a multivariate, general linear model (GLM) analysis of covariance (ANCOVA).

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5.3.7.3 Statistical Parametric Mapping SPECT analysis:

SPECT scans were decompressed, converted to Analyze 7.5 format, and each axial slice was

visually inspected for image quality. Statistical Parametric Mapping version 5 (SPM5,

Wellcome Department of Imaging Neuroscience, University College London) was used to pre-

process and analyze the scans. The images were spatially normalized to a standard SPECT

template in Montreal Neurological Institute (MNI) space [Stamatakis et al. 2001]. This step re-

sampled the voxel dimensions to 2 × 2 × 2 mm. The scans were then smoothed using an

isotropic Gaussian kernel (12 mm FWHM). A thresholded mean voxel value was chosen for

global calculation, and global normalization was achieved by proportional scaling to 50 mL/100

g/min. Voxel-by-voxel t-tests were performed to identify regions with differences in relative

cerebral perfusion between groups. We reported significance using a voxel-wise p-value

threshold (p < 0.05) corrected for multiple comparisons and an extent threshold of at least 20

contiguous voxels. Our correction methodologies included either controlling the family-wise

error (FWE) rate [Worsley et al. 1996] or controlling the false discovery rate (FDR) [Genovese

et al. 2002]. A whole brain mask was used to exclude extracranial voxels from the analysis. The

maximal peak coordinates of the perfusion differences were converted to Talairach space using

the Yale Non-linear MNI to Talairach Converter [Lacadie et al. 2008]

(http://www.bioimagesuite.org/Mni2Tal/index.html). These converted coordinates were

translated into anatomical brain regions and Brodmann Areas (BAs) using Talairach Daemon

Client [Lancaster et al. 2000] (http://www.talairach.org/client.html).

http://www.bioimagesuite.org/Mni2Tal/index.html

http://www.talairach.org/client.html

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5.4 Results

5.4.1 CBS cases vs. controls

5.4.1.1 Neuropsychological, behavioural and functional assessment

Figure 1. Normalized (z-) scores of neuropsychological measures in patients with CBS compared to control

group.

Z-score cut-off of -2.8 corresponds to a p-value of ≤ 0.0026 (Bonferroni-correction for multiple testing). WCST-per

= Wisconsin Card Sort Test-perseverative errors (n=22); WCST-cat = Wisconsin Card Sort Test-categories (n=22);

TMT = Trail Making Test (TMT-A, n=19; TMT-B, n=13); FAS = F-, A-, S-word phonemic fluency (n=21); Benton

Judgement of Line Orientation (n=21); Rey Osterieth Complex Figure Copy (n=20); Digit span B = Backward (23);

F = Forward (23); Semantic fluency (n=26); Boston Naming (n=22); WAB = Western Aphasia Battery (n=23);

WAB-praxis (n=27); DVR = WMS-III-R delayed visual reproduction (n=19); CVLT = California Verbal Learning

Test-long delay free recall (n=21); Raven‟s progressive matrices (n=22); NART = New Adult Reading Test (n=19);

MDRS = Mattis Dementia Rating Scale (n=26); Clock Drawing Test (n=9); MMSE = Mini-Mental State Exam

(n=31). Missing data is secondary to the inability of the patient to complete the test.

Patients were mildly demented at the time of their initial evaluation based on their mean MMSE

score of 21.7 (Standard Error of the Mean = 1.2) and fell below the cut-off for dementia on the

Mattis Dementia Rating Scale (MDRS); mean score in CBS patients was 113.5 (4.1) /144

(MDRS cut-off for dementia in this age group = 123/144). Cognitive domains most impaired

146

were working memory, executive functions, praxis, visuospatial abilities and language tasks

involving fluency and naming, with relative preservation of comprehension. Although delayed

free recall on the CVLT was impaired, delayed cued recall was better (data not shown). Of the

23 CBS patients for which WAB data was available, the classification was as follows: 26.1%

(6/23) had no aphasia; 8.7% (2/23) were borderline aphasic; 47.8% (11/23) had an anomic

aphasia; 8.7% (2/23) had a Broca‟s aphasia; 4.4% (1/23) had a conduction aphasia; and 4.4%

(1/23) had a Wernicke‟s aphasia.

Patients were moderately impaired on both basic and instrumental activities of daily living

assessed using the Disability Assessment for Dementia. In terms of frequency of

neuropsychiatric symptoms, 24/29 (82.8%) CBS patients had at least one neuropsychiatric

symptom present (Supplementary Table 1). Neuropsychiatric symptoms are presented in order

from most common to least common: apathy (58.6%), depressive symptoms (41.4%), abnormal

appetite and eating behaviour (41.4%), irritability (34.5%), agitation (31.0%), anxiety (27.6%),

aberrant night-time behaviour (24.1%), disinhibition (17.2%), aberrant motor behaviour (6.9%),

and delusions (3.5%; Supplementary Table 1). No patients had hallucinations or euphoria. 13/30

(43.3%) had a CSDD score > 25% supportive of significant depressive symptomatology

(Supplementary Table 1), although none met DSM-IV diagnostic criteria for depression or had a

history of clinically relevant depression before the neurodegenerative presentation.

5.4.1.2 MRI features

Table 1 provides case summaries of clinical, pathological and MRI features of the CBS patients.

Two patients did not have MRI examinations completed due to claustrophobia.

Table 1. Case summaries of clinical, pathological, and MRI features of CBS patients. M = Male; F = Female; FTLD-U/TDP43 = Frontotemporal lobar

degeneration-ubiquitin+/Tar DNA binding protein+; AGD = agyrophilic grain disease; CAA = cerebral amyloid angiopathy; CBD = Corticobasal degeneration;

PSP = Progressive supranuclear palsy; L = Left; R = Right; SYM = Symmetrical; O = occipital; P = Parietal; Fr = frontal; Te = Temporal; Gen = Generalized;

POST = posterior; ANT = anterior

AGE OF

ONSET SEX

TYPE OF

PRESENTATION PATHOLOGY

AFFECTED

SIDE OF

BODY

HEMISPHERIC

ATROPHY

LOBAR

PREDILECTION

WHITE MATTER

HYPERINTENSITY

65 F Dementia FTLD-U/TDP43 L R O,P > Fr,Te R>L POST

63 F Dementia AGD/CBD/CAA R L Gen R=L ANT/POST

77 F Dementia N/A R L Fr,Te,P R=L ANT/POST

74 F Motor CBD L SYM Fr,Te,P R=L ANT

75 M Motor N/A R SYM Gen R=L ANT

58 F Dementia N/A L SYM O,P > Te R=L ANT

86 M Dementia N/A L SYM Fr,Te,P R=L ANT

61 F Dementia CBD R L Fr,Te,P L ANT

75 F Dementia N/A R L Fr,P R=L ANT

63 F Motor CBD L SYM Gen R=L ANT/POST

70 F Dementia N/A R SYM Fr,Te,P R=L ANT

57 F Dementia CBD R L Te,P,O > Fr L>R POST

59 F Dementia N/A L SYM P,O > Fr,Te L>R POST

57 M Dementia N/A L R P > Te,O R=L mild

62 F Dementia N/A L SYM P > Fr,Te R=L mild

54 M Dementia N/A L R Gen Absent

59 M Motor N/A L SYM P R>L POST

71 F Motor N/A L R Fr,Te,P R=L ANT/POST

76 M Motor N/A L SYM P > Fr,Te L>R POST

69 M Dementia N/A L R P,T > F R=L ANT

49 F Dementia N/A R SYM P > Fr,Te,O L>R ANT/POST

80 F Dementia N/A R L Fr,Te,P > O L>R POST

68 F Motor N/A R L Fr,Te,P > O R=L mild

55 F Dementia N/A R SYM P > Fr R=L mild

69 F Dementia CBD L R Fr,P > Te R=L mild

71 F Motor N/A R SYM Te,P R=L ANT/POST

46 M Dementia N/A R L Gen R=L mild

62 M Dementia PSP R SYM P Absent

58 M Dementia N/A R L P > Te Absent

67 M Dementia N/A R No MRI N/A N/A

65 M Motor N/A L No MRI N/A N/A

Table 2. MRI atrophy patterns in CBS cases stratified according to body side most affected by motor

symptoms.

All percentages are calculated based on total of 29 CBS patients who had MRI scans.

In terms of lobar predilection, parietal>temporal>frontal atrophy was most commonly seen with

only eight (27.6%) patients having evidence for occipital involvement. Generalized lobar atrophy

was observed in five patients; three with maximal involvement contralateral to the most affected

side of the body, while two had symmetrical generalized atrophy.

The majority of patients (89.7%; 26/29) demonstrated subcortical T2/PD white matter

hyperintensities (WMH) on MRI; 10.3% (3/29) of patients showed no WMH. Of the patients

with WMH, 15.4% (4/26) had maximal WMH contralateral to the most affected side of the body

corresponding to the region of maximal atrophy. 7.7% (2/26) of patients with symmetrical

cortical atrophy had WMH contralateral to the most affected side of the body. 7.7% (2/26) of

patients with symmetrical atrophy had WMH ipsilateral to the most affected side of the body.

69.2% (18/26) of patients had symmetrical WMH independent of the most affected side of the

body and of cortical atrophy.

MRI atrophy pattern

Asymmetric Symmetric

Right Left

Motor

side

Left 6 (20.7%) 0 (0%) 8 (27.6%)

Right 0 (0%) 9 (31%) 6 (20.7%)

Column Totals 15 (51.7%) 14 (48.3%)

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5.4.2 Early dementia vs. early motor presentations

5.4.2.1 Demographic and clinical characteristics

There were no statistically significant differences between the early motor and dementia groups

in terms of gender, handedness, years of education, age of onset, and body side most affected

(Table 3).

Demographic variable CBS-dementia (n=22) CBS-motor (n=9)

Gender 14 (63.6%) F

8 (36.4%) M

5 (55.6%) F

4 (44.4%) M


1 (4.5%)L

8 (88.9%) R

1 (11.1%) L

Site of recruitment* 19 (86.4%) Cog

3 (13.6%) MD

1 (11.1%) Cog

8 (88.9%) MD

Age of Onset

(mean SEM years)

63.6 2.1

69.1 1.9

Age at Investigation

(mean SEM years)

66.8 2.1

72.6 2.0

Duration of symptoms

(mean SEM years)

3.2 0.4

3.4 0.7

Years of Education

(mean SEM years)

12.2 0.7

12.9 0.9

Body side most affected 13 (59.1%) R

9 (40.9%) L

3 (33.3%) R

6 (66.6%) L Table 3. Demographic features of CBS groups presenting with early dementia versus early motor features.

F = Female; M = Male; Cog = Cognitive Neurology Clinic; MD = Movement Disorders Clinic; R = Right; L = Left;

*Fisher‟s Exact test, p<0.0005.

In terms of clinical characteristics (Figure 2), CBS patients presenting with early motor features

were statistically more likely to have cortical sensory loss (defined as occurrence of

astereognosis, agraphesthesia and/or sensory extinction) as compared to the early dementia group

(Fisher‟s Exact Test [2-tailed], p=0.005; Figure 2B). This association was driven by the higher

occurrence of astereognosis in the CBS-M group (77.8% [7/9 cases]; Fisher‟s Exact Test [2-

tailed], p=0.01) compared to CBS-D (22.7% [5/22]). There were no differences between the

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CBS-M and -D groups in terms of agraphesthesia or extinction to double simultaneous stimuli.

Because sensory extinction is known to localize to the right parietal region, the sample was

stratified into those presenting with their left side of the body most affected vs. those involving

mainly their right body. Consistent with this localization, there was a trend for CBS-L patients to

have higher rates of sensory extinction than CBS-R patients (CBS-L: 33.3% [5/15] vs. CBS-R:

6% [1/16 cases]; Fisher‟s Exact test, p=0.08). There were no significant differences between the

CBS-L and CBS-R groups in terms of occurrence of agraphesthesia or astereognosis. Of the 11

patients who died, time to death was not significantly different between the eight patients who

presented with early dementia (80 ± 17 months) and the three patients with early motor features

(91 ± 9 months).

Figure 2. Frequency of (A) extrapyramidal and (B) cortical features of CBS patients presenting with early

dementia vs. early motor symptoms.

CSL = Cortical sensory loss; *Fisher‟s Exact Test, p = 0.005

5.4.2.2 Neuropsychological, behavioural and functional evaluation

CBS patients presenting with early dementia had statistically significant lower scores on MMSE;

WAB aphasia quotient (AQ) and subscores including content, fluency, repetition, and

2A 2B

*

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comprehension; Boston naming; forward digit span; and Benton line orientation than those

presenting with early motor features (See Table 4). This indicates relative difficulties in general

cognition, language function, and tasks involving sustained attention and visuospatial orientation

and attention.

Psychometric Measures CBS-D (n) CBS-M (n)

General cognition

MMSE /30 [n=31]¥ 19.8 ± 1.5 (22) 26.6 ± 1.0 (9)

Clock Drawing Test /10 [n=9] 6.8 ± 1.1 (6) 7.0 ± 1.7 (3)

NART /127.8 [n=19] 104.4 ± 2.5 (11) 111.7 ± 2.6 (8)

Raven‟s Progressive Matrices /36 [n=22]* 18.8 ± 1.3 (14) 27.3 ± 3.0 (8)

MDRS /144 [n=26] 108.0 ± 5.3 (17) 123.9 ± 4.7 (9)

Memory

CVLT Long Delay Free Recall /16 [n=21] 5.9 ± 0.7 (13) 7.6 ± 1.2 (8)

Delayed Visual Reproduction /41 [n=19] 8.7 ± 2.8 (12) 13.7 ±4.1 (7)

Language

WAB total /100 [n=23]* 82.7 ± 3.0 (17) 94.5 ± 1.9 (6)

WAB content /10* 7.8 ± 0.4 9.1 ± 0.4

WAB fluency /10* 8.0 ± 0.5 9.3 ± 0.2

WAB comprehension /10¥ 8.9 ± 0.3 9.9 ± 0.1

WAB repetition /10¥ 8.5 ± 0.3 9.8 ± 0.1

WAB naming /10 8.1 ± 0.3 9.0 ± 0.4

Boston Naming /30 [n=22]* 22.2 ± 1.3 (14) 27.1 ± 0.8 (8)

Semantic Fluency /20 [n=26] 8.3 ± 1.2 (18) 13.3 ± 2.8 (8)

Praxis

WAB praxis /60 [n=27] 51.7 ± 2.4 (18) 56.1 ± 0.9 (9)


Digit span - forward /12 [n=23] ¥

5.9 ± 0.6 (15) 9.1 ± 0.7 (8)

Digit span - backward /12 [n=23] 3.9 ± 0.8 (15) 5.8 ± 0.9 (8)


Rey Osterieth Complex Figure – Copy /36 [n=20] 13.8 ± 3.6 (12) 21.7 ± 4.9 (8)

Benton Line Orientation /30 [n=21]* 9.5 ± 2.6 (13) 19.3 ± 4.2 (8)

Executive functions

Phonemic fluency (FAS) [n=21] 16.6 ± 2.8 (13) 23.1 ± 5.4 (8)

Trail Making Test A (time in seconds) [n=19] 119.9 ± 14.3 (12) 89.6 ± 31.7 (7)

Trail Making Test B (time in seconds) [n=13] 273.6 ± 63.3 (7) 145.5 ± 28.5 (6)

WCST categories /6 [n=22] 1.6 ± 0.4 (13) 2.1 ± 0.4 (9)

WCST perseverative errors [n=22] 12.5 ± 3.8 (13) 10.6 ± 4.7 (9) Table 4. Mean scores (± SEM) on neuropsychological measures in CBS patients presenting with early

dementia vs. early motor symptoms.

The number of patients (n) tested is listed next to individual measures. Missing data is secondary to the inability of

the patient to complete the test. CBS-D = Early dementia; CBS-M = Early motor; MMSE = Folstein‟s Mini-Mental

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State Exam; NART = National Adult Reading Test; MDRS = Mattis Dementia Rating Scale; CVLT = California

Verbal Learning Test; WAB = Western Aphasia Battery; FAS = F-, A-, and S-phonemic fluency; WCST =

Wisconsin Card Sort Test. Independent samples t-tests were used to compare MMSE, NART, Clock, MDRS,

Boston naming, semantic fluency, visual reproduction, forward and backward digit span, CVLT, Benton, Trails A

and B mean scores between groups. Mann Whitney U test was used to compare scores on FAS, WAB, Rey and

WCST between groups. *p≤0.05; ≦≤0.005

The early dementia group was statistically more likely to have a language disturbance based on

combined aphasia categories on the WAB as compared to those presenting with early motor

features (Fisher‟s Exact Test [2-tailed], p=0.02). Specifically, 33.3% of the early motor group

had an anomic aphasia, while the rest had no aphasia. Contrast this to 88.2% of the early

dementia group having a language disturbance (borderline aphasia: 11.8%; anomic aphasia:

52.9%; Broca‟s aphasia: 11.8%; conduction aphasia: 5.9%; Wernicke‟s aphasia: 5.9%) while

11.8% had no aphasia (Figure 3). This difference was even more striking when aphasia

classification was stratified according to side of maximal motor involvement. Specifically, 100%

(10/10) of patients presenting with their right side of the body most affected had evidence for an

aphasic disturbance, whereas only 46% (6/13) of those with left sided motor symptoms were

classified as having aphasia (Fisher‟s Exact Test [2-tailed], p = 0.007). Additionally, mean WAB

AQ scores were lower in those presenting with the right side of the body most affected (78.9 ±

3.8) compared to the left (91.0 ± 2.6) [Mann-Whitney U test (2-tailed), p = 0.005].

153

Figure 3. Frequency of CBS patients with early dementia vs. early motor presentation stratified according to

category on the Western Aphasia Battery (WAB). None of the functional or behavioural measures were statistically different between the early dementia and early

motor groups (Table 5).

154

Behavioural/Functional Measures CBS-D (n) CBS-M (n)


DAD (%) [n=30] 65.7 ± 7.0 (21) 75.8 ± 9.9 (9)

DAD ADL (%) 77.0 ± 7.6 76.0 ± 9.1

DAD iADL (%) 58.6 ± 7.8 75.7 ± 10.5


NPI total /144 [n=29] 14.4 ± 3.4 (20) 7.1 ± 2.7 (9)

NPI apathy /12 4.1 ± 1.0 2.1 ± 1.3

NPI appetite and eating behaviour /12 2.3 ± 0.7 1.8 ± 1.2

NPI dysphoria/depression /12 1.7 ± 0.6 0.6 ± 0.2

NPI night-time behaviour /12 1.1 ± 0.5 1.7 ± 1.1

NPI irritability/lability /12 1.3 ± 0.5 0.4 ± 0.3

NPI agitation/aggression /12 1.1 ± 0.6 0.3 ± 0.2

NPI aberrant motor behaviour /12 0.8 ± 0.6 0.0 ± 0.0

NPI disinhibition /12 0.7 ± 0.3 0.0 ± 0.0

NPI anxiety /12 0.7 ± 0.3 0.2 ± 0.2

NPI delusions /12 0.3 ± 0.3 0.0 ± 0.0

NPI hallucinations /12 0.0 ± 0.0 0.0 ± 0.0

NPI euphoria /12 0.0 ± 0.0 0.0 ± 0.0

NPI caregiver distress /12 7.9 ± 1.9 4.2 ± 1.6

Cornell Depression Scale (%) [n=30] 24.3 ± 3.1 (21) 19.3 ± 4.9 (9) Table 5. Mean scores (± SEM) on behavioural and functional measures in the CBS group.

The number of patients (n) tested is listed next to individual measures. Missing values are secondary to the inability

of the caregiver to complete the test. CBS-D = Early dementia; CBS-M = Early motor; DAD = Disability

Assessment for Dementia; ADL = Activities of daily living; iADL = Instrumental activities of daily living; NPI =

Neuropsychiatric Inventory. There were no statistically significant differences between groups.

5.4.2.3 MRI features

There were no significant differences between the early dementia and motor groups in terms of

symmetry/asymmetry of atrophy on MRI. Stratifying CBS patients with asymmetric MRI

atrophy into those with and without aphasia using the WAB, there was a trend for aphasic

patients to have left hemispheric atrophy compared to those without aphasia (Fisher‟s Exact Test,

p=0.06; Refer to Table 6).

MRI atrophy pattern

Asymmetric* Symmetric

Right Left

Aphasia

Present (n=17) 2 6 7

Absent (n=6) 3 0 3 Table 6. MRI atrophy patterns in CBS cases stratified by the presence or absence of aphasia as determined

by the WAB. Fisher‟s exact test, p=0.06*

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5.4.2.4 SPM and ROI SPECT

Figure 4 and tables 7 and 8 demonstrate results of SPM and ROI SPECT analysis comparing

perfusion in the CBS patients presenting with early dementia to those with early motor features.

In the early dementia versus early motor groups, cortical areas of relative hypoperfusion were

identified in the left fusiform gyrus (uncorrected p<0.001); this result did not survive correction

for multiple testing using FDR or FWE methods. However, employing ROI MANCOVA

analysis, CBS patients presenting with early motor symptoms had relatively reduced perfusion in

the right precentral gyrus and right paracentral lobule (supplementary motor area) as well as in

the left middle posterior cingulate region compared to those with early dementia.

Figure 4. Statistical parametric maps overlaid on multi-slice brain MRI template showing decreased

perfusion in left fusiform gyrus (uncorrected p<0.001) in CBS cases presenting with early dementia versus

early motor features.

156


(Brodmann area)

Talairach

Coordinates

No. of

voxels

SPM t-score

(p-value)

X y Z

CBS-d Occipitotemporal region - SPM

vs. Left fusiform gyrus (19) -34 -76 -11 215 4.5 (p<0.001)

CBS-m Table 7. Areas of relative hypoperfusion on SPECT in CBS patients presenting with early dementia (CBS-d)

versus those presenting with early motor features (CBS-m).

Data are shown for only SPM analysis since no areas of hypoperfusion were shown with ROI; Uncorrected p-value

of p<0.001 used.


(Brodmann area)

No. of

voxels

ROI F-score

(p-value)

CBS-m Frontal regions – ROI

vs. Right paracentral lobule 741 7.0 (p<0.01)

CBS-d Right precentral gyrus 2723 4.9 (p<0.04)

Limbic regions - ROI

Left posterior cingulate - middle 512 5.1 (p<0.03) Table 8. Areas of relative hypoperfusion on SPECT in CBS patients presenting with early motor (CBS-m)

versus those presenting with early dementia (CBS-d).

Data are shown for ROI analyses only since SPM results failed to demonstrate any relative perfusion differences in

this comparison. All p-values for the ROI analysis are derived from a multivariate ANOVA running under a general

linear model.

5.4.3 Description of pathological series and relation to MRI findings

Of the 31 CBS patients in our series, 11 patients died from their neurodegeneration usually from

malnutrition and/or aspiration pneumonia secondary to severe dysphagia. The average time to

death was 82.7 (SEM 12.2; range 32 to 159) months or approximately 7 years. Pathological

analysis was performed on 8 of the 11 patients who died. 5 patients met pathological criteria for

CBD, one had PSP, one had FTLD-U/TDP43 proteinopathy and one had combined dementia

with agyrophilic grains, CBD and cerebral amyloid angiopathy (AGD/CBD/CAA). The average

time to death in the pathologically-confirmed CBD group was 79.2 (22.5) months with a range of

32 to 159 months. The patient with pathologically-proven PSP died after approximately 94

months, while the time to death in the AGD/CBD/CAA and FTLD-U/TDP43 patients were 117

157

and 112 months, respectively. The CBS cases with FTLD-U/TDP43 (left motor symptoms),

AGD/CBD/CAA (right motor symptoms), or PSP (right motor symptoms) pathology presented

with an early dementia syndrome. 60% (3/5) of the CBD cases presented with an early dementia

syndrome; two of these cases had right-sided motor symptoms while one had left-sided

involvement. 40% (2/5) of the CBD cases presented with early motor features and both had the

left side prominently affected by motor symptoms.

There was a relatively good association between the severity and lateralization of cortical

atrophy/subcortical white matter changes observed on MRI in vivo with that of the underlying

pathology. Of the CBS cases with a pathological diagnosis of CBD, the pattern of cortical

atrophy detected on MRI matched that detected by pathological investigation in 60% (3/5) of

cases; in the two cases that did not match, MRI-detected atrophy was asymmetrical while the

macroscopic brain pathology showed symmetrical atrophy. Also in 100% (5/5) of the CBD

group, the severity of white matter changes on MRI correlated well with the severity of

underlying Tau-positive threads and glial coils observed in the white matter with associated

pallor and gliosis. The case with FTLD-U/TDP43 demonstrated marked right > left-sided

cortical atrophy worse in the parieto-occipital region, but also involving the frontotemporal

regions on MRI [Masellis et al. 2006]. There was also severe underlying white matter

hyperintensities worse on the right in the posterior regions. These MRI findings are strongly

correlated with pathological changes of underlying cortical and white matter atrophic and gliotic

changes. The generalized left > right-sided atrophy seen on MRI in the AGD/CBD/CAA case

was discordant with the symmetrical atrophic changes noted on pathology. However, MRI white

matter hyperintensities did correlate with white matter neuropil threads and coiled bodies.

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Finally, the mild symmetrical atrophy on MRI detected in the PSP case and absence of white

matter changes correlated well with that of the cortical atrophy seen on pathology as well as the

minimal white matter gliotic changes. None of the cases had evidence for clasmatodendrosis in

the white matter.

5.5 Discussion

This study provides a comprehensive and integrated multi-modal assessment of a prospective

cohort of CBS patients using clinical data, standardized neuropsychological, neuropsychiatric

and functional measures, as well as brain MRI and SPECT data. Patients were ascertained from

both university-based cognitive and movement disorders clinics overcoming the important

limitation of selection bias in prior studies. Many prior studies did not stratify patients based on

most affected motor side of the body as well as presenting syndrome – dementia or motor – and

our study significantly adds to the literature that has studied lateralization in this syndrome.

Finally, our CBS cohort was followed prospectively over time with about 25% of our sample

coming to autopsy, which was performed in 72% (8/11) who died during the course of the study.

We will now discuss highlights and novel findings of this study.

5.5.1 CBS presenting with early dementia vs. early motor features

There have been very few studies that directly compare the clinical and neuropsychological

profiles of CBS presenting with early dementia (CBS-D) vs. early motor features (CBS-M)

[Kertesz et al. 2000b;McMonagle et al. 2006]. The current study represents the first attempt to

159

compare these CBS subtypes in terms of their relative brain perfusion on SPECT as a biomarker

of brain dysfunction. Compared to previous studies, it also provides a more comprehensive,

comparative neuropsychological assessment of these CBS subtypes that cover all domains of

cognitive functioning.

A highly significant finding was that all CBS patients presenting with early motor features

demonstrated cortical sensory loss on clinical examination in comparison to less than half of

those presenting with early dementia. To our knowledge, this is the first study to demonstrate

this finding. Although sensory extinction can be represented bilaterally in the parietal regions, it

is more commonly associated with lesions involving the right (non-dominant) superior parietal

lobe, specifically, areas 5 and 7 in the inferior part [Rizzo and Eslinger 2004;Ropper and Brown

2005]. Although we could not localize precisely to this region based on the nature of our data,

the CBS patients with prominent symptoms involving the left side of the body had higher rates of

sensory extinction. Agraphesthesia (ability to recognize figures drawn on the hand) has been

associated with lesions of the left intraparietal sulcus [Rizzo and Eslinger 2004]. However, in our

sample, we did not observe any association of left hemispheric/parietal atrophy with presence of

agraphesthesia. This may be due to low power to detect a difference using cateogorical data

(presence/absence of left hemispheric atrophy on MRI) secondary to a relatively small sample

size. Astereognosis (an inability to tactually perceive both texture [ahylognosis] and shape

[amorphognosis] of an object with the hands) localizes to parietal areas 1 (ahylognosis), and 2

through 5 (amorphognosis) and is bilaterally represented [Rizzo and Eslinger 2004]. Our

findings support the bilateral localization of this clinical phenomenon in that there was no

lateralization of MRI-rated atrophy observed.

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Lateralization in our sample is further supported by a careful examination of language function

within the CBS-D vs. -M groups. Specifically, patients with language dysfunction classified

according to the WAB were more likely to have their right side of the body most affected, which

was more commonly the case in the early dementia group. Similar to prior studies [Kertesz et al.

2000b;McMonagle et al. 2006], our CBS patients presenting with early dementia tended to

perform more poorly on the WAB as exemplified by lower mean aphasia quotients and were

more likely to be classified as having severe aphasic disturbances. Furthermore, the CBS-D

group had significant impairments in naming on the Boston Naming Test compared to the CBS-

M group. This association was even stronger when examining for the presence of aphasic

disturbance based on the most affected motor side of the body with 100% of right side afflicted

patients demonstrating aphasia compared to less than half of those with their left body side

affected. Compared to a trend towards this finding in McMonagle et al. [McMonagle et al.

2006], our study achieved high statistical significance. This difference may be due to patient

selection biases – all of our patients had to have substantial asymmetric rigidity and/or apraxia at

some point during the disease course to increase the specificity of the CBS diagnosis whereas 5

patients in McMonagle et al [McMonagle et al. 2006] had clinical findings that were

symmetrical and “atypical” for CBS; our patients were recruited prospectively from both a

cognitive or movement disorders clinic; or this discrepancy may be due to other unknown biases

in the data due to small sample sizes across both patient series.

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Although right-sided motor involvement was strongly associated with aphasia in this study, an

examination of MRI data did not show such a predictable relationship with side of maximal

atrophy. Patients with aphasia tended to have either symmetric or left hemispheric atrophy on

MRI compared to those without aphasia who had symmetric or right hemispheric atrophy. These

results did not achieve statistical significance, but were based on a small sample size. This lack

of an association of aphasia with side of atrophy is consistent with the study of McMonagle et al

[McMonagle et al. 2006]. Asymmetric atrophy on MRI was not found to predict the CBS-D vs.

CBS-M presentation. Few studies have directly compared these two subtypes of CBS utilizing

MRI. One MRI study demonstrated that pathologically-proven CBD patients (n = 11) had more

cortical and subcortical grey matter atrophy on MRI if they presented with dementia symptoms

(CBD-D) compared to a higher degree of subcortical white matter atrophy in motor-onset CBD

(CBD-M) [Josephs et al. 2008]. Similar to our study, their analysis comparing CBD-M and

CBD-D cases was completed qualitatively by visual inspection even though they used voxel-

based morphometric analysis when comparing CBD patients to controls [Josephs et al. 2008].

The lack of association between MRI-rated atrophy and pattern of CBS presentation is likely due

to the fact that even though one hemisphere may be more selectively vulnerable at an earlier

stage of disease, ultimately the pathology underlying CBS is bilateral and frequently does not

always correlate perfectly with the clinical syndrome. Perfusion deficits may be more sensitive to

detect asymmetry in CBS presenting with cognitive- versus motor-onset.

We hypothesized that the CBS-D group would show reduced perfusion in the left peri-Sylvian

region compared to CBS-M given that aphasia was more common and severe in this group.

Conversely, we hypothesized that the CBS-M group would show reduced perfusion in the right

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parietal region compared to the CBS-D group because of the high preponderance of cortical

sensory loss. There were no significant areas of reduced perfusion between the CBS-D and CBS-

M groups identified that survived correction for multiple testing. This was likely on the basis of

the small sample size in each CBS group (CBS-D, n = 22; CBS-M, n = 9) so there was

insufficient power to detect any differences using the conservative SPM method. Even when the

stringency of a whole brain SPM analysis was reduced to explore the data, the only area which

showed reduced perfusion in CBS-D versus -M was the left fusiform gyrus. The fusiform gyri

have been identified as important neural correlates of both facial and word recognition and

perception [Rizzo and Eslinger 2004]. Damage to the right fusiform gyrus is associated with

prosopagnosia, an inability to recognize faces [Rizzo and Eslinger 2004]. The left fusiform gyrus

plays an important role in recognition and processing of visual word forms and as such is also

known as the „visual word form area‟ important in processing strings of letters [Cohen et al.

2000]. However, a more recent study has also shown its importance in perception and memory of

faces [Mei et al. 2010]. To our knowledge, there have been no prior studies demonstrating

reduced perfusion or atrophy in this region in CBS nor has prosopagnosia or impaired processing

of letter strings been observed as a feature of CBS. However, in a combined group of

neurodegenerative disorders including a small subsample with CBS, empathy loss was associated

with atrophy in the right fusiform gyrus among other frontotemporal regions [Rankin et al.

2006]. The authors suggest that the importance of this region in facial perception may be related

to its association with empathy. Future studies will need to clarify whether or not this structure is

indeed important in CBS, especially given that our result could be a false positive association.

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Interestingly, in the ROI analysis, the supplementary motor area on the right showed reduced

perfusion in CBS-M compared to CBS-D. This may be an important neural correlate of the

motor disorder associated with CBS and the observation that it lateralizes to the right in CBS-M

patients is consistent with the finding of an increased number of left-sided motor presenters in

the CBS-M group. In the longitudinal study of McMonagle et al. [McMonagle et al. 2006], there

was a shorter onset to the development of motor symptoms in patients with prominent right

hemispheric atrophy or left-sided akinesia in support of our perfusion findings. Our finding is

further substantiated by a voxel-based morphometry (VBM) MRI study demonstrating that

pathologically-proven CBD patients presenting with a prominent extrapyramidal syndrome, as

opposed to dementia, had atrophy compared to controls involving the superior premotor cortex

extending into the posterior superior, middle and inferior frontal lobes [Josephs et al. 2008].

Grey matter involvement of the supplementary motor area and parietal lobes was also observed

[Josephs et al. 2008]. Although the atrophy pattern was bilateral, it was slightly more

pronounced in the right hemisphere [Josephs et al. 2008]. A limitation of this study was that it

did not document the most affected side of the body in their CBS sample [Josephs et al. 2008].

Consistent with our patients meeting DSM-IV criteria for a diagnosis of dementia, MMSE scores

were, as expected, found to be significantly lower in the CBS-D versus CBS-M group. The CBS-

D group performed significantly worse on the forward digit span indicating a lower primary

attention span suggestive of fronto-subcortical dysfunction. The CBS-D group also showed

significantly more deficits on the Benton judgement of line orientation indicative of right

parieto-occipital dysfunction. These tests do not rely on intact motor function so the differences

are likely not related to the degree of motor impairment or apraxia.

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To our knowledge, no studies have compared neuropsychiatric features in CBS-D versus CBS-M

case series using the Neuropsychiatric Inventory. Although our results did not achieve statistical

significance, the CBS-D group had higher rates of apathy, irritability, and depressive symptoms

which typically involve the limbic-prefrontal circuit. Abnormal appetite and eating behaviours

were also seen in CBS-D versus CBS-M suggestive of more temporal lobe involvement in this

group. Using the Frontal Behavioural Inventory (FBI), a validated, caregiver-administered rating

scale of frontotemporal behavioural symptoms, Kertesz et al. [Kertesz et al. 2000b]

demonstrated variable, but higher scores on the FBI in cognitive-onset CBS patients compared to

motor-onset CBS consistent with this disorder having symptoms occurring along the spectrum of

FTLD.

5.5.2 Pathology

CBS is not only a clinically heterogeneous disorder, but it is also highly variable in terms of the

underlying pathologies that can cause the syndrome. It is the location and burden of the

pathology that produces the clinical syndrome with likely a lesser contribution coming from the

specific pathological changes [Lang 2003]. As such, the clinical syndrome of CBS does not

always predict the specific underlying pathology of CBD [Ling et al. 2010]. Wadia & Lang

[Wadia and Lang 2007] reviewed several studies (total of 83 cases) demonstrating that the CBS

predicts CBD pathology approximately 55% of the time. The second most common pathology

was PSP (21%) followed by Pick‟s disease (7%) [Wadia and Lang 2007]. McMonagle et al.

[McMonagle et al. 2006] followed 19 CBS patients prospectively until autopsy. Specifically,

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they found that a clinical diagnosis of CBS predicted CBD pathology in 58% of the cases, and

predicted underlying Tau histology in 84% of cases. Other pathologies included Alzheimer‟s

disease, FTLD-U pathology, and Gerstmann Straussler Scheinker disease [McMonagle et al.

2006]. Our study also followed patients prospectively until autopsy and found that the CBS

predicted CBD histology in 63% of cases. The rates of CBD pathology in our CBS sample are

similar to prior studies [Boeve et al. 1999;McMonagle et al. 2006]. Other Tau based pathologies

(25%) included mixed CBD with agyrophilic grains and PSP. Although the majority of our cases

were sporadic, one of our patients with CBS had a strong family history of CBS and ended up

having FTLD-U/TDP43 pathology due to PGRN mutation [Masellis et al. 2006]. With the

identification of mutations in PGRN as a major cause of FTLD spectrum disorders and in

particular because the FTLD-U/TDP43 pathology often affects the parietal lobes, CBS related to

PGRN mutation has been more commonly identified in recent years [Benussi et al. 2008;Benussi

et al. 2009;Gabryelewicz et al. 2010;Gass et al. 2006;Ghetti et al. 2008;Guerreiro et al.

2008;Kelley et al. 2009;Le, I et al. 2008;Lopez de et al. 2008;Moreno et al. 2009;Rademakers et

al. 2007;Rohrer et al. 2009;Spina et al. 2007;Yu et al. 2010].

We observed an association between atrophy patterns and white matter hyperintensities found on

MRI with findings on neuropathological examination. For the most part, the white matter

changes on MRI correlated with underlying pathological white matter atrophy and gliosis. In the

two CBD cases, who did not show corresponding asymmetry on the pathological examination,

we suspect that by the time of death, the asymmetrical cortical atrophy observed on MRI had

progressed to symmetrical involvement.

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5.5.3 MRI investigation

Assuming that 1) the presenting lateralized clinical syndrome correlates most strongly with the

area of maximal brain pathology and 2) brain atrophy on MRI is a biomarker of the underlying

burden of pathology, then why do only half of our cases demonstrate asymmetrical brain atrophy

contralateral to the most affected body side? One possibility is that the heterogeneous nature of

pathology underlying CBS may cause varying degrees of atrophy and of hemispheric/lobar

asymmetry. This may be even the case within subtypes of the same pathological substrate; a

study that examined MRI atrophy patterns in patients with diagnoses occurring along the FTLD

spectrum stratified according to type 1, 2, or 3 FTLD-U/TDP43 pathological subtypes

demonstrated that type 1 and 3 pathology were associated with asymmetrical atrophy whereas

type 2 pathology was associated with symmetrical atrophy [Rohrer et al. 2010a]. Similarly,

Whitwell et al. [Whitwell et al. 2010] stratified patients presenting with CBS based on their

underlying pathological diagnosis and compared the subgroups based on their MRI patterns of

atrophy. Although they did not comment on this specifically, it can be extrapolated from their

paper that CBS patients with FTLD-U/TDP43 and AD pathology tended to have more

asymmetrical MRI atrophy whereas those with CBD pathology had more symmetrical atrophy

patterns [Whitwell et al. 2010].

Another possible explanation to account for the lack of asymmetrical atrophy in half of our CBS

patients is that assessment of grey matter loss on MRI may not be a sensitive enough measure.

As such, SPECT perfusion or FDG-PET hypometabolism may be more sensitive in detecting

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hemispheric asymmetries in CBS compared to MRI. Consistent with this, Mendez et al [Mendez

et al. 2007] found that the use of SPECT/PET increased the sensitivity of establishing the correct

diagnosis of FTD (90.5%) compared to MRI atrophy patterns (63.5%). We have also recently

shown in a case that initially presented with PNFA and later evolved to CBS with underlying

FTLD-U/TDP43 pathology due to a novel PGRN mutation that longitudinal SPECT perfusion

loss in the less affected hemisphere occurred before atrophy had progressed on that side

[Gabryelewicz et al. 2010]. Similarly, in two cases of very early stage FTD followed

longitudinally, neuroimaging can be initially normal, but when perfusion abnormalities and

atrophy are eventually shown on SPECT and MRI, respectively, the perfusion abnormalities are

more extensive than the atrophy patterns [Gregory et al. 1999].

Subcortical white matter disease may also be contributing to some of the clinically lateralized

dysfunction and should also be considered as a potential biomarker underlying asymmetry in

CBS. Examining our MRI data, close to 90% of CBS cases demonstrated hyperintensities in the

white matter on T2/PD sequences, and, in about 23%, these were localized contralateral to the

most affected side of the body correlating with side of maximal atrophy (15%) or occurred

contralateral to the motor deficits independent of atrophy in symmetrical cases (8%). This data is

descriptive, but supports the idea that some white matter hyperintensities do not just represent

age-associated microangiopathy [Levy-Cooperman et al. 2008b], but instead may be the result of

white matter glial damage related to CBD [Tan et al. 2005;Forman et al. 2002]. Our MRI-

pathological correlative data confirm this is indeed the case in CBS due to a variety of

pathologies. Recent diffusion tensor imaging (DTI) studies examining the integrity of white

matter tracts adds stronger evidence to this argument. Specifically, two studies have shown

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reduced fractional anisotropy and increased mean diffusivity in CBS occurring contralateral to

the most affected side of the body compared to controls, suggesting that damage to the integrity

of white matter tracts may account for some of the contralateral clinical findings [Boelmans et al.

2009;Bozzali et al. 2008]. Another DTI study found that mean diffusivity was elevated in the

motor thalamus in CBD ipsilateral to the most affected hemisphere (i.e., contralateral to most

affected side of the body), while mean diffusivity was elevated bilaterally in anterior and medial

thalamic areas in PSP [Erbetta et al. 2009].

5.5.4 Limitations

In terms of limitations, although our sample size would be considered reasonably large given the

rarity of the CBS phenotype, from a statistical perspective, the sample was indeed small with a

low power to detect differences especially with the use of non-parametric measures as well as

with multivariate analyses. This was particularly evident in the SPM analyses that attempted to

correct for multiple testing. Other limitations include the fact that not all patients were able to

complete the neuropsychological tests given that their degree of dementia or alternatively degree

of motor disability may have precluded this. Along the same lines, it is difficult to know if

impaired performance on tasks that involve writing and drawing were affected by the cognitive

disruption, motor disability or both. As such, our neuropsychological data is likely biased in

favour of those with milder forms of CBS and this will further reduce the power of the analysis.

Another limitation is that we employed a qualitative instead of a quantitative analysis of the MRI

data since there were only 21 patients who completed MRIs that were not degraded by motion

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artifact. Therefore, only about 2/3 of the sample had useable MRI data for a quantitative analysis,

which is currently in progress in an extended sample.

5.5.5 Conclusions

The current study provides a cross-sectional examination of neuropsychological, MRI and

SPECT features of a prospectively ascertained sample of CBS patients around the time of their

initial diagnosis with a subset followed until autopsy. It highlights the importance of having a

phenotypically well-characterized sample of patients diagnosed with CBS and provides new

insights into the neuropsychological and neuroimaging correlates of lateralized brain dysfunction

in the syndrome. Future studies will include analyzing neuropsychological, SPECT and MRI

changes longitudinally as the disease progresses with correlation to underlying pathology. Only

this kind of study will yield a true incidence of CBD and related pathological subtypes of the

syndrome. Finally, we believe that one of the issues that makes identification and replication of

genomic risk factors for neurodegenerative syndromes challenging is the significant

heterogeneity across these conditions. It will be increasingly necessary that groups share their

data in order to conduct larger studies, and, more importantly, efforts are made to develop novel

endophenotypes of these syndromes that will facilitate the identification of genomic and

epigenomic risk factors for CBS [Masellis et al. 2010].

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Behavioural/Functional Measures Scores


DAD (%) [n=30] 68.8 ± 5.7

DAD ADL (%) 76.7 ± 5.9

DAD iADL (%) 63.9 ± 6.4


NPI total /144 [n=29] 12.1 ± 2.5

NPI apathy /12 3.5 ± 0.8

NPI appetite and eating behaviour /12 2.1 ± 0.6

NPI dysphoria/depression /12 1.3 ± 0.5

NPI night-time behaviour /12 1.2 ± 0.5

NPI irritability/lability /12 1.0 ± 0.4

NPI agitation/aggression /12 0.9 ± 0.4

NPI aberrant motor behaviour /12 0.6 ± 0.4

NPI disinhibition /12 0.5 ± 0.2

NPI anxiety /12 0.5 ± 0.2

NPI delusions /12 0.2 ± 0.2

NPI hallucinations /12 0.0 ± 0.0

NPI euphoria /12 0.0 ± 0.0

NPI caregiver distress /12 6.7 ± 7.5

Cornell Depression Scale (CSDD; %) [n=30] 22.8 ± 2.6 Supplementary Table 1. Mean scores (± SEM) on behavioural and functional measures in the CBS group.

The number of patients (n) tested is listed next to individual measures. Missing values are secondary to the inability

of the patient or caregiver to complete the test. DAD = Disability Assessment for Dementia; ADL = Activities of

daily living; iADL = Instrumental activities of daily living; NPI = Neuropsychiatric Inventory. CSDD = Cornell

Scale for Depression in Dementia.

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6.0 Summary and General Discussion

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6.0 Summary and General Discussion

This thesis has examined several aspects of CBS through the comprehensive study of 31 patients

who met criteria for the clinical syndrome [Boeve et al. 2003]. Multi-modal assessments were

used to characterize the patients including a detailed neurological examination by a movement

disorder and/ or cognitive neurologist with experience in diagnosing the condition,

comprehensive neuropsychological and neuropsychiatric testing, qualitative visual assessment of

MRI scans, and semi-quantitative assessment of SPECT perfusion images using both region of

interest and voxel-wise approaches to data analysis. Additionally, a subset of eight patients

underwent neuropathological examination in order to identify the underlying pathological

substrate of the syndrome and also to correlate imaging features with the burden of observed

pathology. Finally, two families that segregated CBS and related FTD spectrum disorders due to

PRGN mutation are discussed in terms of their heterogeneity in clinical and neuroimaging

findings. This thesis provides many new insights into aspects of CBS from both the genetic and

brain-behaviour correlative perspective and also confirms many findings of previous studies. The

overall thesis findings will now be discussed in the context of prior literature. Limitations and

future recommendations will be also be reviewed.

6.1 Representative sample

6.1.1 Demographic features

The mean age of onset in our CBS sample was approximately 65 years, and 61% of our sample

was female. Our basic demographics compare well to those of previously published case series

of CBS that have shown a mean age of onset of approximately 63 [Wenning et al. 1998] and 61

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years [Murray et al. 2007] with a predominance of affected females in several studies

[Mahapatra et al. 2004]. Differences in age of onset between our study and others might reflect

the fact that these studies included only pathologically confirmed cases of CBD, some of whom

did not meet clinical criteria for CBS, while our study included clinically-diagnosed cases with a

small proportion of pathologically-confirmed ones. When we examined the age of onset in our 5

pathologically-confirmed cases of CBD, it was 64.8 (standard error of the mean 3.0) years

similar to that found in our entire patient sample.

6.1.2 Clinical and neuropsychological features

In terms of clinical features, about half of the patients presented with right-sided predominant

symptoms, while the left was most severely affected in the other half. This distribution of

asymmetry is consistent with other large studies [Huey et al. 2009a;Riley et al. 1990;Rinne et al.

1994;Shelley et al. 2009]. Overall, there does not seem to be a predilection for one hemisphere

over another, although this has been seen perhaps as an artifact in smaller samples by chance

alone [Chang et al. 2007]. At the time of their initial presentation, on average, about three years

into the course of the disease, the most common symptoms/signs were asymmetric rigidity and

apraxia, which eventually occurred in all patients. This was very similar to several large studies

[Kompoliti et al. 1998;Riley and Lang 2000;Rinne et al. 1994;Wenning et al. 1998].

Patients presenting with early dementia before the onset of motor features was also relatively

frequent affecting approximately 71% of the sample. The frequent occurrence of an early

dementia presentation is similar to previous studies [Bergeron et al. 1998;Grimes et al.

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1999b;Mathuranath et al. 2000], although it may have been biased in our study because close to

2/3 of patients were recruited from a cognitive neurology clinic. However, there were some cases

with early dementia who presented to a movement disorders clinic initially. The presence of a

language or speech disturbance was also very common in our study. Cortical sensory loss

manifested by extinction to double blind tactile stimuli, agraphesthesia and/or astereognosis was

also relatively frequent followed by dystonia. Contrary to previous studies, alien limb

phenomenon in our cohort was uncommon, although limb levitation was relatively common.

This likely reflects the fact that many prior studies did not distinguish between these two

phenomena, as there is debate as to the true definition of alien-limb phenomenon (reviewed in

Boeve et al., 2003 Lang). Apart from the differences highlighted above, our cohort presented

with similar clinical features as previous cohorts [Bergeron et al. 1998;Grimes et al.

1999b;Mathuranath et al. 2000] suggesting our sample is representative.

As expected, compared to the normal control group, the CBS patients were significantly

impaired across all domains of cognitive functioning and were mildly demented based on MMSE

and MDRS scores. Most pronounced deficits were noted on tests of working memory, executive

functions, praxis, and visuospatial abilities. This cognitive profile is quite typical of prior studies

in CBS, which have shown frontal subcortical and visuospatial dysfunction as well as significant

apraxia, the latter being the most common finding [Graham et al. 2003b].

Another study compared several tasks of executive function using the Delis-Kaplan Executive

Function System (D-KEFS) in CBS to an FTD group [Huey et al. 2009a]. This study found that

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although both groups exhibited prominent executive dysfunction, performance on most executive

tests tended to be worse in FTD except for tasks such as Trail Making and timed measures of the

Tower test. These tests require intact motor and visuospatial function that are more impaired in

CBS than in FTD [Huey et al. 2009a].

Episodic memory disturbance has also been documented, although this tends to be variable

across individual patients and based on severity of illness [Graham et al. 2003b]. It is thought to

reflect dysfunction of frontal subcortical circuits, rather than primary hippocampal involvement,

and, as such, episodic memory function in CBS also tends to be less affected than that seen in

AD [Graham et al. 2003b]. Two other studies have also confirmed relative preservation of

episodic memory function in CBS [Huey et al. 2009a;Murray et al. 2007]. Although we did not

compare episodic memory performance in our cohort directly with that of an AD cohort,

performance on delayed cued recall on the CVLT was better than delayed free recall supporting

the premise that it may be associated with poor use of strategic processes in encoding and

retrieval (i.e., frontal-subcortical dysfunction) as proposed by Pillon and Dubois [Pillon and

Dubois 2000].

Aphasia was commonly associated with CBS in our sample. Based on purely a clinical

assessment, 77% of patients had an observed language disturbance. This finding was strongly

supported using the WAB, a formal rating instrument of language function, as approximately

74% of the sample was identified as having an aphasic disturbance at the time of their initial

neuropsychological testing. Based on the data available, we were not able to determine how

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many individuals had an associated apraxia of speech, which along with progressive non-fluent

aphasia, have been proposed to be clinical markers of both CBD and PSP pathology [Josephs and

Duffy 2008]. The most common language disturbances seen in our sample were anomic and non-

fluent aphasia subtypes, which are consistent with findings of previous studies [Ferrer et al.

2003;Frattali et al. 2000;Graham et al. 2003a;Kertesz et al. 2000b;McMonagle et al.

2006;Murray et al. 2007]. One patient had a Wernicke‟s aphasia, which has previously been

observed [McMonagle et al. 2006], while another patient had a conduction aphasia with

reasonably intact comprehension and fluency, but impaired repetition. Graham et al. [Graham et

al. 2003a] found that phonologic processing was impaired in a series of 10 unselected CBS

patients. Only two of their patients had a full syndrome of progressive non-fluent aphasia. In a

review of the literature that included 399 patients with CBS, 34% had aphasia, and, of 39

patients with sufficient language characterization to allow for stratification into different aphasic

groups, 56% of these patients had a non-fluent presentation [Graham et al. 2003b]. They

proposed that patients with early troubles in phonologic processing may represent part of the

same spectrum with PNFA being at the more severe extreme.

6.1.3 Neuropsychiatric features

The majority of CBS patients in our sample experienced neuropsychiatric symptoms. Apathy

was both the most frequent and severe symptom in CBS patients followed by abnormal

appetite/eating behaviour and depressive symptoms. Litvan et al [Litvan et al. 1998] used the

NPI 10-item version, which does not assess for abnormal appetite/eating behaviour or aberrant

night-time behaviour, to compare behavioural symptoms in 15 CBS and 34 PSP patients. In

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contrast to our results, they found that depressive symptoms in CBS were more common than

apathy, the latter being more severe in PSP [Litvan et al. 1998]. Their CBS sample was smaller

than ours, which may have accounted for the difference seen between our sample and theirs.

Alternatively, it may reflect differences between the two groups in the caregivers‟ interpretation

of observed patient signs. Irrespective of these inter-group differences, both depression and

apathy have been shown to localize to overlapping regions of the limbic-prefrontal circuit, which

are affected in CBS.

To our knowledge, there have been no studies which directly correlate apathy or depressive

symptoms to specific brain areas in CBS. Dorsolateral prefrontal and anterior cingulate regions

have been shown to be hypometabolic in FDG-PET studies of primary depression [Liotti and

Mayberg 2001] and hypoperfused in ECD-SPECT studies of depressive symptoms associated

with AD [Levy-Cooperman et al. 2008a]. With respect to apathy, MRI studies have

demonstrated that atrophy in the dorsolateral prefrontal cortex and anterior cingulate gyrus was

associated with apathy in FTD [Zamboni et al. 2008]. Similarly, in Alzheimer‟s disease, apathy

was associated with reduced blood flow on SPECT in the anterior cingulate gyrus and

orbitofrontal regions [Lanctot et al. 2007]. Therefore, it is likely that these regions also play a

role in mediating apathy and depressive symptoms in CBS.

Similar to our results, Litvan et al. [Litvan et al. 1998] also found that irritability and agitation

were also relatively frequent in CBS. Symptoms of anxiety and disinhibition were more frequent

in our CBS patients than theirs; this difference likely occurred by chance due to small sample

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sizes. A retrospective review of 36 pathologically-proven cases of CBD found that eight of these

patients had well-documented neuropsychiatric problems including: behavioural dyscontrol,

depression, compulsive behaviour, irritability and disinhibition [Geda et al. 2007]. The majority

of these eight patients had clinical diagnoses occurring along the spectrum of FTD, while only

two patients clinically had CBS [Geda et al. 2007]. The symptoms were identified

retrospectively and there was no use of formal psychometric measures to detect symptoms. Both

of these factors likely account for the lower occurrence of neuropsychiatric symptoms in this

study from the Mayo clinic compared to ours. However, we cannot exclude that the specificity of

identifying true CBD pathology may also be contributing to this discrepant finding between

samples. Other studies have confirmed the occurrence of frontal behaviours in CBS [Borroni et

al. 2009;Kertesz et al. 2000b]. None of our prospectively ascertained CBS patients experienced

visual hallucinations similar to findings of a retrospective review of 36 pathologically-proven

cases of CBD [Geda et al. 2007] and extending on the findings of a prospective study of 11

patients with CBS [Cooper and Josephs 2009] in a larger sample.

6. 2 Apraxia in CBS

As discussed in chapter 4, there have been inconsistent findings with respect to subtypes of limb

apraxia observed in CBS. Ideomotor apraxia is the most commonly documented apraxia with all

patients developing this at some point during the course of the disease followed by limb-kinetic

apraxia, although many studies did not examine specifically for this subtype. However, the

occurrence of conceptual/ ideational apraxia appears to be highly variable with several studies

not demonstrating this phenomenon, while others demonstrated it in 30 to 60% of the CBS

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patients studied [Kertesz et al. 2000b;Leiguarda et al. 1994;Spatt et al. 2002]. There are several

reasons why conceptual/ ideational apraxia appears to be so variable in CBS and important

reasons include the way that this subtype is defined and also the fact that different rating

instruments have been used to assess for this phenomenon. In addition, one predicts that there

would be inconsistency in our ability to map conceptual/ ideational apraxia to specific brain

regions based on this variable nosology. However, other difficulties with characterizing this type

of apraxia may be even more fundamental in nature as discussed below.

Phenomenological confusion obviously exists because of the fact that „artificial‟

neuropsychological constructs, such as conceptual/ ideational apraxia, have been synthesized by

neuropsychologists, cognitive neurologists and behavioural scientists somewhat independently in

order explain observed behaviours. The reality is that praxis, especially relating to tool use, has

slowly evolved over time in response to evolutionary forces to which humans (and their

primitive ancestors) have been subjected. Therefore, definitions that we create and simplistic

models of clinical localization to discrete brain regions are likely to be inadequate in the study of

brain-behaviour relationships [Masellis et al. 2010], as emerging functional connectivity

methods are now revealing [Greicius et al. 2004].

Notwithstanding these issues, many questions remain unanswered with respect to limb praxis in

general and apraxia in neurodegenerative disease: Why does praxis appear to reside mainly in the

dominant hemisphere? What evolutionary forces (environmental factors) combined with

individual heritability (genes) caused the phenomenon to localize there? What disease specific

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factors cause apraxia to manifest? Questions relating to the latter include: what specific

hemispheric, cortical and subcortical lobar involvement predisposes to the different subtypes of

apraxia and what are the specific effects of the underlying neurodegenerative pathology in

determining apraxia? Answers to these questions will have to come from well-designed

prospective and longitudinal neuroimaging and neuropsychological studies of corticobasal

syndrome followed to death with subsequent histopathological, genomic and epigenomic

analyses.

6.3 Comment on the neuroimaging methods

The current study is unique in that SPECT imaging was analyzed using two different approaches.

This included the unbiased, „top-down‟ SPM method, which is more stringent, in addition to the

hypothesis-driven, „bottom-up‟ ROI method, which examines mean perfusion differences

referenced to the cerebellum across select brain regions hypothesized to be involved in the

pathophysiology of disease. Although both methods often identify the same regions affected in

the between group comparisons, occasionally there was no overlap seen. This is because the

methods look at the data in different ways. Statistical parametric maps are image processes based

on voxel values relating to intensity in the case of SPECT, that, under the null hypothesis, fall

under a known distribution such as the Student‟s t- or F distributions

(http://www.fil.ion.ucl.ac.uk/spm/doc/intro/). Multivariate analysis of covariance is performed

on a voxel-by-voxel basis using the general linear model and Gaussian Random Field theory to

make inferences about the spatially extended data (http://www.fil.ion.ucl.ac.uk/spm/doc/intro/).

Voxels of altered intensity that cluster together and survive correction for multiple testing

181

identify areas of altered perfusion between or within groups depending on the type of association

being performed. Contrast this to the ROI method that looks at the mean perfusion in a defined

region of interest (ROI) referenced to the cerebellum. The number of voxels within that region is

pre-defined and the ROIs are based on a template map that is usually traced on a structural MRI.

The use of the ROI method may facilitate the identification of potentially „false negative‟ areas

(type II errors) of reduced perfusion missed by the stringent SPM analysis, while at the same

time the SPM method may either identify important smaller regions of reduced perfusion

„washed out‟ in the larger ROI or alternatively emphasize the most strongly hypoperfused

regions in the CBS group. In this way, the methods are used in synergy to capture the most

salient regions of reduced perfusion in CBS.

As described in the introduction, two main functional neuroimaging techniques, PET and

SPECT, have been used to characterize CBS from the perspective of alterations in cerebral

metabolism and perfusion, respectively. A brief discussion of these two nuclear medicine

imaging modalities will now be provided in order to contrast strengths and weaknesses of both

techniques. The literature will also be reviewed in terms of how they compare with respect to

ability to assist in the accurate diagnosis of dementia. The focus of the discussion will be on

FDG-PET compared to HMPAO- or ECD-SPECT.

The basic underlying principles of PET and SPECT neuroimaging are similar: a radionuclide-

labeled tracer is given intravenously and is taken up by the brain and based on its kinetic

properties (absorption, distribution and metabolism) combined with the decay of the radionuclide

182

and the detection of the latter, images of the brain can be obtained that demonstrate relative

distribution of the tracer in different cerebral regions. For the most part, cerebral glucose

metabolism parallels cerebral perfusion, but occasionally this relationship breaks down in the

presence of cerebrovascular disease [Silverman 2004]. The most commonly used PET tracer is

18F-fluorodeoxyglucose (FDG), which serves as a marker of cerebral glucose metabolism [Bailey

et al. 2005;Frackowiak and Friston 1994]. The radioactively-labeled 18F isotope is synthesized

in a cyclotron by accelerating protons into the nuclei of fluorine atoms. This results in fluorine

with an extra proton in the nucleus producing an unstable isotope, 18F, with a relatively short

half-life. The 18F isotope is then incorporated into the deoxyglucose molecule forming FDG.

FDG is then administered to the subject intravenously and gets distributed regionally in the brain

reflecting cerebral glucose metabolism. After a delay to ensure the appropriate uptake of FDG in

the brain, the subject is placed in a PET scanner, which is comprised of arrays of gamma ray

detectors that encircle the subject‟s head. As the isotope undergoes positron emission decay, the

emitted positron travels a very short distance usually in the range of millimeters dissipating

energy until it encounters an electron. The interaction between the positron and the electron

annihilate each other and results in two gamma photons that travel in opposite directions from

each other. The gamma detectors that encircle the subject‟s head are set up in such a way as to

only detect coincident gamma photons, that are, ones detected simultaneously by two detectors

oriented directly across from each other. The scanner, therefore, detects the site of the

annihilation event and the distance between this and the emitting nucleus limits the spatial

resolution of the technique. During the scanning process, multiple detections are obtained all

oriented along lines each in one plane or slice and from these reconstructed images of the three-

dimensional brain can be derived.

183

Brain SPECT has some important fundamental differences that result in practical advantages and

important limitations of this technique compared to PET [Rahmim and Zaidi 2008]. SPECT also

uses radionuclide tracers to study CBS and other dementias with the most common ones being,

Tc99-HMPAO and -ECD, and these are taken up at a rate that is proportional to cerebral blood

flow [Matsuda et al. 1995]. Therefore, SPECT provides a measure of regional cerebral perfusion

to the brain as opposed to glucose metabolism. SPECT tracers also emit gamma radiation.

However, the gamma photon is detected directly by a camera comprised of a series of physical

collimators lying over detection crystals and photomultiplier arrays. The collimators reject

photons that are not within a small angular range thereby facilitating the localization of the origin

of the gamma ray [Rahmim and Zaidi 2008]. The gamma camera usually rotates 360 degrees

around the patient‟s head allowing for two dimensional images that can then be reconstructed

into a three-dimensional view of the brain. Because the camera only detects one gamma photon

for every emission event, the spatial resolution of SPECT is much lower than that of PET and

this is a relative weakness of the technique [Rahmim and Zaidi 2008]. On the other hand, the

main advantage of this technique has to do with cost and availability. This is because the

radionuclide tracers typically used are more stable in terms of their gamma decay and can

therefore be synthesized off-site and transported to the imaging centre. In other words, there is

no need for a cyclotron on site to synthesize the compounds. In addition, the cameras used are

typically less expensive than the PET scanner technology. As a result of these factors, SPECT

imaging can be acquired more quickly, is significantly cheaper and is more widely available

[Colloby and O'Brien 2004].

184

There have been a few studies that have compared SPECT and PET in the same group of patients

with Alzheimer‟s disease in order to determine the ability of these investigations to improve

diagnostic accuracy [Silverman 2004]. In general, the degree of hypometabolism detected by

FDG-PET is usually greater than the magnitude of hypoperfusion abnormalities seen with

perfusion SPECT although the regions of deficit observed by both methods are similar for AD

[Silverman 2004]. Side-by-side FDG-PET and perfusion SPECT studies of AD have

demonstrated higher sensitivity and diagnostic accuracy of FDG-PET [Silverman 2004]. Studies

using high-resolution SPECT cameras have shown that perfusion SPECT has 15 to 20% reduced

accuracy in the diagnosis of AD compared to FDG-PET [Mielke and Heiss 1998]. An important

study that compared 26 patients with AD to six healthy controls using both HMPAO-SPECT and

FDG-PET data in a voxel-wise analysis found that the correlation across the whole brain using

both methods achieved statistical significance, but the strength of the association was weak

(average correlation coefficient [r] across all patients = 0.43) [Herholz et al. 2002]. However,

this correlation improved substantially when the analysis was restricted to clusters of abnormal

voxels in the temporoparietal and the posterior cingulate association cortices (r=0.90) [Herholz et

al. 2002]. Despite this improved correlation, the tracer uptake reductions using FDG-PET were

substantially more pronounced than that observed with HMPAO-SPECT indicating the former to

be more sensitive in detecting abnormalities [Herholz et al. 2002]. In addition, although tracer

uptake reductions correlated with severity of the dementia using both methods, the correlation

was stronger for PET compared to SPECT [Herholz et al. 2002]. Finally, this study also showed

that distinction of AD patients from controls was better over a wider range of z-thresholds for

FDG-PET than HMPAO-SPECT indicating increased sensitivity of the former [Herholz et al.

2002]. A more recent study compared the sensitivities of mesiotemporal atrophy on MRI,

185

reduced perfusion/hypometabolism in temporoparietal and posterior cingulate cortices on ECD-

SPECT/FDG-PET, respectively, and CSF biomarkers of beta-amyloid 1-42, total tau and

phosphorylated tau in 207 AD patients of varying severity [Morinaga et al. 2010].

Mesiotemporal atrophy on MRI was identified in 77.4%, reduced perfusion and metabolism in

the pre-defined regions in 81.6% (ECD-SPECT) and 93.1% (FDG-PET), and the typical CSF

profile for AD (that is, reduced beta-amyloid and increased total and phosphorylated tau) in 94%

of all the AD patients [Morinaga et al. 2010]. Using the Clinical Dementia Rating (CDR) scale,

used to assess severity of dementia with higher scores being more severe, they observed that all

investigations were sensitive at a CDR of 2, whereas at a CDR of 1 only the FDG-PET and CSF

biomarkers showed high sensitivity [Morinaga et al. 2010]. Finally at the mildest stages of

disease (CDR of 0.5), usually corresponding to cases of amnestic mild cognitive impairment,

only CSF biomarkers showed high sensitivity [Morinaga et al. 2010]. Limitations of this study

were that there was no control group so a discriminant function analysis could not be done, and

also that cases were not pathologically confirmed.

6.4 Can CBS serve as a model of etiology for common sporadic disorders?

Although CBS is a rare syndrome, it can serve as a good model of complex disease due to a

variety of observations. As discussed throughout this thesis, there is substantial pathological

heterogeneity that can produce the syndrome and this can make it difficult to identify genetic and

environmental factors that increase risk for the disease in clinically diagnosed cases. As shown

by recent studies in FTD and PSP, increasing the homogeneity of the sample by inclusion of only

one specific pathological subtype, for example, recent genome wide association studies (GWAS)

186

of FTLD-U/ TDP43 and PSP pathological cases, makes it easier to identify genetic risk factors

for the disease [Hoglinger et al. 2011;Van Deerlin et al. 2010]. In CBS, the pathological

heterogeneity can account for the multiple disease susceptibility loci observed (i.e., genetic

heterogeneity), for example, MAPT and PGRN mutations. However, within patients having a

pathological diagnosis of CBD, there are likely other disease susceptibility loci that have not yet

been discovered that can produce the typical pathology. The reason for this may be that different

pathways in the processing of the MAPT gene and the tau protein might yield the final common

pathology of CBD and therefore a systems biology approach will be helpful in sorting out the

genetic heterogeneity of the disease [Noorbakhsh et al. 2009]. Genetic modifiers may also

contribute to the heterogeneity of the CBS as demonstrated by a study identifying that common

variants in the TMEM106B gene can increase risk for FTLD-U/ TDP43 pathology even within

those harbouring PGRN mutations [Van Deerlin et al. 2010]. Clinical phenotypic variability is

due mainly to the area of the brain most affected by the underlying pathology and this may also

lead to misdiagnosis, which will further confound genetic studies of CBS. Finally, using PRGN

mutation as an example, the age of onset of individuals with FTD and/ or CBS due to PGRN

mutation is highly variable with some not developing disease until their late 80s [Kelley et al.

2009] and this may result in apparent incomplete penetrance of the disease-causing gene

mutation. All of these features of CBS can be observed in complex, non-Mendelian diseases.

Common neurodegenerative diseases, such as Alzheimer‟s and Parkinson‟s disease, are generally

considered sporadic disorders although rarely Mendelian segregation within families is observed

due to identified disease-causing mutations. This is similar to that observed in CBS and given the

rarity of CBS as a clinical entity, one can assume that there are even more numerous pathways

187

leading to AD and PD pathology and that these routes are even more complex in nature.

Recently, through the application of GWAS in very large, multicentre case-control cohorts,

several genes for many of the common neurodegenerative disorders have been identified, each of

which confers an incremental risk to individuals possessing the gene variants [2011;Do et al.

2011;Hollingworth et al. 2011;Naj et al. 2011;Nalls et al. 2011]. The genes include ones already

known to be involved in the underlying pathology of these disorders, for example, apolipoprotein

E (APOE) for Alzheimer‟s disease and alpha-synuclein (SNCA) for Parkinson‟s disease, as well

as many novel genes. The exact role that these genes play and the regional effects that their

variants have on the brain and secondarily on neurological and cognitive functions are mostly

unknown. Based on what we have learned about CBS, it is hypothesized that certain variants,

alone or in combination, strongly increase or decrease risk for these common neurodegenerations

in smaller subsets of the large GWAS samples rather than conferring a small risk to the entire

patient sample, that is, genetic heterogeneity. Other mechanisms of disease within these

identified candidate genes flagged by the GWAS, such as epigenomic factors, for example, DNA

methylation, must also be considered. In order to sort out the complexities of the

neurodegenerations in the post-genomics era, novel approaches such as “reverse phenotyping”

will be required in order to understand the regional brain effects that these genomic and

epigenomic factors have [Joober 2011;Schulze and McMahon 2004].

188

7.0 Conclusions and future directions

189

7.0 Conclusions and future directions

Corticobasal syndrome (CBS) is a unique cognitive and motor disorder. Better understanding of

this rare clinical entity with respect to etiology, clinical and neuroimaging features will provide

new insights that should improve our research approach to other more common, complex

neurodegenerative disorders, such as AD and PD. This thesis work has demonstrated genetic and

pathological heterogeneity underlying the CBS with discovery that PGRN mutation – producing

FTLD-U/ TDP43 pathology – can manifest as a fairly classical picture of the syndrome from the

clinical, cognitive, and neuroimaging perspective. It underscores the importance of obtaining

brain tissue for histopathological study as identification of the underlying pathology will

facilitate subgrouping of clinical cases based on pathology so that heterogeneity is reduced in the

attempts to discover underlying genetic factors.

Furthermore, the well-characterized clinical sample of CBS patients used in this study has

allowed us to expand our understanding of the different phenotypic presentations of the CBS and

associated clinical, cognitive and neuroimaging features. Those presenting with early dementia

tended to have the right side of the body most affected and also had more significant language

dysfunction indicating that the burden of underlying pathology was most pronounced in the left

hemisphere although the SPECT and MRI studies did not confirm this. Apart from issues related

to reduced power, we hypothesize that the reason leftwards asymmetry on MRI and SPECT was

not seen may be because, at the stage that the patients were studied, the microscopic pathology

that might have been asymmetrically more pronounced on the left did not appear to translate into

changes that could be detected in vivo using neuroimaging. The contrary was seen in the early

motor group who tended to have the left side of the body most affected with significant

reductions in perfusion involving the right supplementary and premotor areas.

190

We have also shown that even within the same family, there can be substantial heterogeneity in

clinical presentation due to the same PGRN mutation. The reason for this is that although the

predicted pathology in the brain will be the same in the affected family members, the

hemispheric and lobar localization of the pathology and its severity will determine the observed

clinical syndrome. Therefore, studies that focus on attempting to discern early imaging and other

biomarkers of disease that can predict a specific underlying pathological type in vivo will be

challenged by this heterogeneity in clinical presentation. We propose that future studies

attempting to look for early biological risk factors of CBS will need to examine subgroups of

CBS patients that are classified according to genetic or pathological type, as well as classified

according to in vivo hemispheric and lobar localization on neuroimaging, or even cognitive

endophenotypes, comparing “OMIC” measures across these subgroups. Since the sample size

within each subgroup will likely be small, multicentre studies will be necessary in order to

enhance the sample and thereby power of this approach.

Brain-behavioural correlative studies in CBS are an important line of research that can further

enhance our understanding of the phenomenon observed in the syndrome. This is exemplified by

our study examining severity of ideomotor apraxia and its association with hypoperfusion in the

left inferior parietal lobule. The inconsistencies across studies in terms of localization of limb

apraxia in CBS stem largely from differences in the tools used to assess this phenomenon and

also from the different nosologies used to describe the subtypes of apraxia to date. Investigators

working in this field will have to decide upon accepted diagnostic tools and definitions such that

191

studies are performed in a consistent fashion. We anticipate that this will resolve many of the

inconsistencies in the apraxia literature.

As our CBS sample size increases, we anticipate completing multi-modal neuroimaging analyses

including MRI brain with assessment of both grey matter atrophy and quantification of

underlying white matter disease using our on-site developed program, Lesion Explorer [Ramirez

et al. 2011], diffusion tensor imaging in a subset of patients, combined with brain SPECT

perfusion. We also plan on correlating this imaging dataset with a comprehensive assessment of

apraxia using a conceptual model in order to better identify the neuroanatomical correlates of the

different apraxia subtypes [Stamenova et al. 2011]. Finally, in a subset of the CBS cases,

longitudinal neuropsychological and neuroimaging data is available and further analysis of this

data will allow us to track the progression of the disease in terms of the most affected brain

regions and how these correlate with the neuropsychological and pathological measures over

time. Only this kind of longitudinal study will allow us to develop better in vivo biomarkers of

underlying pathology.

192

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