drosophila melanogaster as a model organism to study human ... · disease-causing genes. using the...

Drosophila melanogaster as a model organism to study human

neurodegenerative diseases

by

Kinga Maria Michno

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Department of Molecular Genetics

University of Toronto

© Copyright by Kinga Maria Michno (2009)

ii

Drosophila melanogaster as a model organism to study human

neurodegenerative diseases

Kinga Maria Michno

Doctor of Philosophy

Department of Molecular Genetics

University of Toronto

2009

Abstract

A great deal of our current understanding about the biology of neurodegenerative diseases has

come from studying the function of genes linked to inherited forms of these disorders. Work

performed in animal models, vertebrates as well as invertebrates, has been instrumental in

deciphering the cellular, physiological and behavioural deficits arising from the expression of

disease-causing genes. Using the fruit fly, Drosophila melanogaster, as a model we examined

the normal and aberrant function of two genes linked to the onset of neurodegeneration in

humans, presenilin and superoxide dismutase-1. Drosophila is an extremely versatile model and

in many ways is ideal for studying the genetic basis of human disease. The high degree of

genetic conservation coupled with low genetic redundancy make this model particularly well

suited for studying the function of disease causing genes. We demonstrate a novel genetic,

physical and physiological interaction between presenilin and calmodulin and describe how this

interaction impacts a very early cellular defect associated with Alzheimer’s Disease, intracellular

iii

calcium dyshomeostasis. We also describe progressive locomotory deficits in flies expressing

mutant alleles of the superoxide dismutase gene, which have been linked to the onset of familial

amyotrophic lateral sclerosis. Collectively, our work demonstrates that Drosophila can be used

to study the cellular, physiological and behavioural basis of human neurodegenerative diseases

and may provide a model to identify novel therapeutic avenues for neurodegenerative diseases.

iv

Acknowledgments

This thesis is a small part of a much bigger story. The story began many years ago when a young

couple decided to escape the constraints of communism to ensure a successful future for their

daughter—me. Having a graduate degree in Slavic Literature my Mother sacrificed her own

future so that I could pursue mine. For this reason I'd like to begin by thanking my mother for

her steadfast support, commitment and engagement in my education. I am thankful to both my

parents for insisting that I pursue my education and for supporting me both emotionally and

financially along the way. I would also like to thank my Father for being a living example of

how to think outside of the box and for teaching me to always look for creative solutions to

problems.

No matter how bad my day was at the lab, a single thought could make all the built up anxiety

and stress disappear. I would think of my husband Artur, and reflect on how lucky I am to be

able to share my life with such a fantastic individual and loving partner. Artur, your are my

foundation, my rock, my reality check and I couldn't have arrived at this point without your

support and patience—thank you.

Graduate research is much like passing a driving exam on an insane obstacle course. Each of us

gets behind the wheel and eagerly pushes the pedal to the metal; determined to be the first to

cross the finish line without any scratches or bruises. But at some point we all crash, and when

we do we do it those who drive with us who pick up the pieces and encourage us to get back

behind the wheel. It has been a true privilege to work along side my fellow Boulianners; to share

all my good and bad science days. I would also like to thank Tania Alexson who has been my

long-time friend, research colleague and confidant. Tania has helped me to re-analyze my data

from so many different angles my head is still spinning. I would also like to thank Shirley Liu

for being generous with her time in helping me ensure the timely completion of the final

experiment necessary to complete the final data chapter of this thesis.

v

A decisive milestone of this dissertation was a very productive collaboration with Diane

O'Dowd’s laboratory at the University of California. I have profound gratitude to Diane for her

guidance and for welcoming me into her lab. Diane provided me unrestricted access to her

equipment without which a considerable portion of the first data chapter would have been very

difficult to complete. By the same token I would like to thank Jorge Campusano and Betty

Siscero for taking the time to train me and for offering much appreciated technical assistance. I

would also like to acknowledge and thank Joel Levine for helping me design and execute the

locomotory behavioural analysis described in the second chapter.

I have been privileged in having excellent mentorship during my training and in chronological

order I would like to thank my mentors. First, thank you to Paul Hamel who took a chance on an

undergraduate student, completely wet behind the ears. As a master's supervisor Paul was

demanding and relentlessly pushed me until I developed a strong work ethic. I’d like to sincerely

thank my PhD supervisor; Gabrielle Boulianne. Gabrielle welcomed me into her lab even though

I had no experience working with Drosophila. Over the years Gabrielle has made a significant

impact on my scientific development. Gabrielle’s mentorship has been instrumental in helping

me to develop my critical thinking and communication skills. I am also very grateful to

Gabrielle for allowing me a great deal of ownership over my research and for being supportive

during my various collaborations. I would also like to thank my committee members Freda

Miller and Marla Sokolowski. Thanks to Gabrielle, Marla and Freda I always left my

committee meetings motivated and determined to reach my research goals. Thank you for your

insightful suggestions and encouragemen; it has been a true privilege to learn from each of you.

Finally, I’d like to thank the Creator for providing me with sufficient aptitude and resolve to

preservere. Believe; achieve.

vi

Table of Contents

Acknowledgments .......................................................................................................................... iv

Table of Contents ........................................................................................................................... vi

List of Tables ................................................................................................................................. ix

List of Figures ................................................................................................................................. x

List of Abbreviations .................................................................................................................... xii

CHAPTER 1 ................................................................................................................................... 1

1.1 Introduction to human neurodegenerative diseases ............................................................ 1

1.2 Alzheimer’s Disease ........................................................................................................... 2

1.2.1 Genetics of Alzheimer’s Disease ............................................................................ 3

1.2.2 Alzheimer’s Disease aetiology ............................................................................... 8

1.2.3 Therapeutic treatments .......................................................................................... 13

1.3 Amyotrophic Lateral Sclerosis ......................................................................................... 14

1.3.1 Genetics of Amyotrophic Lateral Sclerosis .......................................................... 15

1.3.2 Amyotrophic lateral sclerosis aetiology ................................................................ 17

1.3.3 Therapeutic treatments .......................................................................................... 19

1.4 Drosophila as a model of human neurodegenerative diseases ......................................... 20

1.4.1 Drosophila models of Alzheimer’s Disease ......................................................... 23

1.4.2 Drosophila models of Amyotrophic Lateral Sclerosis ......................................... 25

1.5 Purpose of our studies ....................................................................................................... 27

CHAPTER 2 ................................................................................................................................. 28

2 Analysis of the interaction of presenilin with the intracellular calcium stores machinery ...... 28

2.1 Abstract ............................................................................................................................. 29

2.2 Introduction ....................................................................................................................... 30

vii

2.3 Materials and Methods ...................................................................................................... 32

2.4 Results ............................................................................................................................... 36

2.4.1 Intracellular calcium dynamics in primary cholinergic Drosophila neurons ....... 36

2.4.2 Psn-induces deficits in intracellular calcium stores content ................................. 38

2.4.3 Loss-of-function mutations in Cam suppress Psn-induced wing scalloping ........ 41

2.4.4 Cam suppresses Psn-induced deficits in intracellular calcium stores content ...... 43

2.4.5 Psn and Cam physically interact ........................................................................... 45

2.4.6 Primary neurons expressing wild type Psn have lower incidence of apoptosis ... 47

2.4.7 Cholinergic expression of fAD-mutant Psn results in shortened lifespan. ........... 50

2.5 Discusion ........................................................................................................................... 55

CHAPTER 3 ................................................................................................................................. 60

3.1 Abstract ............................................................................................................................. 61

3.2 Introduction ....................................................................................................................... 62

3.3 Methods and materials ...................................................................................................... 64

3.4 Results ............................................................................................................................... 67

3.4.1 Analysis of transgenic flies expressing human wild type and fALS-SOD1 ......... 67

3.4.2 Ubiquitous expression of fALS-SOD1 gives rise to progressive deficits in

adult fly locomotory activity ................................................................................. 69

3.4.3 Survival analysis of flies expressing wild type or fALS-SOD1 ........................... 77

3.4.4 fALS-SOD1 does not appear to form aggregates in adult flies ............................ 81

3.5 Discussion ......................................................................................................................... 84

CHAPTER 4 ................................................................................................................................. 89

4.1 Discussion ......................................................................................................................... 89

4.1.1 Are aggregates toxic? ............................................................................................ 89

4.1.2 What role does stress play? ................................................................................... 90

4.1.3 Behavioural Genetics in Drosophila ..................................................................... 93

viii

4.1.4 Concluding thoughts ............................................................................................. 94

References ..................................................................................................................................... 95

Appendices .................................................................................................................................. 113

ix

List of Tables

Table 1. Survival Analysis ........................................................................................................... 51

Table 2. Average day time activity of 42-45 day old flies ubiquitously expressing wild type or

fALS-mutant human SOD1. ......................................................................................................... 71





Table 5. Survival Analysis ........................................................................................................... 79

x

List of Figures

Figure 1. Schematic of intramembraneous APP proteolysis. ........................................................ 4

Figure 2. The γ-secretase complex ................................................................................................. 6

Figure 3. Intracellular calcium signalling .................................................................................... 11

Figure 4. The GAL4/UAS system ............................................................................................... 22

Figure 5. Evaluation of calcium content in internal stores in Drosophila primary neuronal

cultures .......................................................................................................................................... 37

Figure 6. Expression of wild type and FAD-mutant Psn. ............................................................ 39

Figure 7. Calcium content in internal calcium stores is affected in cholinergic neurons

expressing Psn. ............................................................................................................................. 40

Figure 8. Psn-induced wing notching is suppressed by loss-of-function mutations in Cam. ...... 42

Figure 9. Psn-induced effects on intracellular calcium stores is suppressed by a loss-of-function

mutation in Cam mutations. .......................................................................................................... 44

Figure 10. Cam binds to full-length as well as the N-terminal fragment of Psn. ........................ 46

Figure 11. Total incidence of apoptosis in cultures expressing wild type or FAD-mutant Psn. . 48

Figure 12. Expression of wild type Psn in cholinergic neurons facilitates cell-autonomous cell

survival. ......................................................................................................................................... 49

Figure 13. Survival analysis of flies expressing wild type Psn in cholinergic neurons. .............. 52

Figure 14. Survival analysis of flies expressing FAD-Psn in cholinergic neurons. .................... 53

Figure 15. Survival analysis for flies expressing FAD-Psn in cholinergic neurons with or

without the loss of a single Cam allele. ........................................................................................ 54

xi

Figure 16. Expression of wild type and fALS-mutant human SOD1. ......................................... 68

Figure 17. Day activity levels of 42-45 day old flies expressing wild type or fALS-SOD1. ...... 70



Figure 20. Survival analysis of flies ubiquitously expressing fALS-mutant SOD1. ................... 78

Figure 21. Survival analysis of flies ubiquitously expressing wild type SOD1. ......................... 80

Figure 22. Misfolded human SOD1 is not detected in lysates generated from 30 day old flies

expressing human fALS-SOD1. ................................................................................................... 82

Figure 23. Misfolded human SOD1 is not detected in lysates generated from 56-59 day old flies

expressing human fALS-SOD1. ................................................................................................... 83

Figure 24. Misfolded human SOD1 is not detected in lysates generated from 56-59 day old flies

expressing two copies of human fALS-SOD1. ............................................................................. 85

xii

List of Abbreviations

A amyloid peptide

AD Alzheimer’s disease

AICD APP intracellular domain

ALS Amyotrophic Lateral Sclerosis

ANOVA Analysis of variance

APP Amyloid precursor protein

Appl Drosophial Amyloid precursor protein-like gene or protein

BACE site APP cleaving enzyme

BAK Bcl-2 homologous antagonist/killer

BAX Bcl-2–associated X protein

BSA Bovine serum albumin

bZIP Basic Leucine Zipper

C/EBP CCAAT-enhancer-binding proteins

CaKII Calmodulin kinase II

CALHM1 calcium homeostasis modulator 1

Cam Calmodulin

CBP Calcium binding protein

CHOP CCAAT/enhancer binding protein

CNS Central nervous system

Creb cAMP response element binding

DNA Deoxyribose nucleic acid

DSM Diagnostic and Statistical Manual of Mental Disorders

DTT Dithiothreitol

http://en.wikipedia.org/wiki/Cyclic_adenosine_monophosphate

xiii

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid

EMG Electromyography

ER Endoplasmic reticulum

ERAD ER-associated

fAD familial Alzheimer’s Disease

fALS familial Amyotrophic Lateral Sclerosis

FBS Fetal bovine serum

GADD153 Growth Arrest and DNA Damage

GFP Green fluorescent protein

HBSS Hank's buffered salt solution

HEK 293 Human Embryonic Kidney cell line 293

HRP Horse radish peroxidase

HS Human SOD1

HSP Mammalian heat shock protein

Hsp Drosophila heat shock protein

IHC Immunohistochemistry

IP3R nositol triphosphate receptor

LTP Long term potentiation

MND Motor neuron disease

NDS Normal donkey serum

NEP2 Mammalina neprilysin 2 gene or protein

Nep2 Drosophila neprilysin 2 gene or protein

NGS Normal goat serum

PAG proliferation associated gene

PBS Phosphate buffer saline

xiv

PI3K Phosphoinositide 3-kinases

PS Mouse presenilin gene or protein

PSEN Human presenilin gene

Psn Drosophila presenilin gene or protein

PVDF Polyvinylidene Fluoride

RPM Rotations per minute

RyR Ryanodine receptor

sAD sporadic Alzheimer’s Disease

sALS sporadic Amyotrophic Lateral Sclerosis

SDS Sodium Dodecyl Sulfate

SERCA Sarco/Endoplasmic Reticulum Ca2+

-ATPase

Sod Drosophila superoxide dismutase gene or protein

SOD1 Human superoxide dismutase gene or protein

TACE Tumor necrosis factor-α-converting enzyme

TBST Tris-Buffered Saline Tween-20

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

UAS Upstream activating sequence

UPR Unfolded protein response

WT Wild type

http://en.wikipedia.org/wiki/Sarcoplasmic_reticulum

http://en.wikipedia.org/wiki/Endoplasmic_reticulum

http://en.wikipedia.org/wiki/Calcium

http://en.wikipedia.org/wiki/ATPase

http://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase

1

CHAPTER 1

1.1 Introduction to human neurodegenerative diseases

Today, the aging Canadian population is faced with the grim reality that there are no effective

treatments let alone a cure for many of the diseases that target the elderly brain. Affected

individuals inevitably lose the ability to care for themselves causing significant emotional stress

to both patients and their family. The high costs of long-term palliative care and therapeutics,

which only partially alleviate some symptoms, poses a considerable economic burden on the

elderly, their caregivers as well as on our social health care system. It is estimated that

Canadians spend 5.5 billion dollars annually treating and caring for persons with dementia alone

(Ostbye and Crosse. 1994). Though staggering, the current expenditures pale in comparison with

what’s projected to come. By 2041 nearly a quarter of the Canadian population will be 65 years

of age or older, relative to the current proportion of 12% (Lindsay. 1999). Given that age is the

single most easily identifiable risk factor for neurodegenerative diseases, as the proportion of

seniors continues to increase, so will the prevalence of neurodegenerative diseases in our society

(Nelson. 1995;Anonymous1994).

Research has turned to animal models to explore the cellular and molecular mechanisms

involved in human disease pathogenesis in hopes of one day finding a cure. Modelling human

neurodegenerative diseases in animal models has been instrumental to our current understanding

about the biology of neurodegenerative disease. In addition, work performed in animal models is

indeed guiding the development of new therapeutics. In the proceeding sections two human

neurodegenerative diseases, Alzheimer’s Disease (AD) and Amyotrophic Lateral Sclerosis

(ALS), will be described and explored based on work performed in both vertebrate as well as

invertebrate animal models. In the final section the advantages of using Drosophila

melanogaster as a model for human neurodegenerative diseases will be specifically addressed.

2

1.2 Alzheimer’s Disease

Alzhiemer’s Disease (AD) is the most prevalent form of dementia and affects an estimated 5-8%

of the Canadian population (Anonymous1994;Lindsay et al. 2004). At onset, AD symptoms

include mild cognitive impairment that gradually progresses to severe, incapacitating memory

loss. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM IV) AD

may be suspected if a patient presents with an impaired ability to learn new information or recall

previously learned information concomitant with other cognitive disturbances affecting

language, recognition or motor ability. The duration of disease can range from 1-25 years and

the cause of death is usually associated with malnutrition or pneumonia (Bird. 2008). A positive

AD diagnosis relies both on clinical as well as postmortem neuropathological assessments by

autopsy examination of the brain. AD brains exhibit gross cerebral cortex atrophy as well as

microscopic extracellular amyloid-β (Aβ) neuritic plaques and intraneuronal neurofibrillary

tangles (NFT) (Bird. 2008). NFTs are composed of the hyperphosphorylated form of the

microtubule-associated protein tau. Neuritic plaques, on the other hand, are composed primarily

of a 42- and to a lesser extent a 40-residue β-amyloid peptide (Aβ1-42 and Aβ1-40), amyloidogenic

cleavage products of the amyloid precursor protein (APP).

Synaptic loss in the frontal cortex has been documented in AD brains by electron microscopy

studies and correlates well with cognitive impairment highlighting the importance of preserved

synaptic connectivity for normal brain function (DeKosky and Scheff. 1990).

Immunohistochemical analysis (IHC) has revealed the specific loss of cholinergic neurons which

utilize acetylcholine as a neurotransmitter in AD brains (Geula and Mesulam. 1989). IHC has

also demonstrated intracellular NFT accumulation in AD affected brains (Giannakopoulos et al.

2003) and the deposition of NFTs also correlates with the degree of clinical impairment

(Giannakopoulos et al. 2003). Conversely, although some studies suggest a positive correlation

between increasing numbers of neuritic plaques and cognitive impairment (Hyman. 1997) others

studies show poor correlation between both the distribution and quantity of plaques and clinical

symptoms (Giannakopoulos et al. 2003) as well as neuronal cell loss (Terry et al. 1991). This is

not meant to imply that neuritic plaques do not play a role in AD pathogenesis but rather to

3

suggest that plaque deposition is not a reliable predictor of the degree of cognitive impairment in

AD.

1.2.1 Genetics of Alzheimer’s Disease

Although the majority of AD cases occur sporadically (sAD) approximately 10% are inherited

and are specifically referred to as familial AD (fAD). fAD is clinically and pathologically

indistinguishable from sAD with the exception that the onset of symptoms typically occurs

earlier in fAD (Bird. 2008). To date three genes have been identified that segregate with fAD

(Goate et al. 1991b).

1.2.1.1 The Amyloid Precursor Protein gene

The first fAD–linked gene mutation to be identified was a missense mutation in the amyloid

precursor protein (APP) gene (Goate et al. 1991b). Today over 20 fAD-mutations in the APP

gene have been identified (Gotz and Ittner. 2008). The APP gene encodes a type 1

transmembrane protein that is synthesized in the endoplasmic reticulum (ER), trafficked along

the secretory pathway to the plasma membrane where it undergoes proteolysis. APP proteolysis

is particularly relevant to AD because aberrant APP proteolysis promotes the deposition of β-

amyloid plaques (Figure 1). The extracellular N-terminal of APP can be cleaved by one of two

proteolytic complexes, α-secretase or β-secretase. α-secretase cleavage is catalyzed by the

tumour necrosis factor-α converting enzyme (TACE), which cleaves APP within the β-amyloid

domain precluding Aβ generation. For this reason α-secretase cleavage of APP is referred to as

the non-amyloidogenic pathway. β-secretase activity is attributed to the β-site-APP cleaving

enzyme (BACE). Subsequent to the N-terminal cleavage of APP by either β- or α-secretase,

APP undergoes intramembraneous proteolysis mediated by the γ-secretase complex. The γ-

secretase complex is composed of four proteins, presenilin (Psn) , presenilin enhancer-2 (Pen-2),

nicastrin (Nic) and anterior pharynx defective-1 (Aph-1). γ-secretase cleavage is heterogeneous

4

Figure 1. Schematic of intramembraneous APP proteolysis.

The amyloidogenic pathway consists of the sequential cleavage of APP by β-secretase and γ-

secretase liberating the AICD as well as the Aβ peptide. In the non-amyloidogenic pathway α-

secretase cuts within the Aβ domain thereby precluding the formation of Aβ peptides.

5

and produces various Aβ peptides ranging from 39-43 amino acids in length (Wolfe. 2007).

Importantly, fAD-mutations in APP cluster around proteolytic sites and favour the generation of

Aβ42, a more hydrophobic and aggregation prone Aβ species. The sequential cleavage of APP

by β-secretase followed by γ-secretase is referred to as the amyloidogenic pathway.

In addition to Aβ generation, γ-secretase cleavage also liberates an APP intracellular domain

fragment (AICD) which has been implicated in transcriptional activation (Cao and Sudhof. 2001)

as well intracellular calcium signalling (Leissring et al. 2002). The precise function of the APP

holoprotein is unclear and complete ablation of the APP gene does not give rise to any

substantial phenotypes in mice (Zheng et al. 1996). The lack of deleterious phenotypes in APP

knock-out animals has been attributed to genetic redundancy since the mammalian genome

encodes two APP-like genes which are highly homologous to APP and hence likely to

compensate functionally for the loss of APP.

1.2.1.2 The Presenilin genes

Shortly after mutations in the APP gene were linked to fAD, mutations in two homolgous genes,

presenilin-1 (PSEN1) (Sherrington et al. 1995) and presenilin-2 (PSEN2) (Rogaev et al. 1995)

were also found to segregate with fAD. The vast majority of these mutations are missense

mutations in the PSEN1 gene, which we now know account for over 50% of all fAD cases.

PSEN1 and PSEN2 protein topology includes 8 transmembrane domains with the N- and C-

termini as well as a large cytoplasmic loop residing in the cytoplasm (Figure 2). Presenilins are

broadly expressed in vertebrates including the brain (Lee et al. 1996). At a cellular level

presenilins primarily localize to the ER and Golgi apparatus and to a lesser extent to the plasma

membrane (Berezovska et al. 2003). Along the secretory pathway the presenilin holoprotein

undergoes proteolysis by an unknown protease generating a stable amino (N) and carboxy (C)

terminal heterodimer (Thinakaran et al. 1996). The proteolysis and stability of presenilin is

dependent on its association with the three other molecules composing the γ-secretase complex,

nicastrin, Aph-1 and Pen-2 (Takasugi et al. 2003). Apart from APP, the γ-secretase complex

6

Figure 2. The γ-secretase complex

The γ-secretase complex consists of four proteins, presenilin, nicastrin, anterior pharynx

defective-1 (Aph-1) and presenilin enhancer-2 (Pen-2). Presenilin is believed to be the catalytic

core of the γ-secretase complex since mutating two key aspartate residues (black asterisk) within

the presenilin protein eliminates γ-secretase activity. Together, this complex has proteolytic

activity and is known to cleave a variety of type 1 transmembrane proteins including APP and

Notch.

7

cleaves a variety of other type-1 transmembrane proteins including Notch, a highly conserved

protein that regulates numerous developmental events in most multicellular organisms

(Herreman et al. 2000;Zhang et al. 2000). The proteolytic activity of the γ-secretase complex is

attributed to presenilin since ablation of two aspartate residues in presenilin 1 has been shown to

abolish γ-secretase activity (Wolfe et al. 1999). Importantly, fAD-mutations in PSEN1 and

PSEN 2 promote the generation of the aggregation prone Aβ42 and hence facilitate neuritic

plaque formation.

Mice lacking presenilin 1 (PS1) die shortly after birth and exhibit significant neuronal loss,

impaired neurogenesis as well as hemorrhaging of the CNS (Shen et al. 1997). Mice deficient in

presenilin 2 (PS2), on the other hand, are viable and fertile (Herreman et al. 1999). PS1 and PS2

compound mutants die earlier than PS1 knock-out mice with phenotypes closely resembling

those of Notch-1 deficient mice (Herreman et al. 1999), a key γ-secretase substrate during

development. Hence, while PS2 activity does appear dispensable for normal Notch signalling

and embryonic development, it is not completely redundant to PS1.

Given that elimination of both PS1 and PS2 from the mouse genome is lethal several groups

have taken a conditional knock-out approach to eliminate both PS1 and PS2 specifically in the

postnatal mouse forebrain (Saura et al. 2004;Chen et al. 2008). Conditional inactivation of both

PS1 and PS2 faithfully recapitulates AD phenotypes including progressive neuronal

degeneration, memory deficits as well tau hyperphosphorylation. Although these results appear

to be at odds with the apparent gain-of-function mechanisms responsible for Aβ generation, as

well as the dominant mode of inheritance of fAD, several observations have provided compelling

evidence to account for this apparent paradox. First, mice expressing fAD-PS1 in a PS1-null

heterozygous background exhibit accelerated Aβ plaque deposition suggesting that reduced

presenilin function exacerbates the amyloid cascade (Wang et al. 2006). Second, successive

cleavage of Aβ by γ-secretase from longer to shorter Aβ peptides has been documented and

suggests that accumulation of the longer Aβ42 peptide may actually be indicative of incomplete

Aβ proteolysis, which is consistent with a partial loss of γ-secretase activity (Qi-Takahara et al.

2005). Third, unlike mutations in APP, which cluster around APP cleavage sites, mutations in

PS1 and PS2 are scattered throughout the protein, which is also most consistent with a partial

loss-of-function mechanism, possibly attributed to alterations in protein structure or stability.

8

Taken together, these results are consistent with an autosomal dominant mode of inheritance yet

also support a model where several of the characteristics of AD, including plaque deposition,

memory deficits, tau phosphorylation and neurodegeneration are due to partial loss of presenilin

function.

1.2.2 Alzheimer’s Disease aetiology

For most AD cases the aetiology is unknown however, a combination of genetic and

environmental factors is believed to be involved. Although no single monocausal event is likely

to account for all AD cases, several aberrant cellular processes have been implicated in both

sporadic as well as familial AD aetiology. In this section evidence supporting and opposing two

prevalent theories of AD, the amyloid cascade and intracellular calcium deregulation, will be

summarized and discussed.

1.2.2.1 The amyloid cascade hypothesis

The amyloid cascade hypothesis maintains that aberrant APP proteolysis followed by

progressive Aβ deposition and neuritic plaque formation is the primary cause of AD. For many

years this theory was the dominant model for AD pathogenesis. In order for the amyloid

hypothesis to be valid, the deposition of Aβ must mechanistically be linked to the genesis of all

phenotypic traits associated with AD and it must precede other AD-linked toxic cellular events

such as NFT deposition and neuronal dysfunction. The amyloid hypothesis was first proposed in

1992 and it was supported by several compelling observations (Hardy and Higgins. 1992). First,

Aβ42 and to a lesser extend Aβ40 peptides are the primary components of neuritic plaques, which

are cellular hallmarks of AD. Second, the majority of mutations in known fAD genes alter APP

proteolysis and augment the production of Aβ42 (Tanzi and Bertram. 2005). Third, fAD-

mutations in the APP gene are fully penetrant and hence can induce the onset of AD concomitant

with elevated plaque deposition. Fourth, although the nature of its toxicity is uncertain,

9

microinjection of Aβ into aged rhesus monkey brains results in neuronal loss and tau

phosphorylation (Geula et al. 1998). In addition, individuals with Down’s syndrome (trisomy of

chromosome 21) have three copies of the APP gene and invariably develop early onset AD,

including NFT deposition. Finally, Aβ immunotherapy can decrease Aβ and NFT levels in a

mouse fAD model expressing fAD-APP, fAD-PS1 and a frontal-temporal dementia associated

tau mutation (Oddo et al. 2004).

Equally compelling evidence exists that opposes the validity of the amyloid cascade hypothesis.

For example, while neuritic plaque formation remains a histopathological marker that defines

AD, spatial and temporal plaque deposition does not always correlate well with clinical AD

symptoms (Giannakopoulos et al. 2003). Furthermore, amyloid deposition does not always

precede NFT accumulation (Schonheit, Zarski, and Ohm. 2004) and expression of fAD-APP and

fAD-PS1 in the mouse model fails to recapitulate all AD phenotypic traits including NFT

deposition and neurodegeneration (Wong et al. 2002;Irizarry et al. 1997). Ultimately, while APP

proteolysis is unquestionably linked to AD aetiology it is unlikely that Aβ generation is the

primary and monocausal toxic mechanism responsible for triggering AD pathogenesis.

1.2.2.2 The calcium hypothesis

The calcium hypothesis maintains that defects in intracellular calcium homeostasis are the

primary cause of AD pathogenesis. In order for the calcium hypothesis to be accepted it must be

mechanistically linked to all AD phenotypic traits. The calcium hypothesis was initially

proposed in 1992 based on observations that calcium-dependent neuronal processes, which

regulate synaptic plasticity, learning and memory as well as cell survival, deteriorate with age

(Thibault, Gant, and Landfield. 2007). With the discovery that fAD-mutations in PS1 and PS2

cause profound disruptions in intracellular calcium storage and release, the calcium hypothesis

gained a great deal of credibility and quickly became an intensely studied field.

10

In healthy neurons intracellular calcium homeostasis is tightly regulated (Figure 3). Calcium can

enter the cytosol from the extracellular space through calcium channels present at the plasma

membrane or it can be released from internal stores, the largest of which is the ER. The

concentration of intracellular free calcium in most neurons is about 100 nM, relative to the mM

and μM calcium concentration in the extracellular space and ER, respectively. The ER has two

primary calcium channels the Ryanodine receptor (RyR) and the inositol triphosphate receptor

(IP3R). Calcium entry across plasma membrane calcium channels can stimulate ER calcium

release through RyRs. Alternatively, activation of phospholipase C leads to production of IP3,

which can activate calcium release from the ER through IP3Rs. The activity of both the RyR and

IP3R can be further modulated by calcium binding proteins such as calmodulin (Cam). Calcium

binding proteins, such as Cam, buffer incoming calcium and upon binding calcium, transduce the

calcium signal by impacting the activity of various enzymes and channels. For example, Cam

can directly bind to and impact the activity of both the RyR as well as the IP3R in a calcium

dependent fashion (Balshaw, Yamaguchi, and Meissner. 2002). Excess calcium is cleared from

the cytosol by actively pumping the calcium across the plasma membrane by calcium pumps or

exchangers or into internal stores via the sarco-endoplasmic reticulum ATPase (SERCA) pump.

Changes in cytosolic calcium concentration normally function as a second messenger system,

mediating a wide range of cellular processes relevant to AD including synaptic plasticity as well

as apoptosis. Long term potentiation (LTP) is an example of synaptic plasticity that is

particularly relevant to AD since LTP is believed to be the cellular correlate of learning and

memory. Specifically, LTP describes the long-lasting facilitation of chemical synaptic

transmission after repeated stimulation. LTP is dependent on calcium ions entering the cystosol

since loading of the postsynaptic neuron with a calcium chelator blocks LTP (Nicoll, Malenka,

and Kauer. 1989). Thus, LTP is dependent on transient increases in cytosolic calcium, which in

normal neurons is quickly cleared by mechanisms described in the previous section. However,

during aging and in disease states the ability of a cell to recover from a transient rise in cytosolic

calcium may become compromised and this deficiency can lead to cell death. Apoptosis is a

calcium-dependent programmed cell death mechanism. Apoptosis can be induced by various

intracellular or extracellular signals that result in sustained elevation of intracellular calcium

(Mattson and Chan. 2003). Persistent high levels of cytosolic calcium lead to changes in

11

Figure 3. Intracellular calcium signalling

Intracellular calcium is a tightly regulated process invols the sequestration of calcium ions into

the ER throught the SERCA pump. The reseting calcium concentration in most neurons is in an

nM range relative to the µM and mM range found in the cytosol and extracellular space,

respectively. Release of calcium from internal stores through the RyR and IP3R calcium

channels triggers signalling pathways necessary for coordinating apoptosis, synaptic plasticity as

well as gene transcription. The activity of calcium chanels is modulated by calcium binding

proteins such as calmodulin (Cam) as well as presenilin, which also resides in the ER.

12

mitochondrial membrane permeability, which results in the release of cytochrome C. Once

released, cytochrome C binds to and stimulates more calcium release from the IP3R, which

subsequently activates caspases and nucleases to initiate a proteolytic cascade required for cell

death (Boehning et al. 2003).

Intracellular calcium deregulation has been observed in fibroblasts from fAD patients

(Etcheberrigaray et al. 1998;Zatti et al. 2006) as well as in primary neuronal cultures from fAD-

PS1 transgenic animals (Zatti et al. 2006;Guo et al. 1996;Leissring et al. 2000) and in cells

transiently transfected with fAD-PS1 (Zatti et al. 2006;Chan et al. 2000;Cheung et al. 2008) and

PS2 (Zatti et al. 2006;Cheung et al. 2008;Zatti et al. 2004). The majority of studies suggest that

mutations in PS1 or PS2 potentiate calcium release from both RyR and IP3R sensitive stores.

How they do this remains uncertain. Furthermore, while some groups report overloaded calcium

stores in fAD-presenilin expressing cells, others observe the exact opposite. Those that

subscribe to the calcium-overload hypothesis believe that elevated calcium release in fAD-

presenilin expressing cells is driven by overloaded calcium stores. Conversely, others believe

that deficits in internal calcium stores is a consequence of exaggerated calcium release.

Presenilins have also been implicated in affecting the activity or expression of RyRs (Stutzmann

et al. 2007;Hayrapetyan et al. 2008;Rybalchenko et al. 2008) and IP3R (Cheung et al. 2008). In

addition, a separate but not mutually exclusive hypothesis suggests that presenilins may function

as passive calcium channels and that fAD-mutations in PS1 disable the passive calcium channel

activity leading to overloaded stores (Tu et al. 2006). However, others have been unable to

detect intrinsic calcium channel activity in presenilin (Cheung et al. 2008). Ultimately, although

the specific mechanism remain uncertain, what we do know is that presenilins are involved in

intracellular calcium homeostasis.

Deregulation of intracellular calcium has also been linked to Aβ generation and NFT formation.

Knocking down the activity of IP3R has been shown to reduce Aβ generation (Cheung et al.

2008) and stimulating release of calcium from the RyR has been implicated in increasing Aβ

production (Querfurth et al. 1997). Furthermore, tau is known to be phosphorytlated by a

calmodulin-dependent kinase (CaKII) (Steiner et al. 1990). Hence, intracellular calcium

signalling is linked to the generation of two histological hallmarks of AD. Finally, recently a

polymorphism in a novel calcium channel, calcium homeostasis modulator 1 (CALHM1), has

13

been identified as a risk factor in sporadic AD (Dreses-Werringloer et al. 2008). CALHM1

localizes primarily to the ER but can also be found at the plasma membrane and knocking down

CALHM1 appears to increase Aβ generation. Hence, calcium dysfunction has now also been

implicated in sporadic AD.

1.2.3 Therapeutic treatments

The observation that concomitant with cholinergic cell loss, AD brains also exhibit deficits in

acetylcholine levels prompted the cholinergic deficit hypothesis, which dominated much of the

early 1980’s (van Marum. 2008). Consequently, several drugs were developed that increase

acetylcholine levels in the CNS. These drugs have been shown to have modest clinical benefits

in AD by temporarily slowing down the rate of cognitive decline by 6-12 months (van Marum.

2008). Although measurable, the beneficial effects of these drugs are not large.

Memantine targets the plasma membrane calcium channel and glutamate receptor NMDA (N-

methyl-D-aspartate) by inhibiting calcium influx into the cytosol during aberrant synaptic

transmission. The clinical efficacy of mementine, however, is lower than that of drugs targeting

acetylcholine (van Marum. 2008).

Finally, much effort has been made to develop anti-amyloid therapies. One much anticipated

clinical trial involved an Aβ1-42 vaccination. Unfortunately, this trial did not reveal any benefits

with immunization and ended in disaster with the occurrence of meningeoencephalitis in a subset

of patients receiving vaccination (Gilman et al. 2005). Given that the γ-secretase complex has

many proteolytic substrates general γ-secretase inhibition is not considered to be a good

therapeutic option. Ultimately, despite intense research there are currently no effective

treatments that halt the progression of AD. As researchers unravel the finer mechanistic details

involved in AD aetiology more specific drugs will certainly be developed that target precise AD-

pathogenic processes.

14

1.3 Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease defined by rapid and

progressive muscle weakness leading to paralysis. ALS, also known as Motor Neuron Disease

(MND) or Lou Gehrig’s Disease, is attributed to degeneration of motor neurons in the motor

cortex, brain stem and spinal cord. Today, approximately 3000 Canadians are living with ALS

and 50% of these case are expected to live only 3-4 years after the onset of symptoms

(Mitsumoto. 1997). Death is typically attributed to denervation of respiratory muscles and

diaphragm leading to failure of the respiratory system. ALS involves the progressive spread of

degeneration in lower and upper motor neurons (LMN and UMN, respectively) in at least one of

the four anatomical regions of the body (bulbar, cervical, thoracic or lumbosacral) (Brooks.

1994). Upper motor neurons are neurons that project to motor neurons while lower motor

neurons are neurons that innervate muscle. Clinical signs of ALS include loss of dexterity,

progressive muscle weakness and atrophy, spasticity, as well as the onset of pathological reflexes

each of which reflects progressive muscle denervation and death of motor neurons in any of the

four aforementioned anatomical regions (Mitsumoto. 1997;Brooks. 1994). No specific test exists

to unequivocally diagnose ALS and since ALS symptoms are common to several other

neuromuscular and skeletal disorders, an integral part of diagnosing ALS includes excluding

other possible causes using electromyographs (EMGs), X-rays, neuroimaing as well as muscle

and nerve biopsies. Hence, an ALS diagnosis is supported by the absence of evidence of

neuromuscular abnormalities or other injuries as well as the absence of sensory or cognitive

dysfunction. A positive ALS diagnosis is also supported by the presence of microscopic

ubiquitin-positive cytoplasmic inclusion bodies. Inclusion bodies being cytoplasmic depositions

of misfolded proteins aggregated into ubiquitin-positive proteinaceous bodies.

Hyperphosphorylated neurofilaments concomitant with motor neuron cell loss in the brainstem,

motor cortex and spine are also diagnostic signs of ALS (Mitsumoto. 1997;Brooks. 1994).

15

1.3.1 Genetics of Amyotrophic Lateral Sclerosis

Familial ALS (fALS) occurs in 5-10% of ALS cases and is clinically and neuropathologically

indistinguishable from sporadic ALS (sALS). Approximately 20-25% of fALS cases have been

attributed to missense mutations in the superoxide dismutase 1 gene (SOD1) and the vast

majority of these SOD1-fALS cases are inherited in an autosomal-dominant fashion. Several

other genes have been linked to fALS but validation of their involvement in ALS aetiology is

still pending. In the following section the validated and most intensely studied fALS-associated

gene, SOD1, will be discussed.

1.3.1.1 The superoxide dismutase 1 gene

In 1993 missense mutations in the SOD1 gene were found to segregate with fALS (Rosen et al.

1993). SOD1 encodes a ubiquitously expressed metalloenzyme that catalyzes the dismutation

(conversion) of superoxide radicals (O2˙¯ ) into oxygen (O2) and hydrogen peroxide (H2O2).

Hydrogen peroxide is subsequently converted into water by glutathione peroxidase or catalase.

SOD1 is a critical component of a cell’s anti-oxidant defense system against reactive oxygen

species (ROS), which are a by-product of oxidative metabolism. SOD1 functions as a

homodimer with each subunit containing one copper and zinc atom. The superoxide anion is

guided into the SOD1 catalytic site by the positively charged copper atom (Cu2+

), while the zinc

atom (Zn2+

) stabilizes the dismutation reaction and ensures the rapid purging of hydrogen

peroxide from the active site (Hand and Rouleau. 2002). Within the cell, SOD1 resides primarily

in the cytoplasm.

Today over 100 fALS-SOD1 mutations have been identified and these mutations are distributed

across all five exons of the SOD1 gene (Andersen et al. 2003). The clinical characteristics, age

of onset, and biochemical characteristics of the various fALS-SOD1 mutant proteins vary

greatly. Furthermore, the level of residual dismutase activity does not correlate with disease

severity suggesting that loss of SOD1 function is unlikely to be the primary cause of SOD1-

16

associated pathogenesis (Majoor-Krakauer, Willems, and Hofman. 2003;Boillee, Vande Velde,

and Cleveland. 2006). This theory was corroborated in SOD1 null mice, which do not develop

motor neuron disease and in fact are viable (Reaume et al. 1996;Lino, Schneider, and Caroni.

2002). Ubiquitous overexpression of fALS-SOD1 mutants, on the other hand, does give rise to

many of the characteristic features of ALS including progressive motor deficits, motor neuron

degeneration, formation of microscopic inclusion bodies and premature death (Gurney et al.

1994;Bruijn et al. 1997;Dal Canto and Gurney. 1997;Jaarsma et al. 2000). Importantly, SOD1

toxicity is dependent on gene-dosage hence, higher mutant protein expression levels positively

correlate with earlier disease onset and more severe progression of symptoms (Gurney et al.

1994;Dal Canto and Gurney. 1997).

The selective toxicity of fALS-SOD1 mutations to motor neurons is believed to involve both

cell-autonomous motor neuron defects as well as non-cell autonomous interactions between

motor neurons and glia. In accordance, expression of fALS-SOD1 in motor neurons (Lino,

Schneider, and Caroni. 2002;Lino, Schneider, and Caroni. 2002) or glia (Gong et al. 2000) alone

does not give rise to motor neuron disease. However, in an in vitro model of ALS, co-culturing

wild type motor neurons with fALS-SOD1 glia has been shown to decrease the survival of wild

type motor neurons (Di Giorgio et al. 2007). Furthermore, while diminishing fALS-SOD1

expression exclusively in glia does not have an impact on disease onset, it does slow down

disease progression and extends the lifespan of chimeric fALS-SOD1 transgenic mice (Boillee et

al. 2006). Finally, new evidence has surfaced which suggests that fALS-SOD1 expressing

astrocytes produce a substance that is selectively toxic to motor neurons (Nagai et al. 2007). In

fact, several aberrant processes involving glial cells including defective removal of

neurotransmitters from the synaptic cleft, as well as the production of toxins and inflammatory

factors during disease state, have been implicated in motor neuron degeneration (Bruijn et al.

1997;Di Giorgio et al. 2007;Nagai et al. 2007;Clement et al. 2003). Ultimately, the specific

mechanisms and the cellular origin of SOD1 induced toxicity remain uncertain and are active

areas of research.

17

1.3.2 Amyotrophic lateral sclerosis aetiology

The aetiology of most ALS cases is unknown but is believed to involve both genetic as well as

environmental factors. Multiple theories exist that attempt to account for motor neuron

degeneration in ALS including oxidative stress, glutamate excitoxicity, mitochondrial

dysfunction as well as protein aggregation. In the proceeding section two prevalent theories,

protein aggregation and oxidative stress, will be discussed specifically in the context of SOD1-

associated ALS.

1.3.2.1 The protein aggregation hypothesis

The protein aggregation hypothesis maintains that fALS-SOD1 mutant proteins misfold and

oligomerize into high-molecular-weight species that aggregate into proteinaceous inclusions,

which are selectively toxic to motor neurons. In support of this theory intracellular inclusions

containing SOD1, ubiquitin, heat shock chaperone proteins as well as the proteasome have been

found in tissues taken from fALS patients and ALS mouse models (Watanabe et al. 2001).

Although the precise mechanism by which inclusion bodies selectively damage motor neurons

remains unknown, sequestration of vital chaperones may be involved. Indeed, inclusion bodies

often sequester heat shock protein 70 (Hsp70) and chaperone activity is generally decreased in

fALS-SOD1 transgenic animals (Tummala et al. 2005). In addition, overexpression of Hsp70

has been shown to reduce the formation of SOD1-positive aggregates and to preserve the

viability of cultured primary motor neurons expressing fALS-SOD1 (Bruening et al. 1999).

Inclusion bodies may also impair protein degradation by sequestering the proteasome thus

impairing its ability to degrade misfolded proteins. SOD1 degradation is mediated by the

ubiquitin-proteasome pathway and inhibiting this proteolytic pathway increases fALS-SOD1

aggregate formation in spinal cord slices taken from fALS-SOD1 transgenic animals (Puttaparthi

et al. 2003). fALS-SOD1 aggregate formation, however, can be reversed by restoring

proteasome activity (Puttaparthi et al. 2003).

18

Although aggregates are a cellular hallmark of ALS, whether or not aggregates are actually

cytotoxic is a long standing debate. In support of the aggregation hypothesis, SOD1 aggregates

have been documented to coincide with the onset of neurodegeneration in mouse ALS models

(Bruijn et al. 1997;Jaarsma et al. 2000;Jonsson et al. 2004;Wong et al. 1995). In addition, fALS-

SOD1 proteins aggregate more readily relative to wild type proteins (Wang, Xu, and Borchelt.

2002). Finally, time-lapse studies in live cells have revealed a strong correlation between the

appearance of fALS-SOD1 aggregates and subsequent cell death (Matsumoto et al. 2005).

Conversely, given that aggregate formation is a late-stage event it may actually be a secondary

effect or potentially even represent a cellular defense mechanism, whereby misfolded, toxic

proteins are actively sequestered into inclusion bodies.

1.3.2.2 The oxidative stress hypothesis

The oxidative stress hypothesis maintains that fALS-associated mutant SOD1 proteins have

altered substrate affinity and/or engage in aberrant activity that results in the production of toxic

free-radicals and that these radicals are the primary trigger of the pathogenic process that gives

rise to ALS. In addition to its conventional dismutase activity, SOD1 can also act as a

peroxidase and thus can convert hydrogen peroxide into toxic hydroxyl radicals (Peled-Kamar et

al. 1997;Yim et al. 1996). Normally, access to the wild type SOD1 active site is limited by size

and charge, favouring superoxide as a substrate and excluding larger molecules such as hydrogen

peroxide. However, fALS-SOD1 has a higher affinity for hydrogen peroxide relative to wild

type SOD1 enabling fALS-SOD1 peroxidase function and consequently hydroxyl radical

production (Yim et al. 1996). The hydroxyl radical is highly reactive and can cause oxidative

damage to DNA, lipids and proteins including to SOD1 itself (Andrus et al. 1998). In support of

this theory there is evidence of oxidative pathology in human sporadic and familial ALS patients

including increased oxidative-stress associated damage to DNA, phospholipids and proteins

(Ferrante et al. 1997;Shaw et al. 1995).

An extension of the oxidative stress hypothesis suggests that oxidative damage to SOD1 itself

promotes misfolding, which leads to the oligomerization of misfolded SOD1 into toxic inclusion

19

bodies thus linking oxidative stress to protein aggregation. In support of this theory, SOD1 has

been shown to be one of the most heavily oxidized proteins identified in fALS-SOD1 transgenic

mice as well as in fALS patients (Andrus et al. 1998). Several factors make SOD1 selectively

vulnerable to oxidative stress specifically in motor neurons. First, SOD1 is highly abundant in

motor neurons (Pardo et al. 1995). In addition it has a particularly long half-life in motor

neurons (Borchelt et al. 1998). Finally, motor neurons are very large cells with high energy

demands. This energy is supplied by high metabolic rates and consequently, elevated oxidative

stress. Both fALS-SOD1 and to a lesser extent wild type SOD1 have been shown to be

susceptible to hydrogen peroxide induced oxidative damage, which leads to misfolding and

monomerization of the SOD1 homodimer prior to aggregation (Rakhit et al. 2004). Studies

using an antibody specifically engineered to only detect misfolded-monomeric SOD1 have

revealed the accumulation of misfolded-monomeric fALS-SOD1 in fALS patients as well as in

fALS-SOD1 rodent models specifically in motor neurons prior to neurodegeneration (Rakhit et

al. 2007). However, the degree to which monomer-misfolded SOD1 correlates with ALS disease

progression remains to be determined.

1.3.3 Therapeutic treatments

Currently, ALS treatments focus primarily on relieving symptoms and maintaining an optimum

quality of life. Riluzole is one of the few drugs that appears to have a modest effect on

prolonging the life of ALS patients (Lacomblez et al. 1996). The mechanism of action of

riluzole is uncertain but it may involve alleviating excitotoxic stress by inhibiting glutamate

release (Hand and Rouleau. 2002). A recent study, however, has suggested that riluzole can also

increase the expression of heat shock chaperone proteins and hence the beneficial impact of

riluzole may also, at least in part, include amelioration of protein misfolding and hence

aggregation (Yang et al. 2008).

20

1.4 Drosophila as a model of human neurodegenerative diseases

Numerous features of the fruit fly, Drosophila melanogaster, make it an ideal model organism to

study human neurodegenerative diseases. First, flies have a very short generation time. The life

cycle of a fly is between 10-12 days at 25 °C. Newly laid eggs take 24 hours to undergo

embryogenesis before hatching into first instar larvae, which continue to develop for another 48

hours into second, and then third instar larvae. Two days later, larvae transform into immobile

pupa, undergo metamorphosis and eclose in adult form 5-7 days later. Importantly, adult

females are fertile 12 hours post-eclosion and a single fly can produce hundreds of offspring

within days making it relatively easy to perform analysis on hundreds of flies within a matter of

days.

Many of the genes, cellular processes and basic building blocks of the nervous system are

conserved in flies (Yoshihara, Ensminger, and Littleton. 2001). Cross-genomic comparisons of

the Drosophila and human genomes have revealed a remarkably high degree of conservation

(Adams et al. 2000;Rubin et al. 2000). Over 60% of known human disease causing genes have a

fly orthologue (Rubin et al. 2000). What’s more, there is considerably less genetic redundancy

in Drosophila relative to vertebrate models hence, characterization of disease-causing gene

function is often less complicated. Many of the cellular processes known to be involved in

neurodegeneration including the signalling cascades that orchestrate apoptosis, intracellular

calcium homeostasis as well as oxidative stress are also conserved in flies, as are the proteins

that mediate these processes. The Drosophila nervous system is composed of some 200,000

neurons and supporting glia relative to the millions of neurons found in the mammalian brain.

Although simpler, fly neurophysiology is very similar to it’s mammalian counterpart. For

example, fly neurons exhibit synaptic plasticity and neurotransmission mediated by many of the

same neurotransmitters, synaptic proteins, receptors and ion channels found in the mammalian

brain (Yoshihara, Ensminger, and Littleton. 2001). Flies also exhibit complex behaviours and

like humans, many of these behaviours deteriorate with age including learning, memory and

motor ability (Mockett et al. 2003;Simon, Liang, and Krantz. 2006).

Finally, and perhaps most importantly, the Drosophila genome can be used to perform

sophisticated genetic analyses designed to further elucidate the pathological processes associated

21

with normal as well as aberrant disease gene activity. Genome manipulation in flies includes

exploiting naturally occurring transposable elements referred to as P-elements. Thousands of P-

element lines, representing independent transposon insertions spanning the majority of the fly

genome are available to the fly research community. These insertions often, but not always,

impact the expression or activity of the gene(s) proximal to the insertion site. Mobilization of P-

elements can also give rise to precise as well as imprecise excisions of the transposon, the latter

removing not only the sequence corresponding to the P-element but also the flanking sequence of

the proximal gene(s). Imprecise excision of P-elements is a common method for generating

genetic nulls in flies. P-elements can also be used as vectors to shuttle transgenes into the fly

genome to generate transgenics. The method of choice for targeting exogenous gene expression

in Drosophila is the bi-partite GAL4-UAS (upstream activating sequence) system (Figure 4)

(Brand and Perrimon. 1993). The insertion of the yeast GAL4 transcriptional activator

downstream of an endogenous fly promotor results in the expression of GAL4 in the same

spatial-temporal pattern characteristic of that promoter. Thus, when flies expressing GAL4 are

crossed to flies bearing a transgene downstream of the GAL4 binding UAS-sequence, the

progeny of this cross express the transgene in a spatial-temporal pattern determined by the

promoter driving GAL4 expression. The GAL4/UAS system can thus be used to determine

whether expression of wild type or mutant disease causing genes in various anatomical regions

of the fly results in observable phenotypes. With respect to generating a fly model of a human

neurodegenerative disesase, phenotypes that recapitulate human disease symptoms are in many

ways most desired but, not always necessary. Often studying a phenotype in a structure

unrelated to the human disease or even to human anatomy, for example the fly wing or retina,

can reveal valuable insights into the function of disease causing genes. So long as the relevant

signalling molecules are conserved, such studies may be very relevant to the human disease

process. Additionally, any phenotypic trait associated with the expression of a disease causing

gene can be subject to a genetic screen designed to isolate dominant mutations that suppress or

enhance the original disease gene induced phenotype. In such a way, the power of genetic

analysis in Drosophila can be used to identify novel molecules involved in human disease

processes, which can subsequently be validated in mammalian models. These topics will be

further explored in the proceeding sections with specific focus on how the fruit fly model has

been used to further our understanding of the normal and aberrant function of AD and ALS-

associated genes.

22

Figure 4. The GAL4/UAS system

The GAL4/UAS system is composed of two parts. The first part is the yeast GAL4

transcriptional activator which is under the control of a native promoter of interest. The second

is a short upstream activiating sequence (UAS) to which GAL4 binds and initiates transcription.

Thus the GAL4/UAS system enables tissue specifc transgene expression when flies expessing

GAL4 are crossed to flies bearing an UAS sequence upstream of a transgene. Adapted from:

Brand and Perrimon. 1993.

23

1.4.1 Drosophila models of Alzheimer’s Disease

Drosophila models of AD have included expressing wild type and fAD-mutant forms of APP

and presenilin. Drosophila has a single APP orthologue (Appl) which encodes a β-amyloid

precursor-like protein that notably lacks the Aβ domain. Although γ-secretase activity is

conserved in Drosophila, there is no known β-secretase orthologue hence, to date there is no

clear evidence that Aβ peptides are generated in flies. Overexpression of human Aβ42 in the

Drosophila CNS gives rise to, amyloid deposition, progressive learning defects, extensive

neurodegeneration and ultimately a shortened lifespan (Iijima et al. 2004). Another group took a

different approach and expressed Aβ42 in the Drosophila eye. Here too, overexpression of Aβ42

gave rise to progressive retinal degeneration concomitant with plaque formation (Finelli et al.

2004). The advantage of working with a retinal phenotype is that the eye is a non-essential organ

that is easy to score as a phenotypic trait in a genetic modifier screen, which is precisely what

was done. A random collection of P-element misexpression mutant strains (EP-strains) was used

to screen for suppressors or enhancers of the Aβ42-induced eye phenotype. Overexpression of the

Neprilysin 2 gene (Nep2) was identified as a suppressor of both retinal degeneration as well as

Aβ42 accumulation (Finelli et al. 2004). In addition, overexpression of Nep2 was shown to also

rescue the shortened lifespan observed in flies expressing Aβ42 in the fly CNS raising the

exciting possibility that increasing Nep2 activity could have therapeutic potential for AD (Finelli

et al. 2004). Nep2 encodes a metalloprotease that is known to be involved in Aβ42 degradation.

Importantly, Nep2 is conserved in mammals and transgenic mice overexpressing NEP2 and fAD-

APP exhibited significantly reduced amyloid plaque formation. Moreover, NEP2 expression

also rescued the premature lethality observed in mice expressing fAD-APP alone (Leissring et

al. 2003) thus validating that Drosophila can be used to identify and study the molecular

pathways involved in human neurodegenerative disease process.

The Drosophila genome encodes a single presenilin gene (Psn) (Boulianne et al. 1997). The

structure, proteolysis, trafficking and subcellular localization of Psn is conserved in flies as are

all the components and activity of the γ-secretase complex (Adams et al. 2000;Boulianne et al.

1997;Ye and Fortini. 1998;Fossgreen et al. 1998;Struhl and Greenwald. 1999). In accordance,

like mice, loss of Psn in flies gives rise to Notch-like phenotypes. Specifically, Psn-null flies

24

survive to early pupal stages due to maternal contribution and exhibit defective eye and wing

development as well as incomplete differentiation of central neurons (Ye and Fortini.

1998;Struhl and Greenwald. 1999;Guo et al. 1999). Elevated apoptosis is observed in the

developing eye of Psn-null larvae suggesting that like mice normal Psn activity plays a role in

cell survival (Ye and Fortini. 1999). Lack of maternal or zygotic Psn activity, on the other hand,

results in hyperplasia of the embryonic nervous system hence, loss of Psn can have pleiotropic

effects at different developmental stages (Ye, Lukinova, and Fortini. 1999). Overexpression of

wild type Psn in the fly eye also gives rise to apoptosis and co-expression of an fAD-form of Psn

exacerbates this phenotype (Ye and Fortini. 1999). Hence, both loss of Psn function as well as

its overxpression in the eye give rise to apoptosis. Likewise, both loss of Psn function (Struhl

and Greenwald. 1999), as well as its overexpression (Ye and Fortini. 1999) gives rise to wing

margin scalloping, a classic Notch loss-of-function phenotype. Given that Psn overexpression

can phenocopy Psn loss-of-function, it has been proposed that overexpression of Psn actually

gives rise to a dominant negative effect (Ye and Fortini. 1999). The nature of this dominant

negative effect is unclear but it may involve accumulated Psn holoprotein impairing ER function

or disrupting the assembly of active γ-complexes (Ye and Fortini. 1999).

Work performed in our lab has demonstrated that loss of Psn function in third instar larvae

results in learning deficits concomitant with impaired synaptic plasticity (Knight et al. 2007).

More specifically, Psn-null larvae were tested using olfactory and visual associative-learning

paradigms and in both assays mutant larvae performed significantly worse relative to wild type

controls. In another study, flies were generated that express either wild type or an fAD-mutant

form of Drosophila Psn under the control of the endogenous Psn promoter in a Psn-null genetic

background (Lu et al. 2007). These flies were subjected to heat shock stress and assayed for

long term memory using an olfactory associative learning paradigm. Flies expressing fAD-Psn

were shown to have significant long term memory deficits relative to flies expressing wild type

Psn, but only during the post-stress recovery period. Synaptic activity at the larval

neuromuscular junction (NMJ) was also measured pre- and post heat shock. Both Psn-null and

fAD-Psn but not wild type flies demonstrated prolonged calcium influx through voltage-gated

calcium channels on the plasma membrane but again, only post heat-shock, pointing to a role for

Psn in regulating post-stress calcium-channel activity. Finally, expression of 12/14 fAD-Psn

mutations under the control of the endogenous Psn-promoter only partially rescues Psn-null

25

lethality hence in flies, as in mice, fAD-mutations in Psn appear to be partial loss-of-function

mutations (Seidner et al. 2006). Lifespan analysis was not reported. The effects of wild type or

fAD-mutant Psn expression in the Drosophila CNS has also not been explored.

1.4.2 Drosophila models of Amyotrophic Lateral Sclerosis

The Drosophila genome encodes a single copper-zinc superoxide dismutase gene (Sod). Unlike

mice, flies are very sensitive to reductions in dismutase activity. Sod null flies die soon after

they eclose and exhibit significantly higher levels of oxidative stress relative to wild type

controls (Mockett et al. 2003;Phillips et al. 1989). The lethality of Sod null flies can be rescued

by introducing wild type human SOD1 into the fly genome, demonstrating that the fly and

human gene are functionally homologous (Parkes et al. 1998). Furthermore, there is evidence of

neuropathology in flies bearing a loss-of-function point mutation in the endogenous Sod gene

(Sod n108

) (Phillips et al. 1995). Degeneration of photoreceptor neurons in the retinas of Sod n108

flies was documented as early as 7 days post-eclosion (Phillips et al. 1995). In addition, Sod

loss-of-function mutants have also been shown to have reduced locomotor activity (Mockett et

al. 2003). Hence, unlike mice, loss of Sod function in flies can recapitulate some ALS-like

phenotypes. However, these phenotypes are all recessive, which is unlike the dominant, gain-of-

function mechanisms believed to underlie SOD1-associated ALS pathogenesis in mice and

humans.

Several studies in flies have taken a transgenic approach to express human fALS-SOD1 in

various parts of the fly. The first published study expressed human fALS-SOD1 specifically in

Drosophila motor neurons. The results were quite unexpected since expression of fALS-SOD1

actually extended lifespan and increased resistance to oxidative stress in flies (Elia et al. 1999).

While the precise mechanisms responsible are unclear, the ability of fALS-SOD1 to extend the

lifespan of flies is likely due to the positive outcome of elevating dismutase activity specifically

in motor neurons. In addition, though motor neurons are selectively killed in ALS, we know

from studies in mice that expression of fALS-SOD1 exclusively in motor neurons does not result

26

in motor neuron disease. Hence, perhaps in flies, like in mice, non-cell autonomous fALS-SOD1

toxicity is required to induce ALS-like phenotypes.

Another study used fly transgenics that expressed several different human fALS-SOD1

mutations under the control of the endogenous Drosophila Sod promoter (Mockett et al. 2003).

The longevity and climbing ability of these flies was measured in the context of a Sod null or Sod

wild type genetic background. Expression of fALS-SOD1 had no effect on longevity in a Sod

wild type background. However, in a Sod null background all fALS-SOD1 mutants partially

rescued the short lifespan of Sod null flies. fALS-SOD1 mutants also exhibited progressive

climbing impairments in a Sod null background. Specifically, while the walking speed of young

fALS-SOD1 mutant flies was initially no different to that of wild type flies, within a couple of

weeks the climbing ability of flies expressing fALS-SOD1 had dramatically declined relative to

controls. The climbing ability of fALS-SOD1 transgenics in a Sod wild type background was

not reported. At least two reasons may account for why these studies failed to produce gain-of-

function phenotypes. Both are based on the knowledge that high levels of fALS-SOD1 mutant

protein are often required to induce ALS-like phenotypes in other model systems. In the present

study each fALS-SOD1 transgene was present in only a single copy number. In addition, the

endogenous Sod promoter is not a strong driver of transgene expression. Hence, it is possible

that using a stronger, more robust promoter to drive fALS-SOD1 expression may be necessary to

induce gain-of-function phenotypes in flies.

Recently, a study was published that describes gain-of-function ALS-like phenotypes associated

with the expression of human SOD1 in flies (Watson et al. 2008). In this study, two fALS-SOD1

alleles as well as wild type SOD1 were used to generate independent fly transgenics which were

crossed to a motor neural GAL4 driver. As reported in earlier studies, motor neuron specific

expression of fALS-SOD1 did not impact fly lifespan. However, this study also measured

climbing ability. Flies expressing either wild type or fALS-mutant SOD1 were reported to have

progressive climbing deficits relative to flies expressing Drosophila Sod, although it should be

noted that some important controls were not included in this analysis precluding absolute

certainty that the observed climbing defects are indeed attributed to transgene expression. That

being said, the reported climbing defects were not accompanied by motor neuron loss but

electrophysiological examination did reveal progressive decrements in synaptic transmission.

Both wild type, as well as fALS-SOD1 mutant protein, were observed to accumulate in motor

27

neurons starting as early as day one, however, the SOD1 positive foci did not co-localize with

chaperone proteins indicating that SOD1 accumulation was not inducing a chaperone stress

response. It was noted, however, that a chaperone stress response was being induced in

surrounding glial cells. These studies have thus suggested that expression of fALS-SOD1 or

wild type human SOD1 in fly motor neurons can give rise to progressive climbing and synaptic

defects through cell-autonomous mechanisms. This is in contrast to work performed in the

mouse model, which argues that cell-autonomous mechanisms are necessary but not sufficient at

inducing ALS-like phenotypes (Boillee, Vande Velde, and Cleveland. 2006;Di Giorgio et al.

2007;Boillee et al. 2006;Nagai et al. 2007). What has not been explored, as of yet, is what

consequence fALS-SOD1 expression has on flies when driven by a strong, robust ubiquitous

driver.

1.5 Purpose of our studies

Using Drosophila as a model our ultimate goal is to further decipher the normal and aberrant

function of two genes linked to the onset of neurodegeneration in humans, presenilin and

superoxide dismutase. In the next chapter, we will present data that provides new insights into

the interaction of presenilin with the calcium release machinery. In the third chapter, we will

describe fALS-SOD1-induced locomotory deficits. In the final chapter we will discuss the

implication of our work in the context of the current understanding of the cellular, physiological

and behavioural processes involved in ALS and AD. We will also discuss how future studies

could pave the way towards novel effective treatments against neurodegeneration.

28

CHAPTER 2

2 Analysis of the interaction of presenilin with the intracellular

calcium stores machinery

Kinga Michno, Jorge M. Campussno, Diana van de Hoef , Diane O’Dowd and

Gabrielle L. Boulianne

A modified version of this chapter has been submitted for publication.

Statement of contribution:

A modified version of this chapter has been submitted for publication. Jorge M. Campussano

assisted with the generation of primary cultures and calcium imaging. Diana van de Hoef

constructed the wild type Drosophila presenilin expression construct used in the binding assay.

Commercially obtained reagents are indicated in the materials and methods. Kinga Michno

generated all other reagents and performed all other experiments and analysis as indicated in the

materials and methods. Diane O’Dowd pioneered the Drosophila primary culture protocol.

Calcium imaging analysis was performed under the co-supervision of Diane O’Dowd and

Gabrielle L. Boulianne. All other work was performed under the supervision of Gabrielle L.

Boulianne.

29

2.1 Abstract

Much of our current understanding about neurodegenerative diseases can be attributed to the

study of inherited forms of these disorders. For example, mutations in the presenilin 1 and 2

genes have been linked to early onset familial forms of Azheimer’s Disease (fAD). However, a

clear understanding of the sequence of presenilin-induced toxic events that trigger the onset of

dementia still remains largely unresolved. Using the Drosophila central nervous system as a

model we have investigated the role of presenilin in one of the earliest cellular defects associated

with Alzheimer’s Disease, intracellular calcium deregulation. We show that expression of either

wild type or fAD-mutant presenilin in Drosophila CNS neurons has no impact on resting

calcium levels but does give rise to deficits in internal calcium stores. Furthermore, we show

that a loss-of-function mutation in calmodulin, a key regulator of intracellular calcium, can

suppress the presenilin-induced deficits in intracellular calcium. Our data support a model

whereby presenilin plays a role in regulating intracellular calcium levels and demonstrates that

Drosophila can be used to study the link between presenilin and calcium deregulation.

30

2.2 Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by progressive

dementia and, histopathologically, by the formation of senile neuritic plaques, neurofibrillary

tangles (NFT) and ultimately neuronal cell death. Despite being the most prevalent and intensely

studied form of dementia there is still no effective cure. Although the majority of AD cases are

sporadic, 10% are familial (fAD) and are inherited in an autosomal dominant fashion.

Approximately 50% of fAD cases have been attributed to mutations in three genes, amyloid

precursor protein (APP) (Goate et al. 1991a), presenilin-1 (PSEN1) (Sherrington et al. 1995) or

presenilin-2 (PSEN2) (Rogaev et al. 1995).

Presenilins are integral membrane proteins that are synthesized within the endoplasmic reticulum

(ER) as full-length holoproteins. In the ER, presenilins undergo proteolytic cleavage generating

N- and C-terminal fragments which remain associated. Along the secretory pathway, presenilins

associate with presenilin enhancer-2, nicastrin and anterior pharynx defective-1. Together these

proteins constitute the γ-secretase complex. This complex has proteolytic activity and is known

to cleave several type I transmembrane proteins including Notch and APP. APP proteolysis is

particularly important to AD because aberrant APP proteolysis results in the deposition of Aβ

fragments which are the primary components of senile plaques. However, while Aβ deposition

is a cellular hallmark of AD, it is remains unclear whether or not this process is the primary

cause of AD. In fact, neurodegeneration in the absence of senile plaque formation (Chui et al.

1999;Amtul et al. 2002;Dermaut et al. 2004;Raux et al. 2000) suggests that other toxic processes

in which presenilin could be involved may compromise neuronal function independent of Aβ

generation and ultimately set the stage for the onset of AD pathogenesis. In fact, AD aetiology is

believed to involve several aberrant cellular processes including protein aggregation, oxidative

stress as well as intracellular calcium deregulation.

Deregulation of intracellular calcium signalling is an early event in AD pathogenesis and

precedes any symptoms (Etcheberrigaray et al. 1998). More specifically, internal calcium stores

including the endoplasmic reticulum (ER) and Golgi apparatus have been reported to be either

under, or over-loaded, in cells expressing fAD-mutant forms of PS1 (Zatti et al. 2006;Leissring

31

et al. 2000;Smith et al. 2002;Leissring et al. 2001) or PS2 (Zatti et al. 2006;Zatti et al. 2004).

The apparent discrepancies in published results may be attributed to the use of different fAD-

presenilin mutations, different cell types (often non-neuronal) and different experimental

approaches. Thus further studies focused on understanding the role of presenilins in calcium

dysfunction are needed to resolve these inconsistencies.

Changes in cytosolic calcium concentration normally function as a second messenger system

mediating a wide range of cellular processes, many of which are relevant to AD aetiology

including learning and memory as well as cell death. Internal calcium stores play an important

role in facilitating intracellular calcium homeostasis by regulating calcium release and storage.

The ER contains two main types of calcium release channels, the ryanodine receptor (RyR) and

the inositol 1,4,5-triphosphate receptors (IP3R). Presenilins have been shown to physically

interact with both of these channels and to influence their activity (Cheung et al. 2008;Stutzmann

et al. 2007;Hayrapetyan et al. 2008;Rybalchenko et al. 2008). Presenilins have also been shown

to physically interact with a number of known transducers of calcium signalling including

calmyrin (O'Day and Myre. 2004;Morohashi et al. 2002), sorcin (Zhu et al. 2004) and calsenilin

(Leissring et al. 2001;Ahn et al. 2004). Finally, one study has suggested that presenilins

themselves may function as passive ER calcium channels (Tu et al. 2006). Despite all the

evidence linking presenilin function to intracellular calcium homeostasis, the precise

mechanisms by which presenilins regulate calcium dynamics remain unresolved.

In this study we have investigated the impact of wild type and mutant presenilin expression on

intracellular calcium dynamics in primary Drosophila cholinergic neurons. Importantly, unlike

most presenilin studies performed in Drosophila, our work focuses on presenilin function

specifically in the fly central nervous system (CNS). The genetic tractability of Drosophila

melanogaster makes this organism an ideal model to study the function of presenilin. The

Drosophila genome encodes a single presenilin gene (Psn) (Boulianne et al. 1997)

circumventing genetic redundancy. All the components of the γ-secretase complex are

conserved in flies (Adams et al. 2000) as is the proteolytic specificity and function of this

complex (Struhl and Greenwald. 1999;Ye, Lukinova, and Fortini. 1999). Since flies do not

generate Aβ peptides, Psn function can also be studied without the confounding impact of Aβ

deposition. In addition, studies performed in our laboratory as well as others has implicated Psn

function in synaptic plasticity (Knight et al. 2007), learning and memory (Knight et al. 2007;Lu

32

et al. 2007) as well as ER-stress induced calcium tail currents (Lu et al. 2007) further

demonstrating that Drosophila and mammalian Psn function is highly conserved and that

Drosophila Psn is required for processes that are affected in AD.

Here we demonstrate that Psn expression in primary Drosophila cholinergic neurons causes

deficits in internal calcium stores. Importantly, these deficits occur independent of Aβ

generation. We also describe a novel genetic, physiological and physical interaction between

Psn and calmodulin (Cam), a key regulator of intracellular calcium homeostasis. Specifically,

we show that Psn-induced deregulation of internal calcium stores can be suppressed by the loss

of a single copy of Cam. We also present evidence that Psn and Cam physically interact.

Finally, we also examined whether presenilin expression affects neuronal cell death or fly

survival. Taken together our data support a model whereby Psn plays a role in regulating

intracellular calcium levels that may be mediated by its interaction with Cam and demonstrate

that Drosophila can be used to study the link between presenilin and calcium deregulation.

2.3 Materials and Methods

Fly stocks

Flies bearing both a UAS-wild type Drosophila presenilin (UAS-PsnWT

) transgene as well as the

cut-GAL4 wing margin driver were recombined onto the same third chromosome (cut-Psn). cut-

Psn flies were then crossed at 29°C to flies bearing either a P-element insertion in the Cam gene

(Cam hypomorph) (Wang, Sullivan, and Beckingham. 2003) or a Cam null line (Camn339

)

(Heiman et al. 1996a). The genetic interaction of Cam and Psn at the wing margin was

confirmed by the chi-squared (2) 2 X 2 table method using Statistica software. For the calcium

analysis, full-length wild type UAS-PsnWT

(Guo et al. 1999) or fAD-M146V mutant transgene

(UAS-PsnfAD

) (Ye and Fortini. 1999) Drosophila Psn transgenes both on the third chromosome

were crossed at room temperature to flies bearing both a Cha-GAL4 and UAS-GFP transgene

(Salvaterra and Kitamoto. 2001). Lines bearing both the Camn339

allele as well as the UAS-

33

PsnM146V

(UAS-PsnFAD

) were generated and crossed to the Cha-GAL4 line described above to

assess the physiological interaction between Cam and Psn.

Western Analysis of Cha-GAL4 driven presenilin expression

10 male fly heads were collected and lysed in 50µL of RIPA buffer. Lysates were incubated for

15 minutes on ice, spun at 10,000 x G, supernatant collected and loaded onto a 10%

polyacrylimide gel. Gels were then transferred over-night onto PVDF membranes followed by

blocking in 2.5% each of milk, BSA, FBS, NGS and NDS in 1% Tris-buffer saline tween-20

buffer (TBST – 100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% tween-20) for 1 hour at room

temperature. PVDF membranes were then incubated with a rabbit polyclonal antibody raised

against the N-terminal fragment of Drosophila Psn (Guo et al. 1999) at a 1:1000 dilution in 1%

Tris-buffer saline tween-20 buffer (TBST – 100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1%

tween-20) overnight at 4 °C. The next day membranes were washed three times followed by

incubation with an HRP-conjugated goat ant-rabbit antibody at 1:10,000 in 1% blocking solution

for one hour at room temperature followed by three washes prior to visualization using

chemiluminescence. The blots were then stripped, washed, incubated in 2.5% block as described

above and re-probed using a mouse-anti-actin antibody (Hybridoma bank, JL20) used at 1:1000

in 1% block. Membranes were then washed, incubated with an HRP conjugated goat-anti-mouse

antibody, washed again before exposing using chemiluminescence.

Fly imaging

Whole mount images of the pupal CNS were captured using Zeiss LSM 5 Pascal laser-scanning

confocal microscope using a 20X objective. Whole mount images of fly wings were generated

using the Zeiss Mirax Scan digital imaging platform.

Cell culture, calcium imaging and analysis

Cell culture, calcium imaging and analysis

34

Primary pupal CNS culture, calcium imaging and subsequent analysis was performed according

to previously published methods (Campusano et al. 2007;Sicaeros, Campusano, and O'Dowd.

2007) with the following amendments. After baseline recordings were established in four day

old primary cultures, the cultures were washed three times over a 200 second interval in HBSS

media (Campusano et al. 2007) containing zero calcium, 2 mM EGTA and 4 mM MgCl2. HBSS

media containing 5 µM ionomycin (Sigma) was then added to the cultures and the area under the

curve was calculated using Mini-Analysis (Synaptosoft) to estimate intracellular calcium

content. At least six independent cultures generated on at least three different culturing days

were analyzed for each experimental variation. From each neuronal culture, approximately

fifteen cells were selected based on positive GFP expression (indicating expression of the

cholinergic Cha-GAL4 driver). Each genotype was coded during analysis and not decoded until

all analysis was completed. Statistica software was used for all statistical analysis. Kolmogrov-

Smirnov test was used to analyze raw data distribution. Since the raw data of both the resting

calcium and ionomycin response measurements was not normally distributed the non-parametric

Kruskal-Wallace ANOVA of ranks followed by Mann-Whitney pair-wise comparisons was used

to analyze both the calcium baseline and ionomycin responses.

Binding Assay and Western Analysis

Drosophila S2 cells were maintained at room temperature in Schneider’s media supplemented

with 10% FBS. A construct containing full-length, wild type Drosophila Psn under the control

of the actin promoter was used to transfect a total of approximately 1 x 107 S2 cells using the

Cellfectin reagent (Invitrogen, 10362-010). 48 hours post transfection, microsomal cell fractions

were generated as follows: cells were washed in cold PBS followed by re-suspension in 1.35 mL

of 20 mM Tris pH 7.4 with protease inhibitors and then sheared through a 25 gauge needle with

subsequent sonication (3x30 seconds on ice). Lysates were then incubated on ice for 15 minutes

and spun at 1230 x G for 25 min at 4 °C. The supernatant was collected and spun further at

100,000 x G for 45 min at 4 °C. The microsomal pellet was re-suspended in 1 mL of 50 mM

Tris pH 7.5 plus protease inhibitors and pre-cleared with 100 µL of agarose beads (Sigma, 4B-

200) (pre-washed in 50 mM Tris pH 7.5 buffer) by rotating for 1 hr at 4 °C. The sample was

then split in half and incubated with either 25 µL of Cam-beads (Sigma, P-4385) or 25 µL of

35

beads alone (Sigma, 4B-200) for four hours at 4 °C. Beads were then washed three times with

50 mM Tris pH 7.5 buffer, allowing beads to settle by the force of gravity in between each 10

minute wash. Finally, bound proteins were eluted using 50 µL of 2 x loading buffer with DTT.

Western analysis was performed using a rabbit polyclonal antibody raised against the N-terminal

portion of the wild type Drosophila presenilin protein (Guo et al. 1999) used at 1:1000 and

incubated over-night at 4°C. The anti-calmodulin antibody (Zymed,61-8500) was used at 1:1000

over-night at 4 °C.

Cell Death Analysis

Four day old primary neuronal cultures were fixed using 4% paraformaldehyde at room

temperature in the dark. Cells were subsequently washed three times with PBS, 10 minutes each

wash. Cells were then permeabilized with PBT (PBS/0.2% triton X) for 15 minutes at room

temperature after which the cells were washed three times with PBS, five minutes in between

each wash. TMR red TUNEL reactions were performed according to manufacturers instructions

(Roche, 2 156 792) with the following amendments. The TUNEL reaction was incubated at 37

°C for 45 minutes. Cultures were then washed three times with PBS at room temperature, each

wash lasting five minutes. Nuclei were then stained with the DNA-binding Hoescht dye at 1:500

in PBS for ten minutes and washed three times with PBS, each wash lasting five minutes.

Cultures were imaged using a Zeiss Plan-Apochromat 40X/0.95 NA objective and Zeiss

Axiovert 200 inverted fluorescence microscope equipped with a Sony 3 chip CCD camera

(RGB) and a Hamamatsu Orca ER CCD camera. A total of three brains were imaged for each

genotype tested. Four different fields of view were taken from each culture and used to

determine an average for TUNEL positive and condensed Hoescht stained nuclei for each plate.

Percent of TUNEL positive cells was then quantified for either the total cell population, the

cholinergic GFP positive population or the non-GFP population. A one-way analysis of variance

(ANOVA) was used to test for statistical differences between the groups followed by Tukey

post-hoc analysis.

36

Lifespan Analysis

Male flies were segregated into ten vials housing at least ten individual flies at the beginning of

the experiment and kept at 25 °C. Vials were plugged with a sponge plug and placed

horizontally in racks so as to prevent older flies from getting stuck in the food. Death was

recorded every 3-4 days until the end of the experiment. Statistica software was used for all

statistical analysis. Kaplan-Meier was used to plot survival of each genotype. The Gehan’s

Wilcoxon test was used for two-sample comparisons of life span.

2.4 Results

2.4.1 Intracellular calcium dynamics in primary cholinergic Drosophila neurons

We wanted to investigate the effect of Psn expression on intracellular calcium dynamics in a cell

type relevant to AD and therefore chose to focus on central neurons. The Drosophila pupal CNS

is particularly amenable to culturing and was used for all calcium imaging experiments. The

cholinergic Cha-GAL4 driver was chosen to drive expression of either wild type (PsnWT

) or fAD-

Psn (PsnfAD

). The specific fAD-Psn mutant used is a methionine to valine substitution at amino

acid 146, one of the most intensely studies fAD-Psn mutations. We chose Cha-GAL4 in part

because the Drosophila CNS is primarily cholinergic (Fig. 5A) but also because cholinergic cell

loss is a prominent feature in AD brains (Geula and Mesulam. 1989;Gaburjakova et al. 2001). In

addition, the Cha-GAL4 line used in our studies also contains a UAS-GFP transgene (Salvaterra

and Kitamoto. 2001) enabling us to specifically select cells expressing Psn for calcium analysis

(Fig 5A-C). Calcium dynamics were measured using the calcium binding Fura-2AM fluorescent

dye (Fig. 5D). Plotted over time Fura-2 measurements reveal a calcium trace that can be used to

determine resting cytosolic calcium levels as well as calcium movement from internal stores into

the cytoplasm (Fig. 5D). Since Psn has been shown to impact the calcium content of more than

one internal store (Zatti et al. 2006) we chose to measure the release of calcium from all internal

37

Figure 5. Evaluation of calcium content in internal stores in Drosophila primary neuronal

cultures

A) Whole mount Drosophila CNS 56-72 hour post pupariation expressing GFP in cholinergic

neurons driven by the Cha-GAL4 driver (100 µm scale bar). B) Field of dissociated primary

culture showing cholinergic pupal neurons expressing GFP (50 m scale bar). C) DIC image of

primary pupal cultures overlayed with an inverted image showing GFP fluorescent signal in

cholinergic neurons (50 m scale bar). D) Pseudo-colored 340/380 ratio representation of

intracellular calcium concentration in Fura-2 loaded neurons. Cells showing a red hue indicate

high intracellular calcium levels while cell bodies in blue represent low calcium levels (50 m

scale bar). E) Over time Fura-2 measurements can be translated into estimates of real calcium

levels. Trace here illustrates results obtained in a typical experiment, where after recording in

basal resting conditions, cells are exposed to ionomycin in absence of external calcium.

38

stores using the calcium ionophore ionomycin in zero extracellular calcium conditions (Fig 5E).

Previous studies have shown that ionomycin treatment depletes intracellular calcium stores in

Drosophila cell (Yeromin et al. 2004). Ionomycin treatment causes a rapid increase in cytosolic

calcium concentration during the initial release of calcium from internal stores as can be seen in

Figure 5E. This increased cytosolic calcium concentration gradually returns to baseline as

internal stores are emptied and the calcium is extruded from the cell.

2.4.2 Psn-induces deficits in intracellular calcium stores content

To investigate the role of Psn in intracellular calcium dynamics we expressed wild type or fAD-

mutant Psn in Drosophila cholinergic neurons. Transgene expression was confirmed using

western analysis (Fig. 6). Analysis of basal calcium recordings revealed no significant

differences between neurons expressing wild type (Cha;PsnWT

, median = 90 nM Ca2+

) or mutant

Psn (Cha;PsnFAD

, median = 80 nM Ca2+

) relative to controls (Cha, median = 80 nM Ca2+

)

suggesting that Psn expression is not overtly toxic to these cells (Fig. 7A). Next, we wanted to

determine whether Psn expression could impact internal calcium stores. We measured

intracellular calcium store content using the calcium ionophore ionomycin in a recording

solution that does not contain calcium (zero extracellular calcium) (Fig. 7B). This analysis

revealed that expression of both wild type ( Cha;PsnWT

P < 0.01, median = 3597.780 nM•s) as

well as mutant (Cha;PsnFAD

P < 0.01, median = 3926.490 nM•s) Psn caused a significant

decrease in internal calcium store content relative to controls (Cha, median = 5438.02 nM•s).

There was no significant difference in internal calcium store between neurons expressing wild

type or fAD-Psn.

39

Figure 6. Expression of wild type and FAD-mutant Psn.

Western analysis of lysates generated from adult fly heads reveal lower expression levels of wild

type Psn holoprotein (single asterisk in Cha;PsnWT

lane) relative to that of the FAD-mutant Psn

(single asterisk in Cha;PsnFAD

lane). Loss of a single Cam allele does not appear to alter the

level of Psn holoprotein in flies expressing FAD-mutant Psn (single asterisk in

Cha/Camnull

;PsnFAD

lane). The N-terminal Psn fragment levels appears normal in all lanes

relative to the Cha-GAL4 control (double asterisk in Cha relative to all other lanes). Actin

protein levels serve as loading control (solid black arrow head in all lanes).

40

Figure 7. Calcium content in internal calcium stores is affected in cholinergic neurons

expressing Psn.

A) Expression of wild type or FAD-mutant presenilin protein in cholinergic neurons (Cha;PsnWT

and Cha;PsnFAD

, respectively), does not affect basal calcium levels compared to control strain

(Cha). B) Expression of Cha;PsnWT

or Cha;PsnFAD

results in decrements in calcium content

detected in intracellular reservoirs in these neurons. Data are represented as modified box-

whisker plots with the median indicated by the smaller white box and the 25 and 75 percent

quartiles indicated by the lower and upper margins of the large grey boxes respectively. Each

box represents recordings from cultures generated from at least six independent brains, cultured

on at least three independent culturing days. The area under the response curve was calculated

from baseline to the point of 50 percent return to baseline in Neurons treated with 5µM of

ionomycin

41

2.4.3 Loss-of-function mutations in Cam suppress Psn-induced wing scalloping

Recently, we reported that several known regulators of calcium homeostasis suppressed Psn-

induced phenotypes (Van de Hoef et al, Genesis, in press). Briefly, loss-of-function alleles

generated by P-element insertions in the genes encoding the Ryanodine receptor, cyclic-AMP

response element binding protein A, calcium binding protein as well as calmodulin (Cam)

suppressed the penetrance of either a wing scalloping or thoracic bristle phenotype induced by

Psn expression. Psn has previously been shown to physically interact with, and impact the

activity of, the RyR in vertebrates. Cam is a calcium signal transducer that activates various

enzymes (reviewed in (Stull. 2001)) and modulates the activity of various ion channels,

including the RyR (Balshaw, Yamaguchi, and Meissner. 2002) and IP3R (Cheung et al. 2008).

To date, an interaction between Psn and Cam has not been described however, it could represent

an important mechanism for regulating intracellular calcium stores.

To confirm that Psn and Cam genetically interact we generated a recombinant transgenic line

that carried both a wing margin-GAL4 driver (cut-GAL4) as well as a UAS-wild type Psn

transgene. Overexpression of Psn at the wing margin gave rise to a wing scalloping phenotype

with 58% penetrance (cut-Psn Fig. 8A & C). Of note, others have shown that loss of Psn

function also results in wing scalloping (Struhl and Greenwald. 1999). Overexpression of Psn in

Drosophila is believed to give rise to dominant negative effects since overexpression

phenocopies Psn loss-of-function (Ye and Fortini. 1999). Flies bearing either a P-element

insertion in Cam (characterized elsewhere as a hypomorphic Cam allele) (Wang, Sullivan, and

Beckingham. 2003) or an independent imprecise excision in Cam (Camnull

) (Heiman et al.

1996b), which was not used in the original screen, were crossed to cut-Psn recombinant flies.

Both the Cam hypomorph (33% penetrance, 2: P < 0.05) as well as the Cam null (24%

penetrance, 2: P < 0.05) significantly suppressed the penetrance of the Psn-induced wing

scalloping phenotype (cut-Psn penetrance = 58%, Fig. 8B & C) thereby confirming that Psn and

Cam genetically interact.

42

Figure 8. Psn-induced wing notching is suppressed by loss-of-function mutations in Cam.

A) Overexpression of wild type Psn under the control of cut-GAL4 induces a wing notching

phenotype in flies (500 µm scale bar). B) The loss of a single Cam allele suppresses the Psn-

induced wing phenotype (500 µm scale bar). C) Quantification of the penetrance of Psn-induced

wing notching phenotype and the suppression of this phenotype by two loss-of-function

mutations in Cam (Cam null) and a Cam hypomorph). Penetrance was scored based on the

presence of at least one wing margin notch. Asterisks denote significant differences in expected

penetrance relative to the original cut-Psn recombinant as determined by the 2 test.

43

2.4.4 Cam suppresses Psn-induced deficits in intracellular calcium stores

content

Given that Cam is known to play an important role in the regulation of intracellular calcium

levels, we wanted to examine whether Psn and Cam physiologically interact to regulate internal

calcium stores. We decided to focus on mutant fAD-Psn rather than wild type Psn since

expression of both transgenes gave rise to similar deficits in internal calcium stores and because

the interaction of Cam with an fAD-mutant phenotype would be more relevant to AD aetiology.

The resting calcium levels in Cha/Camnull

trans-heterozygotes (Cha/Camnull

median = 90 nM

Ca2+

) were not significantly different to resting levels in Cha-GAL4 alone (Cha median = 80 nM

Ca2+

) (Fig. 8A). Likewise, resting calcium levels in neuronal cultures generated from flies

expressing fAD-Psn in cholinergic neurons with only a single functional Cam allele

(Cha/Camnull

;PsnfAD

median = 80 nM Ca2+

), also appeared normal relative to Cha-GAL4 controls

(Fig. 9A). We then treated these cells with ionomycin to measure internal calcium stores.

Importantly, loss of a single Cam allele alone did not alter intracellular calcium stores relative to

controls (Fig. 9B). However, as can be seen in Figure 9B, the loss of a single Cam allele

suppressed (Cha/Camnull

;PsnfAD

, median = 5322 nM•s, P=0.01) the Psn-induced calcium store

decrements otherwise observed in neurons expressing fAD-Psn with two functional copies of

Cam (Cha;PsnfAD

, median = 3926 nM•s). There were no significant differences in ionomycin-

induced calcium release between Cha/Camnull

;PsnfAD

and Cha-GAL4 alone.

44

Figure 9. Psn-induced effects on intracellular calcium stores is suppressed by a loss-of-

function mutation in Cam mutations.

A) Resting calcium levels are not affected by the loss of a single Cam allele either in a wildtype

(Cha/Camnull

) or in a Psn mutant background (Cha/Camnull

;PsnfAD

), compared to their respective

controls (Cha and Cha;PsnFAD

, respectively). B) Loss of a single allele in Cam suppresses Psn-

induced deficits in calcium stores content (Cha/Camnull

;PsnfAD

compared to Cha;PsnfAD

, P=0.01).

Neurons were treated with ionomycin and the area under the curve was calculated from baseline

to the point of 50 percent return to baseline. Data are represented by modified box-whisker plots

as described in Figure 2. Light grey boxes identify groups that are statistically different from the

ChaGAL4 control. Each box represents recordings from cultures generated from at least six

independent brains, cultured on at least three independent culturing days.

45

2.4.5 Psn and Cam physically interact

We then sought to determine if the ability of Cam to suppress Psn-induced deficits in

intracellular calcium stores was due to a direct versus indirect interaction between the two

proteins. Presenilins are known to physically interact with other calcium sensing proteins. For

example, mammalian presenilin 2 has been shown to bind to the EF-hand motif of sorcin (Pack-

Chung et al. 2000). Since Cam contains four EF-hand motifs we reasoned that Psn may

physically interact with Cam as well. Cam is highly conserved among species (Fig. 10A-C).

This conservation has enabled us to take advantage of the commercial availability of agarose

beads covalently bound to bovine Cam to perform binding assays. Lysates were generated from

Drosophila S2 cells transfected with full-length wild type Psn. Equal amounts of protein were

incubated with either beads alone or beads covalently bound to Cam. Normally, full-length Psn

is rapidly processed into N- and C-terminal fragments hence full-length Psn is rarely observed.

However, since Psn processing is dependent on limiting factors, when Psn is overexpressed the

full-length holoprotein accumulates (Fig. 10D black arrow head) while the N- and C-terminal

fragment levels remain unaltered. The two N-terminal Psn bands (Fig. 10D asterisk) correspond

to two different isoforms resulting from alternative splicing. Western analysis revealed that

indeed, full-length Psn, and to a lesser extent, the cleaved N-terminal fragment bound to Cam-

beads but not to beads alone (Fig. 10D)

46

Figure 10. Cam binds to full-length as well as the N-terminal fragment of Psn.

A) Sequence alignment of Bovine as well as Drosophila Cam demonstrates the high degree of

conservation of Cam between the two species. Identical sequence homology (asterisk), similar

amino acids (light grey box) and the one different amino acid (black box) are highlighted. B)

Antibodies raised against bovine Cam recognize mammalian Cam (arrow head) in COS cells as

well as Drosophila Cam in S2 cells, highlighting the high degree of conservation of this protein.

C) The specificity of the Cam antibody was confirmed using lysates generated from Camn339

homozygous null larvae (Camnull

) where the band corresponding to Cam is absent, as opposed

lysates generated from Cam heterozygotes (Camnull

/+) and mammalian control COS cells (COS).

D) Equal amounts of protein were incubated with either Cam-beads or beads alone. Full length

(black arrow head) and the N-terminal fragment of Psn in a short (asterisk) or long (pound)

exposure were pulled down by Cam-beads but not beads alone. Two N-terminal Psn bands

indicated in the gels correspond to two isoforms generated by alternative splicing of Psn.

47

2.4.6 Primary neurons expressing wild type Psn have lower incidence of

apoptosis

To determine if the Psn-induced changes in intracellular calcium stores promote the onset of

other cellular defects associated with AD we looked at the incidence of programmed cell death, a

cellular process that is dependent on intracellular calcium signalling. DNA fragmentation in

apoptotic nuclei was assayed using TUNEL staining and nuclear condensationwas confirmed

using Hoescht staining (Fig. 11A). First, we looked at apoptosis in the total neuronal population.

The incidence of TUNEL positive nuclei in neurons expressing wild type Psn was 20.1% which

was significantly lower relative to the 37.9% recorded in control Cha-GAL4 neurons (P=0.02)

(Fig. 11B). There was no significant differences in the incidence of TUNEL positive nuclei in

neurons expressing fAD-Psn relative to Cha-GAL4 or neurons expressing wild type Psn.

Quantification of condensed nuclei revealed similar trends however, statistical analysis did not

reveal significant differences between the three genotypes (Fig 11 C). We then quantified the

incidence of TUNEL positive nuclei in GFP positive, cholinergic neurons (Fig 12 A). Again,

expression of wild type Psn (Cha;PsnWT

= 0.5% TUNEL positive nuclei) resulted in a

significantly lower incidence of TUNEL positive nuclei relative to Cha-GAL4 controls (Cha =

2.3% TUNEL positive nuclei, P=0.04). The incidence of TUNEL positive nuclei in neurons

expressing fAD-Psn (Cha;PsnFAD

= 1.9% TUNEL positive nuclei) was not different to the

incidence observed in Cha-GAL4 or wild type Psn expressing neurons. We reasoned that if there

was less apoptosis occurring in neurons expressing wild type Psn there should also be more GFP

positive neurons surviving in these cultures. Indeed, this is what we observed (Fig 12 B). We

found that 47.7% of the neuronal population was GFP positive in neuronal cultures expressing

wild type Psn, which was significantly higher than the 32.7% observed in Cha-GAL4 control

cultures (P=0.01). Of the neuronal population expressing fAD-Psn, 38.9% was GFP positive

which was not significantly different to either Cha-GAL4 or wild type Psn expressing neuronal

cultures. There were no significant differences in TUNEL reactivity between any of the

genotypes in the non-GFP cell population (data not shown).

48

Figure 11. Total incidence of apoptosis in cultures expressing wild type or FAD-mutant

Psn.

(A) Cells expressing wild type or FAD-mutant Psn driven by the cholinergic Cha-GAL4 driver

are marked by GFP. Red TUNEL positive nuclei depict DNA-nicking. Condensed apoptotic

nuclei are visualized using Hoescht staining. (B) Expression of wild type Psn (Cha;PsnWT

)

significantly decreases the incidence of apoptosis relative to Cha-GAL4 controls (P = 0.02) but

not relative to cells expressing FAD-mutant Psn (Cha;PsnFAD

). (C) Although the same general

trend is observed using Hoescht staining to quantify apoptotic condensed nuclei there were no

significant differences between the three genotypes.

49

Figure 12. Expression of wild type Psn in cholinergic neurons facilitates cell-autonomous

cell survival.

(A) Expression of wild type Psn in cholinergic neurons (Cha;PsnWT

) results in a lower incidence

of TUNEL positive nuclei in cholinergic cells (P = 0.04). The incidence of TUNEL positive

nuclei in GFP positive FAD-mutant Psn (Cha;PsnFAD

) cholinergic neurons was not different to

that of Cha-GAL4 (Cha) or neurons expressing wild type Psn (Cha;PsnWT

) (B) A higher

percentage of GFP positive cholinergic neurons was observed in cultures expressing wild type

Psn relative to Cha-GAL4 cultures (Cha). No differences in the percentage of GFP positive cells

were observed between cultures expressing mutant Psn relative to control Cha-GAL4 (Cha)

neurons or those expressing wild type Psn (Cha;PsnWT

).

50

2.4.7 Cholinergic expression of fAD-mutant Psn results in shortened lifespan.

To determine whether expression of Psn in cholinergic neurons over the entire life time of a fly

could influence lifespan we performed survival analysis (Table 1 & Apendix 1). Flies

expressing wild type Psn had a mean life span of 81.1 days relative to 82.6 days for Cha-GAL4

(Fig. 13). There was no significant difference between the lifespan of flies expressing wild type

Psn and Cha-GAL4 flies. However, flies expressing wild type Psn did live longer than flies

bearing UAS-PsnWT

alone (PsnWT

alone, mean lifespan = 76.8 days, P<0.01) as well as wild type

flies (w1118

, mean lifespan = 73.2 days, P<0.01) (Fig. 13).

The mean lifespan of flies expressing fAD-Psn was 67.9 days which was significantly shorter

than the lifespan of control w1118

flies (w1118

, mean lifespan = 73.2 days, P<0.01), Cha-GAL4

flies (Cha, mean lifespan = 82.6 days, P<0.01) and flies bearing the UAS-PsnFAD

transgene alone

(PsnWT, mean lifespan = 76.8 days, P<0.01) (Fig. 14, Table 1 & Appendix 1). Next we

examined whether one loss-of-function allele in Cam could suppress the short lifespan of flies

expressing fAD-Psn. Indeed, loss of a single Cam allele significantly extended the lifespan of

flies expressing fAD-mutant Psn from 67.9 days to 83.2 days (P<0.001). However, we also

observed that loss of a single Cam allele significantly extended the lifespan of Cha-GAL4 flies

from 82.6 to 97.8 days (P<0.001) (Fig. 15, Table 1 & Apendix 1). Hence, the extension of

lifespan attributed to the loss of a single Cam allele is not specific to Psn expressing flies.

51

Table 1. Survival Analysis

The mean and median lifespan in days for each genotype analyzed plus/minus the standard error

of the mean (SEM) and the standard deviation (SD). N represents the total number of flies

analyzed.

52

Figure 13. Survival analysis of flies expressing wild type Psn in cholinergic neurons.

Flies expressing wild type Psn (Cha;PsnWT

) live longer then wild type flies (w1118

) as well as

flies bearing only the UAS-PsnWT

transgene but not longer then Cha-GAL4 (Cha) control flies.

53

Figure 14. Survival analysis of flies expressing FAD-Psn in cholinergic neurons.

Flies expressing mutant Psn (Cha;PsnfAD

) have a shorter lifespan relative to wild type flies

(w1118

), Cha-GAL4 (Cha) control flies, as well as flies bearing only the UAS-PsnWT

transgene.

54

Figure 15. Survival analysis for flies expressing FAD-Psn in cholinergic neurons with or

without the loss of a single Cam allele.

Flies expressing FAD-mutant Psn (Cha;PsnfAD

) have a shorter lifespan relative to wild type flies

(w1118

), Cha-GAL4 (Cha) control flies, as well as flies bearing only the UAS-PsnWT

transgene.

Loss of a single Cam allele extends the lifespan of both of flies expressing FAD-mutant Psn

(Cam/Cha;PsnfAD

) as well as Cha-GAL4 (Cha) flies.

55

2.5 Discusion

Although the specific cellular mechanisms remain uncertain, increasing evidence suggests that

presenilin plays an important role in regulating intracellular calcium dynamics. We have

investigated Psn function in the context of intracellular calcium homeostasis using the

Drosophila CNS as a model system. Our data demonstrates that expression of wild type or fAD-

mutant Psn in Drosophila cholinergic neurons results in cell-autonomous deficits in calcium

stores. Decrements in calcium stores attributed to wild type or mutant Psn expression have been

documented in human as well as mouse models (Zatti et al. 2006;Cheung et al. 2008;Zatti et al.

2004;Cai et al. 2006). To date, most studies in Drosophila have focused on the role of Psn in

Notch signalling during development. Our data clearly demonstrate that Drosophila can be used

as a model to study additional functions of Psn and specifically, its role in the regulation of

intracellular calcium dynamics. Importantly, unlike mammals Drosophila does not generate Aβ.

Hence, in our model system, any effects on internal calcium stores can be attributed entirely to

presenilin and is not confounded by the production of Aβ peptides.

Interestingly, we found that expression of either wild type or fAD-Psn gave rise to deficits in

intracellular calcium stores. We believe that this is due to a loss of Psn function since previous

studies have shown expression of Psn in Drosophila gives rise to dominant negative effects (Ye

and Fortini. 1999). The mechanistic nature of the dominant negative effect is not known but it

may involve negative repercussions of holoprotein accumulation within the ER or competition

for limiting factors required to generate functional γ-secretase complexes. Moreover, there is

mounting evidence suggesting that loss of Psn function is responsible for some aspects of fAD

pathogenesis. For example, conditional knock-out of both presenilin 1/2 phenocopies AD-like

symptoms including, learning and memory impairments as well as progressive

neurodegeneration (Saura et al. 2004). Notably, these Psn-induced phenotypes were observed in

the absence of Aβ deposition. Another group has also demonstrated memory loss and

degeneration associated with loss of both presenilins in the mouse brain, once again, in the

absence of A generation (Chen et al. 2008). In accordance to these findings, we have

previously shown that loss of Psn function in Drosophila results in defects in synaptic plasticity

56

and learning (Knight et al. 2007). In addition, in both Drosophila (Seidner et al. 2006) and C.

elegans (Baumeister et al. 1997;Levitan et al. 1996), fAD-mutations in Psn fail to completely

rescue loss of wild type Psn function. Since decrements in internal calcium stores have been

documented in mammalian PS1/PS2 null cells (Kasri et al. 2006) our data are consistent with a

dominant negative effect arising from Psn overexpression.

A great deal of effort has been made in several model systems to resolve how presenilin function

impacts intracellular calcium dynamics. The results of one study suggested that wild type, but

not fAD-presenilin, exhibits passive calcium channel activity (Tu et al. 2006). Since our results

indicate that both wild type and fAD-mutant Psn cause decrements in intracellular calcium stores

our data do not support a putative passive calcium channel function for wild type Psn (Tu et al.

2006). Presenilins are not known to bind calcium directly hence their influence on intracellular

calcium signalling may be mediated by interactions with calcium binding proteins and indeed,

presenilins have been shown to bind several calcium binding proteins (Pack-Chung et al.

2000;Stabler et al. 1999;Buxbaum et al. 1998). Using two independent loss-of-function alleles in

Cam we have confirmed that loss-of-function mutations in Cam suppress a Psn-induced wing

phenotype. The mechanism for this suppression may also involve intracellular calcium stores.

Wing scalloping is a classic Notch loss-of-function phenotype. Although we have no direct

evidence to suggest that Psn-induced wing notching is attributed to disruptions of intracellular

calcium at the wing margin, it is known that Notch proteolysis and activity is impacted by

changes in internal calcium stores. For example, loss-of-function mutations in the Drosophila

calcium-ATPase gene Ca-P60A have been shown to cause aberrant Notch trafficking and

secretion due to alterations in internal calcium stores (Periz and Fortini. 1999). Hence, it is

conceivable that Psn-induced deregulation of internal stores is responsible for the observed

Notch phenotypes.

Since Psn has been linked to calcium deregulation and Cam is an important player in

intracellular calcium homeostasis we further investigated the genetic interaction between Psn

and Cam in a cellular context relevant to AD. Using primary Drosophila cholinergic neurons we

found that loss of a single functional Cam allele suppressed calcium store deficits otherwise

observed with the overexpression of fAD-Psn. Furthermore, we showed that Cam physically

interacts with both full-length as well as the N-terminal fragment of Psn, albeit to a lesser extent

relative to the holoprotein. A physical interaction between Cam and Psn has previously been

57

postulated using a Calmodulin Target Database, which identified putative Cam binding sites in

presenilin 1 and 2 (O'Day and Myre. 2004). In fact, this database identified putative Cam

binding sites in all of the components of the γ-secretase complex (O'Day and Myre. 2004). Since

Cam binds both full-length as well as the N-terminal fragments of Psn, Cam may play a role in

regulating Psn proteolysis or protein stability. Cam activity has previously been implicated in

regulating the stability and proteolysis of other integral membrane proteins (Li, Joyal, and Sacks.

2001). The ability of Cam to regulate Psn protein levels could explain how partial loss of Cam

activity can suppress both Psn-induced wing notching and calcium-deficits.

AD-associated neurodegeneration ultimately involves neuronal cell death including the induction

of programmed cell death. Under-loaded calcium stores have been implicated as both pro-

apoptosis (Nguyen, Wang, and Perry. 2002) as well as anti-apoptotic (Scorrano et al. 2003)

triggers. Under-loaded calcium stores are associated with ER stress, which can trigger several

signalling pathways that lead to apoptosis. One of these ER stress signalling cascades involves

the induction of a member of the C/EBP family of bZIP transcription factors referred to as

CHOP (GADD153). CHOP has been shown to promote apoptosis by inhibiting the pro-survival

activity of the Bcl-2 protein (McCullough et al. 2001). Conversely, mouse embryonic fibroblasts

deficient for the pro-apoptotic BAX and BAK proteins exhibit depleted ER stores concomitant

with a decreased incidence of apoptosis (Scorrano et al. 2003). How depletion of internal stores

can coincide with both pro- or anti-apoptotic events is uncertain. Our data demonstrate that

overexpression of Psn in the Drosophila CNS results in enhanced neuronal survival.

Furthermore, since the enhanced survival is only observed in cultures where wild type Psn is

expressed we cannot attribute the enhanced survival to deficits in intracellular calcium stores.

Evidence from both vertebrate and invertebrate model systems suggests that Psn function

involves both pro-apoptotic as well as anti-apoptotic activity. For example, in mammals wild

type but not fAD-presenilin 1 has also been shown to inhibit apoptosis by activating PI3K/Akt

signalling (Baki et al. 2004). Conversely, expression of wild type and to a lesser extent fAD-Psn

in the Drosophila eye results in the induction of apoptosis (Wu, Lu, and Xu. 2001). Elevated

levels of apoptosis are also a feature of Psn loss-of-function in both the fly (Ye and Fortini.

1999) and mammalian (Saura et al. 2004;Chen et al. 2008) CNS arguing that presenilin can act

as a pro-survival factor. Ultimately, Psn function with respect to cell survival and cell death is

likely pleiotropic and context dependent. Our analysis has revealed that expression of wild type

58

but not mutant Psn enhanced cell survival relative to control Cha-GAL4 central neurons.

Furthermore, our data suggests that the enhanced survival of neurons expressing wild type Psn is

cell autonomous since lower levels of apoptosis were observed in GFP positive neurons

expressing wild type Psn concomitant with a larger pool of surviving GFP positive cholinergic

neurons relative to Cha-GAL4. Hence, our data support a pro-survival role for Psn activity in the

fly CNS.

Several reasons may explain why no significant differences in cell survival were observed

between neurons expressing wild type Psn and fAD-mutant Psn. First, fAD-mutant Psn alleles

are believed to be partial loss-of-function alleles hence fAD-Psn may retain sufficient levels of

wild type function to exert some pro-survival activity, at least during the specific developmental

stage from which the neuronal cultures are generated. Second, the expression level of wild type

Psn is lower than that of fAD-Psn. Hence, if both proteins were expressed at equal levels a

difference in survival might be observed between the two genotypes. Third, in the mammalian

system fAD-Psn knock-in mice are asymptomatic until exposed to toxic insult (Guo et al. 1999).

Perhaps exposure of fAD-mutant Psn expressing cholinergic fly neurons to toxic stress is also

necessary to induce apoptosis. Finally, fAD-mutant Psn toxicity may only become observable

with age.

To address what impact expression of wild type or fAD-Psn has on fly lifespan we performed

survival analysis. The mean lifespan of flies expressing wild type Psn was in between the mean

lifespan of Cha-GAL4 flies and flies bearing the UAS-PsnWT

transgene alone. Hence, the pro-

survival capacity of wild type Psn is insufficient to extend lifespan. The lifespan of flies

expressing fAD-mutant Psn in cholinergic neurons, on the other hand, was shortened relative to

all controls. Thus, like AD patients, flies expressing fAD-mutant Psn die prematurely. Future

studies will have to address whether these fly die due to progressive neuronal degeneration and

whether these changes coincide with alterations in intracellular calcium in the aging fly CNS.

Although loss of a single Cam allele did indeed suppress the shortened lifespan of flies

expressing fAD-Psn it did so non-specifically since the lifespan of Cha-GAL4 flies was also

extended by the loss of a single Cam allele. Cam activity is known to change during aging in

both mammals (Hoskins and Ho. 1986) as well as flies (Massie et al. 1989) however, there is no

clear evidence that could explain how loss of Cam function could extend lifespan. Ultimately,

59

these studies should be repeated once all the various transgenic and mutants fly stocks are

backcrossed onto a common genetic background in order to reduce the genetic variability.

Since Cam activity is known to play a role during learning and memory, apoptosis as well as tau

phosphorylation the interaction between Psn and Cam may be very relevant to AD pathogenesis.

Only by characterizing how normal and aberrant Psn activity impact calcium homeostasis can

we begin to resolve how this cellular process contributes to AD pathogenesis. Future studies will

need to address whether presenilins’ effect on intracellular calcium channel activity is mediated

through Cam and what if any impact this interaction has on progressive neuronal cell death in

adult flies. Deciphering the significance of the molecular and neurophysiological interactions

between Psn, Cam, IP3R and Ryr in flies would be greatly facilitated by the fact that unlike

mammals each of these proteins is encode by a single gene in Drosophila. Ultimately, only by

characterizing how normal and aberrant Psn activity impacts calcium homeostasis can we begin

to resolve how this cellular process contributes to AD pathogenesis.

60

3 CHAPTER 3

Expression of human fALS–associated SOD1 in Drosophila induces progressive activity

deficits

Kinga Michno, Shirley Liu, Joel Levine and Gabrielle L. Boulianne

Statement of contributions:

Shirley Liu generated the mouse spinal lysates and provided the antibodies used in the

immunoprecipitation analysis. The adult fly activity analysis was performed in Joel Levine’s

laboratory. Commercially obtained reagents are indicated in the materials and methods. Kinga

Michno generated all other reagents and performed all other experiments and analysis as

indicated in the materials and methods. The activity analysis was performed under the co-

supervision of Joel Levine and Gabrielle L. Boulianne. All other work was performed under the

supervision of Gabrielle L. Boulianne.

61

3.1 Abstract

Amyotrophic lateral Sclerosis (ALS) is a fatal neurodegenerative disease that specifically targets

motor neurons and causes progressive paralysis. Currently, there are no effective treatments for

ALS and as a result most patients die 3-5 years after the onset of symptoms. Most ALS cases are

sporadic (sALS) however, approximately 10% of cases are familial (fALS). Since, sporadic and

familial cases are clinically indistinguishable much effort has been made to understand the

normal and aberrant activity of genes associated with fALS. For example, dominant gain-of-

function mutations in the superoxide dismutase 1 gene have been linked to fALS. fALS-SOD1

associated toxicity has been shown to involve oxidative stress as well as protein aggregation

concomitant with progressive decline in motor ability. In this study, we demonstrate that

ubiquitous expression of fALS but not wild type human SOD1 in Drosophila gives rise to

progressive locomotory deficits in adult flies. This Drosophila model of fALS-SOD1 associated

locomotory deficits can now be used to further characterize the cellular and molecular basis of

the locomotory defects and to search for novel therapeutic interventions.

62

3.2 Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative

disease that selectively targets motor neurons. Most ALS cases occur sporadically (sALS) but

10% are inherited and are specifically referred to as familial ALS (fALS) (Majoor-Krakauer,

Willems, and Hofman. 2003). Histopathologically, ALS is characterized by the presence of

ubiqutinated aggregates of misfolded proteins, referred to as inclusion bodies. These inclusion

bodies form primarily in motor neurons but can also be found in glial cells (Jonsson et al. 2004).

ALS patients succumb to progressive paralysis due to motor neuron death but are spared from

any apparent cognitive decline. Since there is currently no cure for ALS, identifying the unique

molecular events that result in selective motor neuron death has been the subject of intense

research. Such studies have implicated several gene mutations and consequently, several

cytotoxic processes in promoting motor neuron degeneration, including protein aggregation

(Watanabe et al. 2001;Rakhit et al. 2007;Johnston et al. 2000a) as well as oxidative stress (Peled-

Kamar et al. 1997;Yim et al. 1996). Understanding the pathogenic role of fALS-associated

genes is unquestionably valuable, since sALS and fALS are clinically indistinguishable. Thus,

any insights into the pathological processes involved in fALS may also shed light into sALS

pathogenesis.

Insight into the mechanisms underlying ALS has come from studying genes implicated in fALS.

The most intensely studied fALS-associated gene is superoxide dismutase 1 (SOD1). Mutations

in SOD1 account for approximately 20% of all fALS cases and 5% of sporadic ALS (Majoor-

Krakauer, Willems, and Hofman. 2003). To date, over 100 different fALS-associated mutations

have been identified in SOD1 yet how these different mutations give rise to the same motor

neuron pathology remains unresolved. The function of SOD1 provides critical insight into one

possible mechanism involved in motor neuron degeneration. SOD1 catalyzes the dismutation of

superoxide free radicals. Thus, SOD1 functions as a critical cellular antioxidant. Furthermore,

inclusion bodies in fALS patients stain positively for SOD1, suggesting that SOD1 aggregation

may also be toxic to motor neurons (Watanabe et al. 2001). Importantly, given that the level of

SOD1 activity does not correlate with disease severity, it is unlikely that loss of SOD1 function

is the primary cause of motor neuron degeneration (Ratovitski et al. 1999) .

63

To further understand fALS pathogenesis, several groups have explored normal and aberrant

SOD1 activity in mouse models. SOD1 activity appears to be dispensable in mice since SOD1-

null animals are viable and do not succumb to motor neuron disease (Reaume et al. 1996). This

corroborates the theory that loss of SOD1 enzymatic function is unlikely to be a major

contributor to the onset of motor neuron degeneration in mammals (Reaume et al. 1996).

Ubiquitous expression of human fALS-SOD1, on the other hand, does give rise to progressive

paralysis, premature death and other characteristic features of ALS (Gurney et al. 1994;Bruijn et

al. 1997;Wong et al. 1995). Importantly, expression of fALS-SOD1 exclusively in motor

neurons does not give rise to motor neuron disease suggesting that SOD1 pathogenesis involves

non-cell autonomous mechanisms (Lino, Schneider, and Caroni. 2002;Boillee et al. 2006).

Collectively, it is believed that toxic gain-of-function, rather than loss-of-function of SOD1 is

responsible for the vast majority of SOD1-associated fALS pathogenesis.

The function of SOD1 has also been explored in invertebrate model systems, including

Drosophila. Flies encode a single superoxide dismutase gene, Sod. Unlike mice, Sod is required

for viability in flies (Phillips et al. 1989). Interestingly, expression of human SOD1 exclusively

in fly motor neurons results in a significant increase in the otherwise very short lifespan of Sod

null flies (Parkes et al. 1998). This outcome is likely attributed to the positive effects of re-

introducing dismutase activity into Sod null flies (Mockett et al. 2003). Perhaps even more

interesting, in a wild type background, motor neuron expression of human SOD1 results in a

significant increase in the lifespan of flies concomitant with an increased resistance to oxidative

stress (Parkes et al. 1998). These studies suggest that although SOD1 is a ubiquitously expressed

enzyme its function is particularly critical in motor neurons, which are the precise targets of

ALS. Using the endogenous fly Sod promoter, expression of human fALS-SOD1 in flies has

been shown to induce progressive locomotory deficits, but unlike the human disease these

SOD1-induced defects were determined to be recessive (Mockett et al. 2003). More recently, a

study has described dominant, cell-autonomous phenotypes in flies expressing human wild type

as well as fALS-mutant SOD1 in motorneurons (Watson et al. 2008). Specifically, flies

expressing both wild type and fALS-SOD1 were shown to have progressive decrements in

climbing activity, concomitant with defects in synaptic transmission, but notably in the absence

of neurodegeneration (Watson et al. 2008). A limitation to this study is that several important

controls are missing from the analysis. In addition, work performed in the mouse model has

64

suggested that cell-autonomous fALS-SOD1 expression is not sufficient to induce the onset of

motor neuron disease. Ultimately, why expression of fALS-SOD1 in flies has thus far failed to

induce neurodegeneration remains unknown. One reason may be that flies simply do not live

long enough. Alternatively, perhaps using a strong ubiquitous promoter to drive the expression

of fALS-SOD1 is required to induce dominant ALS-like phentoypes.

Modeling motor neuron degeneration in invertebrate models has not been fully exploited and

warrants further investigation. An ideal invertebrate ALS model would exhibit dominant,

progressive locomotor deficits, decreased lifespan, concomitant with ALS-like neuropathology

including protein aggregation, elevated oxidative stress and progressive motor neuron loss. In

this study we show that ubiquitous expression of human fALS-SOD1, but not wild type SOD1 in

Drosophila leads to progressive decrements in adult fly locomotor activity. Importantly, the

observed activity deficits are not accompanied by fALS-SOD1 mutant protein aggregation

suggesting that other toxic mechanisms may be responsible for the progressive decline

locomotory deficits.

3.3 Methods and materials

Fly stocks

The generation of the UAS-wild type human SOD1 transgenes (HS1 and HS2) is described

elsewhere (Elia et al. 1999). Briefly, both the HS1 and HS2 transgenes are inserted on the

second chromosome. UAS-human fALS-SOD1 transgenes bearing a glycine to alanine

substitution at position 93 were inserted onto either the X chromosome (fALS1) or the third

chromosome (fALS2). Ubiquitous expression was accomplished using daughterless-GAL4 (da-

GAL4) to drive UAS-transgene expression. Wild type (w1118

) flies were used as controls. Each

of these stocks were backcrossed eight generations onto a common Canton S genetic background

to alleviate the confounding effects of genetic variability.

65

Adult fly locomotor behaviour

Fly locomotor behaviour was measured in TriKinetics locomotor activity monitors in the

laboratory of Joel Levine at the University of Toronto at Mississagua. A single 42-45 day old

male fly was placed within a narrow glass vial (5 mm in diameter and 65 mm in length)

containing food at one end and a cotton plug at the other. Vials were loaded onto a horizontally

positioned TriKinetics activity monitor housed at 25 °C and set to a 12 hour light, 12 hour dark

cycle. A minimum of 14 individual flies were loaded for each genotype at the beginning of the

experiment. As a fly walks from one end of the vial to the other its activity is monitored by a

beam of infrared light that bisects the tube perpendicular to its axis. The number of times a fly

breaks the beam of light within a 5 minute time frame is then digitally recorded by a computer.

The average activity counts for each genotype aged 42-45, 52-55 and 56-59 days was

determined. An activity count is defined as an event that is triggered by breaking a beam of light

and then recorded digitally.

Life span analysis

Male flies were segregated into ten vials housing at least twenty individual flies at the beginning

of the experiment and kept at 25 °C. Vials were plugged with a sponge plug and placed

horizontally in racks so as to prevent older flies from getting stuck in the food. Death was

recorded every 3-4 days until the end of the experiment. Statistica software was used for all

statistical analysis. Kaplan-Meier was used to plot survival of each genotype. The Gehan’s

Wilcoxon test was used for two-sample comparisons of life span.

66

Immunoprecipitation and western analysis

SOD1G93A mice (B6.Cg-Tg[SOD1-G93A]1Gur/J) were purchased from The Jackson

Laboratory. Mouse spinal lysates were generated from 4 month old mice by adding 10X volume

of RIPA buffer (50 mM Tris-HCl pH 7.5,150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate,

1mM EDTA, 0.1% SDS) + protease inhibitors (PI) to spinal tissue and homogenized for 30

strokes. Lysates were then incubated on ice for 20 min. Samples were spun at 15,000 X G at 4

°C for 15 min. Supernatant was transferred to a clean tube, protein concentration was

determined using BCA assay and frozen at -80 °C until ready to use. Fly lysates were generated

in the same manner as mouse lysates with the following amendments: 30 flies were

homogenized in 600 µL of RIPA + PI using a dounce homogenizer and protein lysates were used

immediately, not frozen. 1 mg of mouse spinal lysate and 1 mg of 30 day old fly lysate or 2 mg

of 56-59 day old fly lysates were used for immunoprecipitation (IP) and protein-A beads alone

incubation. 0.1% (1 µg) of the sample was set aside for the input lanes with the exception of 30

day old fly lysates for which 0.5% (50 µg) of total protein was used for input lanes. Protein A-

agarose beads were washed in RIPA buffer 3X, spun down at 6,000 RPM for 5 min at 4 °C in

between washes. Samples were pre-cleared with 20 µL of pre-washed protein A-agarose for 1hr

at 4 °C. Lysates were then spun down for 10 min at 10,000 X G for 10 min at 4 °C.

Supernatants were transferred to a clean eppendorf tube and 10 µL of rabbit anti-human

superoxide dismutase1 exposed dimer interface antibody (SEDI) (Rakhit et al. 2007) was added

and incubated overnight at 4 °C. The next day, 20 µL of pre-washed protein-A-agarose beads

was added and incubated for 4 hr at 4 °C . The IP was then spun down at 6,000 RPM for 5 min

at 4 °C and the supernatant discarded or frozen. Beads were washed with 1 mL of RIPA 3x

spinning down at 6,000 RPM for 5 min at 4 °C in between washes. Supernatants were

completely removed using a gel-loading tip after the last wash. 12 µL of 2 X SDS sample buffer

plus 5% of β-mercaptoethanol was added and the sample was boiled for 5min. Samples were

spun at 10,000 X G for 10 min at room temperature, transferred to a clean tube with a gel-

loading tip and loaded onto a 10% SDS-page electrophoresis gel. Gels were then transferred

over-night onto PVDF membranes. The membranes were blocked in 2.5% each of milk, BSA,

FBS, NGS and NDS in 1% Tris-buffer saline Tween-20 buffer (TBST – 100 mM Tris-HCl pH

7.5, 150 mM NaCl, 0.1% tween-20) for 1 hour at room temperature. PVDF membranes were

67

then incubated with a sheep antibody raised against human SOD1 (574597,Calbiochem ) diluted

at 1:1000 dilution in TBST overnight at 4 °C. The next day membranes were washed three times

in TBST followed by incubation with an HRP-conjugated rabbit anti-sheep antibody at 1:5,000

in 1% blocking solution for one hour at room temperature followed by three washes prior to

visualization using chemiluminescence.

3.4 Results

3.4.1 Analysis of transgenic flies expressing human wild type and fALS-SOD1

To determine if expression of fALS-SOD1 in flies could give rise to dominant ALS-like

phenotypes we expressed UAS-human SOD1 in transgenic flies. Two independent UAS-human

fALS-SOD1 (fALS1 and fALS2) transgenics both bearing a glycine to alanine substitution at

position 93 and two independent UAS-human wild type SOD1 (HS1 and HS2) transgenics were

crossed to the ubiquitous daughterless-GAL4 driver (da-GAL4). We chose to ubiquitously

express SOD1 since ubiquitous expression of fALS-SOD1 in the mouse model has proven to be

successful at recapitulating ALS-like phenotypes (Gurney et al. 1994;Bruijn et al. 1997;Wong et

al. 1995). Importantly, we backcrossed all UAS-trangenic as well as the da-GAL4 line onto a

common genetic background to minimize the confounding effect of genetic variability. To

ensure that both the wild type and mutant SOD1 transgenes were expressed, western analysis

was performed. As can be seen in Figure 16, wild type SOD1 protein was more abundant

relative to the fALS-SOD1. This is consistent with what has previously been shown in the

mouse model and is attributed to the fact that fALS-SOD1 is less stable than wild type SOD1

(Gurney et al. 1994).

68

Figure 16. Expression of wild type and fALS-mutant human SOD1.

The dark arrow head points to the band corresponding to human SOD1 protein expression driven

by the da-GAL4 driver (da). Human SOD1 protein levels are lower in lysates generated from

flies expressing fALS-mutant SOD1 protein (fALS2/da and fALS1;;da) relative to wild type

SOD1 (HS2;da and HS1;da) protein levels. Relative to the single insert transgenic lines protein

levels of human fALS-SOD1 are higher in flies expressing both fALS1 and fALS2 (fALS1;;

fALS2/da). Absence of a SOD1 band in lysates generated from control w1118

flies or da-GAL4

flies confirms the specificity of the antibody for human SOD1 protein. β-tublin protein levels

serve as loading control (Hollow black arrow head for all lanes).

69

3.4.2 Ubiquitous expression of fALS-SOD1 gives rise to progressive deficits in

adult fly locomotory activity

Progressive decline in locomotor activity is a prominent feature in ALS patients as well as fALS-

SOD1-associated animal models (Gurney et al. 1994;Watson et al. 2008). To determine if

expression of fALS-SOD1 in flies could also induce progressive motor deficits we ubiquitously

expressed wild type or fALS-SOD1 using da-GAL4 and measured adult fly locomotion. Day

time locomotory activity of adult flies was recorded using TriKinetics Drosophila activity

monitors. We focused on day time activity because during the day flies are more active relative

to night and hence more activity measurements can be recorded. As a fly walked from one end

of a narrow vial to the other its activity was monitored by a beam of infrared light that bisected

the tube perpendicular to its axis. The number of times a fly broke the beam of lights within a

five minute time frame was defined as an activity count. The average activity count for each

genotype within a 12 hour light cycle was determined. Since ALS affects older adults we chose

to begin our analysis with 42-45 day old flies. Flies ubiquitously expressing HS1 had higher

locomotor activity relative to flies bearing UAS-HS1 alone, but not higher relative to da-GAL4

flies (HS1;da: mean activity counts = 509.3; HS1: mean activity counts = 363.2, P = 0.04)( da:

mean activity counts = 459.3) (Fig. 17A) (Table 2). There were no differences in the locomotor

activity in flies expressing HS2 relative to their respective controls (Fig. 17 B) (Table 2). Flies

expressing fALS-SOD1 also had normal locomotor activity relative to da-GAL4 flies as well as

flies bearing the UAS-fALS SOD1 transgene alone (fALS1;da: mean activity counts = 347.6 and

fALS2/da: activity counts = 459.3; da: activity counts = 423.8; fALS1: mean activity counts =

361.9; fALS2: mean activity counts = 449.3) (Fig. 17C & D) (Table 2). Hence, at 42-45 days of

age ubiquitous expression of wild type or fALS –SOD1 did not result in locomotory deficits.

Ten days later we measured the locomotory activity of the same flies. At 52-55 days of age the

only differences in activity detected in flies expressing wild type SOD1 was between da-GAL4

flies and the UAS-HS1 line (da: mean activity counts = 264.8; HS1: mean activity counts =

253.4, P = 0.04). Flies expressing fALS1, however, did exhibit significantly lower activity

relative to both the UAS-line alone as well as da-GAL4 flies (fALS1;;da: mean activity counts =

162.8; fALS1: mean activity counts = 239.9, P = 0.04) (fALS1;;da: mean activity counts =

162.8; da: mean activity counts = 264.8, P < 0.01 ) (Fig. 18 D) (Table 3). Although flies

70

Figure 17. Day activity levels of 42-45 day old flies expressing wild type or fALS-SOD1.

A-D) Activity counts were quantified and averaged during a 12 hour day light cycle for each

genotype. A) Flies expressing wild type SOD1 from the HS1 insertion (HS1;da) had higher

activity relative to flies bearing UAS-HS1 alone (HS1) but not relative to the da-GAL4 control

(da). B-D) There were no significant differences between flies expressing HS2, fALS1 and

fALS2 relative to each respective control. Error bars represent the SEM.

71

Table 2. Average day time activity of 42-45 day old flies ubiquitously expressing wild type

or fALS-mutant human SOD1.

Activity counts were quantified and averaged during a 12 hour day light cycle for each genotype.

(Avg. activity). The standard error (SEM), standard deviation (SD) and sample size (N) was

recorded for each genotype. da-GAL4 is the daughterless-GAL4 driver alone, uncrossed. HS1

and HS2 represent independent UAS-transgenic stocks bearing human wild type SOD1 transgene

insertion, uncrossed . HS1;da and HS2;da represent the same UAS-transgenes crossed to the da-

GAL4 driver. fALS1 and fALS2 represent two independent UAS-transgenic stocks bearing a

glycine to alanine substitution at position 93 in human fALS-SOD1, uncrossed. fALS1;;da and

fALS2/da represent the same UAS-transgenes crossed to the da-GAL4 driver

72



genotype. Significant differences are denoted using an asterisk (determined using two-tailed t-

tests). A double asterisks signifies that the activity levels induced by transgene expression was

significantly different to both the da-GAL4 as well as the respective UAS-transgene alone. A)

Flies bearing the UAS-HS1 transgene control alone had lower activity levels relative to da-GAL4

but not to flies expressing the HS1 transgene (HS1;da) B) There were no significant differences

between flies expressing HS2 and its respective controls. C) Flies expressing the fALS2-SOD1

transgene (fALS2/da) had significantly lower activity relative to da-GAL4 (da) flies but not to

flies bearing the fALS2-SOD1 transgene alone (fALS2). D) Flies expressing the fALS1-SOD1

transgene (fAL12;;da) had significantly lower activity relative to both da-GAL4 (da) flies as

well as flies bearing the fALS1-SOD1 transgene alone (fALS1). Error bars represent the SEM

73






and HS2 represent independent UAS-transgenic stocks bearing human wild type SOD1 transgene

insertion, uncrossed . HS1;da and HS2;da represent the same UAS-transgenes crossed to the da-

GAL4 driver. fALS1 and fALS2 represent two independent UAS-transgenic stocks bearing a

glycine to alanine substitution at position 93 in human fALS-SOD1, uncrossed. fALS1;;da and

fALS2/da represent the same UAS-transgenes crossed to the da-GAL4 driver

74

expressing fALS2 had lower activity relative to da-GAL4 flies they were not different to UAS-

fALS2 control flies (fALS2/da: mean activity counts = 187.1; da: mean activity counts = 264.8,

P = 0.02) (fALS2: mean activity counts = 219.8) (Fig. 18 C) (Table 3). Hene, at 56-59 days of

age only flies expressing fALS1 has clear locomotory deficits relative to both respective

controls.

Four days later, we again measured the locomotory activity of the same flies. By 56-59 days of

age the activity of all the flies had considerably decreased relative to the activity levels of the

same flies at day 42-55 (Fig 17 & 19) (Table 2 & 4). da-GAL4 flies also had significantly lower

locomotory activity relative to flies expressing HS1 and flies bearing UAS-HS1 alone (HS1;da:

mean activity counts = 163.1, P < 0.01) (HS1: mean activity counts = 159.1, P = 0.04).

However, there were no significant differences in activity levels between flies expression HS1

and those bearing only the UAS-HS1 transgene (HS1: mean activity counts = 159.1; HS1;da:

mean activity counts = 163.1) (Fig. 18 A). The average activity of flies expressing HS2, was not

different to flies bearing UAS-HS2 alone or da-GAL4 flies (Fig. 19 B) (Table 4). Flies

expressing fALS2, however, did exhibit significantly lower locomotor activity relative to both

da-GAL4 flies as well as to flies bearing UAS-fALS2 alone (fALS2/da: mean activity counts =

134.2; da: mean activity counts = 248.4, P < 0.01) (fALS2/da: mean activity counts = 134.2;

fALS2: mean activity counts = 2236.4, P = 0.03) (Fig. 19 C) (Table 4). Although flies

expressing fALS1 were significantly less active relative to da-GAL4 flies they were no longer

less active relative to flies bearing UAS-fALS1 alone (fALS1;;da; mean activity counts = 111.9;

da: mean activity counts = 248.4, P < 0.01) (fALS1: mean activity counts = 167.1) (Fig. 19 D)

(Table 4). Hence, only flies ubiquitously expressing fALS2 exhibited locomotory deficits as 56-

59 days of age.

75



genotype. Significant differences are denoted using an asterisk (determined using two-tailed t-

tests). A double asterisks signifies that the activity levels induced by transgene expression was

significantly different to both the da-GAL4 as well as the respective UAS-transgene alone. A)

da-GAL4 flies had higher activity levels compared to both flies expressing the HS1 transgene

(HS1;da) as well as flies bearing the UAS-HS1 transgene alone (HS1). B) There were no

significant differences between flies expressing HS2 and respective controls. C) Flies expressing

the fALS2-SOD1 transgene (fALS2/da) had significantly lower activity relative to both da-GAL4

(da) flies as well as flies bearing the fALS2-SOD1 transgene alone (fALS2). D) da-GAL4 flies

had higher activity levels compared to both flies expressing the fALS1-SOD1 transgene

(fALS2/da) as well as flies bearing the fALS2-SOD1 transgene alone (fALS21). Error bars

represent the SEM.

76






and HS2 represent independent UAS-transgenic stocks bearing human wild type SOD1

transgene insertion, uncrossed . HS1;da and HS2;da represent the same UAS-transgenes crossed

to the da-GAL4 driver. fALS1 and fALS2 represent two independent UAS-transgenic stocks

bearing a glycine to alanine substitution at position 93 in human fALS-SOD1, uncrossed.

fALS1;;da and fALS2/da represent the same UAS-transgenes crossed to the da-GAL4 driver

77

3.4.3 Survival analysis of flies expressing wild type or fALS-SOD1

To determine what effects expression of fALS-SOD1 had on fly lifespan we performed survival

analysis. We expressed wild type SOD or fALS-mutant SOD1 using the da-GAL4 driver. Flies

expressing fALS1 did indeed exhibit a significantly reduced lifespan relative to da-GAL4 flies,

wild type flies, as well as flies bearing the UAS-fALS1 transgene alone (fALS1;;da: mean

lifespan = 64.7 days; da: mean lifespan = 69.9 days; w1118

: mean lifespan = 73.4 days; fALS1:

mean lifespan = 74.3) (Fig 20 B) (Table 5 & Apendix 2). Expression of fALS2, on the other

hand, did not give rise to a shorter life span relative to controls (Fig. 20 A) (Table 5 & Apendix

2).

Surprisingly, expression of HS1 also resulted in a shorter lifespan relative to wild type, da-

GAL4, as well as the to flies bearing UAS-HS1 alone (HS1;da: mean lifespan = 67.5 days; da:

mean lifespan = 69.9 days; w1118

: mean lifespan = 73.4 days; HS1: mean lifespan = 76.9 days)

(Fig. 21 A) (Table 5 & Apendix 2). Expression of HS2 did not alter the lifespan of flies relative

to controls (Fig. 21 B) (Table 5 & Apendix 2).

78

Figure 20. Survival analysis of flies ubiquitously expressing fALS-mutant SOD1.

A-B) Survival plots were generated using the Kaplan-Meier method. A) Flies ubiquitously

expressing fALS2 (fALS/da) did not have a shorter life span relative to control w1118

flies, da-

GAL4 flies or flies bearing the UAS-fALS2 transgene alone. B) Flies expressing fALS1-SOD1

had a shorter lifespan relative to control w1118

flies, flies bearing only the UAS-fALS1 transgene

(fALS1) as well as to da-GAL4 flies (da).

79

Table 5. Survival Analysis

The mean and median life span in days for each genotype analyzed plus/minus the standard error

of the mean (SEM) and the standard deviation (SD) and sample size (N). da-GAL4 is the

daughterless-GAL4 driver alone, uncrossed. HS1 and HS2 represent independent UAS-

transgenic stocks bearing human wild type SOD1 transgene insertion, uncrossed . HS1;da and

HS2;da represent the same UAS-transgenes crossed to the da-GAL4 driver. fALS1 and fALS2

represent two independent UAS-transgenic stocks bearing a glycine to alanine substitution at

position 93 in human fALS-SOD1, uncrossed. fALS1;;da and fALS2/da represent the same

UAS-transgenes crossed to the da-GAL4 driver

80

Figure 21. Survival analysis of flies ubiquitously expressing wild type SOD1.

A-B) Survival plots were generated using the Kaplan-Meier method. A) Flies expressing HS1

(HS1;da) had a shorter lifespan relative to control w1118

flies, flies bearing only the UAS-HS1

transgene (HS1) as well as to da-GAL4 flies (da). B) Flies ubiquitously expressing HS2

(HS2;da) had a shorter lifespan relative to control w1118

flies (w1118

), as well to flies bearing only

the UAS-HS1 transgene (HS1) but not shorter relative to da-GAL4 flies (da).

81

3.4.4 fALS-SOD1 does not appear to form aggregates in adult flies

To determine whether expression of fALS-SOD1 in flies could give rise to aggregate formation

as it does in fALS mouse models and fALS patients we evaluated SOD1 misfolding in flies

expressing wild type or fALS-SOD1. We used an antibody specifically engineered to only

recognize misfolded SOD1 (SEDI) to determine whether misfolded fALS-SOD1 protein was

present in Drosophila lysates. First, we looked at lysates generated from 30 day old adult flies

expressing fALS-SOD1. We initially focused on flies expressing fALS1 because this line had

the earliest deficits in locomotor activity as well as the shortest lifespan relative to controls. As a

positive control for the presence for misfolded fALS-SOD1 we used lystates generated from

symptomatic transgenic mouse spinal cords expressing the same fALS-SOD1 mutant as our flies

(a glycine to alanine substitution at position 93) (hSOD1fALS

). Non-transgenic (non-Tg) mice

were used as a negative control. As can be seen in Figure 22, misfolded monomeric fALS-SOD1

was pulled down from lysates generated from fALS-SOD1 but not from non-transgenic mice

(non-Tg) spinal lysates (Fig. 24). We were unable to detect misfolded fALS-SOD1 in lysates

generated from 30 day old flies expressing fALS-SOD1. We reasoned that 30 days of age may

be too early to detect misfolded SOD1 and decided to evaluate fALS-SOD1 misfolding in older

flies. In addition, we doubled the amount of protein used to 2 mg in an effort to facilitate the

detection of potentially very small amounts of aggregates. Again, misfolded fALS-SOD1 was

successfully immunoprecipated with the SEDI antibody from mouse fALS-SOD1 transgenic

lysates (hSOD1fALS

) but not from non-transgenic lystates (Fig. 23). High molecular weight

human SOD1 complexes were visible in the mouse fALS-SOD1 input lane and these were also

pulled down by the SEDI antibody (data not shown). We were unable to detect misfolded SOD1

in lysates generated from 56-59 day old flies expressing either fALS1 or fALS2 despite the fact

that the murine and Drosophila SOD1 protein levels appear similar in respective input (Fig. 23).

However, high molecular weight bands were also observed in lysates generated from transgenic

flies expressing fALS-SOD1 (fALS1;;da and fALS2/da) (Fig. 23).

82

Figure 22. Misfolded human SOD1 is not detected in lysates generated from 30 day old

flies expressing human fALS-SOD1.

Lysates totaling 1 mg of protein generated from either non-transgenic (non-Tg) mice expressing

fALS- mutant SOD1(hSOD1fALS

) or 30 day old whole flies ubiquitously expressing the same

human fALS-SOD1mutant (a glycine to alanine substitution at position 93) (fALS1;;da) were

incubated with an antibody that recognizes misofolded human SOD1 (SEDI) or beads alone. A

total of 10 µg of protein was loaded in both the mouse input lanes. A total of 50 µg of protein

was loaded as the input for the fly lysate. An antibody that recognizes both mouse (double

asterisk) as well as human (single asterisk) SOD1 was used to probe for immunoprecipitated,

misfolded SOD1. Misfolded human SOD1 was immunoprecipitated only from spinal lysates

generated from transgenic mice expressing fALS-mutant SOD1 (hSOD1fALS

). A total of 1 µg of

purified wild type human SOD1 protein was loaded as a positive control for SOD1detection by

western analysis

83

Figure 23. Misfolded human SOD1 is not detected in lysates generated from 56-59 day old

flies expressing human fALS-SOD1.

Lysates totaling 1 mg of protein generated from either non-transgenic (non-Tg) mice expressing

fALS- mutant SOD1(hSOD1fALS

) or 2 mg of protein generated from 56-59 day old whole flies

ubiquitously expressing the same human fALS-SOD1 mutant (a glycine to alanine substitution

at position 93) (fALS2/da and fALS1;;da) were incubated with an antibody that recognizes

misofolded human SOD1 (SEDI) or beads alone. A total of 10 µg of protein was loaded for both

mouse and fly input lanes. An antibody that recognizes both mouse (double asterisk) as well as

human (single asterisk) SOD1 was used to probe for immunoprecipitated, misfolded SOD1.

Misfolded human SOD1 was immunoprecipitated only from spinal lysates generated from

transgenic mice expressing fALS-mutant SOD1 (hSOD1fALS

). High molecular weight, SOD1

positive complex were visible in fly lysates (black arrowhead). The hollow arrow head points to

a non-specific band which is also present in fly lysates not expressing human SOD1 (data not

shown).

84

Finally, we generated a multiple-insert transgenic line that expressed both fALS1 and fALS2 in

an effort to increase the abundance of mutant protein and thus facilitate our ability to detect

misfolded mutant SOD1. Western analysis confirmed that fALS-SOD1 protein is more abundant

in lysates generated from the double-insert fALS-SOD1 line (Fig. 16). Again, we used 2 mg of

total protein to immunoprecipite misfolded human SOD1 using the SEDI antibody, however we

were unable to detect misfolded fALS-SOD1 in lysates generated from flies 56-59 day old flies

expressing both fALS1 and fALS2 (Fig. 24).

3.5 Discussion

4

We have used Drosophila as a model system to study locomotory behaviour, lifespan and

aggregate formation in flies expressing either wild type or fALS-SOD1. We show that

expression of human fALS-SOD1in Drosophila gives rise to locomotory deficits, reduced

lifespan but notably, these phentoypes occur in the absence of fALS-SOD1 misfolding.

Our findings demonstrate that ubiquitous expression of fALS-SOD1 (fALS1 and fALS2) but not

wild type SOD1 (HS1 and HS2) results in progressive deficits in locomotor activity.

Decrements in fly locomotor activity can be reflective of muscle or nervous system defects,

which are the precise targets of ALS. Importantly, locomotor deficits were not detected in 42-45

day old flies indicating that like humans, flies expressing fALS-SOD1 mutant alleles initially

have normal motor ability. By 52-55 days of age, however, flies expressing fALS1-SOD1

exhibit a significant reduction in activity relative to controls. Three days later, at 56-59 days of

age, flies expressing fALS2-SOD1 also exhibit significant deficits in locomotor activity. The

fact that fALS1-SOD1 expressing flies no longer exhibit activity deficits at 56-59 days of age

may be due to the fact that as the activity of flies decreases with age, it becomes more difficult to

detect small differences in activity.

85

Figure 24. Misfolded human SOD1 is not detected in lysates generated from 56-59 day old

flies expressing two copies of human fALS-SOD1.

A total of 2 mg of lysate generated from 56-59 day old whole flies ubiquitously expressing the

human fALS-SOD1mutant from two independent insertion (fALS1;;fALS2/da) was incubated

with an antibody that recognizes misofolded human SOD1 (SEDI) or beads alone. A total of 10

µg of protein was loaded for input lanes. An antibody that recognizes human SOD1 (single

asterisk) was used to probe for immunoprecipitated, misfolded SOD1. Misfolded human SOD1

was not detected in lysates generated from flies expressing the fALS-mutant SOD1

(fALS1;;fALS2/da).

86

In our study, expression of wild type SOD1 did not have an affect on locomotor activity

indicating that the deficits we observe in flies expressing fALS-SOD1 are specific to the

expression of the mutant allele. Previous studies have described climbing deficits in flies

expressing wild type or fALS-SOD1 specifically in motor neurons (Watson et al. 2008). Since,

fALS-SOD1 induced toxicity has been shown to involve non-cell autonomous mechanisms in

promoting motor neuron cell death, ubiquitous expression of SOD1 may more faithfully

recapitulate fALS-SOD1 specific toxicity rather than motor neuron specific expression (Clement

et al. 2003). Further analysis using nervous system or muscle specific GAL4 drivers will need to

be performed to determine if the primary defect caused by fALS-SOD1 expression originates in

neurons, glia, muscle cells or a combination thereof.

Lifespan analysis of flies expressing fALS-SOD1 revealed that ubiquitous expression of the

fALS1 allele but not fALS2 resulted in a shorter lifespan. Although not detectable by western

analysis this discrepancy may be due to slightly different levels of transgene expression. More

sensitive techniques such as real-time RTPCR could determine whether this is indeed the case.

We were unable to test the lifespan of flies expressing two copies of fALS-SOD1 on account of

the fact that this line, unlike all the other fly stocks used in this study, was not backcrossed onto a

common genetic background. Surprisingly, expression of one of the wild type SOD1 alleles

(HS1) also gave rise to a shorter lifespan. Neurodegenerative toxicity as a result of wild type

SOD1 expression has been observed in a SOD1 mouse model where moderate motor neuron

loss, mild motor impairments and SOD1 aggregate formation but no premature death was

reported (Jaarsma et al. 2000). In addition, work performed by others has shown that

overexpression of wild type SOD1 can catalyze surrogate reactions that produce highly toxic

hydroxyl radicals (Peled-Kamar et al. 1997). Hydroxyl free radicals can then go on to devastate

other biological molecules including SOD1 itself. Furthermore, oxidative damage to SOD1 has

been shown to promote SOD1 aggregate formation (Rakhit et al. 2004). It is thus possible that

sustained oxidative damage to SOD1 can lead to SOD1 misfolding and aggregate formation

which ultimately may lead to toxicity and premature death in flies. In such a way, perhaps high

levels of wild type SOD1 in certain cell types, other than motor neurons, actually results in

toxicity.

87

Work performed in the mouse model has shown that very small quantities of misfolded mutant

SOD1 coincide with aggregate formation (Rakhit et al. 2004) and progressive fALS-SOD1-

induced neurodegeneration in vivo (Rakhit et al. 2007). We used an antibody specifically

engineered to recognize misfolded human SOD1 (SEDI) to determine if misfolded SOD1 could

also be detected in lysates generated from flies ubiquitously expressing human fALS-SOD1.

Using, this approach we did not detect misfolded SOD1 in fly lysates expressing fALS-SOD1

from single or multiple-insert transgenic lines. There are several possible reasons that could

explain why we do not see misfolding of human SOD1 in flies. First, perhaps the fALS-SOD1-

induced toxic mechanisms responsible for the activity deficits in flies do not involve protein

misfolding. Second, perhaps minute amounts of misfolded SOD1 are present but are below the

threshold of detection. Although we observed similar fALS-SOD1 protein levels in lysates

generated from flies and mice, the fly lysates were generated from whole flies while the mouse

lysate was exclusively generated from symptomatic mouse spines where misfolded, aggregated

SOD is expected to be most abundant. Third, perhaps the specific human fALS-SOD1 epitope

used to generate the SEDI antibody is not exposed in Drosophila. Although we cannot

mechanistically account for how this could happen we suspect that that presence of high

molecular bands in lysates generated from flies expressing human SOD1 may represent

aggregated SOD1. High molecular weight, SOD1 positive bands were also detected in the

mouse spinal lysates generated in this study as well as by others (Wang, Xu, and Borchelt. 2002)

and importantly these bands too were pulled down by the SEDI antibody, indicating they also

contain misfolded SOD1. In order to confirm whether aggregates are present but perhaps

spatially localized in flies an independent method such as immunohistochemistry (IHC) should

be used. Work performed by others has shown that expression of fALS-SOD1 in the Drosophila

eye gives rise to SOD1 positive aggregates upon cell-autonomous fALS-SOD1 expression

(Watson et al. 2008). Using an antibody specific for human SOD1 to probe for in situ aggregates

in older adult brain and ventral nerve cord would confirm whether SOD1 positive aggregates

accumulate in flies and whether aggregate formation is tissue specific, as it is in mice. If

aggregates are found by IHC it would indicate that either the amount of aggregated SOD1 is

diluted in whole fly lysate to a point that it is undetectable, even by immunoprecipitation, or that

the SEDI antibody does not recognize misfolded SOD1 in flies. If aggregates are not found it

would indicate that aggregate formation does not contribute to the locomotor activity deficits

88

observed with fALS-SOD1 expression and that alternative fALS-SOD1-induced toxicity such as

oxidative stress may be the underlying cause.

We have shown that expression of human fALS-SOD1 in Drosophila results in dominant,

progressive locomotory deficits. We can now express fALS-SOD1 in different cell types

including glial cells, various neuronal populations as well as muscle cells in various

combinations to determine the precise cellular origin of the fALS-SOD1-induced locomotor

defect. Such studies coupled with in situ analysis of aggregate formation will further our

understanding about what role aggregate formation plays in promoting ALS-like phenotypes.

Defining the specific interactions of glia, neurons and muscles during fALS-SOD1-induced

progressive motor deficits in Drosophila will help guide the course of ALS research aimed at

identifying novel treatments and ultimately a cure for ALS.

89

CHAPTER 4

4.1 Discussion

Many important insights into the molecular mechanisms underlying neurodegenerative diseases

have come from studying genes associated with inherited forms of these disorders. However,

several questions remain unanswered. For example, although genetic mutations that cause

neurodegenerative diseases are expressed throughout the lifetime of an organism, their

deleterious activities take decades to clinically manifest. In addition, the factors that determine

the progressive nature of neurodegeneration also remain largely unknown. In the proceeding

section, age-related forms of cumulative cytotoxic stress will be discussed as potential

determining factors that trigger the onset and progressive decline of neuronal function. Cellular

coping strategies in place to counteract these stressors will also be discussed with particular

emphasis on how the Drosophila model can be exploited to further our understanding of these

toxic mechanisms and how to treat them.

4.1.1 Are aggregates toxic?

Aggregate formation is a classical hallmark of most neurodegenerative diseases including AD

and ALS, yet a causative relationship between aggregate formation and the onset of pathogenesis

is still not certain. Are aggregates a cause of disease or a consequence? Are they toxic or

protective? Perhaps cells actively sequester mutant PSEN, APP, SOD1 or other cytotoxic

proteins into inactive aggregates as a means to control the aberrant activity of the soluble mutant

protein? In fact, this mechanism has been documented in several cell types including neurons.

When a cells’ capacity to degrade misfolded or aggregated proteins by conventional ubiquitin-

proteasome mechanisms has been exceeded or compromised it will expend energy to sequester

and actively transport aggregates into large, highly structured proteinacious bodies (Johnston,

90

Ward, and Kopito. 1998;Johnston et al. 2000b). What happens to these inclusion bodies is

unclear, but degradative autophagy may be involved (Keller et al. 2004). Evidence exists linking

Presenilins, SOD1 and Aβ to aggresome formation (Johnston, Ward, and Kopito. 1998;Johnston

et al. 2000b). So long as aggregates are efficiently sequestered or cleared neuronal function

likely continues uninterrupted. However, every cellular coping mechanism has a threshold and it

is likely that at a certain point cells run out of space to safely sequester aggregates, especially if

back-up degradation mechanisms themselves become overwhelmed. Since proteosomal decline

is observed during normal aging, the process of aging itself may be the single most important

determinant of how long a cell can hold-off the negative impact of aggregate accumulation

(Keller et al. 2004). Long-term accumulation of protein aggregates, in turn, may compromise

cell viability by sequestering vital proteins such as chaperones or by physically impairing

intracellular trafficking, which happens to be particularly critical to neuronal physiology.

Intriguingly, within a single day AD patients can exhibit significant fluctuations in functional

abilities, especially memory (Palop, Chin, and Mucke. 2006). Why this happens is uncertain but

it is unlikely to involve a sudden loss or gain of neurons. Instead these fluctuations may actually

represent temporary amelioration of chronic intoxication, such as the clearance of a large

aggregate clogging axonal transport. Whether or not aggregates are the primary cause of the

disease process, however, remains uncertain but is arguably unlikely.

4.1.2 What role does stress play?

Oxidative stress is an unavoidable consequence of oxidative metabolism and as we age, our

cellular defense mechanisms against oxidative stress decline. Oxidative stress is considered one

of the principle causes of progressive decline in cellular function during normal aging (Keller et

al. 2004). What’s more, oxidative damage to proteins can be a primary trigger of protein

aggregation (Rakhit et al. 2007;Rakhit et al. 2004). For example, oxidative damage to SOD1 has

been shown to promote SOD1 misfolding and aggregation (Rakhit et al. 2007;Rakhit et al.

2004). Since, fALS-SOD1 mutants are known to promote oxidative stress, this would exacerbate

oxidative damage and consequently further promote protein aggregation (Peled-Kamar et al.

1997;Yim et al. 1996). In addition, oxidative damage to proteins involved in protein

91

degradation, such as the proteasome, can lead to further accumulation of unfolded, aggregated

proteins. Hence, cumulative oxidative damage over the entire lifespan of a post-mitotic neuron

may be a primary toxic trigger for subsequent aggregate formation.

In our model system, overexpression of fALS-SOD1 was shown to give rise to dominant

locomotory deficits in the absence of detectable aggregates, suggesting that aggregate formation

may not be a primary cause of locomotory decline. However, others have successfully detected

fALS-SOD1 aggregates in flies (Watson et al. 2008). The unanswered question is, whether our

failure to detect aggregates is due to the fact that the SED1 antibody cannot detect misfolded

SOD1 aggregates in flies, or that the aggregates are below the threshold of detection.

Alternatively, it may be that an earlier primary toxic insult, such as oxidative stress, is

responsible for the observed locomotory deficits rather than aggregate formation. This

hypothesis could be tested by challenging flies with oxidative stress and assaying whether flies

expressing fALS-SOD1 are more sensitive to this stress. These experiments would specifically

address whether oxidative stress causes flies expressing fALS-SOD1 to die even earlier or

exhibit even earlier locomotory defects relative to controls. In addition, oxidative insult may

also promote SOD1 aggregation to a level where it becomes detectable. This would argue that

indeed, oxidative stress is the primary trigger of motor deficits in flies and that protein

aggregation is a secondary effect. This model could then be further subjected to several

screening methods. For example, it may be worthwhile to examine whether protein aggregation

could also be induced at earlier larval stages. If so, fALS-SOD1-GFP constructs could be

generated and aggregation visualized through the translucent body walls of larvae. These

transgenenics could then be subjected to a drug screen designed to identify chemicals that slow

down or reduce the accumulation of aggregates. Promising therapeutic compounds could then be

screened further by assaying whether they are also able to ameliorate locomotory deficits or

premature death of fALS-SOD1 expressing flies. The small size, short lifespan and relative low

costs associated with fly husbandry facilitates such large scale analyses, which can be conducted

in a relatively short time frame. Ultimately, however, the efficacy of any findings in flies would

have to be rigorously confirmed in mice before moving into human clinical trial.

Another form of stress related to protein aggregation and relevant to both AD and ALS is ER

stress. The ER is a multifunctional organelle, acting as a calcium storage sink but also as an

important site for protein synthesis, modification and folding. Not surprisingly, disruptions in

92

intracellular calcium homeostasis, and specifically depletion of ER calcium stores, have been

shown to cause ER stress (Nguyen, Wang, and Perry. 2002;Kudo et al. 2002;Verkhratsky and

Petersen. 2002). ER stress disrupts normal protein folding and leads to accumulation of unfolded

proteins. This triggers an evolutionarily conserved cellular response referred to as the unfolded

protein response (UPR). The UPR upregulates the transcription of chaperones and purges

misfolded proteins into the cytoplasm by a processes referred to as ER-associated degradation

(ERAD). Once eradicated, misfolded proteins are ubiquitnated and targeted for protesosomal

degradation. If the UPR fails to clear the back-up, ER stress can lead to apoptosis. What’s more,

fAD-mutations in presenilin have been shown to attenuate the UPR (Kudo et al. 2002).

Collectively, it is possible that in fAD brains aberrant presenilin activity facilitates amyloid

deposition by first inducing intracellular calcium deficits, which sets the stage for ER stress.

Misfolded proteins accumulate, degradation systems start to back-up to a point that the cell has

no choice but to initiate apoptosis. Since all the components of the UPR are conserved in flies it

would be interesting to determine whether the UPR is indeed being induced in our model system.

Our data did not reveal elevated apoptosis associated with fAD-Psn expression, however, pupal

neurons are relatively young and likely efficiently cope with stress, ER stress included. Our data

also suggests that Psn has a pro-survival function in the CNS. This pro-survival function may be

sufficient to counteract any pro-apoptotic signals arising from calcium-deficit induced ER stress.

However, if neurons expressing fAD-Psn were challenged with further stress, elevated levels of

apoptosis may be revealed. This would be consistent with work performed in the mouse model.

Specifically, neurons cultured from fAD-presenilin1 knock-in mice do not exhibit overt cell

death unless challenged with cellular stress (Guo et al. 1999). In future studies, pupal neuronal

cultures expressing fAD-Psn could be challenged with various forms of stress, including

oxidative stress, and apoptosis re-quantified. I hypothesize that any residual pro-survival activity

that fAD-Psn will be insufficient to deal with the additional external cellular stress. In fact,

accumulation of oxidative damage, which we know is a normal function of aging, may be the

reason why fAD patients and flies expressing fAD-Psn die prematurely. It would also be very

informative to determine the status of internal calcium stores and UPR in older flies.

Unfortunately, the adult fly CNS is not amenable to culturing. However, several labs have

pioneered protocols involving whole mount adult CNS preparations which may in the future be

adapted to measure intracellular calcium (Olsen and Wilson. 2008).

93

Mutations in SOD1 are also linked to ER stress. Recent evidence, has suggested that fALS-

SOD1 impairs ERAD, disabling the degradation of misfolded proteins and activating proteins

involved in triggering apoptosis (Nishitoh et al. 2008). This evidence links SOD1 activity with a

primary histopathalogical hallmark of ALS, ubiquitinated aggregates of misfolded proteins.

There is reason to believe that ERAD is conserved in flies since genes known to function in

ERAD coordination are conserved in flies, but await characterization (Ryoo and Steller. 2007).

Drosophila would be an ideal platform to characterize these genes and to investigate whether

mutations in the ERAD machinery can modify fALS-SOD1 or Psn-induced phenotypes. In fact,

since protein aggregation is common to so many neurodegenerative diseases any insights into

how disease-causing genes interact with the components of ER-stress machinery may pave the

way towards novel therapeutic avenues.

4.1.3 Behavioural Genetics in Drosophila

Our work has demonstrated that expression of fAD-presenilin in cholinergic neurons results in

premature death. What we don’t know is whether expression of fAD-mutant or wild type Psn

has any effect on learning and memory. Previous work performed in our lab has determined that

Psn-null larvae exhibit defects in olfactory-associative learning suggesting that indeed Psn does

play a role in learning and memory, at least during larval development (Knight et al. 2007). In

addition, there is some evidence suggesting that expression of fAD-Psn driven by the

endogenous Psn promoter may give rise to learning defects in adults. However, this study did

not include some important controls, including a wild type non-transgenic control. Hence, the

role of Psn in learning and memory in the adult fly brain warrants further investigation.

Specifically, it would be interesting to test the learning and memory abilities of adult flies

expressing wild type or fAD-mutant Psn in cholinergic neurons, especially since these neurons

are affected in AD. Using an olfactory learning paradigm where flies are trained to associate a

specific odour with an electric shock future studies could test learning and memory at various

stages of adult life. In addition, given that wild type Psn has a pro-survival effect in the

Drosophila CNS perhaps flies expressing wild type Psn will exhibit enhanced learning or

memory.

94

4.1.4 Concluding thoughts

Drosophila is an extremely versatile model system and in many ways is ideal for studying the

genetic basis of human disease. The high degree of genetic conservation coupled with low

genetic redundancy make this model particularly well suited for studying the function of disease

causing genes. Importantly, Drosophila is amenable to cellular, physiological as well as

behavioural analysis and unlike mammalian models these studies can be performed in a

relatively short time frame at minimal costs. Pioneering work in Drosophila can then be

validated in mammalian model systems and hopefully one day contribute to the development of

effective treatments against neurodegeneration.

95

References

"Canadian Study of Health and Aging: Study Methods and Prevalence of Dementia." CMAJ :

Canadian Medical Association Journal = Journal De l'Association Medicale Canadienne

150, no. 6 (Mar 15 1994): 899-913.

Adams, M. D., S. E. Celniker, R. A. Holt, C. A. Evans, J. D. Gocayne, P. G. Amanatides, S. E.

(TRUNCATED). "The Genome Sequence of Drosophila Melanogaster." Science 287, no.

5461 (Mar 24 2000): 2185-2195.

Ahn, E. Y., S. T. Lim, W. J. Cook, and J. M. McDonald. "Calmodulin Binding to the Fas Death

Domain. Regulation by Fas Activation." Journal of Biological Chemistry 279, no. 7 (Feb 13

2004): 5661-5666.

Amtul, Z., P. A. Lewis, S. Piper, R. Crook, M. Baker, K. Findlay, A. Singleton, M. Hogg, L.

Younkin, S. G. Younkin, J. Hardy, M. Hutton, B. F. Boeve, D. Tang-Wai, and T. E. Golde.

"A Presenilin 1 Mutation Associated with Familial Frontotemporal Dementia Inhibits

Gamma-Secretase Cleavage of APP and Notch." Neurobiology of Disease 9, no. 2 (Mar

2002): 269-273.

Andersen, P. M., K. B. Sims, W. W. Xin, R. Kiely, G. O'Neill, J. Ravits, E. Pioro, Y. Harati, R.

D. Brower, J. S. Levine, H. U. Heinicke, W. Seltzer, M. Boss, and R. H. Brown Jr. "Sixteen

Novel Mutations in the Cu/Zn Superoxide Dismutase Gene in Amyotrophic Lateral

Sclerosis: A Decade of Discoveries, Defects and Disputes." Amyotrophic Lateral Sclerosis

and Other Motor Neuron Disorders : Official Publication of the World Federation of

Neurology, Research Group on Motor Neuron Diseases 4, no. 2 (Jun 2003): 62-73.

Andrus, P. K., T. J. Fleck, M. E. Gurney, and E. D. Hall. "Protein Oxidative Damage in a

Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis." Journal of

Neurochemistry 71, no. 5 (Nov 1998): 2041-2048.

Baki, L., J. Shioi, P. Wen, Z. Shao, A. Schwarzman, M. Gama-Sosa, R. Neve, and N. K.

Robakis. "PS1 Activates PI3K thus Inhibiting GSK-3 Activity and Tau

Overphosphorylation: Effects of FAD Mutations." The EMBO Journal 23, no. 13 (Jul 7

2004): 2586-2596.

Balshaw, D. M., N. Yamaguchi, and G. Meissner. "Modulation of Intracellular Calcium-Release

Channels by Calmodulin." The Journal of Membrane Biology 185, no. 1 (Jan 1 2002): 1-8.

96

Baumeister, R., U. Leimer, I. Zweckbronner, C. Jakubek, J. Grunberg, and C. Haass. "Human

Presenilin-1, but Not Familial Alzheimer's Disease (FAD) Mutants, Facilitate

Caenorhabditis Elegans Notch Signalling Independently of Proteolytic Processing." Genes

and Function 1, no. 2 (Apr 1997): 149-159.

Berezovska, O., P. Ramdya, J. Skoch, M. S. Wolfe, B. J. Bacskai, and B. T. Hyman. "Amyloid

Precursor Protein Associates with a Nicastrin-Dependent Docking Site on the Presenilin 1-

Gamma-Secretase Complex in Cells Demonstrated by Fluorescence Lifetime Imaging." The

Journal of Neuroscience : The Official Journal of the Society for Neuroscience 23, no. 11

(Jun 1 2003): 4560-4566.

Bird, T. D. "Genetic Aspects of Alzheimer Disease." Genetics in Medicine : Official Journal of

the American College of Medical Genetics 10, no. 4 (Apr 2008): 231-239.

Boehning, D., R. L. Patterson, L. Sedaghat, N. O. Glebova, T. Kurosaki, and S. H. Snyder.

"Cytochrome c Binds to Inositol (1,4,5) Trisphosphate Receptors, Amplifying Calcium-

Dependent Apoptosis." Nature Cell Biology 5, no. 12 (Dec 2003): 1051-1061.

Boillee, S., C. Vande Velde, and D. W. Cleveland. "ALS: A Disease of Motor Neurons and their

Nonneuronal Neighbors." Neuron 52, no. 1 (Oct 5 2006): 39-59.

Boillee, S., K. Yamanaka, C. S. Lobsiger, N. G. Copeland, N. A. Jenkins, G. Kassiotis, G.

Kollias, and D. W. Cleveland. "Onset and Progression in Inherited ALS Determined by

Motor Neurons and Microglia." Science (New York, N.Y.) 312, no. 5778 (Jun 2 2006): 1389-

1392.

Borchelt, D. R., P. C. Wong, M. W. Becher, C. A. Pardo, M. K. Lee, Z. S. Xu, G. Thinakaran, N.

A. Jenkins, N. G. Copeland, S. S. Sisodia, D. W. Cleveland, D. L. Price, and P. N. Hoffman.

"Axonal Transport of Mutant Superoxide Dismutase 1 and Focal Axonal Abnormalities in

the Proximal Axons of Transgenic Mice." Neurobiology of Disease 5, no. 1 (Jul 1998): 27-

35.

Boulianne, G. L., I. Livne-Bar, J. M. Humphreys, Y. Liang, C. Lin, E. Rogaev, and P. St George-

Hyslop. "Cloning and Characterization of the Drosophila Presenilin Homologue."

Neuroreport 8, no. 4 (Mar 3 1997): 1025-1029.

Brand, A. H., and N. Perrimon. "Targeted Gene Expression as a Means of Altering Cell Fates

and Generating Dominant Phenotypes." Development (Cambridge, England) 118, no. 2 (Jun

1993): 401-415.

Brooks, B. R. "El Escorial World Federation of Neurology Criteria for the Diagnosis of

Amyotrophic Lateral Sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic

97

Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular

Diseases and the El Escorial "Clinical Limits of Amyotrophic Lateral Sclerosis" Workshop

Contributors." Journal of the Neurological Sciences 124 Suppl (Jul 1994): 96-107.

Bruening, W., J. Roy, B. Giasson, D. A. Figlewicz, W. E. Mushynski, and H. D. Durham. "Up-

Regulation of Protein Chaperones Preserves Viability of Cells Expressing Toxic Cu/Zn-

Superoxide Dismutase Mutants Associated with Amyotrophic Lateral Sclerosis." Journal of

Neurochemistry 72, no. 2 (Feb 1999): 693-699.

Bruijn, L. I., M. W. Becher, M. K. Lee, K. L. Anderson, N. A. Jenkins, N. G. Copeland, S. S.

Sisodia, J. D. Rothstein, D. R. Borchelt, D. L. Price, and D. W. Cleveland. "ALS-Linked

SOD1 Mutant G85R Mediates Damage to Astrocytes and Promotes Rapidly Progressive

Disease with SOD1-Containing Inclusions." Neuron 18, no. 2 (Feb 1997): 327-338.

Buxbaum, J. D., E. K. Choi, Y. Luo, C. Lilliehook, A. C. Crowley, D. E. Merriam, and W.

Wasco. "Calsenilin: A Calcium-Binding Protein that Interacts with the Presenilins and

Regulates the Levels of a Presenilin Fragment." Nature Medicine 4, no. 10 (Oct 1998):

1177-1181.

Cai, C., P. Lin, K. H. Cheung, N. Li, C. Levchook, Z. Pan, C. Ferrante, G. L. Boulianne, J. K.

Foskett, D. Danielpour, and J. Ma. "The Presenilin-2 Loop Peptide Perturbs Intracellular

Ca2+ Homeostasis and Accelerates Apoptosis." The Journal of Biological Chemistry 281,

no. 24 (Jun 16 2006): 16649-16655.

Campusano, J. M., H. Su, S. A. Jiang, B. Sicaeros, and D. K. O'Dowd. "NAChR-Mediated

Calcium Responses and Plasticity in Drosophila Kenyon Cells." Developmental

Neurobiology 67, no. 11 (Sep 15 2007): 1520-1532.

Cao, X., and T. C. Sudhof. "A Transcriptionally [Correction of Transcriptively] Active Complex

of APP with Fe65 and Histone Acetyltransferase Tip60." Science (New York, N.Y.) 293, no.

5527 (Jul 6 2001): 115-120.

Chan, S. L., M. Mayne, C. P. Holden, J. D. Geiger, and M. P. Mattson. "Presenilin-1 Mutations

Increase Levels of Ryanodine Receptors and Calcium Release in PC12 Cells and Cortical

Neurons." Journal of Biological Chemistry 275, no. 24 (Jun 16 2000): 18195-18200.

Chen, Q., A. Nakajima, S. H. Choi, X. Xiong, and Y. P. Tang. "Loss of Presenilin Function

Causes Alzheimer's Disease-Like Neurodegeneration in the Mouse." Journal of

Neuroscience Research 86, no. 7 (May 15 2008): 1615-1625.

Cheung, K. H., D. Shineman, M. Muller, C. Cardenas, L. Mei, J. Yang, T. Tomita, T. Iwatsubo,

V. M. Lee, and J. K. Foskett. "Mechanism of Ca2+ Disruption in Alzheimer's Disease by

98

Presenilin Regulation of InsP3 Receptor Channel Gating." Neuron 58, no. 6 (Jun 26 2008):

871-883.

Chui, D. H., H. Tanahashi, K. Ozawa, S. Ikeda, F. Checler, O. Ueda, H. Suzuki, W. Araki, H.

Inoue, K. Shirotani, K. Takahashi, F. Gallyas, and T. Tabira. "Transgenic Mice with

Alzheimer Presenilin 1 Mutations show Accelerated Neurodegeneration without Amyloid

Plaque Formation." Nature Medicine 5, no. 5 (May 1999): 560-564.

Clement, A. M., M. D. Nguyen, E. A. Roberts, M. L. Garcia, S. Boillee, M. Rule, A. P.

McMahon, W. Doucette, D. Siwek, R. J. Ferrante, R. H. Brown Jr, J. P. Julien, L. S.

Goldstein, and D. W. Cleveland. "Wild-Type Nonneuronal Cells Extend Survival of SOD1

Mutant Motor Neurons in ALS Mice." Science (New York, N.Y.) 302, no. 5642 (Oct 3

2003): 113-117.

Dal Canto, M. C., and M. E. Gurney. "A Low Expressor Line of Transgenic Mice Carrying a

Mutant Human Cu,Zn Superoxide Dismutase (SOD1) Gene Develops Pathological Changes

that most Closely Resemble those in Human Amyotrophic Lateral Sclerosis." Acta

Neuropathologica 93, no. 6 (Jul 1997): 537-550.

DeKosky, S. T., and S. W. Scheff. "Synapse Loss in Frontal Cortex Biopsies in Alzheimer's

Disease: Correlation with Cognitive Severity." Annals of Neurology 27, no. 5 (May 1990):

457-464.

Dermaut, B., S. Kumar-Singh, S. Engelborghs, J. Theuns, R. Rademakers, J. Saerens, B. A.

Pickut, K. Peeters, M. van den Broeck, K. Vennekens, S. Claes, M. Cruts, P. Cras, J. J.

Martin, C. Van Broeckhoven, and P. P. De Deyn. "A Novel Presenilin 1 Mutation

Associated with Pick's Disease but Not Beta-Amyloid Plaques." Annals of Neurology 55,

no. 5 (May 2004): 617-626.

Di Giorgio, F. P., M. A. Carrasco, M. C. Siao, T. Maniatis, and K. Eggan. "Non-Cell

Autonomous Effect of Glia on Motor Neurons in an Embryonic Stem Cell-Based ALS

Model." Nature Neuroscience 10, no. 5 (May 2007): 608-614.

Dreses-Werringloer, U., J. C. Lambert, V. Vingtdeux, H. Zhao, H. Vais, A. Siebert, A. Jain, J.

Koppel, A. Rovelet-Lecrux, D. Hannequin, F. Pasquier, D. Galimberti, E. Scarpini, D.

Mann, C. Lendon, D. Campion, P. Amouyel, P. Davies, J. K. Foskett, F. Campagne, and P.

Marambaud. "A Polymorphism in CALHM1 Influences Ca2+ Homeostasis, Abeta Levels,

and Alzheimer's Disease Risk." Cell 133, no. 7 (Jun 27 2008): 1149-1161.

Elia, A. J., T. L. Parkes, K. Kirby, P. St George-Hyslop, G. L. Boulianne, J. P. Phillips, and A. J.

Hilliker. "Expression of Human FALS SOD in Motorneurons of Drosophila." Free Radical

Biology & Medicine 26, no. 9-10 (Jun 1999): 1332-1338.

99

Etcheberrigaray, R., N. Hirashima, L. Nee, J. Prince, S. Govoni, M. Racchi, R. E. Tanzi, and D.

L. Alkon. "Calcium Responses in Fibroblasts from Asymptomatic Members of Alzheimer's

Disease Families." Neurobiology of Disease 5, no. 1 (Jul 1998): 37-45.

Ferrante, R. J., S. E. Browne, L. A. Shinobu, A. C. Bowling, M. J. Baik, U. MacGarvey, N. W.

Kowall, R. H. Brown Jr, and M. F. Beal. "Evidence of Increased Oxidative Damage in both

Sporadic and Familial Amyotrophic Lateral Sclerosis." Journal of Neurochemistry 69, no. 5

(Nov 1997): 2064-2074.

Finelli, A., A. Kelkar, H. J. Song, H. Yang, and M. Konsolaki. "A Model for Studying

Alzheimer's Abeta42-Induced Toxicity in Drosophila Melanogaster." Molecular and

Cellular Neurosciences 26, no. 3 (Jul 2004): 365-375.

Fossgreen, A., B. Bruckner, C. Czech, C. L. Masters, K. Beyreuther, and R. Paro. "Transgenic

Drosophila Expressing Human Amyloid Precursor Protein show Gamma-Secretase Activity

and a Blistered-Wing Phenotype." Proceedings of the National Academy of Sciences of the

United States of America 95, no. 23 (Nov 10 1998): 13703-13708.

Gaburjakova, M., J. Gaburjakova, S. Reiken, F. Huang, S. O. Marx, N. Rosemblit, and A. R.

Marks. "FKBP12 Binding Modulates Ryanodine Receptor Channel Gating." Journal of

Biological Chemistry 276, no. 20 (May 18 2001): 16931-16935.

Geula, C., and M. M. Mesulam. "Cortical Cholinergic Fibers in Aging and Alzheimer's Disease:

A Morphometric Study." Neuroscience 33, no. 3 (1989): 469-481.

Geula, C., C. K. Wu, D. Saroff, A. Lorenzo, M. Yuan, and B. A. Yankner. "Aging Renders the

Brain Vulnerable to Amyloid Beta-Protein Neurotoxicity." Nature Medicine 4, no. 7 (Jul

1998): 827-831.

Giannakopoulos, P., F. R. Herrmann, T. Bussiere, C. Bouras, E. Kovari, D. P. Perl, J. H.

Morrison, G. Gold, and P. R. Hof. "Tangle and Neuron Numbers, but Not Amyloid Load,

Predict Cognitive Status in Alzheimer's Disease." Neurology 60, no. 9 (May 13 2003):

1495-1500.

Gilman, S., M. Koller, R. S. Black, L. Jenkins, S. G. Griffith, N. C. Fox, L. Eisner, L. Kirby, M.

B. Rovira, F. Forette, J. M. Orgogozo, and AN1792(QS-21)-201 Study Team. "Clinical

Effects of Abeta Immunization (AN1792) in Patients with AD in an Interrupted Trial."

Neurology 64, no. 9 (May 10 2005): 1553-1562.

Goate, A., M. C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, L. Fidani, L. Giuffra, A.

Haynes, N. Irving, and L. James. "Segregation of a Missense Mutation in the Amyloid

100

Precursor Protein Gene with Familial Alzheimer's Disease." Nature 349, no. 6311 (Feb 21

1991a): 704-706.

"Segregation of a Missense Mutation in the Amyloid Precursor Protein Gene with Familial

Alzheimer's Disease." Nature 349, no. 6311 (Feb 21 1991b): 704-706.

Gong, Y. H., A. S. Parsadanian, A. Andreeva, W. D. Snider, and J. L. Elliott. "Restricted

Expression of G86R Cu/Zn Superoxide Dismutase in Astrocytes Results in Astrocytosis but

does Not Cause Motoneuron Degeneration." The Journal of Neuroscience : The Official

Journal of the Society for Neuroscience 20, no. 2 (Jan 15 2000): 660-665.

Gotz, J., and L. M. Ittner. "Animal Models of Alzheimer's Disease and Frontotemporal

Dementia." Nature Reviews.Neuroscience 9, no. 7 (Jul 2008): 532-544.

Guo, Q., W. Fu, B. L. Sopher, M. W. Miller, C. B. Ware, G. M. Martin, and M. P. Mattson.

"Increased Vulnerability of Hippocampal Neurons to Excitotoxic Necrosis in Presenilin-1

Mutant Knock-in Mice." Nature Medicine 5, no. 1 (Jan 1999): 101-106.

Guo, Q., K. Furukawa, B. L. Sopher, D. G. Pham, J. Xie, N. Robinson, G. M. Martin, and M. P.

Mattson. "Alzheimer's PS-1 Mutation Perturbs Calcium Homeostasis and Sensitizes PC12

Cells to Death Induced by Amyloid Beta-Peptide." Neuroreport 8, no. 1 (Dec 20 1996):

379-383.

Guo, Y., I. Livne-Bar, L. Zhou, and G. L. Boulianne. "Drosophila Presenilin is Required for

Neuronal Differentiation and Affects Notch Subcellular Localization and Signaling." The

Journal of Neuroscience : The Official Journal of the Society for Neuroscience 19, no. 19

(Oct 1 1999): 8435-8442.

Gurney, M. E., H. Pu, A. Y. Chiu, M. C. Dal Canto, C. Y. Polchow, D. D. Alexander, J.

Caliendo, A. Hentati, Y. W. Kwon, and H. X. Deng. "Motor Neuron Degeneration in Mice

that Express a Human Cu,Zn Superoxide Dismutase Mutation." Science 264, no. 5166 (Jun

17 1994): 1772-1775.

Hand, C. K., and G. A. Rouleau. "Familial Amyotrophic Lateral Sclerosis." Muscle & Nerve 25,

no. 2 (Feb 2002): 135-159.

Hardy, J. A., and G. A. Higgins. "Alzheimer's Disease: The Amyloid Cascade Hypothesis."

Science (New York, N.Y.) 256, no. 5054 (Apr 10 1992): 184-185.

Hayrapetyan, V., V. Rybalchenko, N. Rybalchenko, and P. Koulen. "The N-Terminus of

Presenilin-2 Increases Single Channel Activity of Brain Ryanodine Receptors through

Direct Protein-Protein Interaction." Cell Calcium 44, no. 5 (Nov 2008): 507-518.

101

Heiman, R. G., R. C. Atkinson, B. F. Andruss, C. Bolduc, G. E. Kovalick, and K. Beckingham.

"Spontaneous Avoidance Behavior in Drosophila Null for Calmodulin Expression."

Proceedings of the National Academy of Sciences of the United States of America 93, no. 6

(Mar 19 1996a): 2420-2425.

"Spontaneous Avoidance Behavior in Drosophila Null for Calmodulin Expression." Proceedings

of the National Academy of Sciences of the United States of America 93, no. 6 (Mar 19

1996b): 2420-2425.

Herreman, A., D. Hartmann, W. Annaert, P. Saftig, K. Craessaerts, L. Serneels, L. Umans, V.

Schrijvers, F. Checler, H. Vanderstichele, V. Baekelandt, R. Dressel, P. Cupers, D.

Huylebroeck, A. Zwijsen, F. Van Leuven, and B. De Strooper. "Presenilin 2 Deficiency

Causes a Mild Pulmonary Phenotype and no Changes in Amyloid Precursor Protein

Processing but Enhances the Embryonic Lethal Phenotype of Presenilin 1 Deficiency."


(Oct 12 1999): 11872-11877.

Herreman, A., L. Serneels, W. Annaert, D. Collen, L. Schoonjans, and B. De Strooper. "Total

Inactivation of Gamma-Secretase Activity in Presenilin-Deficient Embryonic Stem Cells."

Nature Cell Biology 2, no. 7 (Jul 2000): 461-462.

Hoskins, B., and I. K. Ho. "Effects of Maturation and Aging on Calmodulin and Calmodulin-

Regulated Enzymes in various Regions of Mouse Brain." Mechanisms of Ageing and

Development 36, no. 2 (Oct 1986): 173-186.

Hyman, B. T. "The Neuropathological Diagnosis of Alzheimer's Disease: Clinical-Pathological

Studies." Neurobiology of Aging 18, no. 4 Suppl (Jul-Aug 1997): S27-32.

Iijima, K., H. P. Liu, A. S. Chiang, S. A. Hearn, M. Konsolaki, and Y. Zhong. "Dissecting the

Pathological Effects of Human Abeta40 and Abeta42 in Drosophila: A Potential Model for

Alzheimer's Disease." Proceedings of the National Academy of Sciences of the United States

of America 101, no. 17 (Apr 27 2004): 6623-6628.

Irizarry, M. C., M. McNamara, K. Fedorchak, K. Hsiao, and B. T. Hyman. "APPSw Transgenic

Mice Develop Age-Related A Beta Deposits and Neuropil Abnormalities, but no Neuronal

Loss in CA1." Journal of Neuropathology and Experimental Neurology 56, no. 9 (Sep

1997): 965-973.

Jaarsma, D., E. D. Haasdijk, J. A. Grashorn, R. Hawkins, W. van Duijn, H. W. Verspaget, J.

London, and J. C. Holstege. "Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression

in Mice Causes Mitochondrial Vacuolization, Axonal Degeneration, and Premature

Motoneuron Death and Accelerates Motoneuron Disease in Mice Expressing a Familial

102

Amyotrophic Lateral Sclerosis Mutant SOD1." Neurobiology of Disease 7, no. 6 Pt B (Dec

2000): 623-643.

Johnston, J. A., M. J. Dalton, M. E. Gurney, and R. R. Kopito. "Formation of High Molecular

Weight Complexes of Mutant Cu, Zn-Superoxide Dismutase in a Mouse Model for Familial

Amyotrophic Lateral Sclerosis." Proceedings of the National Academy of Sciences of the

United States of America 97, no. 23 (Nov 7 2000a): 12571-12576.

"Formation of High Molecular Weight Complexes of Mutant Cu, Zn-Superoxide Dismutase in a

Mouse Model for Familial Amyotrophic Lateral Sclerosis." Proceedings of the National

Academy of Sciences of the United States of America 97, no. 23 (Nov 7 2000b): 12571-

12576.

Johnston, J. A., C. L. Ward, and R. R. Kopito. "Aggresomes: A Cellular Response to Misfolded

Proteins." The Journal of Cell Biology 143, no. 7 (Dec 28 1998): 1883-1898.

Jonsson, P. A., K. Ernhill, P. M. Andersen, D. Bergemalm, T. Brannstrom, O. Gredal, P. Nilsson,

and S. L. Marklund. "Minute Quantities of Misfolded Mutant Superoxide Dismutase-1

Cause Amyotrophic Lateral Sclerosis." Brain : A Journal of Neurology 127, no. Pt 1 (Jan

2004): 73-88.

Kasri, N. N., S. L. Kocks, L. Verbert, S. S. Hebert, G. Callewaert, J. B. Parys, L. Missiaen, and

H. De Smedt. "Up-Regulation of Inositol 1,4,5-Trisphosphate Receptor Type 1 is

Responsible for a Decreased Endoplasmic-Reticulum Ca2+ Content in Presenilin Double

Knock-Out Cells." Cell Calcium 40, no. 1 (Jul 2006): 41-51.

Keller, J. N., E. Dimayuga, Q. Chen, J. Thorpe, J. Gee, and Q. Ding. "Autophagy, Proteasomes,

Lipofuscin, and Oxidative Stress in the Aging Brain." The International Journal of

Biochemistry & Cell Biology 36, no. 12 (Dec 2004): 2376-2391.

Knight, D., K. Iliadi, M. P. Charlton, H. L. Atwood, and G. L. Boulianne. "Presynaptic Plasticity

and Associative Learning are Impaired in a Drosophila Presenilin Null Mutant."

Developmental Neurobiology 67, no. 12 (Oct 2007): 1598-1613.

Kudo, T., T. Katayama, K. Imaizumi, Y. Yasuda, M. Yatera, M. Okochi, M. Tohyama, and M.

Takeda. "The Unfolded Protein Response is Involved in the Pathology of Alzheimer's

Disease." Annals of the New York Academy of Sciences 977 (Nov 2002): 349-355.

Lacomblez, L., G. Bensimon, P. N. Leigh, P. Guillet, and V. Meininger. "Dose-Ranging Study of

Riluzole in Amyotrophic Lateral Sclerosis. Amyotrophic Lateral Sclerosis/Riluzole Study

Group II." Lancet 347, no. 9013 (May 25 1996): 1425-1431.

103

Lee, M. K., H. H. Slunt, L. J. Martin, G. Thinakaran, G. Kim, S. E. Gandy, M. Seeger, E. Koo,

D. L. Price, and S. S. Sisodia. "Expression of Presenilin 1 and 2 (PS1 and PS2) in Human

and Murine Tissues." The Journal of Neuroscience : The Official Journal of the Society for

Neuroscience 16, no. 23 (Dec 1 1996): 7513-7525.

Leissring, M. A., Y. Akbari, C. M. Fanger, M. D. Cahalan, M. P. Mattson, and F. M. LaFerla.

"Capacitative Calcium Entry Deficits and Elevated Luminal Calcium Content in Mutant

Presenilin-1 Knockin Mice." The Journal of Cell Biology 149, no. 4 (May 15 2000): 793-

798.

Leissring, M. A., W. Farris, A. Y. Chang, D. M. Walsh, X. Wu, X. Sun, M. P. Frosch, and D. J.

Selkoe. "Enhanced Proteolysis of Beta-Amyloid in APP Transgenic Mice Prevents Plaque

Formation, Secondary Pathology, and Premature Death." Neuron 40, no. 6 (Dec 18 2003):

1087-1093.

Leissring, M. A., F. M. LaFerla, N. Callamaras, and I. Parker. "Subcellular Mechanisms of

Presenilin-Mediated Enhancement of Calcium Signaling." Neurobiology of Disease 8, no. 3

(Jun 2001): 469-478.

Leissring, M. A., M. P. Murphy, T. R. Mead, Y. Akbari, M. C. Sugarman, M. Jannatipour, B.

Anliker, U. Muller, P. Saftig, B. De Strooper, M. S. Wolfe, T. E. Golde, and F. M. LaFerla.

"A Physiologic Signaling Role for the Gamma -Secretase-Derived Intracellular Fragment of

APP." Proceedings of the National Academy of Sciences of the United States of America 99,

no. 7 (Apr 2 2002): 4697-4702.

Levitan, D., T. G. Doyle, D. Brousseau, M. K. Lee, G. Thinakaran, H. H. Slunt, S. S. Sisodia,

and I. Greenwald. "Assessment of Normal and Mutant Human Presenilin Function in

Caenorhabditis Elegans." Proceedings of the National Academy of Sciences of the United

States of America 93, no. 25 (Dec 10 1996): 14940-14944.

Li, Z., J. L. Joyal, and D. B. Sacks. "Calmodulin Enhances the Stability of the Estrogen

Receptor." The Journal of Biological Chemistry 276, no. 20 (May 18 2001): 17354-17360.

Lindsay, Colin. A Portrait of Seniors in Canada. 3rd Ed. Ottawa, Canada: Ottawa: Statistics

Canada, 1999.

Lindsay, J., E. Sykes, I. McDowell, R. Verreault, and D. Laurin. "More than the Epidemiology

of Alzheimer's Disease: Contributions of the Canadian Study of Health and Aging."

Canadian Journal of Psychiatry.Revue Canadienne De Psychiatrie 49, no. 2 (Feb 2004):

83-91.

104

Lino, M. M., C. Schneider, and P. Caroni. "Accumulation of SOD1 Mutants in Postnatal

Motoneurons does Not Cause Motoneuron Pathology Or Motoneuron Disease." The Journal

of Neuroscience : The Official Journal of the Society for Neuroscience 22, no. 12 (Jun 15

2002): 4825-4832.

Lu, Y., Y. Lv, Y. Ye, Y. Wang, Y. Hong, M. E. Fortini, Y. Zhong, and Z. Xie. "A Role for

Presenilin in Post-Stress Regulation: Effects of Presenilin Mutations on Ca2+ Currents in

Drosophila." The FASEB Journal : Official Publication of the Federation of American

Societies for Experimental Biology 21, no. 10 (Aug 2007): 2368-2378.

Majoor-Krakauer, D., P. J. Willems, and A. Hofman. "Genetic Epidemiology of Amyotrophic

Lateral Sclerosis." Clinical Genetics 63, no. 2 (Feb 2003): 83-101.

Massie, H. R., Valerie Aeillo R, Trevor R. Williams, and L. K. DeWolfe. "Calcium and

Calmodulin Changes with Aging in Drosophila." Age 12 (1989): 7-11.

Matsumoto, G., A. Stojanovic, C. I. Holmberg, S. Kim, and R. I. Morimoto. "Structural

Properties and Neuronal Toxicity of Amyotrophic Lateral Sclerosis-Associated Cu/Zn

Superoxide Dismutase 1 Aggregates." The Journal of Cell Biology 171, no. 1 (Oct 10 2005):

75-85.

Mattson, M. P., and S. L. Chan. "Calcium Orchestrates Apoptosis." Nature Cell Biology 5, no.

12 (Dec 2003): 1041-1043.

McCullough, K. D., J. L. Martindale, L. O. Klotz, T. Y. Aw, and N. J. Holbrook. "Gadd153

Sensitizes Cells to Endoplasmic Reticulum Stress by Down-Regulating Bcl2 and Perturbing

the Cellular Redox State." Molecular and Cellular Biology 21, no. 4 (Feb 2001): 1249-1259.

Mitsumoto, H. "Diagnosis and Progression of ALS." 48, no. Suppl 4 (1997): S2.

Mockett, R. J., S. N. Radyuk, J. J. Benes, W. C. Orr, and R. S. Sohal. "Phenotypic Effects of

Familial Amyotrophic Lateral Sclerosis Mutant Sod Alleles in Transgenic Drosophila."


(Feb 7 2003): 301-306.

Morohashi, Y., N. Hatano, S. Ohya, R. Takikawa, T. Watabiki, N. Takasugi, Y. Imaizumi, T.

Tomita, and T. Iwatsubo. "Molecular Cloning and Characterization of CALP/KChIP4, a

Novel EF-Hand Protein Interacting with Presenilin 2 and Voltage-Gated Potassium Channel

Subunit Kv4." Journal of Biological Chemistry 277, no. 17 (Apr 26 2002): 14965-14975.

105

Nagai, M., D. B. Re, T. Nagata, A. Chalazonitis, T. M. Jessell, H. Wichterle, and S. Przedborski.

"Astrocytes Expressing ALS-Linked Mutated SOD1 Release Factors Selectively Toxic to

Motor Neurons." Nature Neuroscience 10, no. 5 (May 2007): 615-622.

Nelson, L. M. "Epidemiology of ALS." Clinical Neuroscience (New York, N.Y.) 3, no. 6 (-1996

1995): 327-331.

Nguyen, H. N., C. Wang, and D. C. Perry. "Depletion of Intracellular Calcium Stores is Toxic to

SH-SY5Y Neuronal Cells." Brain Research 924, no. 2 (Jan 11 2002): 159-166.

Nicoll, R. A., C. Malenka, and J. A. Kauer. "The Role of Calcium in Long-Term Potentiation."

Annals of the New York Academy of Sciences 568 (1989): 166.

Nishitoh, H., H. Kadowaki, A. Nagai, T. Maruyama, T. Yokota, H. Fukutomi, T. Noguchi, A.

Matsuzawa, K. Takeda, and H. Ichijo. "ALS-Linked Mutant SOD1 Induces ER Stress- and

ASK1-Dependent Motor Neuron Death by Targeting Derlin-1." Genes & Development 22,

no. 11 (Jun 1 2008): 1451-1464.

O'Day, D. H., and M. A. Myre. "Calmodulin-Binding Domains in Alzheimer's Disease Proteins:

Extending the Calcium Hypothesis." Biochemical and Biophysical Research

Communications 320, no. 4 (Aug 6 2004): 1051-1054.

Oddo, S., L. Billings, J. P. Kesslak, D. H. Cribbs, and F. M. LaFerla. "Abeta Immunotherapy

Leads to Clearance of Early, but Not Late, Hyperphosphorylated Tau Aggregates Via the

Proteasome." Neuron 43, no. 3 (Aug 5 2004): 321-332.

Olsen, S. R., and R. I. Wilson. "Cracking Neural Circuits in a Tiny Brain: New Approaches for

Understanding the Neural Circuitry of Drosophila." Trends in Neurosciences 31, no. 10 (Oct

2008): 512-520.

Ostbye, T., and E. Crosse. "Net Economic Costs of Dementia in Canada." CMAJ : Canadian

Medical Association Journal = Journal De l'Association Medicale Canadienne 151, no. 10

(Nov 15 1994): 1457-1464.

Pack-Chung, E., M. B. Meyers, W. P. Pettingell, R. D. Moir, A. M. Brownawell, I. Cheng, R. E.

Tanzi, and T. W. Kim. "Presenilin 2 Interacts with Sorcin, a Modulator of the Ryanodine

Receptor." The Journal of Biological Chemistry 275, no. 19 (May 12 2000): 14440-14445.

Palop, J. J., J. Chin, and L. Mucke. "A Network Dysfunction Perspective on Neurodegenerative

Diseases." Nature 443, no. 7113 (Oct 19 2006): 768-773.

Pardo, C. A., Z. Xu, D. R. Borchelt, D. L. Price, S. S. Sisodia, and D. W. Cleveland. "Superoxide

Dismutase is an Abundant Component in Cell Bodies, Dendrites, and Axons of Motor

106

Neurons and in a Subset of Other Neurons." Proceedings of the National Academy of

Sciences of the United States of America 92, no. 4 (Feb 14 1995): 954-958.

Parkes, T. L., A. J. Elia, D. Dickinson, A. J. Hilliker, J. P. Phillips, and G. L. Boulianne.

"Extension of Drosophila Lifespan by Overexpression of Human SOD1 in Motorneurons."

Nature Genetics 19, no. 2 (Jun 1998): 171-174.

Peled-Kamar, M., J. Lotem, I. Wirguin, L. Weiner, A. Hermalin, and Y. Groner. "Oxidative

Stress Mediates Impairment of Muscle Function in Transgenic Mice with Elevated Level of

Wild-Type Cu/Zn Superoxide Dismutase." Proceedings of the National Academy of

Sciences of the United States of America 94, no. 8 (Apr 15 1997): 3883-3887.

Periz, G., and M. E. Fortini. "Ca(2+)-ATPase Function is Required for Intracellular Trafficking

of the Notch Receptor in Drosophila." The EMBO Journal 18, no. 21 (Nov 1 1999): 5983-

5993.

Phillips, J. P., S. D. Campbell, D. Michaud, M. Charbonneau, and A. J. Hilliker. "Null Mutation

of copper/zinc Superoxide Dismutase in Drosophila Confers Hypersensitivity to Paraquat

and Reduced Longevity." Proceedings of the National Academy of Sciences of the United

States of America 86, no. 8 (Apr 1989): 2761-2765.

Phillips, J. P., J. A. Tainer, E. D. Getzoff, G. L. Boulianne, K. Kirby, and A. J. Hilliker.

"Subunit-Destabilizing Mutations in Drosophila copper/zinc Superoxide Dismutase:

Neuropathology and a Model of Dimer Dysequilibrium." Proceedings of the National

Academy of Sciences of the United States of America 92, no. 19 (Oct 12 1995): 8574-8578.

Puttaparthi, K., C. Wojcik, B. Rajendran, G. N. DeMartino, and J. L. Elliott. "Aggregate

Formation in the Spinal Cord of Mutant SOD1 Transgenic Mice is Reversible and Mediated

by Proteasomes." Journal of Neurochemistry 87, no. 4 (Nov 2003): 851-860.

Qi-Takahara, Y., M. Morishima-Kawashima, Y. Tanimura, G. Dolios, N. Hirotani, Y. Horikoshi,

F. Kametani, M. Maeda, T. C. Saido, R. Wang, and Y. Ihara. "Longer Forms of Amyloid

Beta Protein: Implications for the Mechanism of Intramembrane Cleavage by Gamma-

Secretase." The Journal of Neuroscience : The Official Journal of the Society for

Neuroscience 25, no. 2 (Jan 12 2005): 436-445.

Querfurth, H. W., J. Jiang, J. D. Geiger, and D. J. Selkoe. "Caffeine Stimulates Amyloid Beta-

Peptide Release from Beta-Amyloid Precursor Protein-Transfected HEK293 Cells." Journal

of Neurochemistry 69, no. 4 (Oct 1997): 1580-1591.

Rakhit, R., J. P. Crow, J. R. Lepock, L. H. Kondejewski, N. R. Cashman, and A. Chakrabartty.

"Monomeric Cu,Zn-Superoxide Dismutase is a Common Misfolding Intermediate in the

107

Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis." The Journal of

Biological Chemistry 279, no. 15 (Apr 9 2004): 15499-15504.

Rakhit, R., J. Robertson, C. Vande Velde, P. Horne, D. M. Ruth, J. Griffin, D. W. Cleveland, N.

R. Cashman, and A. Chakrabartty. "An Immunological Epitope Selective for Pathological

Monomer-Misfolded SOD1 in ALS." Nature Medicine 13, no. 6 (Jun 2007): 754-759.

Ratovitski, T., L. B. Corson, J. Strain, P. Wong, D. W. Cleveland, V. C. Culotta, and D. R.

Borchelt. "Variation in the biochemical/biophysical Properties of Mutant Superoxide

Dismutase 1 Enzymes and the Rate of Disease Progression in Familial Amyotrophic Lateral

Sclerosis Kindreds." Human Molecular Genetics 8, no. 8 (Aug 1999): 1451-1460.

Raux, G., R. Gantier, C. Thomas-Anterion, J. Boulliat, P. Verpillat, D. Hannequin, A. Brice, T.

Frebourg, and D. Campion. "Dementia with Prominent Frontotemporal Features Associated

with L113P Presenilin 1 Mutation." Neurology 55, no. 10 (Nov 28 2000): 1577-1578.

Reaume, A. G., J. L. Elliott, E. K. Hoffman, N. W. Kowall, R. J. Ferrante, D. F. Siwek, H. M.

Wilcox, D. G. Flood, M. F. Beal, R. H. Brown Jr, R. W. Scott, and W. D. Snider. "Motor

Neurons in Cu/Zn Superoxide Dismutase-Deficient Mice Develop Normally but Exhibit

Enhanced Cell Death After Axonal Injury." Nature Genetics 13, no. 1 (May 1996): 43-47.

Rogaev, E. I., R. Sherrington, E. A. Rogaeva, G. Levesque, M. Ikeda, Y. Liang, H. Chi, C. Lin,

K. Holman, and T. Tsuda. "Familial Alzheimer's Disease in Kindreds with Missense

Mutations in a Gene on Chromosome 1 Related to the Alzheimer's Disease Type 3 Gene."

Nature 376, no. 6543 (Aug 31 1995): 775-778.

Rosen, D. R., T. Siddique, D. Patterson, D. A. Figlewicz, P. Sapp, A. Hentati, D. Donaldson, J.

Goto, J. P. O'Regan, and H. X. Deng. "Mutations in Cu/Zn Superoxide Dismutase Gene are

Associated with Familial Amyotrophic Lateral Sclerosis." Nature 362, no. 6415 (Apr 4

1993): 59-62.

Rubin, G. M., M. D. Yandell, J. R. Wortman, G. L. Gabor Miklos, C. R. Nelson, I. K. Hariharan,

M. E. Fortini, P. W. Li, R. Apweiler, W. Fleischmann, J. M. Cherry, S. Henikoff, M. P.

Skupski, S. Misra, M. Ashburner, E. Birney, M. S. Boguski, T. Brody, P. Brokstein, S. E.

Celniker, S. A. Chervitz, D. Coates, A. Cravchik, A. Gabrielian, R. F. Galle, W. M. Gelbart,

R. A. George, L. S. Goldstein, F. Gong, P. Guan, N. L. Harris, B. A. Hay, R. A. Hoskins, J.

Li, Z. Li, R. O. Hynes, S. J. Jones, P. M. Kuehl, B. Lemaitre, J. T. Littleton, D. K. Morrison,

C. Mungall, P. H. O'Farrell, O. K. Pickeral, C. Shue, L. B. Vosshall, J. Zhang, Q. Zhao, X.

H. Zheng, and S. Lewis. "Comparative Genomics of the Eukaryotes." Science 287, no. 5461

(Mar 24 2000): 2204-2215.

108

Rybalchenko, V., S. Y. Hwang, N. Rybalchenko, and P. Koulen. "The Cytosolic N-Terminus of

Presenilin-1 Potentiates Mouse Ryanodine Receptor Single Channel Activity." The

International Journal of Biochemistry & Cell Biology 40, no. 1 (2008): 84-97.

Ryoo, H. D., and H. Steller. "Unfolded Protein Response in Drosophila: Why another Model can

make it Fly." Cell Cycle (Georgetown, Tex.) 6, no. 7 (Apr 1 2007): 830-835.

Salvaterra, P. M., and T. Kitamoto. "Drosophila Cholinergic Neurons and Processes Visualized

with Gal4/UAS-GFP." Brain Research.Gene Expression Patterns 1, no. 1 (Aug 2001): 73-

82.

Saura, C. A., S. Y. Choi, V. Beglopoulos, S. Malkani, D. Zhang, B. S. Shankaranarayana Rao, S.

Chattarji, R. J. Kelleher 3rd, E. R. Kandel, K. Duff, A. Kirkwood, and J. Shen. "Loss of

Presenilin Function Causes Impairments of Memory and Synaptic Plasticity Followed by

Age-Dependent Neurodegeneration." Neuron 42, no. 1 (Apr 8 2004): 23-36.

Schonheit, B., R. Zarski, and T. G. Ohm. "Spatial and Temporal Relationships between Plaques

and Tangles in Alzheimer-Pathology." Neurobiology of Aging 25, no. 6 (Jul 2004): 697-711.

Scorrano, L., S. A. Oakes, J. T. Opferman, E. H. Cheng, M. D. Sorcinelli, T. Pozzan, and S. J.

Korsmeyer. "BAX and BAK Regulation of Endoplasmic Reticulum Ca2+: A Control Point

for Apoptosis." Science (New York, N.Y.) 300, no. 5616 (Apr 4 2003): 135-139.

Seidner, G. A., Y. Ye, M. M. Faraday, W. G. Alvord, and M. E. Fortini. "Modeling Clinically

Heterogeneous Presenilin Mutations with Transgenic Drosophila." Current Biology : CB 16,

no. 10 (May 23 2006): 1026-1033.

Shaw, P. J., P. G. Ince, G. Falkous, and D. Mantle. "Oxidative Damage to Protein in Sporadic

Motor Neuron Disease Spinal Cord." Annals of Neurology 38, no. 4 (Oct 1995): 691-695.

Shen, J., R. T. Bronson, D. F. Chen, W. Xia, D. J. Selkoe, and S. Tonegawa. "Skeletal and CNS

Defects in Presenilin-1-Deficient Mice." Cell 89, no. 4 (May 16 1997): 629-639.

Sherrington, R., E. I. Rogaev, Y. Liang, E. A. Rogaeva, G. Levesque, M. Ikeda, H. Chi, C. Lin,

G. Li, and K. Holman. "Cloning of a Gene Bearing Missense Mutations in Early-Onset

Familial Alzheimer's Disease." Nature 375, no. 6534 (Jun 29 1995): 754-760.

Sicaeros, B., J. M. Campusano, and D. K. O'Dowd. "Primary Neuronal Cultures from the Brains

of Late Stage Drosophila Pupae." Journal of Visualized Experiments : JoVE (4), no. 4

(2007): 200.

109

Simon, A. F., D. T. Liang, and D. E. Krantz. "Differential Decline in Behavioral Performance of

Drosophila Melanogaster with Age." Mechanisms of Ageing and Development 127, no. 7

(Jul 2006): 647-651.

Smith, I. F., J. P. Boyle, P. F. Vaughan, H. A. Pearson, R. F. Cowburn, and C. S. Peers. "Ca(2+)

Stores and Capacitative Ca(2+) Entry in Human Neuroblastoma (SH-SY5Y) Cells

Expressing a Familial Alzheimer's Disease Presenilin-1 Mutation." Brain Research 949, no.

1-2 (Sep 13 2002): 105-111.

Stabler, S. M., L. L. Ostrowski, S. M. Janicki, and M. J. Monteiro. "A Myristoylated Calcium-

Binding Protein that Preferentially Interacts with the Alzheimer's Disease Presenilin 2

Protein." The Journal of Cell Biology 145, no. 6 (Jun 14 1999): 1277-1292.

Steiner, B., E. M. Mandelkow, J. Biernat, N. Gustke, H. E. Meyer, B. Schmidt, G. Mieskes, H.

D. Soling, D. Drechsel, and M. W. Kirschner. "Phosphorylation of Microtubule-Associated

Protein Tau: Identification of the Site for Ca2(+)-Calmodulin Dependent Kinase and

Relationship with Tau Phosphorylation in Alzheimer Tangles." The EMBO Journal 9, no.

11 (Nov 1990): 3539-3544.

Struhl, G., and I. Greenwald. "Presenilin is Required for Activity and Nuclear Access of Notch

in Drosophila." Nature 398, no. 6727 (Apr 8 1999): 522-525.

Stull, J. T. "Ca2+-Dependent Cell Signaling through Calmodulin-Activated Protein Phosphatase

and Protein Kinases Minireview Series." Journal of Biological Chemistry 276, no. 4 (Jan 26

2001): 2311-2312.

Stutzmann, G. E., I. Smith, A. Caccamo, S. Oddo, I. Parker, and F. Laferla. "Enhanced

Ryanodine-Mediated Calcium Release in Mutant PS1-Expressing Alzheimer's Mouse

Models." Annals of the New York Academy of Sciences 1097 (Feb 2007): 265-277.

Takasugi, N., T. Tomita, I. Hayashi, M. Tsuruoka, M. Niimura, Y. Takahashi, G. Thinakaran,

and T. Iwatsubo. "The Role of Presenilin Cofactors in the Gamma-Secretase Complex."

Nature 422, no. 6930 (Mar 27 2003): 438-441.

Tanzi, R. E., and L. Bertram. "Twenty Years of the Alzheimer's Disease Amyloid Hypothesis: A

Genetic Perspective." Cell 120, no. 4 (Feb 25 2005): 545-555.

Terry, R. D., E. Masliah, D. P. Salmon, N. Butters, R. DeTeresa, R. Hill, L. A. Hansen, and R.

Katzman. "Physical Basis of Cognitive Alterations in Alzheimer's Disease: Synapse Loss is

the Major Correlate of Cognitive Impairment." Annals of Neurology 30, no. 4 (Oct 1991):

572-580.

110

Thibault, O., J. C. Gant, and P. W. Landfield. "Expansion of the Calcium Hypothesis of Brain

Aging and Alzheimer's Disease: Minding the Store." Aging Cell 6, no. 3 (Jun 2007): 307-

317.

Thinakaran, G., D. R. Borchelt, M. K. Lee, H. H. Slunt, L. Spitzer, G. Kim, T. Ratovitsky, F.

Davenport, C. Nordstedt, M. Seeger, J. Hardy, A. I. Levey, S. E. Gandy, N. A. Jenkins, N.

G. Copeland, D. L. Price, and S. S. Sisodia. "Endoproteolysis of Presenilin 1 and

Accumulation of Processed Derivatives in Vivo." Neuron 17, no. 1 (Jul 1996): 181-190.

Tu, H., O. Nelson, A. Bezprozvanny, Z. Wang, S. F. Lee, Y. H. Hao, L. Serneels, B. De

Strooper, G. Yu, and I. Bezprozvanny. "Presenilins Form ER Ca2+ Leak Channels, a

Function Disrupted by Familial Alzheimer's Disease-Linked Mutations." Cell 126, no. 5

(Sep 8 2006): 981-993.

Tummala, H., C. Jung, A. Tiwari, C. M. Higgins, L. J. Hayward, and Z. Xu. "Inhibition of

Chaperone Activity is a Shared Property of several Cu,Zn-Superoxide Dismutase Mutants

that Cause Amyotrophic Lateral Sclerosis." The Journal of Biological Chemistry 280, no. 18

(May 6 2005): 17725-17731.

van Marum, R. J. "Current and Future Therapy in Alzheimer's Disease." Fundamental & Clinical

Pharmacology 22, no. 3 (Jun 2008): 265-274.

Verkhratsky, A., and O. H. Petersen. "The Endoplasmic Reticulum as an Integrating Signalling

Organelle: From Neuronal Signalling to Neuronal Death." European Journal of

Pharmacology 447, no. 2-3 (Jul 5 2002): 141-154.

Wang, B., K. M. Sullivan, and K. Beckingham. "Drosophila Calmodulin Mutants with Specific

Defects in the Musculature Or in the Nervous System." Genetics 165, no. 3 (Nov 2003):

1255-1268.

Wang, J., G. Xu, and D. R. Borchelt. "High Molecular Weight Complexes of Mutant Superoxide

Dismutase 1: Age-Dependent and Tissue-Specific Accumulation." Neurobiology of Disease

9, no. 2 (Mar 2002): 139-148.

Wang, R., B. Wang, W. He, and H. Zheng. "Wild-Type Presenilin 1 Protects Against Alzheimer

Disease Mutation-Induced Amyloid Pathology." The Journal of Biological Chemistry 281,

no. 22 (Jun 2 2006): 15330-15336.

Watanabe, M., M. Dykes-Hoberg, V. C. Culotta, D. L. Price, P. C. Wong, and J. D. Rothstein.

"Histological Evidence of Protein Aggregation in Mutant SOD1 Transgenic Mice and in

Amyotrophic Lateral Sclerosis Neural Tissues." Neurobiology of Disease 8, no. 6 (Dec

2001): 933-941.

111

Watson, M. R., R. D. Lagow, K. Xu, B. Zhang, and N. M. Bonini. "A Drosophila Model for

Amyotrophic Lateral Sclerosis Reveals Motor Neuron Damage by Human SOD1." The

Journal of Biological Chemistry 283, no. 36 (Sep 5 2008): 24972-24981.

Wolfe, M. S. "When Loss is Gain: Reduced Presenilin Proteolytic Function Leads to Increased

Abeta42/Abeta40. Talking Point on the Role of Presenilin Mutations in Alzheimer Disease."

EMBO Reports 8, no. 2 (Feb 2007): 136-140.

Wolfe, M. S., W. Xia, B. L. Ostaszewski, T. S. Diehl, W. T. Kimberly, and D. J. Selkoe. "Two

Transmembrane Aspartates in Presenilin-1 Required for Presenilin Endoproteolysis and

Gamma-Secretase Activity." Nature 398, no. 6727 (Apr 8 1999): 513-517.

Wong, P. C., H. Cai, D. R. Borchelt, and D. L. Price. "Genetically Engineered Mouse Models of

Neurodegenerative Diseases." Nature Neuroscience 5, no. 7 (Jul 2002): 633-639.

Wong, P. C., C. A. Pardo, D. R. Borchelt, M. K. Lee, N. G. Copeland, N. A. Jenkins, S. S.

Sisodia, D. W. Cleveland, and D. L. Price. "An Adverse Property of a Familial ALS-Linked

SOD1 Mutation Causes Motor Neuron Disease Characterized by Vacuolar Degeneration of

Mitochondria." Neuron 14, no. 6 (Jun 1995): 1105-1116.

Wu, L. J., Y. Lu, and T. L. Xu. "A Novel Mechanical Dissociation Technique for Studying

Acutely Isolated Maturing Drosophila Central Neurons." Journal of Neuroscience Methods

108, no. 2 (Jul 30 2001): 199-206.

Yang, J., K. Bridges, K. Y. Chen, and A. Y. Liu. "Riluzole Increases the Amount of Latent HSF1

for an Amplified Heat Shock Response and Cytoprotection." PLoS ONE 3, no. 8 (Aug 6

2008): e2864.

Ye, Y., and M. E. Fortini. "Apoptotic Activities of Wild-Type and Alzheimer's Disease-Related

Mutant Presenilins in Drosophila Melanogaster." The Journal of Cell Biology 146, no. 6

(Sep 20 1999): 1351-1364.

"Characterization of Drosophila Presenilin and its Colocalization with Notch during

Development." Mechanisms of Development 79, no. 1-2 (Dec 1998): 199-211.

Ye, Y., N. Lukinova, and M. E. Fortini. "Neurogenic Phenotypes and Altered Notch Processing

in Drosophila Presenilin Mutants." Nature 398, no. 6727 (Apr 8 1999): 525-529.

Yeromin, A. V., J. Roos, K. A. Stauderman, and M. D. Cahalan. "A Store-Operated Calcium

Channel in Drosophila S2 Cells." The Journal of General Physiology 123, no. 2 (Feb 2004):

167-182.

112

Yim, M. B., J. H. Kang, H. S. Yim, H. S. Kwak, P. B. Chock, and E. R. Stadtman. "A Gain-of-

Function of an Amyotrophic Lateral Sclerosis-Associated Cu,Zn-Superoxide Dismutase

Mutant: An Enhancement of Free Radical Formation due to a Decrease in km for Hydrogen

Peroxide." Proceedings of the National Academy of Sciences of the United States of America

93, no. 12 (Jun 11 1996): 5709-5714.

Yoshihara, M., A. W. Ensminger, and J. T. Littleton. "Neurobiology and the Drosophila

Genome." Functional & Integrative Genomics 1, no. 4 (Mar 2001): 235-240.

Zatti, G., A. Burgo, M. Giacomello, L. Barbiero, R. Ghidoni, G. Sinigaglia, C. Florean, S.

Bagnoli, G. Binetti, S. Sorbi, P. Pizzo, and C. Fasolato. "Presenilin Mutations Linked to

Familial Alzheimer's Disease Reduce Endoplasmic Reticulum and Golgi Apparatus Calcium

Levels." Cell Calcium 39, no. 6 (Jun 2006): 539-550.

Zatti, G., R. Ghidoni, L. Barbiero, G. Binetti, T. Pozzan, C. Fasolato, and P. Pizzo. "The

Presenilin 2 M239I Mutation Associated with Familial Alzheimer's Disease Reduces Ca2+

Release from Intracellular Stores." Neurobiology of Disease 15, no. 2 (Mar 2004): 269-278.

Zhang, Z., P. Nadeau, W. Song, D. Donoviel, M. Yuan, A. Bernstein, and B. A. Yankner.

"Presenilins are Required for Gamma-Secretase Cleavage of Beta-APP and Transmembrane

Cleavage of Notch-1." Nature Cell Biology 2, no. 7 (Jul 2000): 463-465.

Zheng, H., M. Jiang, M. E. Trumbauer, R. Hopkins, D. J. Sirinathsinghji, K. A. Stevens, M. W.

Conner, H. H. Slunt, S. S. Sisodia, H. Y. Chen, and L. H. Van der Ploeg. "Mice Deficient

for the Amyloid Precursor Protein Gene." Annals of the New York Academy of Sciences 777

(Jan 17 1996): 421-426.

Zhu, J., S. M. Stabler, J. B. Ames, I. Baskakov, and M. J. Monteiro. "Calcium Binding

Sequences in Calmyrin Regulates Interaction with Presenilin-2." Experimental Cell Research

300, no. 2 (Nov 1 2004): 440-454.

113

Appendices

Apendix 1. Gehan’s Wilcoxon Survival Analysis

5

UAS-PsnWT

represents a UAS-transgenic stock bearing a full length copy of the Drosophila

presenilin gene. UAS-PsnFAD

represents a UAS-transgenic stock bearing a methionine to valine

substigution at position 146 in the full length Drosophila presenilin protein. Camnull

is a loss of

function calmodulin allele. w1118

flies are wild type, non-trangenic flies.

114

Apendix 2. Gehan’s Wilcoxon Survival Analysis.

HS1 and HS2 represent independent UAS-transgenic stocks bearing human wild type SOD1

transgene insertion, uncrossed . HS1;da and HS2;da represent the same UAS-transgenes crossed

to the da-GAL4 driver. fALS1 and fALS2 represent two independent UAS-transgenic stocks

bearing a glycine to alanine substitution at position 93 in human fALS-SOD1, uncrossed.

fALS1;;da and fALS2/da represent the same UAS-transgenes crossed to the da-GAL4 driver

drosophila melanogaster as a model organism to study human ... · disease-causing genes. using the...

Documents