The Role of BERP in Mammalian Systems

by

Carol C. Cheung

A thesis submitted in conformity with the requirements for the degree ofDoctor of Philosophy

Department of Medical BiophysicsFaculty of Medicine

School of Graduate StudiesUniversity of Toronto

Copyright by Carol C. Cheung 2009

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Thesis Abstract

The Role of BERP in Mammalian SystemsCarol C. Cheung

Doctor of Philosophy, Department of Medical Biophysics, University of Toronto, 2009

p53 functions as an important tumour suppressor through its ability to regulate a number of

important cellular processes such as cell cycle arrest, apoptosis, DNA repair, senescence, and

angiogenesis. An in vivo genetic modifier screen performed using Drosophila melanogaster

resulted in the identification of D. melanogaster brain tumour (brat) as a putative modifier of

the p53 small eye phenotype. Mammalian homologs of brat are members of the tripartite

motif family that contain a c-terminal NHL domain. We focus on elucidating the in vivo role

of one such homolog, BERP, through the generation and characterization of a classical gene-

deletion mouse mutant. We report that BERP-deficient mice exhibit enhanced

learning/memory, increased fear, impaired motor coordination, and increased resistance to

PTZ -induced seizures. Electrophysiological and biochemical studies show a decrease in

mIPSC amplitude along with a decrease in cell surface expression of gamma2 subunit-

containing GABA A receptors in the brains of BERP-deficient mice. In addition, no effect of

genotype is apparent when examining BERP mRNA levels in the brain. This suggests that

the decreased cell surface expression of gamma2 subunit-containing GABA A receptors is

likely a posttranscriptional phenomenon and supports the possibility that BERP may be

involved in the intracellular trafficking of GABA A receptors. In investigating the possible

relationship between BERP and p53, we identify the presence of a transcriptionally

competent p53 response element within the first intron of the human BERP genomic locus

and demonstrate that the BERP expression is up regulated in a p53-dependent manner both in

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vitro and in vivo. These results support the interpretation that BERP is a novel p53-regulated

gene and suggest a new role for p53 in the regulation of GABA A receptor trafficking and

epileptogenesis.

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Acknowledgements

My eternal gratitude to the people who made this journey possible:

To Dennis, Harrison and Leonardo…for being the reasons that make life worth living.

To my parents, my brother, and my parents-in-law…for their constant support and love.

To all the members of the Mak Lab for their help and friendship over the years. My sincerethanks to Scott Pownall who pointed the way for me during the earliest stages of this project.And to Thorsten Berger, Andrew Elia, Jillian Haight, Dave McIlwain, Patrick Reilly, CaimeiYang, and Kathrin Zaugg...I cannot even begin to thank them for their scientific guidance, theirunwavering support, and their unconditional friendship.

To Qi Wan, Ning Chang, and Lijun Li...for sharing their expertise on ion channel functionswith me.

To members of the Van der Kooy lab, especially Sue Runciman and Brenda Coles-Takabe, forsharing their expertise on neural stem cell biology with me.

To Tom Ferraro...for sharing his expertise in seizure susceptibility testing with me.

To Avrum Gottlieb and the Canadian Instititues of Health Research...for their financialsupport that has made this work possible.

To Yvonne Bedford, Runjan Chetty, Andrew Evans, Roni Sambas, Joan Sweet, and Theo vander Kwast, of the Department of Pathology at the Universitiy Health Network...for theirsupport in allowing me to complete the work of this thesis.

To Sylvia Asa, Pathologist-in-Chief and clinician-scientist extraordinaire, for her mentorshipover the years and for being a truly inspiring real-life example of “The Triple Threat“.

To the members of my supervisory committee, Sam Benchimol and James Woodgett...forsharing their time and scientific ideas with me. I realize just how lucky I was to have twosuch incredible minds guiding my scientific progress. I shall miss their wise counsel.

And last but certainly not least, to my supervisor Tak Mak…for his support, encouragement,and guidance…for teaching me the importance of perseverance….and for allowing me theopportunity, if only for a little while, to walk in the land of the giants.

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Table of Contents

THESIS ABSTRACT................................................................................................................... II

ACKNOWLEDGEMENTS.......................................................................................................... IV

TABLE OF CONTENTS...............................................................................................................V

LIST OF ABBREVIATIONS.....................................................................................................VIII

LIST OF FIGURES................................................................................................................... XII

1 INTRODUCTION................................................................................................................. 1

1.1 OPENING REMARKS ......................................................................................................... 11.2 P53 - MOLECULE OF THE YEAR (1993, SCIENCE MAGAZINE) ................................................... 2

1.2.1 p53 – The protein ....................................................................................................... 21.2.2 p53 and Cancer.......................................................................................................... 31.2.3 p53-deficient mice ....................................................................................................... 41.2.4 p53 - The Transcription Factor ..................................................................................... 41.2.5 The p53 Network ........................................................................................................ 51.2.6 In search of novel p53 targets: In vivo genetic screen using Drosophila melanogaster ............. 9

1.3 B RAIN E XPRESSED R ING FINGER P ROTEIN (BERP)...............................................................111.3.1 D. melanogaster Brain Tumour (brat) suppresses the eye phenotype of dp53 ........................111.3.2 Mammalian homologs of D. melanogaster brat ...............................................................11

1.3.2.1 BERP/TRIM3........................................................................................................................................................151.3.2.2 NARF/TRIM2.......................................................................................................................................................161.3.2.3 HT2A/TRIM32.....................................................................................................................................................17

1.4 OVERVIEW OF NEURONAL FUNCTION IN THE MAMMALIAN CNS.............................................181.4.1 Anatomy of the Neuron ...............................................................................................181.4.2 Neuronal membrane potentials .....................................................................................21

1.4.2.1 Resting potential ................................................................................................................................................211.4.2.2 Action potential ..................................................................................................................................................21

1.4.3 Overview of the synapse ..............................................................................................221.4.4 Overview of general neuroreceptor functions...................................................................221.4.5 Overview of Neurotransmitter Systems...........................................................................23

1.5 GABAA RECEPTOR SIGNALING WITHIN THE MAMMALIAN CNS.............................................241.5.1 GABA - The Neurotransmitter ......................................................................................241.5.2 GABA Receptor Family ...............................................................................................251.5.3 GABA A Receptors .....................................................................................................25

1.5.3.1 The GABAA Receptor in Human Disease......................................................................................................301.5.3.1.1 Epilepsy...........................................................................................................................................................301.5.3.1.2 Schizophrenia ................................................................................................................................................311.5.3.1.3 Anxiety Disorders.........................................................................................................................................31

1.5.3.2 The GABA A Receptor Lifecycle.....................................................................................................................321.5.3.2.1 Assembly of GABAARs...............................................................................................................................321.5.3.2.2 Trafficking of the GABAAR to the cell surface.....................................................................................331.5.3.2.3 Synpatic Clustering of GABAARs...........................................................................................................341.5.3.2.4 Endocytosis of GABAARs..........................................................................................................................351.5.3.2.5 Post-endocytic recycling and degradation of GABAARs..................................................................36

1.5.4 GABAB Receptors ......................................................................................................36

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1.5.5 GABAC Receptors......................................................................................................371.6 THESIS RATIONALE AND OBJECTIVES.................................................................................37

2 BERP IS A NOVEL P53 TARGET GENE.............................................................................38

2.1 INTRODUCTION..............................................................................................................382.2 MATERIALS AND METHODS..............................................................................................40

2.2.1 DNA sequences and prediction of p53-binding sites .........................................................402.2.2 Cell culture...............................................................................................................402.2.3 ChIP assay ...............................................................................................................402.2.4 Luciferase assay ........................................................................................................412.2.5 Real time RT-PCR......................................................................................................412.2.6 Genotyping of p53-deficient mice ..................................................................................422.2.7 In-situ hybridization...................................................................................................42

2.3 RESULTS.......................................................................................................................442.3.1 The BERP promoter region contains multiple potential p53 response elements .....................442.3.2 p53 can bind BERP p53REs in vivo and regulate transcription..........................................472.3.3 BERP expression is p53-dependent in vitro .....................................................................502.3.4 BERP expression is p53-dependent in vivo......................................................................53

3 THE ROLE OF BERP IN THE MAMMALIAN CNS .............................................................56

3.1 INTRODUCTION..............................................................................................................563.2 MATERIALS AND METHODS..............................................................................................57

3.2.1 ES cell culture ...........................................................................................................573.2.2 Generation of BERP-deficient mice................................................................................573.2.3 Genotyping of BERP-deficient mice by PCR and Southern Blotting.....................................583.2.4 Generation and genotyping of BERP-/-;p53-/- mice ..........................................................593.2.5 Histological analysis ..................................................................................................593.2.6 Western blot analysis..................................................................................................603.2.7 Behavioural characterization .......................................................................................603.2.8 Pentylenetetrazol seizure susceptibility testing.................................................................643.2.9 Hemi-brain slice preparation .......................................................................................653.2.10 Electrophysiology...................................................................................................663.2.11 Murine cortical neuron cultures................................................................................663.2.12 Biotinylation Assay ................................................................................................673.2.13 Real time RT-PCR..................................................................................................683.2.14 Neurite Outgrowth Assay ........................................................................................683.2.15 Neurosphere formation and differentiation assays .......................................................693.2.16 Neurosphere differentiation assay.............................................................................703.2.17 Thymidine incorporation assay.................................................................................71

3.3 RESULTS.......................................................................................................................723.3.1 Generation of BERP-deficient mice................................................................................723.3.2 Behavioural characterization of BERP-/- mice.................................................................77

3.3.2.1 SHIRPA Neurological Screen in BERP-deficient mice .............................................................................773.3.2.2 BERP-/- mice exhibit increased freezing in fear conditioning tests ....................................................803.3.2.3 BERP-/- mice exhibit abnormalities in the open field test for exploration and generallocomotion; however, no effect of BERP is seen in the elevated plus maze test ......................................................843.3.2.4 BERP-/- mice exhibit abnormalities in the accelerating rotarod test...................................................923.3.2.5 No effect of BERP in tests for sensorimotor gating ..................................................................................953.3.2.6 No effect of BERP in tail suspension test (depression-related behaviour). .......................................983.3.2.7 No effect of BERP in grip test (neuromuscular function)......................................................................1013.3.2.8 No effect of BERP in tail flick test (nociception) ....................................................................................101

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3.3.3 Increased resistance to PTZ-induced seizures in Berp-/- mice........................................... 1013.3.4 The amplitude of mIPSCs is decreased in BERP-/- mice.................................................. 1053.3.5 Decreased surface expression of GABAAR gamma2 subunit in BERP-/- mice ...................... 1083.3.6 GABA A receptor gamma2 subunit mRNA levels are unchanged in BERP-/- mice................. 1113.3.7 Cortical neurons from BERP-/- embryos show an increase in total neurite length ............... 1113.3.8 BERP-deficiency has no impact on the self-renewal, differentiation, proliferation, viability andsize of murine neural stem cells .............................................................................................. 114

4 DISCUSSION.................................................................................................................... 118

4.1 WHAT IS THE NEUROLOGICAL PHENOTYPE OF BERP-DEFICIENT MICE?.................................... 1184.2 INVOKING OCCAM'S RAZOR: THE BERP-DEFICIENT MOUSE AS A MODEL FOR NON-ATAXICCEREBELLAR DYSFUNCTION?....................................................................................................... 1224.3 WHAT IS THE POSSIBLE ROLE OF BERP IN THE CNS?........................................................... 1274.4 IS BERP INVOLVED IN NEURITE OUTGROWTH?.................................................................. 1284.5 IS BERP A FUNCTIONAL HOMOLOG OF D. MELANOGASTER BRAT? ........................................... 1294.6 WHY DO KNOCKOUT MICE FROM THE TRIM-NHL FAMILY NOT DEVELOP BRAIN TUMOURS? ........ 1314.7 THE P53/BERP /GABAAR RELATIONSHIP - A NOVEL ROLE FOR P53 IN THE BRAIN?................. 1334.8 PTZ – A NOVEL THERAPY IN THE BATTLE AGAINST CANCER? ............................................... 1344.9 CLOSING REMARKS ...................................................................................................... 136

5 REFERENCES.................................................................................................................. 137

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List of Abbreviations

A322D alanine to aspartic acid at position 322A adenineABP actin binding proteinACSF artificial cerebral spinal fluidAMPA alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionateAP2 adapter protein 2ASR acoustic startle reflexATP adenosine triphosphateATM ataxia telangiectasia mutatedB2m beta-2-microglobulinBax bcl-2-associateed protein XBERP brain expressed ring finger proteinBLASTP basic local alignment search tool for proteinsbp base pairsBRH best reciprocal hitBWS Beckwith-Wiedemann syndromeCaCl2 calcium chlorideCART cytoskeleton-asssociated recycling or transportCCAS cerebellar cognitive affective syndromeC cytosinecDNA complementary deoxyribonucleic acidCHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonateChIP chromatin immunoprecipitationChk2 checkpoint kinase 2cm centimetreCMHD Centre for Modeling Human DiseaseCNQX 6-cyano-7-nitroquinoxaline-2,3-dioneCNS central nervous systemCO2 carbon dioxideCsCl cesium chlorideCS conditioned stimulusCsOH cesium hydroxideD-APV D-2- amino-5-phosphonovaleratedB decibelDIC differential interference contrastDMEM Dulbecco's modified Eagle culture medium

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DMSO dimethyl sulfoxideDNA deoxyribonucleic aciddp53 D. melangaster p53DVAMC Department of Veterans Affairs Medical CenterEGF epidermal growth factorEGTA ethylene glycol tetraacetic acidEPSP excitatory postsynaptic potentialERAD endoplasmic reticulum-associated degradationER endoplasmic reticulumES embryonic stemGABAAR GABA A receptorGABABR GABA B receptorGABACR GABA C receptorGABA gamma amino butyric acidGABARAP GABAAR-associated proteinGabrg2 GABA A receptor gamma2 subunitGAD glutamic acid decarboxylaseGADD45 growth arrest and DNA damage-inducible protein 45GAPDH glyceraldehyde-3-phosphate dehydrogenaseGAT GABA transporterGDP guanosine diphosphateGFAP glial fibrillary acid proteinG guanineGMR glass multimer reporterGPCR G-protein-coupled receptorGTP guanosine triphospateHBSS Hank's balanced salt solutionH&E hematoxylin and eosinH&E/LFB hematoxylin and eosin with luxol fast blueHEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acidHIV human immunodeficiency virusHprt1 hypoxanthine phosphoribosyltransferase 1Hsp53 H. sapiens p53Hz hertzIAP inhibitor of apoptosisIPSP inhibitory postsynaptic potentialK289M lysine to methionine mutation at position 289K2ATP adenosine 5’-triphosphate dipotassium salt

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KCl potassium chloridekDA kilodaltonkg kilogramK+ potassiumLOH loss of heterozygosityLTD long-term depressionLTP long-term potentiationmA milliampMAP2 microtubule-associated protein 2mCi millicurieMdm2 mouse double minute 2MgCl2 magnesium chloridemg milligrammIPSC miniature inhibitory postsynaptic currentmL millilitremm millimetremM millimolarmOsm milliosmolmRNA messenger ribonucleic acidmV millivoltNa3VO4 sodium orthovanadateNaCl sodium chlorideNaF sodium flourideNaH2PO4 sodium dihydrogenphosphateNaHCO3 sodium bicarbonateNARF neural activity-related RING finger proteinNa+ sodiumNCBI National Center for Biotechnology InformationNHL ncl-1, HT2A, lin41NMDA N-methyl-D-aspartic acidO2 oxygenp53RE p53 response elementPAC P1-derived artificial chromosomepA pulse amplitudePBS phosphate buffered salinePBS phosphate buffered salinePCR polymerase chain reactionPKA protein kinase A

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PKC protein kinase CPNS peripheral nervous systemPP1alpha protein phosphatase 1 alphaPPIA peptidylprolyl isomerase APPI prepulse inhibitionPRIP phospholipase-C-related catalytically inactive proteinPTZ pentylenetetrazolPu purinePy pyrimidineQ351X glutamine to unspecified amino acid at position 351R43Q arginine to glutamine mutation at position 43RBCC RING finger, b-box, coiled-coilRING really interesting new geneRNA ribonucleic acidrpm revolutions per minuteSEM standard error of the meanSFM serum free mediaSHIRPA Smithkline Beecham, MRC Harwell, Imperial College, the Royal London hospital Phenotype AssessmentSVZ sub-ventricular zoneTBP TATA binding proteinTLE temporal lobe epilepsyTRIM tripartite motifTris 2-amino-2-(hydroxymethyl)-1,3-propanediolT thymineTTX tetrodotoxinUAS upstream activating sequenceuM micromolarUS unconditioned stimulusUTP uridine triphosphateVGAT vesicular neurotransmitter transporterv/v volume to volume5FU 5-fluorouracil

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List of Figures

Chapter 1Figure 1 Structure and Functions of p53…………………………..………………. 8Figure 2 Mammalian homologs of brat…………………………………………… 14Figure 3 Anatomy of the neuron……………………………………………………20Figure 4 The GABA A Receptor……………………………………………….…..28Chapter 2Figure 5 Predicted p53REs within the BERP genomic locus……………………… 46Figure 6 In vivo binding and transcriptional competence of predicted BERP p53REs…………………………………………………………… 49Figure 7 BERP expression in HCT116 p53-/- and HCT116 p53+/+ cells………… 52Figure 8 BERP expression in p53-/- and p53+/+ mice…………………………….. 55Chapter 3Figure 9 Targeted disruption of the murine BERP locus…………………….…….. 74Figure 10 General phenotypic assessment of BERP-deficient mice…………………76Figure 11 SHIRPA neurological assessment for body weight in BERP-deficient mice…………………………………………………... 79Figure 12 Fear conditioning in BERP-deficient mice……………………………….. 83Figure 13 Open field exploration in BERP-deficient mice………………………….. 87Figure 14 Elevated plus maze test in BERP-deficient mice………………………….89Figure 15 General locomotor activity in BERP-deficient mice……………………... 91Figure 16 Accelerating rotarod test in BERP-deficient mice………………………... 94Figure 17 Prepulse inhibition of the acoustic startle response in BERP-deficient mice…………………………………………………... 97Figure 18 Tail suspension test in BERP-deficient mice……………………………...100Figure 19 PTZ seizure susceptibility testing in BERP-deficient mice……………….104Figure 20 Electrophysiological measurements in BERP-deficient mice…………….. 107Figure 21 GABA A receptor expression in BERP-deficient mice…………………...110Figure 22 Neurite outgrowth measurements in BERP-deficient mice………………. 113Figure 23 Analysis of neural stem cells in BERP-deficient and p53-deficient mice... 117Chapter 4Figure 24 Neuroanatomical structures………………………………………………..125

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1 Introduction

1.1 Opening Remarks

BERP, p53, GABA - three apparent strangers in an incredibly small yet infinitely complex

domain known as the cell. Now trapped together in the same thesis, their confusion is

understandable. However, in the realm of scientific endeavour anything is possible. And

soon, these strange bedfellows will realize they are headed for a collision course and that the

world as they know it will change forever. So read on, for in this study of BERP, we will

venture from the fruit fly to the field mouse and encounter famous names such as 5FU and

PTZ. The remainder of chapter 1 is a review of the background information necessary to

comprehend the scientific foundations upon which this work is based. Chapter 2 is an

examination of the evidence to determine whether BERP will be admitted as a new member

into the esteemed club of p53-regulated genes. In chapter 3, the role of BERP in the

mammalian central nervous system and its possible relationship to the GABAergic signaling

pathway is examined. Chapter 4 involves a discussion that will integrate the new knowledge

gained from chapters 2 and 3 with the foundations presented in chapter 1. And if BERP is

indeed a novel p53-regulated gene, then its functions may point to an entirely new and

previously unreported role for one of the most famous molecules in present-day biomedical

research.

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1.2 p53 - Molecule of the Year (1993, Science Magazine)

1.2.1 p53 – The protein

The human p53 gene (TP53) is located on chromosome 17p13.1. It contains 11 exons and

encodes a protein that is 393 amino acids in length and has a number of highly conserved

functional domains (Figure 1A) (Olsson et al. 2007). Located at the N-terminal region is the

transactivation domain, which is required for transcriptional regulation of p53-target genes.

This is followed by a proline-rich domain, which is involved in protein-protein interactions.

The core DNA binding domain is located at the centre of the protein and is required for the

recognition and binding of p53 to specific p53-response elements (p53REs). The significance

of this domain is evident given the fact that the vast majority of mutations in human cancers

are found within this region (Joerger and Fersht 2007). The oligomerization domain is

required for the oligomerization of p53; this is important since p53 performs its in vivo

functions as a tetramer. In addition, a nuclear export signal is located in this domain. Finally,

there is the C-terminal domain that regulates the binding of p53 to damaged DNA and also

houses nuclear localization signals.

After translation, p53 remains in the cytoplasm during the G1 phase of the cell cycle. It

translocates to the nucleus for S phase and then returns to the cytoplasm. In unstressed cells,

the half-life of p53 is very short (ie. 20 minutes) due to the fact that it undergoes rapid

degradation. Mdm2, an E3 ubiquitin ligase, is a major negative regulator of p53. It can control

cellular p53 levels in two ways: i) by binding to and partially obscuring the transactivation

domain of p53, thus impairing p53 transactivation functions, and more importantly, ii) by

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targeting p53 for degradation via the ubiquitin-proteasome pathway (Momand et al. 1992;

Kubbutat et al. 1997; Daujat et al. 2001). Therefore, disrupting the interaction between

Mdm2 and p53 results in the stabilization and subsequent increase in cellular p53 levels. One

way that this occurs is by alteration of the p53 protein via various posttranslational

modifications (Figure 1A). For example, in response to gamma irradiation-induced DNA

damage, activation of ATM leads to phosphorylation of amino acid residue serine-15 (and

also of serine-20 via ATM activation of checkpoint kinase 2) in the N-terminus of human

p53. These and other modifications to p53 interfere with its binding to Mdm2, thus

increasing its stability and half-life. Another example illustrating the importance of Mdm2 in

maintaining low levels of p53 can be found after oncogene activation, which results in the

upregulation and activation of ARF. ARF has the ability to bind and inhibit the ubiquitin

ligase activity of Mdm2, thus resulting in stabilization of p53 (Levine et al. 2006).

1.2.2 p53 and Cancer

In humans, known aberrations of p53 manifest primarily as increased cancer susceptibility.

TP53 is the single most common target for genetic alteration in human cancers. Somatic

mutations of TP53 are present in half of all human malignancies. Of these mutations, 95%

occur in the DNA binding domain and 75% are single missense mutations (Hollstein et al.

1991; Hainaut et al. 1998; Hollstein et al. 1999; Hainaut and Hollstein 2000). Germline

mutations of TP53 results in the Li-Fraumeni syndrome characterized by a 25-fold increased

risk of developing one or more malignancies by age 50; the spectrum of tumours seen in these

patients are variable and include breast carcinoma, adreno-cortical carcinoma, brain neoplasms,

sarcomas, and leukemia (Evans and Lozano 1997; Kleihues et al. 1997). Many of the

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remaining tumours that retain wild-type p53, have defects in their ability to activate p53. In

addition to its role in the development and progression of cancer, p53 status also influences

the tumour’s ability to evade treatment. The therapeutic effects of chemotherapy and

radiotherapy are mediated by their ability to cause DNA damage, which then in turn triggers

p53-dependent apoptosis. Therefore, tumours that exhibit loss of p53 functions will be more

resistant to such treatment (Igney and Krammer 2002).

1.2.3 p53-deficient mice

p53-deficient mice have been generated and exhibit high rates of spontaneous tumour

development (Donehower et al. 1992; Harvey et al. 1993; Jacks et al. 1994). Approximately

75% of p53-/- mice developed tumours by 6 months. By 9-10 months of age, all p53-null

mice either developed tumours or had perished. The most common tumour types were

lymphomas (lymphoid tissue origin) and sarcomas (mesenchymal tissue origin); occasional

carcinomas (epithelial tissue origin) also developed. Similar to p53-/- mice, p53+/- mice are

also prone to tumour formation but with increased latency (9-18 months of age) and a slightly

different frequency of tumour type (sarcomas were most common, followed by lymphoma).

Furthermore, p53-/- mouse embryonic fibroblasts exhibit many characteristics of cancer cells

such as anchorage independence, resistance to apoptotic stimuli, accelerated growth, and

abnormal cell cycle profiles (Harvey et al. 1993; Lowe et al. 1994).

1.2.4 p53 - The Transcription Factor

The major function of p53 lies in its ability to regulate the transcription of target genes. In

order to do this, p53 must recognize and bind, via its central DNA binding domain, specific

5

DNA segments located within or near its target genes. These specific DNA segments, called

p53 response elements (p53REs), consist of two decamers (each of which is referred to as a

"half site") separated by a spacer that can range from 0-13 nucleotides. The p53RE

consensus sequence has the following structure: 5'-PuPuPuC(A/T)(T/A)GPyPyPy(N)0-

13PuPuPuC(A/T)(T/A)GPyPyPy-3', where Pu = purine, Py = pyrimidine, N = any

nucleotide (el-Deiry et al. 1992). In trying to predict the strength of a potential p53RE

identified by sequence analysis, several factors may serve as guides. The presence of 1) the

"C" and "G" at positions 4 and 7 of each half site, 2) a spacer that has either 0 nucleotides or

1 nucleotide, 3) no more than 3 mismatches to the consensus sequence, all suggest a likelihood

that the putative p53RE is functional (Resnick et al. 2005; Tomso et al. 2005).

1.2.5 The p53 Network

Based on a recent review by Levine et al. (Levine et al. 2006), the p53 pathway can be

divided into 5 parts: 1) triggers (eg. DNA damage, hypoxia, oncogene expression) that are

detected by 2) upstream mediators (eg. ATM) which act on either the p53 protein itself or

one of its 3) core regulators (eg. MDM2) in order to stabilize p53 and modulate its function;

this most often involves posttranslational modifications of the p53 protein. Activated p53

then influences 4) downstream mediators (eg. p21) that ultimately leads to various 5) cellular

responses (eg. cell cycle arrest).

Triggers that activate the p53 pathway include a myriad of insults such as DNA damage,

hypoxia and oncogene expression. However, knowing the entire contents of the extensive

trigger list is less important than realizing that all of the triggers have in common their ability

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to cause stress to an otherwise unstressed cell. Therefore, despite the already impressive

bestowed title of "guardian of the genome" (Lane 1992), it seems that p53 can also more

expansively be described as "guardian of genetic and metabolic homeostasis". To date, many

molecules in the p53 network have been identified. For example, MDM2, which functions as

an E3 ubiquitin ligase, is a major negative regulator of p53 levels in the cell (Momand et al.

1992; Kubbutat et al. 1997; Daujat et al. 2001). Other target genes that are transcriptionally

activated by p53 include p21 (cell cycle arrest), GADD45 (DNA repair), and Bax (apoptosis)

(Vogelstein et al. 2000; Vousden and Lu 2002). It has been shown that the promoter of

PTEN, another important tumour suppressor gene, contains a p53 binding element and that

p53 can activate transcription of PTEN (Stambolic et al. 2001). Cellular responses to p53

activation involve cell cycle arrest, apoptosis, senescence, DNA repair, angiogenesis, exosome

mediated secretion, and the IGF-1/mTOR pathway. However, despite these and other

advances, our understanding of the role of the p53 network in human disease development

and progression is far from complete. In fact, this multifunctional protein's involvement in

such a wide variety of cellular processes underscores the importance of understanding the true

nature and scope of its functions (Figure 1B).

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Figure 1. Structure and Functions of p53. (A) Schematic representation of the p53

protein. TAD, transactivation domain. PRD, proline-rich domain. DBD, DNA-binding

domain. OD, oligomerization domain. CTD, C-terminal domain. Relative frequencies and

positions of mutational hotspots are indicated above the protein. Domains affected by

various posttranslational modifications are idicated below the protein. Based on Olsson et al.,

2007; Joerger and Fersht, 2007. (B) Examples of triggers (orange), upstream mediators

(yellow), core regulators and downstream mediators (gray), and cellular responses (green) of

the p53 network. Please refer to text for details. Based on Levine et al., 2006.

8

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1.2.6 In search of novel p53 targets: In vivo genetic screen usingDrosophila melanogaster

Studies of homologous molecules in the p53 pathways have shown that apoptotic pathways

are well conserved from D. melanogaster to mammals. Drosophila p53 (dp53), the D.

melanogaster homolog of human p53 (hp53), has recently been cloned and characterized (Jin

et al. 2000; Ollmann et al. 2000). There is significant structural homology between the two

molecules in the important DNA binding domain, dp53 can bind hp53 consensus binding

sites, and overexpression of dp53 can induce apoptosis. In addition, ectopic expression of

hp53 in the D. melanogaster eye has previously been shown to inhibit entry into S-phase and

to induce apoptosis (Yamaguchi et al. 1999). These data indicate that p53 pathways are

conserved from flies to mammals and that D. melanogaster provides a simple and powerful

system in which to further study the molecules involved in the p53 network.

Therefore, in order to identify novel molecules within the p53 signaling pathway, we have

utilized an in vivo genetic screen using a binary vector system in D. melanogaster (Brand and

Perrimon 1993). The screen takes advantage of the fact that overexpression of dp53 in the D.

melanogaster eye imaginal disc results in a small rough eye phenotype. The first part of this

binary system involved the creation of a vector that contains the Gal4-dependent upstream

activating sequence (UAS) promoter driving the expression of a dp53 cDNA. This UAS-dp53

vector was then used to make a transgenic fly in which every cell has the potential to express

dp53, if a source of Gal4 is present. For the second part of the binary vector system, the eye-

specific glass multimer reporter (GMR) promoter was used to drive the expression of Gal4

only in the fly eye. Mating GMR-Gal4 transgenic flies with UAS-dp53 transgenic flies

resulted in progeny that express dp53 specifically in the fly eye. In effect then, these flies

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contain a dp53 transgene driven by the GMR promoter. GMR-Gal4-dp53 flies have smaller,

rougher eyes when compared with wild-type flies. This eye phenotype is mainly due to

increased apoptosis (Ollmann et al. 2000). To identify genetic modifiers of the dp53

pathway, both the GMR-Gal4-dp53 and GMR-Gal4-hp53 flies (which also yields a small

rough eye phenotype) were crossed with mutant fly strains each containing one of 1150

distinct P-element insertions which disrupts one copy of a vital D. melanogaster gene. The

progeny of these crossings were collected and analyzed according to phenotypic changes in

eye size. Progeny flies with a larger eye phenotype than those of GMR-Gal4-dp53 flies were

scored as phenotypic suppressors of dp53. Progeny flies with smaller eyes than those of the

GMR-Gal4-dp53 flies were scored as phenotypic enhancers of dp53. Progeny flies exhibiting

no phenotypic change were scored as non-interacters. There are several advantages in

conducting in vivo genetic screens using D. melanogaster. Firstly, a functional screen allows

for the identification of genes important for the observed phenotype irrespective of their

location within any given signaling pathway. For example, a physical interaction is not

necessarily required. Secondly, the relatively short generation time of D. melanogaster

permits the analysis of a large number of mutants. Lastly, genomic information on D.

melanogaster is readily available through public databases and hence allows for timely

identification of P-element inactivated genes.

11

1.3 Brain Expressed RING finger Protein (BERP)

1.3.1 D. melanogaster Brain Tumour (brat) suppresses the eye phenotypeof dp53

Based on results of the D. melanogaster genetic screen, brain tumour (brat) was identified as a

suppressor of the p53 eye phenotype. Brat is a D. melanogaster tumour suppressor gene

first identified in 1976 during a screen for lethal mutations (Wright et al. 1976; Wright et al.

1981). The brat gene, first cloned by Arama et al., encodes a 1037 amino acid protein

consisting of two B-box domains, a coiled-coil domain, and a C-terminal beta-propeller

domain composed of NHL repeats; these domains have all been implicated in protein-protein

interactions. In the larval brain, brat is a negative regulator of cell growth. Recessive loss of

function mutations of brat result in greatly enlarged brain hemispheres (due to neoplastic

proliferation of optic neuroblasts) and lethality during the third instar larval and pupal stages

(Arama et al. 2000). More recently, it has been shown that brat inhibits self-renewal of larval

neuroblasts (Betschinger et al. 2006). In the early embryo, brat appears to be a translational

repressor of hunchback mRNA via its interactions with nanos and pumilio (Sonoda and

Wharton 2001). In addition, brat has been shown to be a negative regulator of cell growth and

ribosomal synthesis (Frank et al. 2002).

1.3.2 Mammalian homologs of D. melanogaster brat

Mammalian brat homologs contain a N-terminal RING finger, a B-box, a coiled coil, and a C-

terminal domain composed of NHL repeats (Figure 2); the relative order of the domains

remains the same as that for brat (Arama et al. 2000). Three mammalian molecules fit this

12

pattern: BERP/TRIM3, NARF/TRIM2, HT2A/TRIM32. They all belong to the TRIM

(TRIpartite Motif) family of proteins which consists of 37 members and are defined by the

presence of an N-terminal RING finger, followed by one or two B-boxes, and a coiled-coil

domain; the C-terminus is variable (Reymond et al. 2001). TRIM proteins have diverse

functions ranging from development and cell growth to oncogenesis. Reymond et al. (2001)

showed that 1) through their coiled coil regions, TRIM proteins are able to form homo and

hetero-oligomers, and 2) the RING finger and B-box(es) are important for proper subcellular

localization of the TRIM proteins. The three putative mammalian homologs of brat are

TRIM proteins that have a C-terminal beta-propeller domain composed of NHL repeats.

13

Figure 2. Mammalian homologs of brat. Schematic representation of the brat protein and

its mammalian homologs: BERP/TRIM3, NARF/TRIM2, HT2A/TRIM32. Hs, Homo

sapiens. Dm, Drosophila melanogaster. Red, RING finger domain. Dark blue, B-box 1

domain. Light blue, B-box 2 domain. Green, coiled-coil domain. Yellow, NHL repeats

domain. Based on Arama et al., 2000 and Reymond et at., 2001.

14

15

1.3.2.1 BERP/TRIM3

BERP (Brain Expressed Ring finger Protein), was first cloned and characterized in the rat by

El-Husseini et al. (El-Husseini and Vincent 1999; El-Husseini et al. 2000). It is most strongly

expressed in the brain, especially in the cerebellum. Other sites of expression include heart,

breast, lung, pancreas, kidney, and liver. Its expression in the murine brain is upregulated

following chemically induced seizures (Ohkawa et al. 2001). The BERP protein is 744 amino

acids in length and consists of an N-teminal RBCC domain (RING finger, b-box, coiled-coil), a

central ABP-like domain, and a C-terminal beta-propeller domain. In response to nerve

growth factor, PC12 cells overexpressing a BERP truncation mutant lacking the NHL beta-

propeller domain fail to stop proliferation and do not exhibit neurite outgrowth (El-Husseini

and Vincent 1999). Yeast-two-hybrid and immunoprecipitation experiments indicate that

BERP interacts with two proteins of the actin cytoskeleton, alpha-actinin-4 and myosin V,

via its N-terminal and C-terminal domains respectively (El-Husseini and Vincent 1999; El-

Husseini et al. 2000). Alpha-actinin-4, an isoform of the actin binding protein alpha-actinin,

has been implicated in cell motility and cancer (Honda et al. 1998; Nikolopoulos et al. 2000).

Class V myosins are implicated in organelle trafficking and cell motility (Provance and Mercer

1999; Yan et al. 2005; Wang et al. 2008). BERP has been identified as a member of the

cytoskeleton-associated recycling or transport (CART) complex along with the endosome

associated protein hrs, alpha-actinin-4, and myosin V. In this complex, which localizes to the

early endosome, the proteins are bound to each other in a linear fashion: hrs binds to alpha-

actinin-4, which binds to BERP, which binds to myosin V. The authors show that the CART

complex is involved in the constitutive recycling of the transferrin receptor and that this

process utilizes the actin cytoskeleton (Yan et al. 2005). Large-scale phosphoproteome

16

characterizations have identified BERP as a possible phosphorylated protein in epidermal

growth factor (EGF)-stimulated HeLa cells (Olsen et al. 2006) and in preparations isolated

from murine postsynaptic densities (Trinidad et al. 2006).

BERP is well conserved in rat, mouse and human. Rat BERP shares 99% identity with

murine BERP and 98% with human BERP (Arama et al. 2000; El-Husseini et al. 2001). As of

this time, no studies involving murine BERP have been published. In humans, BERP, also

known as RNF22 (El-Husseini et al. 2001) or TRIM3 (Reymond et al. 2001), maps to

chromosome 11p15.5, a region that has been named “multiple tumour-associated region-1”

because deletions of this region can be found in many human malignancies including those of

the brain, breast, bladder, pancreas, testes, liver, and striated muscle (El-Husseini et al., 2001).

Interestingly, the Beckwith-Wiedemann syndrome (BWS) also maps to 11p15.5. BWS is a

congenital overgrowth syndrome with an incidence of approximately 1:15000. Clinical

manifestations are variable and include pre and post-natal somatic overgrowth, organomegaly,

hemihypertrophy, abdominal wall defects, and a predisposition to childhood malignancies

(Steenman et al. 2000). In a recent shotgun proteomics analysis, BERP was one of the

proteins shown to be up-regulated in the dorsolateral prefrontal cortex of schizophrenic

patients (Martins-de-Souza et al. 2009). In addition, Boulay et al. recently identified BERP

(as TRIM3) as the candidate tumour suppressor gene at the 11p15.5 locus after examining 70

human gliomas for loss of heterozygosity (Boulay et al. 2009).

1.3.2.2 NARF/TRIM2

NARF (Neural Activity-related RING Finger protein; also known as TRIM2), a second

mouse homolog of rat BERP has been cloned and characterized (Ohkawa et al. 2001).

17

Ohkawa et al. chemically induced seizures in mice and then used a PCR-based cDNA

subtraction method in order to identify genes whose expressions were upregulated in the

hippocampus. NARF and BERP are closely related (67% identity) and contain the same

protein domains. NARF is reported to be predominantly expressed in the brain, especially

the hippocampus. Similar to BERP, NARF also binds to myosin V via its beta-propeller

domain. Balastik et al. recently reported that NARF (as TRIM2) is an ubiquitin ligase and

that TRIM2-deficient mice develop tremor and ataxia beginning at 4 months with subsequent

spontaneous seizures and that this is due to neurodegeneration secondary to axonal

accumulation of neurofilament light chain (Balastik et al. 2008).

1.3.2.3 HT2A/TRIM32

HT2A/TRIM32 was first identified as a novel human protein that interacts with the Tat

proteins of HIV-1 and HIV-2 (Fridell et al. 1995). It is an E3 ubiquitin ligase (Horn et al.

2004) that can ubiquitinate actin (Kudryashova et al. 2005) and is highly expressed in skeletal

muscle. Mutations of TRIM32 have been identified several human conditions: limb-girdle

muscular dystrophy type 2H (Frosk et al. 2002), sarcotubular myopathy (Schoser et al.

2005) and Bardet-Biedl syndrome (Chiang et al. 2006). It is also highly expressed in

squamous cell carcinomas and has proposed oncogenic functions via its ability to mediate the

ubiquitination and degradation of the tumour suppressor Abl-interacter 2 (Kano et al. 2008).

The phenotype of TRIM32-deficient mouse confirms its utility as a model for limb-girdle

muscular dystrophy type 2H and sarcotubular myopathy (Kudryashova et al. 2009).

Recently, it has been shown that TRIM32 is a positive regulator of microRNAs and that it is

a negative regulator of murine neural stem cell renewal (Schwamborn et al. 2009).

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1.4 Overview of Neuronal Function in the Mammalian CNS

1.4.1 Anatomy of the Neuron

Neurons account for approximately 10% of the cells present in the mammalian CNS. The

main function of neurons is to transmit information. The neuron is composed of a cell body

from which radiates a number of neurites (Figure 3). The cell body contains the nucleus along

with other organelles typically found in non-neuronal cells such as mitochondria, endoplasmic

reticulum, and Golgi apparatus. Neurites, specialized structures that carry information in the

form of electrical impulses towards and away from the neuronal cell body, are divided into

dendrites and axons. The axon begins at the axon hillock next to the cell body and ends some

distance away at the axon terminal. The axon transmits nerve impulses in a unidirectional

fashion from the hillock towards the axon terminal. The nerve impulse is then transmitted

from the axon terminal of the originating neuron to a dendrite or cell body of the target neuron

via a specialized inter-neuronal communication structure called the synapse. (Bear et al. 2001;

Purves et al. 2004)

19

Figure 3. Anatomy of the neuron. Please refer to text for details. Adapted from www.web-

books.com/Free/Images/Synapse.jpg.

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21

1.4.2 Neuronal membrane potentials

1.4.2.1 Resting potential

The resting membrane potential in mammalian neurons is always negative and depending on

the species, usually ranges from about -40mV to -90mV; in humans, it is -70mV. The resting

potential is maintained by membrane pumps, such as the Na+K+ATPase pump, which cause

an imbalance of ions across the plasma membrane. This imbalance results in a potential

difference (voltage) between the inner and outer surfaces of the cell membrane. (Bear et al.

2001; Purves et al. 2004)

1.4.2.2 Action potential

The action potential is a brief reversal of the membrane potential that results in the

propagation of a nerve impulse along the axon of a neuron. The two phases of an action

potential are depolarization and repolarization. Depolarization occurs when there is enough

of a change in the membrane potential such that it reaches the threshold value of 15-20mV

depolarized from membrane potential. If threshold is reached, then voltage-gated Na+

channels in that area of the membrane open, allowing Na+ to flood into the cell.

Depolarization ends when the Na+ channels close. During repolarization, K+ channels open

so that K+ can leave the cell; the membrane potential returns to -70mV. The resting

concentrations of Na+ and K+ ions are then restored by membrane pumps. (Bear et al. 2001;

Purves et al. 2004)

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1.4.3 Overview of the synapse

The synapse is a specialized communication junction that allows neurons to pass information

to other cells. There are several different types of synapses including electrical synapses in

which cells are physically connected via gap junctions, chemical synapses in which electrical

impulses from one cell is transferred to another cell via chemicals messengers across a small

space separating the cells, and neuromuscular junctions which are chemical synapses found in

the PNS between motor neurons and muscle cells. For the purposes of this thesis, only the

classical chemical synapse occurring between two neurons within the CNS will be considered.

There are three basic components to the classical chemical synapse: the presynaptic element,

the synaptic cleft, and the postsynaptic density. The presynaptic axon terminal contains

mitochondria and vesicles, which contain neurotransmitters. In response to upstream

electrical signals, synaptic vesicles fuse with the pre-synaptic membrane and release

neurotransmitter molecules into the synaptic cleft. The neurotransmitter molecules then

diffuse across the synaptic cleft where they bind to and activate neurotransmitter receptors

located on the post-synaptic membrane. (Bear et al. 2001; Purves et al. 2004)

1.4.4 Overview of general neuroreceptor functions

There are two basic types of neuroreceptors: ionotropic neurotransmitter-gated ion channels

and metabotropic G-protein-coupled receptors (GPCRs). Neurotransmitter-gated ion

channels are composed of multiple polypeptide units that come together to form a

transmembrane pore. When the receptor is bound by its neurotransmitter, it undergoes a

conformational change that allows the passage of ions through its central pore. The functional

effects of activating neurotransmitter-gated ion channels are dependent on which ions are

23

involved. Generally, if a Na+ channel is involved, then the net effect will be excitatory due to

a local depolarization of the postsynaptic membrane. In other words, activation of a

neurotransmitter-gated Na+ channel results in the generation of an excitatory postsynaptic

potential (EPSP). If a Cl- channel is involved, then the net effect will be inhibitory, due to a

local hyperpolarization of the postsynaptic membrane. Similar to the above example,

activation of a neurotransmitter-gated Cl- channel results in the generation of an inhibitory

postsynaptic potential (IPSP). If the sum of all the EPSPs and IPSPs results in a net

excitatory effect, then an action potential is generated; if the net effect is inhibitory, then no

action potential is generated. While ion channels mediate fast synaptic transmission, GPCRs

mediate slower synaptic transmission with more diverse and longer lasting postsynaptic

effects. Upon binding of neurotransmitter molecules, the receptor activates G-proteins

within the membranes, which in turn activates effector proteins such as G-protein-gated ion

channels and second messengers. (Bear et al. 2001; Purves et al. 2004)

1.4.5 Overview of Neurotransmitter Systems

Neurotransmitters can be classified based on biochemical composition into three categories:

(1) amino acids, (2) amines, and (3) peptides. Amino acid and amine neurotransmitters are

small molecules that are released from synaptic vesicles; peptide neurotransmitters are larger

molecules that are released from secretory granules. Amino acid neurotransmitters include

gamma-amino butyric acid (GABA), glutamate, and glycine. Amine neurotransmitters include

acetylcholine, dopamine, epinephrine, histamine, norepinephrine, and serotonin. Peptide

neurotransmitters include cholecystokinin, dynorphin, enkephalins, N-

acetylaspartylglutamate, neuropeptide Y, somatostatin, substance P, thyrotropin-releasing

24

hormone, vasoactive intestinal polypeptide. Within the CNS, amino acid neurotransmitters

mediate the majority of fast synaptic transmissions. Generally speaking, glutamate and

GABA are considered to be the major excitatory and inhibitory neurotransmitters in the

mature mammalian CNS, respectively. (Bear et al. 2001; Purves et al. 2004)

1.5 GABAA Receptor signaling within the Mammalian CNS

1.5.1 GABA - The Neurotransmitter

GABA, present in approximately 15-20% of neurons, is the primary inhibitory

neurotransmitter in the mature mammalian CNS. It is synthesized in presynaptic neurons

from glutamate using the enzyme glutamic acid decarboxylase (GAD). There are two

different isoforms of GAD: GAD65 and GAD67 (Erlander et al. 1991). The gene for GAD65

is located on human chromosome 2 and encodes a 65 kDa isoform of the protein, most of

which is in the inactive apo-GAD65 form. Therefore, although GAD65 comprises the

majority of GAD present in the brain, it synthesizes only about 30% of the brain's GABA.

In GAD65-deficient mice, levels of apo-GAD65 were lower compared to wildtype controls;

however, levels of active holo-GAD65 and GABA were unchanged in the CNS. In addition,

they had increased susceptibility for developing spontaneous seizures (Asada et al. 1996).

The gene for GAD67 is located on human chromosome 10 and encodes a 67kDa isoform of

GAD. Unlike GAD65, most of the GAD67 in the brain is in the active holo-GAD67 form

and synthesizes approximately 70% of the brain's GABA. GAD67-deficient mice have

severe cleft palate and die shortly after birth (Asada et al. 1997). Mice deficient for both

GAD65 and GAD67 contain 0.02% of normal GABA levels. They have a similar phenotype

25

to the GAD67 knockout mice in that they die shortly after birth from severe cleft palate and

show no gross or microscopic abnormalities in brain development (Ji et al. 1999).

Once synthesized, GABA is loaded into synaptic vesicles by the vesicular neurotransmitter

transporter VGAT (Cherubini and Conti 2001). Release of GABA from synaptic vesicles

into the synaptic cleft is mediated by calcium-dependent exocytosis. Re-uptake of GABA

into the axon terminal and surrounding glial cells occurs via GABA transporters (GATs)

(Schousboe et al. 2004). GABA transaminase metabolizes GABA into succinic

semialdehyde, which is then metabolized into succinic acid by succinic semialdehyde

dehydrogenase or into gamma-hydroxybutyric acid by succinic semialdehyde reductase

(Wong et al. 2003).

1.5.2 GABA Receptor Family

There are 3 classes of GABA receptors: the ionotropic GABA A and GABA C receptors

(GABAARs and GABACRs, respectively), which are ligand-gated chloride channels, and the

metabotropic G-protein-coupled GABA B receptors (GABABRs). Although the focus will

be on GABAARs, the other two classes will also be reviewed briefly.

1.5.3 GABA A Receptors

GABA A receptors are heteropentameric ligand-gated chloride channels that mediate the

majority of fast synaptic inhibitory signals within the mature mammalian CNS. Structurally,

each receptor is composed of five polypeptide subunits drawn from a pool of 16 different

subunits (alpha 1-6, beta 1-3, gamma 1-3, delta, epsilon, theta, pi) that are encoded by seven

26

different gene families (Olsen and Sieghart 2008). The presence of certain polypeptide

subunits tends to confer particular functional characteristics to the assembled receptor. For

example, the presence of the gamma2 subunit is required for benzodiazpine sensitivity and

synaptic clustering (Pritchett et al. 1989; Essrich et al. 1998). Although there are

approximately 20 native GABA A receptors in the human CNS, the majority of them are

composed of 2 alpha subunits, 2 beta subunits and 1 gamma subunit (Figure 4) (Chang et al.

1996; Sieghart and Sperk 2002). Receptors containing the gamma2 subunit are the most

prevalent (Vicini and Ortinski 2004; Whiting 2006); specifically, the alpha1/beta2/gamma2

combination represents approximately 43% of GABAARs within the CNS (McKernan and

Whiting 1996). Each polypeptide subunit is composed of approximately 500 amino acids

and contains an N-terminal domain, a cys-loop domain, and a C-terminal domain (Schofield et

al. 1987; Macdonald and Olsen 1994). The N-terminal domain, composed of approximately

200 amino acids, is extracellular and sometimes forms the agonist binding sites for the

receptor. This is followed by a 15 amino acid, disulphide-linked, extracellular cysteine-loop.

The C-terminal domain contains 4 alpha-helical, hydrophobic, membrane-spanning segments

(M1, M2, M3, M4) with a large intracellular loop located between M3 and M4; this is

followed by a short, extracellular C-terminus. The M3/M4 intracellular loop, an important

site for phosphorylation and synaptic localization, is the most divergent region among the

various receptor subtypes.

27

Figure 4. The GABA A Receptor. Shown is a schematic representation of a receptor

composed of two alpha subunits, two beta subunits, and 1 gamma subunit. Select GABAAR

modulators and their binding sites are also depicted. Please refer to text for details. Adapted

from Purves et al., 2004.

28

29

Pharmacologically, compounds that enhance GABAAR function include muscimol,

benzodiazapines, barbiturates, neuroactive steroids, alcohol, and general anesthetics.

Compounds that inhibit GABAAR function include bicuculline, picrotoxin, and

pentylenetetrazol (Macdonald and Olsen 1994; Huang et al. 2001).

Gene-deficient mice have been generated for the following GABAAR subunits: alpha1-6,

beta2, beta3, gamma2, and delta. The most severe phenotypes are seen in mice lacking the

gamma2 or beta3 subunits. Gamma2 subunit knockout mice show perinatal lethality, have

decreased channel conductance of GABAARs, and show defects in postsynaptic clustering of

GABAARs (Gunther et al. 1995). Gamma2 subunit heterozygotes are viable and fertile but

show decreased synaptic clustering of GABAARs especially in the hippocampus.

Behaviourally, they show increased chronic anxiety with aversion towards stressful situations

and enhanced responses to fear conditioning (Crestani et al. 1999). In beta3 subunit knockout

mice, 57% develop cleft palate while 90% (including mice without cleft palate) die shortly

after birth (Homanics et al. 1997). In addition, they exhibit hyperactivity, spontaneous

seizures, abnormalities on electroencephalograms, impaired learning and memory, impaired

motor coordination, and repetitive stereotypical movements; these findings are similar to

Angelman syndrome in humans (DeLorey et al. 1998). Alpha1 subunit knockout mice have

decreased body weight, exhibit tremors when handled, have increased susceptibility to

bicuculine-induced seizures and show a >50% loss of GABAAR receptors; however, no

motor deficits or spontaneous seizures were apparent (Sur et al. 2001; Vicini et al. 2001).

Most of the remaining mutants display no overt phenotype but rather exhibit more subtle

behavioural or biochemical abnormalities. For example, alpha5 subunit knockout mice exhibit

enhanced hippocampus-dependent spatial learning (Collinson et al. 2002); alpha3 subunit

30

knockout mice exhibit decreased prepulse inhibition of the acoustic startle reflex, which is an

abnormality of sensorimotor gating often found in patients with schizophrenia (Yee et al.

2005).

Some of the GABAAR polypeptide subunits are encoded by gene clusters on human

chomosomes 4p12 (alpha2, alpha4, beta1, gamma1), 5q34 (alpha1, alpha6, beta2, gamma2),

15q13.2 (alpha5, beta3, gamma3) and Xq28 (alpha3, epsilon, theta); however, the genes

encoding the delta and pi subunits do not belong in a cluster and can be found on

chromosomes 1p36.3 and 5q35.1, respectively (Olsen and Sieghart 2008).

1.5.3.1 The GABAA Receptor in Human Disease

GABAAR dysfunction has been implicated in a number of human diseases including

epilepsy, anxiety disorders, schizophrenia, drug abuse, autism, and Angelman syndrome.

1.5.3.1.1 EpilepsySeizures result from uncontrolled electrophysiological discharge of neurons within the CNS

due to an increase in excitatory (glutaminergic) signals or a decrease in inhibitory

(GABAergic) signals. These imbalances of excitatory and inhibitory neurotransmission have

been correlated with altered expression of GABAAR subunits (Brooks-Kayal et al. 1998)

and/or altered GABAAR trafficking (Coulter 2001). For example, induction of status

epilepticus in animal models lead to increased GABAAR endocytosis (Wagstaff et al. 1991).

Gene-deletion mutants involving GABAAR subunits exhibit altered seizure thresholds (such

as the increased sensitivity to the GABAAR antagonist bicuclline seen in the alpha1-subunit

knockout mouse) and outright seizures (such is seen in the beta3-subunit knockout mouse).

Mutations have been identified in families with seizure syndromes (Jones-Davis and

31

Macdonald 2003) that all involve the gamma2 subunit of the GABAAR (R43Q, K289M,

Q351X). A mutation in the alpha1 subunit (A322D) has been found in patients with juvenile

myoclonic epilepsy (Jones-Davis and Macdonald 2003). In addition, the genomic locus of

the beta3 subunit has been implicated in Angelman Syndrome, which is characterized, by

mental retardation and epilepsy (Wagstaff et al. 1991).

1.5.3.1.2 SchizophreniaThree lines of evidence implicate the involvement of GABAARs in schizophrenia: 1)

Changes in expression of GABAAR subunits are present in post mortem brain tissue from

schizophrenic patients (Akbarian et al. 1995), 2) Drugs, such as benzodiazpines, that affect

GABAAR function improve symptoms of schizophrenia (Hosak and Libiger 2002), and 3)

The alpha3 subunit knockout mouse shows decreased prepulse inhibition, an abnormality in

sensorimotor gating that can be found in schizophrenic patients, which can be reversed by

treatment with the antipsychotic medication haloperidol (Yee et al. 2005).

1.5.3.1.3 Anxiety DisordersAnxiety disorders include generalized chronic anxiety, panic disorder and posttraumatic stress

disorder. GABAARs are implicated since the mainstay of treatment for these conditions are

benzodiazepines, which enhance GABAAR function. The gamma2 subunit heterozygous

mouse, which exhibits behavourial traits consistent with chronic anxiety, has decreased

binding of benzodiazepines in several regions of the brain (Crestani et al. 1999). This is

similar to patients with panic attacks that exhibit decreased GABAAR-benzodiazepine

binding in the brain (Tiihonen et al. 1997). In addition, while benzodiazepines (enhancers of

GABAAR function) inhibit anxiety, low doses of PTZ (antagonist of GABAAR function)

causes anxiety in human patients (Kalueff and Nutt 1996).

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1.5.3.2 The GABA A Receptor Lifecycle

GABAARs are constantly in a dynamic state of flux. Therefore molecules and pathways that

regulate GABAAR trafficking have significant influence on the inhibition of synaptic activity

within the CNS. The GABAAR lifecycle can be divided into several stages: 1) assembly of

receptors from component subunits, 2) trafficking of receptors to the cell surface, 3)

clustering of receptors on the postsynaptic membrane, 4) endocytosis, 5) post-endocytic

sorting resulting in either receptor recycling or lysosomal degradation.

1.5.3.2.1 Assembly of GABAARsAssembly, via oligomerization, of GABAARs from component subunits occurs in the

endoplasmic reticulum (ER). This process is characterized by its dependence on N-terminal

sequences of the polypeptide subunits, its speed of occurence (within 30 minutes of

translation), and its inefficiency (less than 25% of translated polypeptide subunits are

assembled into receptors) (Gorrie et al. 1997). Receptors that are improperly assembled

undergo endoplasmic reticulum-associated degradation (ERAD) in which subunits are

ubiquitylated and then degraded via the ubiquitin-proteosome system. In addition,

GABAARs are subject to activity-dependent ubiquitylation in which the level of neuronal

activity can affect the ubiquitylation of the receptors and thus regulate cell surface levels

(Saliba et al. 2007). A decrease in neuronal activity leads to an increase in ERAD that

ultimately results in decreased insertion of GABAARs into the surface membrane. Likewise,

increased neuronal activity results in decreased ERAD of GABAAR and increased expression

of receptors at the cell surface. In this way, the level of neuronal activity can affect the cell

surface expression of GABAARs and thus regulate synaptic inhibition. PLIC1 (protein

linking IAP to the cytoskeleton), a ubiquitin-like protein that prevents the degradation of

33

ubiquitylated substrates, binds to the intracellular domains of alpha and beta subunits which

blocks ERAD and results in increased numbers of receptors expressed on the surface of the

cell (Bedford et al. 2001).

1.5.3.2.2 Trafficking of the GABAAR to the cell surfaceAssembled receptors that successfully exit the ER proceed to the Golgi apparatus where they

are packaged into vesicles and transported to the cell surface for insertion into the plasma

membrane. Several molecules are thought to be involved in this process.

GABAAR-associated protein (GABARAP) localizes to the Golgi apparatus and binds to

intracellular domain of the GABAAR gamma2 subunit (Wang et al. 1999). Overexpression of

GABARAP along with GABAARs result in increased surface expression of receptors (Chen

et al. 2000). These data, along with the fact that it is not present at synaptic sites, suggests

that GABARAP may be involved in trafficking of the GABAAR to the cell surface (Leil et al.

2004). Although GABARAP-deficient mice show no change in levels of gamma2-containing

GABAARs; this may indicate redundancy or other compensatory mechanisms (O'Sullivan et

al. 2005).

Phospholipase-C-related catalytically inactive proteins (PRIPs) have been show to bind

beta1-3 subunits, the gamma2 subunit, and GABARAP (Kanematsu et al. 2002). PRIP2 is

expressed ubiquitously while PRIP1 is expressed mainly in the brain (Uji et al. 2002).

Possible functions for PRIPs in the GABAAR lifecycle include trafficking of receptors to the

cells surface, modulation of receptor phosphorylation, and internalization of receptors.

PRIPs may be involved in trafficking by acting as a bridge between GABAARs and

GABARAP; this is supported by the decreased association between GABAARs and

34

GABARAP in PRIP1/PRIP2 double knockout mice (Kanematsu et al. 2002). PRIPs may

also be involved in the phosphorylation-dependent modulation of GABAARs through the

inactivation of protein phosphatase 1 alpha (PP1alpha) (Yoshimura et al. 2001). While PKC

and PKA can phosphorylate the beta subunits of GABAARs and modulate its functions

(Kittler and Moss 2003), PP1alpha can terminate this effect by dephosphorylating the

receptor (Terunuma et al. 2004); therefore, the inactivation of PP1alpha by PRIP1 can lead to

enhanced phosphorylation of GABAARs.

Golgi-specific DHHC zinc-finger-domain protein (GODZ) has been identified as the principal

enzyme responsible for the palmitylation of GABAARs, a process that is required for

postsynaptic clustering (Keller et al. 2004; Fang et al. 2006). Brefeldin-A-inhibited

GDP/GTP exchange factor 2 (BIG2) binds beta subunits and transports GABAARs to the

cell surface (Charych et al. 2004). GABAAR-interacting factor 1 (GRIF1) binds the beta2

subunit of GABAARs as well as to the motor protein kinesin; it is thought to be involved

regulating motor-dependent transport of GABAARs (Beck et al. 2002; Smith et al. 2006).

1.5.3.2.3 Synpatic Clustering of GABAARsReceptors that are successfully assembled and trafficked to the cell surface are typically

inserted into the cell membrane at extrasynaptic sites (Bogdanov et al. 2006). Then,

depending on their subunit composition, they reach their postsynaptic locations through

lateral diffusion and trapping (Thomas et al. 2005). For example, gamma2 subunit-containing

GABAARs tend to be present in synaptic clusters while receptors containing alpha5 and

delta subunits are primarily extrasynaptic. Currently, two mechanisms for clustering are

thought to be possible: gephyrin-dependent clustering and gephyrin-independent clustering.

As the name implies, gephyrin-dependent clustering of GABAARs involves the protein

35

gephyrin, which localizes to synaptic sites on GABAergic neurons and binds the alpha2

subunit of GABAARs (Tretter et al. 2008). Loss of gephryin results in loss of synaptic

clusters that contain GABAARs with alpha2 and/or gamma2 subunits (Kneussel et al. 1999;

Jacob et al. 2005). Neuronal cultures derived from the GABAAR gamma2 subunit knockout

mouse shows a lack of postsynaptic GABAARs and gephrin (Essrich et al. 1998). However,

due to the fact that synaptic GABAAR clusters are still present in gephyrin knockout mice, a

gephyrin-independent mechanism of GABAAR synaptic clustering has been proposed

(Kneussel et al. 2001; Levi et al. 2004). Radixin, an ERM (ezrin, radixin, moesin) family

protein, has been implicated in this process. Radixin localizes to the plasma membrane, and

binds the alpha5 subunit of GABAARs as well as F-actin. Loss of radixin results in

decreased synaptic clustering of alpha5-containing GABAARs while maintaniing overall cell

surface receptor levels (Loebrich et al. 2006).

1.5.3.2.4 Endocytosis of GABAARsNeuronal GABAARs at the cell surface undergo constitutive internalization via clathrin-

dependent endocytosis. When this process is blocked, an increase in mini inhibitory

postsynaptic current (mIPSC) amplitude is seen; this is consistent with increased numbers of

cell surface receptors due to decreased internalization. Adapter protein 2 (AP2) of the

clathrin-AP2 complex recruits GABAARs into clathrin-coated pits via its ability to bind

beta1-3 and gamma2 subunits (Kittler et al. 2000). Phosphorylation at AP2-binding sites of

GABAAR subunits decreases AP2 binding which leads to decreased receptor endocytosis

and increased mIPSC amplitudes (Kittler et al. 2005; Kittler et al. 2008). This suggests a

mechanism by which pathways that regulate protein kinases and phosphatases may be able

to influence synaptic inhibition.

36

1.5.3.2.5 Post-endocytic recycling and degradation of GABAARsOnce GABAARs are internalized via endocytosis they can either be recycled to the cell

surface or undergo lysosomal degradation. Currently, little is known about the molecules or

mechanisms that influence this process. However, overexpression of Huntingtin-associated

protein (HAP1), a protein shown to bind GABAAR beta subunits, results in decreased

degradation and increased recycling of GABAARs, increased surface expression of

GABAARs, and increased mIPSC amplitude (Kittler et al. 2004).

1.5.4 GABAB Receptors

In contrast to the ionotropic GABAA and GABAC receptors, GABABRs are metabotropic

G-protein-coupled receptors. Pharmacologically, they are characterized by their sensitivity

to the GABA analog baclofen and insensitivity to the GABAAR antagonist bicuculline (Hill

and Bowery 1981). Structurally, they are heterodimers composed of 2 seven-transmembrane

subunits: GABAB1 and GABAB2. Gene deletion mouse models for GABAB1 (Prosser et

al. 2001; Schuler et al. 2001) and GABAB2 (Gassmann et al. 2004; Thuault et al. 2004) have

been generated and show similar phenotypes: spontaneous seizures, hyperalgesia, increased

locomotor activity, impaired memory. The genes for the human GABAB1 and GABAB2

subunits are mapped to chromosomes 6p21.3 and 9q22.1-q22.3, respectively. Abnormalities

of GABABRs have been implicated in a number of human conditions including spasticity,

pain, cognitive impairments, anxiety and depression, schizophrenia, absence epilepsy, and

drug addiction (Bowery 2006).

37

1.5.5 GABAC Receptors

Like GABAARs, GABACR are also pentameric ligand-gate chloride channels.

Pharmacologically however, they are characterized by their insensitivity to the GABAAR

antagonist bicuculline and to GABAAR modulators such as benzodiazapines and barbiturates

(Bormann 2000). GABACRs are composed of 3 different rho subunits and are expressed

primarily in the retina (Enz and Cutting 1998). Mice that are deficient in the rho1 subunit

have altered visual and olfactory processing (McCall et al. 2002; Chen et al. 2007). The genes

encoding human rho1 and rho2 are located on chromosome 6q14-q21 (Cutting et al. 1992);

human rho3 is located on chromosome 3q11-q13.3 (Bailey et al. 1999).

1.6 Thesis Rationale and Objectives

The tumour suppressor p53 is a key player in the cell's response to a variety of stress

signals. In order to identify novel molecules in the p53 network, the Mak lab performed an in

vivo genetic screen using Drosophila melanogaster and identified the D. melanogaster tumour

suppressor Brain Tumour (brat) as a putative interacter of p53. . In mammals, there exists

three homologs to brat: TRIM3/BERP, TRIM2/NARF and TRIM32/HT2A. Using NCBI's

BLASTP program, BERP was identified as the best mammalian hit for brat and was thus

chosen as the molecule upon which the work of this thesis would be based.

The two main objectives for this thesis are:

1) To determine the role of BERP in the p53 network.

2) To characterize the role of BERP in the mammalian CNS through the generation and

analysis of a gene-deficient mutant mouse.

2 BERP is a Novel p53 Target Gene

2.1 Introduction

Normal p53 function is vital for maintaining the integrity of our genome in the face of DNA

damage and other abnormal stresses such as aberrant proliferative signals, telomere erosion,

hypoxia, loss of adhesion, and loss of survival signals. It is a multifunctional protein involved

in a variety of cellular processes including cell cycle arrest, senescence, apoptosis, DNA

repair, and angiogenesis. The ability of p53 to prevent the propagation of damaged or

potentially transformed cells is key to its function as a tumour suppressor (Vousden and Lu

2002; Levine et al. 2006). Although many molecules in the p53 pathway have been identified

including transcriptional targets p21 (cell cycle arrest), GADD45 (DNA repair), Bax

(apoptosis), and major negative regulator MDM2, our understanding of this complex network

is incomplete (Momand et al. 1992; Kubbutat et al. 1997; Vogelstein et al. 2000; Daujat et al.

2001; Vousden and Lu 2002). As more molecules within the p53 network are identified and

characterized, the apparent reach of this transcription factor increases accordingly. For

example, it has been shown that the promoter of PTEN, a major negative regulator of the PI3

kinase pathway, contains a p53 response element and that p53 can activate transcription of

PTEN (Stambolic et al. 2001). This discovery revealed a previously unknown connection

between these two important tumour suppressors and their respective signaling pathways. It

follows then that the identification of novel molecules in the p53 network would allow us not

only to better understand the role(s) of this tumour suppressor in already defined pathways

(eg. cell cycle arrest, apoptosis, senescence...etc...), but may also provide clues that point to

its involvement in novel functions that have not yet been described.

39

Therefore, in order to identify novel molecules within the p53 network, the laboratory

performed a genetic modifier screen in D. melanogaster using fly lines that overexpress dp53

in the developing eye (GMR-Gal4-dp53); these flies have a small, rough eye phenotype

compared to wild type flies. To identify genetic modifiers of the p53 pathway, GMR-Gal4-

dp53 flies were crossed with mutant fly strains each containing one of 1150 distinct P-

element insertions which disrupts one copy of a vital D. melanogaster gene. The progeny of

these crossings were scored according to phenotypic changes in the eye. Progeny with larger

eyes than those of GMR-Gal4-dp53 flies were phenotypic suppressors; those with smaller

eyes were phenotypic enhancers. Results of this screen identified D. melanogaster brain

tumour (brat) as a suppressor of the p53 eye phenotype and hence, putative interacter of

p53. Using NCBI's BLASTP program, we determined that of the three known mammalian

brat homologs, BERP was the best hit and therefore would be the focus of our studies.

Because the D. melanogaster screen is a genetic screen and not a biochemical screen, three key

possibilities should be considered: i) BERP functions upstream of p53, ii) BERP functions

downstream of p53, and iii) BERP functions in parallel to p53. However, due to the nature

of the screen performed (ie. in which p53 was overexpressed in the fly eye), a hit that

functions upstream of p53 is unlikely. Therefore it is most likely that BERP is either

downstream of p53 or functions in a parallel pathway. Since p53 exerts many of its cellular

effects by regulating the transcription of downstream genes, we wondered whether BERP

could be a transcriptional target of p53. This chapter describes several lines of evidence

supporting the interpretation that BERP is a novel p53-regulated gene.

40

2.2 Materials and Methods

2.2.1 DNA sequences and prediction of p53-binding sites

DNA sequences and potential p53-binding sites were obtained from NCBI

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and TFBIND (http://tfbind.hgc.jp/)

(Tsunoda and Takagi 1999) respectively.

2.2.2 Cell culture

The isogenic human colon cancer cell lines HCT116 p53+/+ and HCT116 p53-/-, a kind gift

from Dr. B. Vogelstein (Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD),

were cultured in McCoy's 5A media supplemented with 10% fetal calf serum and L-

glutamine.

2.2.3 ChIP assay

HCT116 p53+/+ and HCT116p53-/- cells were treated with 5FU for 24 hours, cross-linked

with formaldehyde and sonicated (4 x 20s pulses, duty cycle 30%, output control 3, Branson

Sonifier® S-450A) into fragments ranging in size from 200bp to 1000bp. Chromatin

immunoprecipitation was performed on cell extracts using EZ ChIP Kit (Millipore) and anti-

p53 antibody (DO-1, Santa Cruz). PCR amplification on the immunoprecipitated chromatin

was performed using primers specific for potential p53REs in the region upstream of the

BERP gene. Primer sequences were as follows. p21, 5'-GTGGCTCTGATTGGCTTTCTG-

3' and 5'-CTGAAAACAGGCAGCCCAAGG-3'; BERP B, 5'-

41

GGGTGTGGGAGATTGGACATGGTAC-3' and 5'-

CCAGGAAGATAGACAGGGCAGCAAC-3'; BERP EFG, 5'-

GGGACAGATGGTTGCAGGTTGGAG-3' and 5'-

CTGCTAGGGCCAGTCCTGAGTTTC-3'.

2.2.4 Luciferase assay

pGL3-p21, a kind gift from Dr. S. Benchimol (Lin et al. 2000), contains a p53-binding site

from the Cdkn1a promoter and the E1B core promoter sequence subcloned into the pGL3-

Basic vector (Promega) just upstream of the luc+ gene. pGL3-TATA is the pGL3-p21

vector in which the Cdkn1a p53-binding site is removed. The Cdkn1a p53-binding site of

pGL3-p21 was replaced by PCR-amplified fragments, from HCT116 cell genomic DNA, of

potential p53 response elements upstream of the BERP gene in order to generate pGL3-

BERPA, pGL3-BERPB, and pGL3-BERPE vectors. The constructs were cotransfected

using Fugene 6 (Roche) into HCT116 p53-/- cells with a beta-galactosidase vector and vectors

expressing either wildtype or a DNA binding-mutant form p53 (Baker et al. 1990; Kern et al.

1992). Cells were harvested and lysed 24 hours post transfection using the Luciferase Assay

System (Promega). Luciferase activity was normalized to beta-galactosidase activity and

expressed relative to pGL3-TATA.

2.2.5 Real time RT-PCR

Total RNA was extracted using RNeasy (Qiagen) and reverse transcribed using Superscript

first strand RT-PCR cDNA synthesis system (Invitrogen) both according the manufacturers'

42

protocols. Quantitative PCR was performed on cDNA using TaqMan Gene Expression

Assays (BERP: Hs00197678_m1, P21: Hs00355782_m1, NARF: Hs00209620_m1, HT2A:

Hs00705875_s1, PPIA: Hs99999904_m1, GAPDH: Hs99999905_m1, HPRT1:

Hs99999909_m1, TBP: Hs99999910_m1, Applied Biosystems) and the 7900HT Fast Real-

Time PCR System (Applied Biosystems).

2.2.6 Genotyping of p53-deficient mice

p53-deficient mice, originally created in the laboratory of Dr. Tyler Jacks (Jacks et al. 1994),

were generated from heterozygous intercrosses. Genotyping of the mice was performed as

per the protocol from the Jacks laboratory. Briefly, this consitsted of multiplex PCR

performed on genomic DNA extracted from mouse tails using the following primers: 5'-

CAGCGTGGTGGTACCTTAT-3', 5'-TATACTCAGAGCCGGCCT-3', 5'-

CTATCAGGACATAGCGTTGG-3'. The PCR conditions used were 95ºC x 15' followed

by 30 cycles of 94ºC x 1', 60ºC x 1', 72ºC x 1' with a final extension at 72ºC x 10' (GeneAmp

9600, Perkin Elmer). The presence of the wild-type and mutant alleles were denoted by PCR

products of ~ 450 and ~ 615 base pairs, respectively.

2.2.7 In-situ hybridization

The in situ hybridization procedure was performed similar to that previously described (Hui

and Joyner, 1993; Skinnider et al., 2001). Briefly, p53 +/+ and p53 -/- adult male mice (8-12

weeks old) were treated with PTZ (45 mg/kg, Sigma) or PBS via a single intra-peritoneal

injection. At 6 and 24 hours post-injection, the brains were harvested, fixed for 24 hours in

43

10% neutral buffered formalin, dehydrated, and embedded in paraffin. RNAse-free sections

(4 µm thick) were placed onto glass slides, de-paraffinized, acetylated, dehydrated, and

hybridized with sense or antisense -[33P]-UTP-labeled RNA probes that were synthesized

from linearized template with T3 or T7 RNA polymerase. The BERP DNA template was a

428bp fragment subcloned into pBluescript SK (Stratagene). C- (cardiac) actin, a DNA

template known to consistently work with a well-defined expression pattern, was run in

parallel as a positive control for the procedure. Once hybridization and washes were

completed, the slides were dehydrated and a sheet of x-ray film was exposed overnight (to

gain a rough sense of expression level). Slides were then coated with liquid Kodak NTB 2 or

NTB photographic emulsion (Eastman Kodak) and stored at 4°C. After exposure for 5 to

14 days (depending upon the strength of the signal determined from the x-ray film exposure),

the slides were developed in Kodak D19 developer (4min at 14ºC, Eastman Kodak), fixed

(Kodak Fixer) and counterstained with toluidine blue. The specificity of the hybridization

signal was verified by the corresponding lack of signal when sense RNA probes were used.

Cells/areas were scored as positive if the number of silver grains was at least 2-fold over

background.

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2.3 Results

2.3.1 The BERP promoter region contains multiple potential p53 responseelements

The 14.8kb genomic fragment upstream of the BERP translational start site was examined for

the presence of potential p53 response elements. Using sequence analysis, multiple potential

sites similar to the p53RE consensus sequence as described by El-Deiry et al., 1992 were

identified. Of these, three sites (B, E, G) that had “C “and “G” nucleotides at positions 4 and

7 of each half site, and that had no more than 3 mismatches to the consensus sequence were

chosen for testing (Figure 5); site F, although it had >3 mismatches to the consensus sequence

was also included due to its proximity to both sites E and G. Site B

(GGACATGGTAcatcctttgTCACTTGCCC) is located between bases -11349 to -11321

relative to the BERP translational start site (the nucleotide immediately 5' to the ATG is -1).

It contains two half-sites with 17 of 20 nucleotide matches to the p53RE consensus sequence;

there is a 9 bp spacer between the two half-sites. Site E is located within intron 1 between

bases -7374 to -7354; it contains two half-sites separated by a 1 bp spacer, with 19 of 20

nucleotide matches to the p53RE consensus sequence. Site F is located within intron 1

between bases -7258 to -7235; it contains two half-sites separated by a 4 bp spacer, with 15

of 20 nucleotide matches to the p53RE consensus sequence. Site G is located within intron 1

between bases -7151 to -7128; it contains two half-sites separated by a 4 bp spacer, with 17

of 20 nucleotide matches to the p53RE consensus sequence.

45

Figure 5. Predicted p53REs within the BERP genomic locus. (A) Genomic localization of 4

potential human BERP p53REs. Gray ovals represent the location of four putative BERP

p53REs (B, E, F, G) as indicated. Residue positions are expressed relative to the translation

start site; the "A" of the ATG is +1. (B) Sequences of the putative p53REs within the

BERP genomic locus. The p53RE consensus sequence is shown for comparison; Pu = A/G,

Py = C/T, N = A/G/C/T. Capitalized letters, nucleotides from half-site sequences. Small

letters, nucleotides from the spacer sequence. Asterisks, mismatches to the p53RE consensus

sequence. The position of each potential site is given relative to the start of translation. For

each potential site, the number of residues matching the p53RE consensus sequence is

indicated.

46

47

2.3.2 p53 can bind BERP p53REs in vivo and regulate transcription

ChIP analysis was performed in order to determine whether p53 could bind to the potential

BERP p53REs in vivo. HCT116 p53+/+ and HCT116 p53-/- cells were treated with 5FU in

order to induce p53. Cells were harvested 24 hours later and chromatin immunoprecipitation

was performed using anti-p53 antibody. This was followed by PCR with primers specific for

the potential p53REs. Results indicate that p53 binds to BERP site B as well as to BERP

sites EFG in vivo (Figure 6A). Note that due to proximity of BERP sites E, F and G, it is

impossible to determine the exact location to which p53 binds. In order to determine whether

the potential BERP p53REs can regulate transcription, oligonucleotides containing the

sequences for BERP sites B, E, F and G were cloned into the pGL3-TATA construct

upstream of the luciferase reporter gene. The constructs were then co-transfected into

HCT116 p53-/- cells along with a vector expressing either wild-type p53 or a DNA-binding-

domain mutant form of p53. pGL3-p21 was used a positive control. Of the four potential

sites tested, only BERP E resulted in an increase in luciferase activity in the presence of wild-

type p53; no increase was seen in the presence of mutant p53 (Figure 6B). Additionally,

a C → A point mutation at position 4 of the BERP E site results in loss of luciferase activity

(Figure 6C). These findings suggest that the BERP E p53RE is bound by p53 and can

regulate transcription in a p53-dependent manner.

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Figure 6. In vivo binding and transcriptional competence of predicted BERP p53REs.

(A) Chromatin immunoprecipitation assay in HCT116 cells showing the in vivo binding of

p53 with potential BERP p53REs (sites BERP B and BERP EFG). The p21 p53RE was

used as a positive control. Cells were treated with 5FU to induce p53; untreated controls

received DMSO only. Immunoprecipitations were performed using non-specific mouse

immunoglubulin ("IgG" lanes) and DO-1 anti-p53 antibody ("anti-p53" lanes). 1% of the

total sonicated chromatin was removed prior to immunoprecipitation and used for input

DNA ("Input" lanes). H2O, water control. PCR products were resolved on a 1% agarose gel

stained with ethidium bromide. (B) Luciferase assay showing the ability of wild-type (light

blue bars) and mutant p53 (orange bars) to activate transcription of luciferase reporter

constructs containing potential BERP p53REs (pGL3-BerpB, pGL3-BerpE, pGL3-BerpF,

pGL3-BerpG). pGL3-p21 was used as a positive control. pGL3-TATA, empty vector

control. Luciferase activity was normalized to beta-galactosidase activity and expressed

relative to pGL3-TATA levels. Results shown are the means of four independent

experiments performed in triplicate; error bars represent 1 SEM. (C) Luciferase assay

showing the ability of wild-type (light blue bars) and mutant p53 (orange bars) to activate

transcription of luciferase reporter constructs containing site BERP E (pGL3-BerpE) and a

mutant version of site BERP E containing a C → A point mutation at position 4 (pGL3-

BerpE*). pGL3-p21, positive control. pGL3-TATA, empty vector control. pGL3-BerpG,

a construct containing a putative BERP p53RE that is unable to support transcriptional

activation by p53. Luciferase activity was normalized to beta-galactosidase activity and

expressed relative to pGL3-TATA levels. Results shown are the means of 3 independent

experiments performed in quadruplicate; error bars represent 1 SEM.

49

50

2.3.3 BERP expression is p53-dependent in vitro

To determine whether BERP expression is p53 dependent in vitro, we treated the isogenic

HCT116 p53+/+ and HCT116 p53-/- cancer cell lines with increasing doses of 5FU (a

compound known to activate p53 in this system) and measured the levels of BERP mRNA

using real-time RT-PCR. Results show that BERP expression is upregulated 4 to 5-fold in a

p53-dependent manner at both 24 (Figure 7B) and 48 (Figure 7A) hours after treatment with

5FU. In addition, BERP homologs NARF and HT2A did not show p53-dependent

upregulation (Figure 7B), which suggests that this effect is specific to BERP under these

conditions. Similar results were obtained using additional endogenous controls (please refer to

materials and methods).

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Figure 7. BERP expression in HCT116 p53-/- and HCT116 p53+/+ cells. (A) Real-time

RT-PCR analysis of BERP expression in HCT116 cells. RNA extracted from HCT116

p53+/+ and HCT116 p53-/- cells 48 hours after treatment with increasing doses of 5FU.

Real-time RT-PCR was performed in order to determine the relative expression levels of

target genes. Values were normalized to HPRT1 and expressed relative to untreated controls.

P21 was used as a positive control for p53 activation. HCT116-/BERP (light red bars),

BERP expression in HCT116 p53-/- cells. HCT116+/BERP (dark red bars), BERP

expression in HCT116 p53+/+ cells. HCT116-/P21 (light blue bars), P21 expression in

HCT116 p53-/- cells. HCT116+/P21 (dark blue bars), P21 expression in HCT116 p53+/+

cells. Results shown are the means of three independent experiments performed in triplicate;

error bars represent 1 SEM. (B) Real-time RT-PCR analysis of BERP, NARF and HT2A in

HCT116 cells. RNA was extracted from HCT116 p53+/+ and HCT116 p53-/- cells 24 hours

after treatment with increasing doses of 5FU. Real-time RT-PCR was performed to

determine the relative expression levels of the indicated target genes. HCT116mut/BERP

(light red bars), BERP expression in HCT116 p53-/- cells. HCT116wt/BERP (dark red bars),

BERP expression in HCT116 p53+/+ cells. HCT116 HCT116mut/HT2A (light green bars),

HT2A expression in HCT116 p53-/- cells. HCT116wt/HT2A (dark green bars), HT2A

expression in HCT116 p53+/+ cells. HCT116mut/NARF (light orange bars), NARF

expression in HCT116 p53-/- cells. HCT116+/NARF (dark orange bars), NARF expression

in HCT116 p53+/+ cells. HCT116mut/P21 (light blue bars), P21 expression in HCT116

p53-/- cells. HCT116wt/P21 (dark blue bars), P21 expression in HCT116 p53+/+ cells.

P21expression was used as a positive control for p53 activation. Values were normalized to

HPRT1 and expressed relative to untreated controls. Results shown are the means of three

independent experiments performed in triplicate; error bars represent 1 SEM.

52

53

2.3.4 BERP expression is p53-dependent in vivo

Results from section 2.3.2 suggest that site E within intron 1 of the human BERP genomic

locus is indeed a functional p53 response element. Sequence analysis of the murine BERP

genomic locus also reveals multiple putative p53 response elements (data not shown).

Although no site identical to Site E of the human genomic locus was identified within the

murine genomic locus, it is interesting that one of the putative murine sites (AGGCAGGCCC

t GTGCATGTTC) is located at a similar relative position within intron 1 (7121 bp upstream

of the translational start site compared to 7374 bp for human Site E), and has a high number

of residues matching the consensus sequence (18/20 matches compared to 19/20 matches for

human Site E). In addition, both are composed of half-sites that are separated by a spacer

region consisting of a single nucleotide. Therefore, in order to determine whether BERP

expression is p53-dependent in mouse tissue, p53 +/+ and p53 -/- adult male mice between 8

- 12 weeks old were injected with PTZ (3-5 mice per genotype (p53 +/+ and p53-/-) per

condition (PTZ and PBS) per time point (6 and 24 hours). The brains were harvested at 6

hours (Figure 8) and 24 hours (data not shown) post injection, then subjected to in situ

hybridization using a 33P-labeled BERP riboprobe. At 6 hours, endogenous levels of BERP

were present in the brains of p53 -/- mice (Figures 8A and 8C). In contrast, strong up-

regulation of BERP was seen in the brains of p53 +/+ mice (Figures 8B and 8D). The brains

of sham injected p53 -/- and p53 +/+ mice showed endogenous levels of BERP (Figures 8E

and 8F). Taken together, these data indicate that BERP is upregulated in a p53-dependent

manner in response to PTZ in vivo.

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Figure 8. BERP expression in p53-/- and p53+/+ mice. In response to PTZ injection, BERP

expression is up-regulated in the cerebellum (B) and hippocampus (D) of p53 +/+ mice

compared to levels present in the cerebellum and hippocampus of p53 -/- mice (A and C,

respectively). BERP expression in the p53 -/- mice (A and C) is comparable to the

endogenous levels present in sham injected p53 -/- and p53 +/+ mice (E and F). Scale bar:

200µm.

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56

3 The Role of BERP in the Mammalian CNS

3.1 Introduction

BERP (Brain Expressed Ring finger Protein), a molecule that initially drew our interest for its

homology to a D. melanogaster tumour suppressor, was first cloned and characterized in the

rat by El-Husseini et al. (El-Husseini and Vincent 1999; El-Husseini et al. 2000). It binds to

alpha-actinin-4 and myosin V, two members of the actin cytoskeleton that have been

implicated in cell motility, cancer and organelle trafficking (Honda et al. 1998; Provance and

Mercer 1999; Nikolopoulos et al. 2000; Yan et al. 2005; Wang et al. 2008). In PC12 cells,

overexpression of a BERP truncation mutant prevented NGF-induced differentiation and

neurite outgrowth (El-Husseini and Vincent 1999). It is strongly expressed in the brain and is

upregulated in the murine brain following chemically induced seizures (Ohkawa et al. 2001).

In humans, BERP (as TRIM3) was recently identified as the candidate tumour suppressor

gene at the 11p15.5 locus after examining 70 human gliomas for loss of heterozygosity

(Boulay et al. 2009). In addition, a recent shotgun proteomics analysis suggested that BERP

is up-regulated in the brains of schizophrenic patients (Martins-de-Souza et al. 2009). These

findings, along with the discovery that BERP is a novel p53-regulated gene (as described in

Chapter 2) suggest that BERP may an important role in the mammalian CNS. However, an in

vivo role for BERP is yet to be described. Therefore, we sought to determine the possible

physiologic function of BERP through the generation of a gene-deficient mutant mouse model.

This chapter describes the generation of a BERP-deficient mouse using targeted mutagenesis

and its characterization based on the clues provided from the published literature.

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3.2 Materials and Methods

3.2.1 ES cell culture

E14K embryonic stem cells derived from 129/OLA mice were grown on a monolayer of

mitomycin C-treated primary mouse embryonic fibroblasts. The culture medium consisted of

DMEM with 15% FCS (HyClone, Logan, Utah), leukemia inhibitory factor, L-glutamine, and

beta-mercaptoethanol.

3.2.2 Generation of BERP-deficient mice

The full-length mouse BERP cDNA was PCR amplified from a mouse brain cDNA library

(Clontech) using specific primers flanking the BERP open reading frame. The BERP cDNA

was then used to probe a P1-derived artifical chromosome (PAC) clone library in order to

identify clones that carry a genomic fragment containing the mouse BERP locus. Two such

PAC clones were successfully obtained (3M16 and 143K22). A targeting construct designed

to replace coding exons 2-5 of BERP with a pGK promoter driven neomycin resistance gene

cassette in reverse orientation of BERP transcription was created. The final targeting

construct contained both a short arm and a long arm of sequences derived from the PAC clone

3M16, which flanked the neomycin cassette. The short arm introduced a stop codon

immediately after the 5’ splice site of coding exon 2. The targeting construct (20ug) was

linearized with NotI, electroporated into 5x106 E14K mouse embryonic stem cells (Bio-Rad

Gene Pulser; 0.34 kV, 250 F) and treated with 300µg/mL of G418 (Sigma) for 10 days. ES

cells were screened for successful homologous recombination by PCR using primers specific

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for the neomycin resistance gene and the flanking BERP locus (5'-

CATACCCGAATCCAGGTCTATCTC-3’ and 5'-

CCTCCCACTCATGATCTATAGATCGG-3’). PCR-positive clones were confirmed by

Southern hybridization of NcoI-digested genomic DNA with radio-labeled probes for both the

5’ region immediately flanking the targeted BERP locus (which detects a 13.5 kb wild-type

allele and a 4.5 kb mutant allele) and the neomycin resistance gene (to confirm single

integration). ES clones that contain the correctly targeted allele were micro-injected into

blastocysts harvested from day 3.5 pregnant C57BL/6 female mice and implanted into the

uteri of day 2.5 pseudopregnant CD1 foster mother mice in order to generate chimeras. All

resultant chimeras were mated with C57BL/6 mice and Agouti-coloured offspring were

assessed for successful germline transmission of the mutant allele (BERP+/-) by tail genomic

DNA extraction followed by PCR and Southern hybridization analysis. Heterozygous mice

(BERP +/-) were intercrossed to produce BERP-null mice (BERP -/-). Two lines from

independently targeted ES clones were successfully generated and backcrossed to C57BL/6.

3.2.3 Genotyping of BERP-deficient mice by PCR and Southern Blotting

PCR was performed on genomic DNA extracted from mouse tails using the following primer

sets: 5'-CCTGCTTCTCTCACTGTCACCACC-3' and 5'-

CACCCTTCTCCCTTCCACGACTAC-3' for the wild-type allele; 5'-

CAGTCATAGCCGAATAGCCTCTCC-3' and 5'-CACCCTTCTCCCTTCCACGACTAC-

3' for the mutant allele. The PCR conditions used were 94ºC - 15' followed by 40 cycles of

94ºC - 30", 58ºC - 30", 72ºC - 1', with a final extension at 72ºC - 7' (GeneAmp 9600, Perkin

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Elmer). The presence of the wild-type and mutant alleles was denoted by PCR products of

632 and 1155 base pairs, respectively.

Southern blot analysis was performed on 10µg of tail-derived genomic DNA. After digestion

with NcoI, the DNA was fractionated on a 1% agarose gel and transferred to a positively

charged nylon membrane (Hybond N+, Amersham). Membranes were hybridized with a

radio-labeled probe from the 5' flanking region, which detects a 13.5 kb band representing the

wild type allele and a 4.5 kb band representing the mutant allele.

3.2.4 Generation and genotyping of BERP-/-;p53-/- mice

BERP+/- mice (section 3.2.2) were crossed with p53+/- mice (Jacks et al. 1994) in order to

generate BERP+/-;p53+/- mice. Mice with simultaneous deficiencies of BERP and p53 were

generated by intercrossing pairs of BERP+/-;p53+/- mice. Genotyping of the BERP locus

was performed by PCR as described in section 3.2.3. Genotyping of the p53 locus was

performed by PCR as described in section 2.2.6.

3.2.5 Histological analysis

Tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin.

Sections were cut (approximately 4 µm thick), placed onto glass slides and stained with either

H&E/LFB (central nervous system tissues) or H&E (all other tissues). Sectioned material was

also used for in situ hybridization analysis of mRNA (see below).

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3.2.6 Western blot analysis

Tissues were homogenized in lysis buffer containing 0.5% CHAPS w/v, 10mM Tris (pH7.5),

1mM MgCl2, 1mM EGTA, 10% glycerol v/v, 50µM NaF, 100µM Na3VO4, and protease

inhibitor cocktail tablets (Roche). Total protein concentration was determined using the

Bradford assay (Bio-Rad). Protein lysates were fractionated by sodium dodecyl sulfate

polyacrylamide gel electrophoresis on an 8% tris-glycine polyacrylamide gel (Invitrogen) and

blotted onto a polyvinylidene difluoride membrane (Millipore). Blots were incubated with

mouse anti-BERP antibody (BD Biosciences) at a dilution of 1:500 or rabbit anti-Actin

antibody (Sigma) at a dilution of 1:1000. Bound antibodies were visualized by incubation

with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary

antibodies followed by enhanced chemiluminescence according to the manufacturer's protocol

(Amersham).

3.2.7 Behavioural characterization

All behavioural tests were performed at the Samuel Lunenfeld Research Institute's Centre for

Modeling Human Disease (CMHD) Mouse Physiology Facility, Toronto, Canada. Test

subjects consisted of BERP+/+ and BERP-/- male mice backcrossed at least 6 generations into

a C57BL/6 genetic background. All mice were housed in groups of 3-5 per cage and were

between 10-13 weeks old at the time of testing. Food and water were available ad

libitum.

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SHIRPA Neurological Screen

A general neurological screen was performed on test subjects according to the modified

SHIRPA (Smithkline Beecham, MRC Harwell, Imperial College, the Royal London hospital

Phenotype Assessment) protocol (Rogers et al. 1997).

Locomotor Activity and Open-Field Test

Subjects were individually placed into the centre of a chamber (42cm wide x 42cm long x

33cm high) equipped with horizontal and vertical infrared sensors (Ugo Basile, Italy). For the

first 5 minutes, the following parameters were recorded using The Observer (Noldus

Information Technology): time spent in and number of entries into the border area, time spent

in and number of entries into the centre area, number of on wall- and off wall-rearings,

horizontal and vertical activity (beam breaks). After the first 5 minutes, measurement of

horizontal and vertical activity continued for an additional 25 minutes. The test chamber was

cleaned with a 70% ethanol solution between subjects.

Elevated plus maze test

The testing apparatus consisted of two opposed open arms (25cm x 5.5cm) and two opposed

closed arms (25cm x 5.5cm x 25cm) extending at right angles from a centre platform; the entire

apparatus was elevated 50 cm above the ground. Test subjects were individually placed in

the centre of the maze and the following parameters were recorded by The Observer (Noldus

Information Technology) during a 5 minute observation period: time spent on the open arms,

closed arms, and on the central platform. Total number of entries into open and closed arms

was used as a measure of overall motor activity. The test apparatus was cleaned with a 70%

ethanol solution between subjects.

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Contextual and cued fear conditioning

The test apparatus consisted of a test chamber (25cm high x 30cm wide x 25cm deep) with a

grid floor connected to a current generator and a speaker. On day 1 (conditioning training),

test subjects were placed individually into the chamber and observed without stimulus for

120 seconds. This was followed by 30s of an audible tone (3600Hz, 85dB) and a 2s foot

shock (1mA) that co-terminated with the tone. The subject was removed from the chamber

30s later. Baseline freezing activity was recorded using automated fear conditioning software

(FreezeFrame, Actimetrics Software). On day 2, approximately 24 hours later, the test

subjects were individually returned to the test chamber; freezing activity was monitored for

300s during which time no stimulus was applied (contextual fear test). Test subjects were

then removed from the test chamber. Two hours later, test subjects were individually placed

into an altered version of the test chamber (grid floor was covered with a plastic sheet, two

pieces of fiberglass were used to create triangle-shaped chamber, door was covered with black

cardboard) and observed without stimulus for 180s. This was followed by an audible tone

(3600Hz, 85dB) for 180s (cued fear test). Freezing activity was recorded throughout the test.

The test apparatus was cleaned with a 70% ethanol solution between subjects.

Prepulse Inhibition of the Acoustic Startle Response

The testing apparatus consists of 4 sound-attenuating chambers (Startle Reflex System, MED

Associates Inc.) that could confine and detect the movement of the test subjects. Five

different trial types were used: (i) A "pulse-only" trial in which only an acoustic startle pulse

of 120dB was presented for 40 milliseconds. (ii-iv) "Prepulse-pulse" trials in which a

prepulse tone (69, 73, 81dB, 20 milliseconds) is followed by a pulse (120dB, 40 milliseconds)

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at 100 millisecond intervals. (v) A "no stimulus" trial in which only background noise of 65dB

was presented to measure baseline movement of the test subject in the chamber. The trials

were performed in the following order: (1) A 15 minute "no stimulus" trial for acclimation, (2)

five "pulse-only" trials, (3) 10 combination blocks that contained all the five trial types in

pseudo-random order, (4) five "pulse-only" trials. Inter-trial intervals varied between 12 and

30 seconds. The maximum force intensity for each trial was recorded as startle level. The

PPI level is defined as the average percent reduction in startle intensity between prepulse-

pulse and pulse-only trials at all three prepulse levels (69, 73, 81dB). The test apparatus was

cleaned with a 70% ethanol solution between subjects.

Accelerating Rotarod

The testing apparatus consisted of a rod that was suspended 30cm above a plastic floor and

was divided into 4 sections by opaque plastic dividers, thus allowing 4 subjects to be tested

simultaneously (Economex Rotarod, Columbus Instruments). Test subjects were placed onto

the rod facing away from the observer. Initially, subjects were observed for 60 seconds while

the rod remained stationary. Next, the rod was rotated at a constant speed of 5 revolutions

per minute (rpm) for 90 seconds. The rotational speed was then gradually increased by

0.1rpm per second for 300 seconds; the maximum speed reached was 30rpm. The latency to

falling off the rod was recorded. Test subjects underwent 3 consecutive trials per day for 3

consecutive days. The mean latency was calculated by averaging the latency for three

consecutive trials.

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Tail suspension test

Test subjects were suspended within the tail suspension chamber from a small metallic hook

by their tails using adhesive tape. The cumulative duration of immobility over a 6 minute

observation period was recorded using The Observer (Noldus Information Technology)

Grip strength test

Subjects were held by the tail and allowed to grip a metal grid. Slow and steady traction was

applied until the grip was released down the complete length of the grid. The maximum grip

strength was recorded in grams. Eight trials in total were performed per subject (4 forelimb

trials and 4 fore-hindlimb trials).

Tail flick test

The distal portion of the subject's tail was submerged in 50º degree Celsius water bath; the

latency to removal was measured.

3.2.8 Pentylenetetrazol seizure susceptibility testing

Mice were shipped to the Research Service at the Department of Veterans Affairs Medical

Center (DVAMC) in Coatesville, Pennsylvania in the USA in six batches. Each batch

contained both wild type (N ~ 10) and knock out mice (N ~ 10) and batches were shipped

approximately 6 weeks apart. Upon arrival to the DVAMC, mice were housed in a

quarantine room of the animal facility in the basement of Building #11 for at least 1 week

prior to testing. Mice were maintained on a 14 hr (light) 10 hr (dark) circadian light cycle and

had food and water available ad libitum. All mice were male and were between the ages of 8-

12 weeks at the time of seizure testing. Seizure tests were conducted between the hours of 9

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a.m. and 12 noon. Mice were given a single injection of pentylenetetrazole (PTZ, Sigma)

dissolved in physiological saline at pH 7.0. Injections were given subcutaneously in a volume

equal to 1% of body weight. Drug solutions were prepared fresh on the day of each

experiment. A range of doses was studied (40, 60, 80, 90 and 100 mg/kg) and doses were

staggered such that several doses were utilized in testing each batch of mice. Individuals

performing seizure tests were blind with respect to the genotypes of the mice. Following

PTZ injection, mice were placed into a plexiglass chamber (20mm wide, 30 mm deep, 40 mm

high) with mesh floor and observed for 45 minutes during which time seizure responses were

scored. Three endpoints, as well as the latencies to each endpoint were recorded: partial

seizure (PS), generalized seizure (GS), maximal seizure (MS). Partial seizure is characterized

by a sudden whole body spasm or a sudden and rapid clonus involving the head, neck or one

forelimb. Generalized seizure is characterized by loss of upright posture and bilateral clonus

of fore and hind limbs. Maximal seizure is characterized by bilateral tonic extension of the

hind limbs; it is sometimes fatal in susceptible mice.

3.2.9 Hemi-brain slice preparation

Male mice 3-4 weeks old were anesthetized with halothane and decapitated. The brain was

rapidly extracted and placed into ice-cold, oxygenated (95%O2, 5%CO2) artificial cerebral

spinal fluid (ACSF) that contained 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM

NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, and 25 mM glucose and was titrated to pH 7.3–7.4

with osmolarity of 300–320 mOsm. Hemi-brain coronal slices (300 µm) were cut using a

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vibratome (Vibratome Co.) and placed in oxygenated ACSF at room temperature for 1 hour

before recording.

3.2.10 Electrophysiology

Slices were transferred into a recording chamber on the stage of an infrared-DIC E600FN

microscope (Nikon). The chamber was continuously perfused with oxygenated ACSF at

room temperature at a rate of 2 mL/minute. The whole cell patch-clamp technique was used

to record from cortical pyramidal neurons. Recording electrode resistance was 3–5 M_ when

filled with solution containing 145 mM CsCl, 1mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10

mM HEPES, 4 mM K2ATP, titrated to pH 7.3 with CsOH and the osmolarity was 280–290

mOsm. An Axopatch 200B (MDC) was used to record mIPSCs. Data acquisition and

analysis were performed using DigiData 1322A (MDC) and the analysis software pClamp 10

(MDC). Signals were filtered at 2 kHz and sampled at 10 kHz. The resistance of the patch

pipette was monitored throughout the whole-cell recording and data were discarded if the

resistance of the electrode changed by more than 20%. For recording mIPSCs, neurons were

voltage-clamped at -70 mV in the presence of TTX (1.0 µM), CNQX (20 µM) and D-APV

(50 µM). All recordings were performed at room temperature.

3.2.11 Murine cortical neuron cultures

Cultures of primary neurons were derived from the cerebral cortex of embryonic day 17.5

mice (E17.5). Pregnant females from timed matings were sacrificed by cervical dislocation and

the uteri were dissected and the embryos were removed. The cortex from the embryo brains

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were excised, placed in ice-cold HBSS, and gently triturated with fire-polished Pasteur

pipettes. The dissociated cell suspension was pelleted by centrifugation at 300g x 5' at 4ºC,

re-suspended in plating medium, and plated on poly-D-lysine coated Petri dishes (day 0). On

day 3, half of the plating medium was replaced with maintenance medium. Maintenance

medium was exchanged every 3 days thereafter. Plating medium consisted of Neurobasal

medium (Invitrogen), 2% B-27 supplement (Gibco), 10% FBS (Hyclone), 0.5 mM L-

glutamine, and 25 µM glutamic acid. Maintenance medium consisted of Neurobasal medium,

2% B-27 supplement, and 0.5 mM L-glutamine.

3.2.12 Biotinylation Assay

After 10 days in culture, cell surface proteins were isolated using the Pierce Cell Surface

Protein Isolation Kit (Thermo Scientific) according to the manufacturer's protocol. Biotin-

tagged protein was quantified using the Bradford assay (Bio-Rad). Protein lysates

(20µg/lane) were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis

on a 10% tris-glycine polyacrylamide gel (Invitrogen) and transferred onto a polyvinylidene

difluoride membrane (Millipore). Blots were probed with anti-GABA-A-receptor-gamma-2

antibody (ab16213, Abcam) at a dilution of 1:1000 and anti-ionotropic-Glutamate-receptor-2

antibody (ab40878, Abcam) at a dilution of 1:5000. Bound antibodies were visualized by

incubation with horseradish peroxidase-conjugated secondary antibodies followed by

enhanced chemiluminescence according to the manufacturer's protocol (Amersham). Blots

were subsequently stripped and incubated with anti-Actin antibody (Sigma) at a dilution of

1:5000 in order to confirm that the biotin-tagged surface proteins were not contaminated with

cytoplasmic proteins. Densitometry analysis was performed using NIH ImageJ software

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(Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,

http://rsb.info.nih.gov/ij/, 1997-2008).

3.2.13 Real time RT-PCR

Total RNA was extracted using RNeasy (Qiagen) and reverse transcribed using Superscript

first strand RT-PCR cDNA synthesis system (Invitrogen) both according the manufacturers'

protocols. Quantitative PCR was performed on cDNA using TaqMan Gene Expression

Assays (Gabrg2: Mm00433489_m1, Hprt1: Mm01324427_m1, TBP: Mm01277042_m1,

B2m: Mm00437762_m1, Applied Biosystems) and the 7900HT Fast Real-Time PCR

System (Applied Biosystems).

3.2.14 Neurite Outgrowth Assay

Neurons from d17.5 embryos were isolated as described above (see "Mouse cortical neuron

cultures"). Cells were then plated on poly-D-lysine coated coverslips in 12-well plates and

fixed 18, 48, or 72 hours later with 4% paraformaldehyde. The cells were stained with a

mouse monoclonal anti-MAP2 antibody (Sigma, M4403), which was visualized using a Cy3

conjugated affinity purified donkey anti-mouse antibody (Jackson Immuno Labs, 115-165-

146). Images were captured using an OrcaER (Hamamatsu), Micropublisher MP5

(QImaging), or Leica DC500 (Leica Microsystems) camera attached to a DM6000 or DMRE

Leica Research microscope and analyzed using Volocity (Improvision Inc) or LAS (Leica)

software. Measurements of neurites were made using differential interference contrast (DIC)

optics on the Leica DM6000 upright microscope with Volocity (Improvision Inc.) acquisition

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and measurement software. The total neurite length for each neuron was the sum of all

neurites extending from the cell body. A minimum of 50 neurons were examined per genotype

per experiment.

3.2.15 Neurosphere formation and differentiation assays

Adult male mice, 8-12 weeks old, were killed by cervical dislocation. All subsequent steps

were performed using aseptic techniques. The brain was removed and placed into ice cold,

oxygenated artificial cerebral spinal fluid containing 124mM NaCl, 5mM KCl, 1.3 mM

MgCl2, 26mM NaHCO3, 10mM glucose, 2mM CaCl2. The cortex was removed in order to

expose the lateral ventricles. A small strip of tissue from the medial and lateral walls of the

lateral ventricles from both brain hemispheres was removed. The dissected tissue was

digested using the Papain Dissociation Kit (Worthington Biochemical Corporation) as per the

manufacturer's instructions for 60 minutes at 37ºC. The tissue was mechanically dissociated

into a single cell suspension by trituration using fire-polished Pasteur pipettes. Viable cells,

as determined by trypan blue exclusion, were counted and plated on 24-well plates at a

density of 20 cells/µL (10000 cells per well). Cells were cultured in chemically defined serum

free medium (SFM) in the presence of 20 ng/mL epidermal growth factor (Sigma), 10 ng/mL

fibroblast growth factor 2 (Sigma), 2 µg/mL heparin (Sigma). SFM consisted of DMEM/F12

(1:1) (Invitrogen), 5 mM HEPES buffer, 0.6% glucose, 3 mM NaHCO3, 2 mM L-glutamine,

25 mg/ml insulin, 100 mg/ml transferrin, 20 nM progesterone, 60 mM putrescine, and 30 nM

sodium selenite. After 7 days in culture, the number of neurospheres (“primary”

neurospheres) in each well was counted. To determine self-renewal capacity, primary

neurospheres in each well were collected, mechanically dissociated into a single cell

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suspension and re-plated on new 24-well plates at a density of 10 viable cells/µL (5000 cells

per well; 8 wells per animal). After 7 days in culture, the number of secondary neurospheres

in each well was counted. For continued passaging, spheres were collected, dissociated and

re-plated as described above. Cell size and viability for single cell suspensions from

dissociated neurospheres were determined using a Beckman Coulter Vi-CELL XR Viability

Analyzer.

3.2.16 Neurosphere differentiation assay

To determine multipotentiality of the neurospheres, single spheres were transferred to poly-

D-lysine/laminin-coated 8 -well chamber slides (BD BioCoat, BD Biosciences) containing

SFM with 1% fetal bovine serum. After 7 days, the spheres were fixed in 4%

paraformaldehyde treated with 0.5% Triton X-100 for 30 minutes prior to

immunocytochemistry. Spheres were preblocked in histoblock containing 5% goat serum,

10% donkey serum, 10% FCS, and 0.5% Triton X-100. Neurospheres were incubated with

primary antibody overnight at 4ºC and with secondary antibodies at room temperature for 1.5

hours. Anti-beta-III-tubulin (R&D Systems, clone TuJ-1, mouse monoclonal) was used as a

neuron-specific marker at a dilution of 1:1000. Glial fibrillary acid protein (rabbit anti-cow

GFAP, DAKO,) was used as a specific marker for astrocytes at a dilution of 1:1000.

Secondary antibodies (Jackson Immuno Labs) were donkey anti-mouse Cy3 (1:200 dilution)

and goat anti-rabbit Cy2 (1:200 dilution). Chambers were washed with PBS (-Ca2+, -Mg2+)

before dehydrating and mounting in a xylene-based mounting medium (Entellan, EMD). Slides

were then examined using a Leica DM6000 microscope with epifluorescence and appropriate

filter sets. The proportion of neurons was determined by counting beta-III-tubulin positive

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cells and expressing this number as a percentage of the total number of cells in 5 random fields

per neurosphere.

3.2.17 Thymidine incorporation assay

Proliferation assays of neural stem/progenitor cells were performed in flat-bottom 96-well

plates. 2500 viable cells per well were plated in triplicate and cultured in chemically defined

serum free medium in the presence of growth factors (as described in Neurosphere Formation

and Differentiation Assays). Plates were pulsed with 1 mCi [3H] thymidine per well and

harvested after 6 hours. [3H] Thymidine uptake was measured using a scintillation counter

(Topcount, Canberra Packard).

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3.3 Results

3.3.1 Generation of BERP-deficient mice

BERP-deficient mice were generated using homologous recombination in ES cells to disrupt

the BERP gene (Figure 9A). The targeting construct was designed to replace coding exons 2-5

of BERP with a pGK promoter driven neomycin resistance cassette. After several cycles of

electroporation and G418 selection, independent ES cell clones that contained the mutant

BERP allele were injected into C57BL/6 blastocysts in order to generate chimeras. Agouti-

coloured pups (F1) resulting from matings between chimeras and C57BL/6 mice were

assessed for successful germline transmission of the mutant BERP allele by PCR and

Southern blotting of tail-extracted genomic DNA (Figure 9B). The absence of BERP protein

in BERP -/- mice was confirmed by Western blotting of whole brain lysates using an anti-

BERP antibody (Figure 9C). F1 heterozygotes were backcrossed six generations into the

C57BL/6 background. F1 heterozygous intercrosses yielded the expected sex and Mendelian

ratios. BERP -/- mice were viable, healthy, and fertile, and weight of pups at weaning age of

the same sex were not significantly different (Figure 10). No gross or histological

abnormalities were identified. Aged BERP+/- and BERP-/- mice (>1 year) showed no

differences in lifespan or tumour formation compared with wild-type controls (data not

shown).

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Figure 9. Targeted disruption of the murine BERP locus. (A) Schematic representations

of the wild-type BERP allele (top), the targeting construct showing the replacement of most

of coding exons 2-5 with a neomycin cassette along with the introduction of an additional

NcoI site (middle), and the mutant BERP allele after successful homologous recombination

(bottom). The 5' flanking probe (fp) located external to the targeting construct was used to

identify successful recombinant ES clones as well as to confirm the genotypes of mice by

Southern hybridization. (B) PCR and Southern blot analysis of genomic tail DNA derived

from Agouti mice. PCR performed using primers a and c (arrowheads under wild type allele

in panel A) yields a 632 bp band indicating the presence of the wild-type allele; primers b and

c (arrowheads under mutant allele in panel A) detects the presence of a 1155 bp band

representing the mutant allele (left panel). After digestion with NcoI followed by Southern

hybridization using the radiolabeled 5' flanking probe, the wild-type allele yields a 13.5 kb

fragment while the mutant allele yields a 4.5 kb fragment (right panel). (C) Western blot

analysis of BERP protein expression. Protein lysates from the brains of BERP+/+ and

BERP-/- mice were probed with anti-BERP and anti-actin antibodies.

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Figure 10. General phenotypic assessment of BERP-deficient mice (129ola/C57BL/6

mixed background). (A) Genotype analysis of BERP mice. PCR of genomic tail DNA was

performed using primers specific for the BERP wild-type and mutant alleles. Results shown

are from one litter of a heterozygous intercross. (B) Chart showing the sex and genotype

breakdown of 296 offspring from heterozygous intercrosses. (C) Mean weights at weaning of

BERP+/+, BERP+/-, and BERP-/- mice (n=15-41) for each genotype of each sex; error bars

represent 1 SEM.

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3.3.2 Behavioural characterization of BERP-/- mice

3.3.2.1 SHIRPA Neurological Screen in BERP-deficient mice

To assess gross neurological function, a modified SHIRPA neurological screen was performed

on the mice. This battery of tests is a semi-quanititative observational method of assessing

basic phenotypic, behavioural and sensorimotor characteristics (Rogers et al. 1997). Of note,

BERP-/- mice had decreased body weight (21.7g±0.7 vs. 24.3g±0.4; p<0.0001, Student's t-

test) and body length (93.4mm±0.5 vs. 95.6mm±0.6; p<0.05, Student's t-test) relative to

wild-type controls (Figure 11). No other significant differences between the genotypes were

seen in the rest of the SHIRPA screen.

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Figure 11. SHIRPA neurological assessment for body weight in BERP-deficient mice.

(A) and body length (B) in BERP+/+ (yellow bars) and BERP-/- (blue bars) mice. Results

shown are the means of 9 BERP+/+ and 12 BERP-/- mice; error bars represent 1 SEM.

*p<0.05 and ***p<0.001, Student's t-test.

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3.3.2.2 BERP-/- mice exhibit increased freezing in fear conditioning tests

Fear conditioning is used to determine a subject's fear-driven learning ability. In the

contextual test, the subject is placed into a chamber and observed for fear as measured by the

amount of freezing behaviour; freezing is defined as the absence of all observable movement

except respiration. The subject then undergoes a single conditioning session in which a 30-

second neutral tone (the auditory cue/conditioned stimulus, CS) is paired with a 2-second

foot-shock (the unconditioned stimulus, US) that co-terminates with the tone. After 24 hours

the subject is placed into the same conditioning chamber and its fear response is measured.

Contextual fear conditioning relies on hippocampus and amygdala-dependent memory

(Anagnostaras et al. 2001), and measures the ability of subjects to form a negative association

between the contextual cues of the conditioning chamber and the unconditioned stimulus. In

the cued test, 24 hours after undergoing the conditioning session, the subject is placed into an

altered version of the chamber (in which the visual, auditory, olfactory, and tactile contextual

cues have been changed) during which time the neutral tone (the auditory cue/conditioned

stimulus) from the previous day's conditioning session is played; fear response is measured

before and after the presentation of the tone. Cued fear conditioning is amygdala-dependent

(Anagnostaras et al. 2001), and measures the ability of subjects to form a negative association

between the auditory cue of the conditioned stimulus and the unconditioned stimulus.

Baseline measurements show a slight increase in freezing in BERP-/- mice compared to

BERP+/+ mice (Figure 12, baseline); however, this was not statistically significant. In the

context test, BERP-/- mice exhibited significantly increased freezing in response to the

conditioning context compared to wild-type controls (45.4%±3.8 for BERP-/- vs. 23.2%±4.0

for BERP+/+; p<0.01, Student's t-test; Figure 12, 24hour context). In the cued test, BERP-/-

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exhibited increased freezing in response to the conditioned stimulus compared to wild-type

controls (56.6%±5.9 for BERP-/- vs. 31.9%±5.6 for BERP+/+; p<0.01, Student's t-test;

Figure 12, post-CS). Interestingly, BERP-/- mice also exhibited increased freezing prior to the

presentation of the conditioned stimulus during the cued fear test (11.4%±1.9 for BERP-/- vs.

1.6%±0.5 for BERP+/+; p<0.01; Student's t-test; Figure 12, pre-CS). These data suggest that

BERP-/- mice have enhanced contextual and cued fear conditioning compared to wild-type

controls. The enhanced freezing of the BERP-/- mice to the altered context (pre-CS) but not

at baseline, raises the possibility that after the conditioning session, BERP-/- mice retained a

higher level of fear and anxiety leading to increased freezing even during the pre-CS phase of

the cued test.

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Figure 12. Fear conditioning in BERP-deficient mice. (A) Contextual fear conditioning

test showing the amount of freezing for BERP+/+ (yellow bars) and BERP-/- (blue bars) mice

with no stimulus (baseline), and with being placed in the same environment 24 hours after

receiving a tone/shock trial (24 hour context). (B) Cued fear conditioning test showing the

amount of freezing for BERP+/+ (yellow bars) and BERP-/- (blue bars) mice when placed in

an altered environment 24 hours after receiving a tone/shock trial (pre-CS), and when being

presented with a tone while in the altered environment (post-CS). Results shown are means

from 9 BERP+/+ and 12 BERP-/- mice; error bars represent 1 SEM. **p<0.01, Student's t-

test.

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3.3.2.3 BERP-/- mice exhibit abnormalities in the open field test forexploration and general locomotion; however, no effect of BERP isseen in the elevated plus maze test

The open-field and elevated plus maze tests were used to assess anxiety in the mice. In the

open-field test, which is used to assess the response to being placed in a novel, open

environment, fewer entries into the centre zone, decreased time spent in the centre zone,

increased time spent in the border zone and fewer number of rearings are all parameters

indicative of increased anxiety. These parameters are all measured during the first five

minutes of the subject being placed in the open-field because it represents the period of

greatest anxiety (see Materials and Methods). In the elevated plus maze test, increased

anxiety is manifested by a decrease in the amount of time spent in the open arms and an

increase in the amount of time spent in the closed arms. Results show that during the open-

field test (Figure 13), BERP-/- mice had fewer numbers of rearing (22.3±2.2 for BERP-/- vs.

32.0±3.6 for BERP+/+; p<0.05, Student's t-test; Figure 13C), and chose to spend more time

in the border zone (1435s±26 for BERP-/- vs. 1306s±29 for BERP+/+; p<0.01, Student's t-

test; Figure 13B). However, there was no difference between the genotypes for the length of

time spent in the centre zone or the number of entries into the centre zone (Figures 13A and

13D). Although BERP-/- mice seemed to spend slightly less time in the open arms of the

elevated plus maze than wild-type controls (Figure 14), this difference was not statistically

significant (p=0.18). Overall horizontal (Figure 15A) and vertical (Figure 15B) activity did

not differ between BERP-/- and BERP+/+ mice suggesting that there is no effect of genotype

on general locomotor activity. However, in the first five minutes of the test, which represents

the time of greatest anxiety, there was a significant decrease in vertical activity for BERP-/-

mice compared to wild-type controls 40.3±6.0 for BERP-/- vs. 75.8±9.8 for BERP+/+;

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p<0.01, Student's t-test; Figure 15B). This supports the decreased number of rearings seen in

the BERP-/- mice and is another parameter indicative of increased anxiety. Taken together,

these data suggest that the BERP-/- mice are slightly more anxious than wild-type controls.

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Figure 13. Open field exploration in BERP-deficient mice. (A) Time spent in the centre

zone. (B) Time spent in the border zone. (C) Number of rearings. (D) Number of entries

into centre zone. Results shown are the means for 9 BERP+/+ (yellow bars) and 12 BERP-/-

mice (blue bars); error bars represent 1 SEM. *p<0.05 and **p<0.01, Student's t-test.

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Figure 14. Elevated plus maze test in BERP-deficient mice. (A) Percentage of time spent

on open arms. (B) Percentage of time spent on closed arms. (C) Total number of entrances.

Results shown are the means for 9 BERP+/+ (yellow bars) and 12 BERP-/- mice (blue bars);

error bars represent 1 SEM.

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Figure 15. General locomotor activity in BERP-deficient mice. (A) Horizontal activity

as measured by the number of beam breaks. (B) Vertical activity as measured by the number

of beam breaks. Results shown are the means for 9 BERP+/+ and 12 BERP-/- mice; error

bars represent 1 SEM. Yellow denotes BERP+/+ values; blue denotes BERP-/- values.

**p<0.01, Student's t-test.

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3.3.2.4 BERP-/- mice exhibit abnormalities in the accelerating rotarod test

BERP-/- mice were deficient in rotarod test for motor coordination (Figure 16) compared to

wild-type controls (p<0.05 for days 1 and 3; p<0.01 for day 2, Student’s t-test). This effect

was present in all 3 days of the test. Of note is the fact that despite consistently falling off

the rotarod faster than wild-type controls, BERP-/- mice appear to improve each day, which

suggests that they may have the capacity for ongoing motor learning. This is in contrast to

the wild-type controls, which showed an improvement from day 1 to day 2, but no

improvement from day 2 to day 3.

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Figure 16. Accelerating rotarod test in BERP-deficient mice. Results shown are the

mean latencies to fall of three trials for the indicated test days in 9 BERP+/+ (yellow graph)

and 12 BERP-/- mice (blue graph); error bars represent 1 SEM. *p<0.05 and **p<0.01,

Student's t-test.

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3.3.2.5 No effect of BERP in tests for sensorimotor gating

Sensorimotor gating was assessed by measuring prepulse inhibition (PPI) of the acoustic

startle response (ASR). In this test, the ability of a prepulse to inhibit the acoustic startle

response is measured. Patients with schizophrenia often show deficits in PPI compared to

control subjects and so a deficit in PPI can be used as a test for psychosis in mice (Braff and

Geyer 1990). There was no significant difference in acoustic startle response between the

two genotypes (Figure 17A). Although not statistically significant, there was a trend that the

prepulse inhibition was decreased in BERP-/- mice for all three (69dB, 73dB, 81dB) prepulse

sound levels (Figure 17B).

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Figure 17. Prepulse inhibition of the acoustic startle response in BERP-deficient mice.

Acoustic startle response (A) and prepulse inhibition of acoustic startle response (B) at the

indicated prepulse sound levels in BERP-/- (blue bars) and BERP+/+ (yellow bars) mice.

Results shown are means from 9 BERP+/+ and 12 BERP-/- mices; error bars represent 1

SEM.

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3.3.2.6 No effect of BERP in tail suspension test (depression-relatedbehaviour).

The tail suspension test is a widely used model for assessing depression-like behaviour in

mice. As shown in (Figure 18), no difference was seen in the tail suspension test between

BERP+/+ and BERP-/- mice; this suggests that BERP does not play a role in depression-like

behaviour.

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Figure 18. Tail suspension test in BERP-deficient mice. Tail suspension test illustrating

the total duration of immobility for BERP+/+ (yellow bar) and BERP-/- (blue bar). Results

shown are means of 9 BERP+/+ and 12 BERP-/- mice; error bars represent 1 SEM.

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3.3.2.7 No effect of BERP in grip test (neuromuscular function)

There was no difference in the grip test between BERP-/- and BERP+/+ mice. This suggests

that neuromuscular function and muscle strength are normal in BERP-/- mice compared to

wild-type controls (data not shown).

3.3.2.8 No effect of BERP in tail flick test (nociception)

No difference was seen in the tail flick test between BERP-/- and BERP+/+ mice. This

suggests that sensitivity to pain is normal in BERP-/- mice compared to wild-type controls

(data not shown).

3.3.3 Increased resistance to PTZ-induced seizures in Berp-/- mice

Since BERP was shown to be upregulated in the brains of mice subjected to injections of the

chemoconvulsant PTZ, we decided to examine susceptibility to PTZ-induced seizures in

BERP-deficient mice. Injection of PTZ elicits a constellation of sequence-specific behavioural

seizure responses. Susceptibility to PTZ-induced seizures can be measured semi-

quantitatively by documenting the presence and progression of seizure responses during a

defined observation period. A more quantitative measure of susceptibility may be derived

from the latency to specific seizure response endpoints. Our initial tests were performed in

mice of mixed (129Ola x C57BL/6) genetic background in order to determine if further testing

was warranted. Male mice 8-12 weeks of age were treated with 45mg/kg of the

chemoconvulsant PTZ via intraperitoneal injection and observed over a period of 60 minutes

for the occurrence of a generalized seizure (which was defined in the methods section); the

observer was blinded to the genotypes of the test subjects. Results show that 67% of wild-

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type mice (n=36) and 51% of BERP-/- mice (n=33) experienced generalized seizures (p=0.2,

chi square) (Figure 19A). Although not statistically significant, the data indicated the

presence of a biological trend suggesting that BERP-/- mice may be more resistant to PTZ-

induced seizures compared to wild-type controls. Several factors made these initial tests less

than ideal: the mixed genetic background of the mice, the inability to provide a consistently

uniform testing environment, and the use of intraperitoneal injections, which while easier to

administer, is more variable in its effects. Therefore, to test whether BERP-/- mice indeed

exhibit increased resistance to PTZ-induced seizures, we backcrossed the mice 6 generations

onto a C57BL/6 genetic background and repeated the experiments in a specialized testing

facility using multiple doses of PTZ delivered via subcutaneous injection (see Materials and

Methods for details). At 60mg/kg, there was a significant decrease in the proportion of

BERP-/- mice that exhibited generalized seizures compared to wild-type controls (9 of 13 for

BERP-/- vs. 15 of 15 for BERP+/+; p<0.05 Fisher's exact test and Chi square) (Figure 19F).

The difference in mean latencies to generalized seizure for BERP-/- vs. BERP+/+ mice was

also statistically significant (p<0.05, Student's t-test) at this dose (Figure 19C). Although

results for the other doses did not reach statistical significance, the data trends indicate that in

general, BERP-/- were more resistant to PTZ-induced seizures compared to wild-type

controls. For each dose, the proportion of knockout mice that experienced seizure activity

was consistently lower than wild-type controls; this was true for partial, generalized and

maximal seizures. Similarly, the latency to each seizure type was consistently longer in

BERP-/- mice than wild-type controls. Taken together, these results suggest that BERP-

deficient mice are more resistant to PTZ-induced seizures compared to wild-type controls.

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Figure 19. PTZ seizure susceptibility testing in BERP-deficient mice. Unless otherwise

specified, all mice were 8-12 week old males backcrossed 6 generations onto a C57BL/6

genetic background; PTZ at the indicated doses was administered by subcutaneous injection

after which the mice were monitored for a period of 3600s. Blue bar/lines, BERP-/- mice.

Yellow bar/lines, BERP+/+ mice. (A) Proportion of mixed genetic background (129Ola x

C57BL/6) BERP+/+ (n=36) and BERP -/- (n=33) mice exhibiting generalized seizure in

response to 45mg/kg of PTZ by intraperitoneal injection. (B-D) Latencies to PTZ-induced

partial seizures (PS - panel B), generalized seizures (GS - panel C), and maximal seizures (MS

- panel D) in BERP+/+ and BERP -/- mice. PTZ was administered at doses of 40mg/kg (n=21

for BERP+/+, n=19 for BERP-/-), 60mg/kg (n=15 for BERP+/+, n=13 for BERP-/-), 80mg/kg

(n=11 for BERP+/+, n=9 for BERP-/-), 90mg/kg (n=10 for BERP+/+, n=10 for BERP-/-), and

100mg/kg (n=7 for BERP +/+, n=7 for BERP-/-). Each data point represents the mean from

the indicated number of BERP+/+ and BERP-/- mice; error bars are 1 SEM. PS, partial

seizure. GS, generalized seizure. MS, maximal seizure. *p<0.05, Student's t-test. (E-G)

Dose response curves for PTZ-induced partial seizures (panel E), generalized seizures (panel

F), and maximal seizures (panel G) in BERP+/+ and BERP -/- mice. PTZ was administered at

doses of 40mg/kg (n=21 for BERP+/+, n=19 for BERP-/-), 60mg/kg (n=15 for BERP+/+,

n=13 for BERP-/-), 80mg/kg (n=11 for BERP+/+, n=9 for BERP-/-), 90mg/kg (n=10 for

BERP+/+, n=10 for BERP-/-), and 100mg/kg (n=7 for BERP +/+, n=7 for BERP-/-). Each

data point represents the percentage of mice exhibiting partial, generalized, or maximal

seizures at the indicated dose of PTZ. *p<0.05, Fisher's exact test and Chi square.

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3.3.4 The amplitude of mIPSCs is decreased in BERP-/- mice

Recording mIPSCs allows us to assess abnormalities in the function of ionotropic GABA-

mediated synaptic transmission. In general, changes in the frequency and amplitude of mean

mIPSCs are correlated with presynpatic (neurotransmitter release) and postsynpatic

(neuroreceptor function) factors, respectively. Since PTZ is an antagonist of GABAAR

signaling and our data indicate that BERP-deficient mice show increased resistance to PTZ-

induced seizures, we decided to examine whether BERP may be related to GABAAR

function. In order to determine whether BERP plays a role in GABAergic synaptic

transmission, we examined GABAAR-mediated mIPSCs using whole cell, patch-clamp

electrophysiology. To record mIPSCs from neurons of brain slices prepared from BERP+/+

and BERP-/- mice, NBQX (an AMPA/kainate receptor antagonist), D-APV (an NMDA

receptor antagonist) and TTX (a Na+ channel blocker) were applied to the external solution.

Results show that the mean mIPSC amplitude is reduced in BERP-/- mice compared with

BERP+/+ mice (Figure 20D, right). No difference was seen in the mean mIPSC frequency

between the two genotypes (Figure 20D, left). Since it has been suggested that changes in the

amplitude of mIPSCs are mediated by the number of postsynaptic GABAARs (Nusser et al.

1997), our data suggest that BERP-/- mice have fewer GABAARs compared with wild-type

controls.

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Figure 20. Electrophysiological measurements in BERP-deficient mice.

(A) Representative mIPSCs traces recorded from BERP+/+ (top) and BERP-/- mice (bottom).

(B) An example of averaged mIPSC from BERP+/+ (n=1899 events, left) and BERP-/- mice

(n=3122 event, right). (C) Cumulative probability plots of mIPSC peak amplitude (bin size

0.5 pA). The mIPSC amplitude distribution significantly shifted towards smaller values in

BERP-/- mice than BERP+/+ mice (p < 0.001, Kolmogorov-Smirnov test). (D) Mean

mIPSCs frequency (left) and amplitude (right) in BERP-/- mice compared to BERP+/+ mice.

Data shown are the means from 11 BERP+/+ and 13 BERP-/- mice; error bars represent 1

SEM. **p <0.01, Student’s t-test.

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3.3.5 Decreased surface expression of GABAAR gamma2 subunit inBERP-/- mice

To determine whether BERP plays a role in the number of surface GABAARs present in the

brain, we performed biotinylation assays on cortical neuron cultures from BERP+/+,

BERP+/-, and BERP-/- embryos. This was followed by Western blot analyses for the

gamma2 subunit of the GABAAR. Results show that cell surface expression of GABAARs

is decreased in BERP-/- mice compared to BERP+/+ and BERP+/- mice (Figure 21). In

addition, probing with anti-GluR2 antibody showed no change in expression between BERP

wild-type, heterozygous and knockout mice, suggesting that BERP is not involved in

regulating the number of surface glutamate receptors.

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Figure 21. GABA A receptor expression in BERP-deficient mice. (A) Western blot

analysis of biotinylated surface proteins extracted from pooled cortical neuron cultures of day

17.5 BERP +/+, BERP +/-, BERP -/- embryos. Results shown are a single trial representative

of 4 independent experiments, probed with anti-GABA-A-receptor-gamma-2 (Gamma2) and

anti-ionotropic-Glutamate-receptor-2 (GluR2) antibodies. (B) Densitometry analysis of

surface GABA A receptor gamma 2 subunit expression in cortical neuron cultures from BERP

+/+, BERP+/- and BERP -/- embryos (day 17.5). Results shown are the means of 4

independent experiments; error bars represent 1 SEM. **p<0.01, Student’s t-test. (C) Real-

time RT-PCR analysis of GABA A receptor expression in BERP+/+ and BERP-/- embryos.

Real-time RT-PCR was performed on RNA extracted from the brains of day 17.5 BERP+/+

and BERP-/- embryos in order to determine the relative expression levels of GABA A

receptor (gamma subunit) mRNA. Results shown are the means from 17 wild-type and 19

knockout embryos performed in triplicate; error bars represent 1 SEM. Values were

normalized to HPRT1 and expressed relative to wild-type controls.

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3.3.6 GABA A receptor gamma2 subunit mRNA levels are unchanged inBERP-/- mice

To determine whether mRNA levels of GABA A receptor gamma2 subunit is decreased in

BERP-/- mice, we performed real time RT-PCR on RNA extracted from the brains of day

17.5 embryos (Figure 21C). Results show similar expression levels of GABAARg2 mRNA

in BERP+/+ and BERP-/- embryos, suggesting that the decrease in GABAAR surface protein

expression seen in BERP-/- neurons is a post-transcriptional phenomenon.

3.3.7 Cortical neurons from BERP-/- embryos show an increase in totalneurite length

In order to determine whether BERP plays a role in neuronal proliferation, a neurite

outgrowth assay was performed using cortical neurons isolated from day 17.5 embryos. Cells

were plated and allowed to proliferate for 18 hours (48 and 72 hours) before being fixed,

stained (MAP2) and analyzed for total neurite length. Measurements of neurites were made

using DIC. The MAP2 staining confirmed the neuronal identity of each neurite measured.

Measurements were made at later time points (48 and 72 hours) but by this time neurites

were too long and of rather convoluted morphology to be accurately assessed. Results

showed that BERP -/- neurons have a greater average total neurite length compared to

wildtype controls (15.28 µm ± 1.45 vs. 19.65 µm ± 0.65, p<0.05, Student’s t-test) (Figure

22). Although the difference is small, it is statistically significant at a 5% confidence limit. It

is also clear from these data that BERP deficiency was not a detriment or inhibitor of cortical

cell growth in culture.

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Figure 22. Neurite outgrowth measurements in BERP-deficient mice.

(A) Representative images of BERP +/+ (left panel) and BERP -/- (right panel) cortical

neurons derived from day 17.5 embryos at 18h after plating. Cells were stained for MAP2

(not visible) and imaged/measured using DIC optics (see text). Neurites were colour-marked

and tagged once measured to avoid duplication. Scale bar: 40mm. (B) Graphical

representation of the neurite outgrowth assay showing the mean total neurite length for BERP

+/+ (yellow column) and BERP -/- (blue column) neurons. Results shown are the means of

three independent experiments with at least 5 animals of each genotype; error bars represent 1

SEM. *p<0.05 (Student's t-test).

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3.3.8 BERP-deficiency has no impact on the self-renewal, differentiation,proliferation, viability and size of murine neural stem cells

In order to determine whether BERP plays a role in neural stem cell biology, we isolated

neural stem cells from the subventricular zone (SVZ) of the lateral ventricles of wild-type and

BERP-/- mice and examined their self-renewal capacity, proliferation, size, viability, and

differentiation potential. p53 has been shown to be a negative regulator of neural stem cell

self-renewal (Meletis et al. 2006) and since our laboratory has identified BERP as a possible

novel p53 interacter, we decided to also isolate neural stem cells from p53-/- and

BERP-/-;p53-/- double mutant mice for our studies. Results from neurosphere formation

assays (Figure 23A) indicate that neural stem cells derived from BERP-/- mice showed no

difference in self-renewal capacity compared to wild-type controls. Neural stem cells derived

from p53-/- mice showed an increase in self-renewal capacity compared to wild-type

controls, which supports the findings of Meletis et al. (2006). Neural stem cells from BERP-

/-;p53-/- double knockout mice also showed an increase in self-renewal capacity compared to

wild-type controls; however, no significant difference was seen when compared to p53-/-

mice. The proliferative capacity of dissociated stem cells was determined by thymidine

uptake (Figure 23B). Neural stem cells derived from BERP-/- mice showed no difference in

thymidine incorporation compared to wild-type controls. Neural stem cells derived from

both p53-/- and BERP-/-;p53-/- mice showed increased proliferative capacity compared to

wild-type controls; however, no significant difference was seen between cells derived from

p53-/- mice and those derived from BERP-/-;p53-/- mice. No difference in cell diameter

(Figure 23C) or viability (Figure 23D) was detected for any of the knockout mice compared

to wild-type controls. No significant difference in neuronal differentiation potential was seen

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in neural stem cells derived from any of the genotypes compared to wild-type controls

(Figure 23E). Thus, BERP-deficiency has no impact on the self-renewal, differentiation,

proliferation, viability and size of murine neural stem cells derived from the SVZ.

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Figure 23. Analysis of neural stem cells in BERP-deficient and p53-deficient mice. For

all panels, yellow bar = wild-type (WT); blue bar = BERP-/-; pink bar = p53-/-; green bar =

BERP-/-;p53-/-. (A) Neurosphere formation assay. Results shown are the average number of

secondary neurospheres per well relative to wild-type controls for each genotype (n=8 for

WT; n=7 for BERP-/-; n= 7 for p53-/-; n= 6 for BERP-/-;p53-/-); error bars represent 1 SEM.

*p<0.05 and **p<0.01, Student’s t-test. (B) Thymidine incorporation assay for dissociated

neural stem/progenitor cells from secondary neurospheres Results shown are the mean

thymidine uptake at 92h after plating for each genotype (n=7 for WT; n=7 for BERP-/-; n= 5

for p53-/-; n= 5 for BERP-/-;p53-.-) from 4 independent experiments each performed in

triplicate; error bars represent 1 SEM. *p<0.05 and **p<0.01, Student’s t-test. (C) Cell

viability assay. Secondary neurospheres were dissociated into single cells and viability was

determined using trypan blue exclusion. Results shown are the mean number of viable cells

expressed as a percent of total cell number for each genotype (n=5 for WT; n=4 for BERP-/-;

n= 6 for p53-/-; n= 6 for BERP-/-;p53-/-); error bars represent 1 SEM. (D) Cell size

determination. Results shown are the mean diameter (µm) of viable cells from dissociated

secondary neurospheres for each genotype (n=5 for WT; n=4 for BERP-/-; n= 6 for p53-/-;

n= 6 for BERP-/-;p53-.-); error bars represent 1 SEM. (E) Neurosphere differentiation assay.

Results shown are the proportion of _-III-tubulin positive cells (neurons) in secondary

neurospheres from each genotype (n=3 for WT; n=3 for BERP-/-; n=4 for p53-/-; n=3 for

BERP-/-;p53-/-); error bars represent 1 SEM. (F) Photomicrograph depicting an example of

beta-III-tubulin positive (arrowhead) and GFAP+ (arrow) cells from a differentiated

neurosphere.

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4 Discussion

4.1 What is the neurological phenotype of BERP-deficient mice?

BERP-/- mice are viable indicating that BERP is dispensable for normal development (Figure

10). In post-natal mice, BERP is highly expressed in the brain, especially in the cerebellum

and hippocampus. BERP-/- mice survived to maturity, were able to breed, and their brains

appear morphologically normal compared to wild-type controls. However, the fact that no

obvious morphological abnormalities were identified by gross and histological examiniation

does not preclude the possibility of neurological dysfunction. Therefore, we performed a

battery of tests in a cohort of BERP-deficient and BERP wild-type mice in order to assess

possible abnormalities in neurological function.

BERP-deficient mice exhibit enhanced learning ability

This was seen in fear conditioning tests as well as the accelerating rotarod test. In both the 24

hour context (which is hippocampus and amygdala-dependent) and cued (which is only

amygdala-dependent) fear conditioning tests, BERP-deficient mice exhibited increased

freezing behaviour (Figure 12). The possibility that pain perception may impact these results

was eliminated with the tail flick test that showed no effect of genotype on nociception

(section 3.3.2.8). A difference in hearing ability was also eliminated as a possible confounding

factor since no effect of genotype was seen in the acoustic startle response (Figure 17A). In

the rotarod test, BERP-deficient mice showed improvement in performance (as determined by

latency to fall) from day 1 to day 2 as well as from day 2 to day 3. In contrast, BERP wild-

type mice showed improvement only between day 1 and day 2; no further improvement was

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seen between day 2 and day 3 (Figure 16). It would be interesting to see whether with

continued testing, the BERP-deficient mice would be able to surpass the wild-type controls in

their ability to remain on the rotarod. These results suggest that BERP may be involved in

more than one type of learning: hippocampus and amygdala-dependent fear learning, as well

as motor learning. It also suggests that BERP is a negative regulator of these processes and

that removing BERP somehow enhances learning and memory.

BERP-deficient mice are more fearful and anxious

Factors that support increased anxiety in BERP-deficient mice include more time spent in the

border zone, fewer numbers of rearings, and decreased vertical activity during the first 5

minute period in the open field test (Figures 13B, 13C, 15B). An additional supporting factor

can be found in the cued fear conditioning test (Figure 12). No significant difference in

freezing between BERP-deficient and BERP wild-type mice was seen at baseline. However,

during the cued test, BERP-deficient mice showed increase freezing in the altered context

prior to the presentation of the conditioned stimulus. This would support a generally

heightened sense of fear in the BERP-deficient mice after receiving a foot shock due to a

general increase in anxiety. Factors that do not support an interpretation of increased anxiety

in BERP-deficient mice include no difference the time spent in the centre zone and in the

number of entries into the centre zone of the open field test (Figures 13A and 13D), and no

difference in the elevated plus maze test (Figure 14). Taken together, these results suggest

that BERP likely plays a role mediating anxiety-like behaviour.

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BERP-deficient mice have decreased motor coordination

This was well demonstrated in the accelerating rotarod test where BERP-deficient mice had

shorter latencies to fall compared to wild-type controls (Figure 16). A difference in muscle

strength was eliminated as a possible cause of the rotarod deficit since no effect of genotype

was seen in the grip test for muscle strength (section 3.3.2.7). This suggests that the decrease

motor coordination exhibited by BERP-deficient mice is likely cerebellar in origin.

BERP-deficient mice have increased resistance to PTZ-induced seizures

This trend was seen for all seizures types both in the proportion of mice succumbing to

seizures and in the latency to seizure (Figure 19). In addition, both these readout parameters

reached statistical significance for generalized seizures at a dose of 60mg/kg. This suggests

that BERP likely plays a role in epileptogenesis.

BERP-deficient mice may have decreased prepulse inhibiition of the acoustic startle reflex

The tendency for BERP-deficient mice to have decreased prepulse inhibition was seen at all

three prepulse sound levels (Figure 17B). Since the responses to the acoustic startle were

very similar between the two genotypes (Figure 17A), it is unlikely that issues involving

hearing impairment would confound the results. Therefore, this raises the possibility that

although the differences did not reach statistical significance, the observed tendencies may

have physiological significance. In light of the PTZ data supporting the increased resistance

to seizures, it is not surprising to find that the level of arousal in BERP-deficient mice is

lower than that found in the wild-type mice.

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In summary, we find that BERP-deficient mice exhibit enhanced learning ability, increased

fear and anxiety, impaired coordination, increased resistance to PTZ-induced seizures, and

possibly decreased prepulse inhibition of the acoustic startle reflex compared to wild-type

controls.

In light of these findings, future experiments using BERP-deficient mice may offer additional

illumination on the molecular basis of, for example, learning and memory. Results from both

the fear conditioning and accelerating rotarod tests suggest that BERP-deficient mice exhibit

enhanced abilities in emotional and motor forms of learning/memory. This implies that BERP

is a negative regulator of these processes and that removing BERP somehow enhances learning

and memory. Further exploration of BERP's role in this area can be pursued by examining

whether other components of memory (spatial, working, and reference) is also affected in

BERP-deficient mice. The biological basis for learning/memory formation in BERP-deficient

mice can be examined by studying synaptic plasticity, the ability to increase or decrease the

strength of synaptic connections, which can be demonstrated experimentally as long term

potentiation (LTP) and long term depression (LTD) in the classic hippocampal and cerebellar

paradigms, respectively. Therefore, a further dissection of BERP's role in memory formation

and retention can be pursued by examining whether LTP or LTD is enhanced in hippocampal

and cerebellar brain slice preparations derived from BERP-deficient and BERP wild-type

mice.

Interestingly, the regulation of emotions such as fear and anxiety has been shown to involve

the cerebral cortex, hippocampus, amygdala, and cerebellum (Schutter and van Honk 2005),

all areas that have high levels of BERP expression. As well, there is evidence indicating that

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those suffering from posttraumatic stress disorder have decreased hippocampal volume due to

the loss of dentrites (Radley et al. 2004; Bonne et al. 2008). Therefore, it would be

interesting to more closely examine the neuronal architecture of these structures in BERP-

deficient mice using Golgi staining of 100µm-thick brain sections. This is especially true

since the “classic” dendritic arbour architecture of the cerebellum lends itself quite well to the

examination by Golgi staining and any differences will likely reveal itself most easily in this

area of the brain.

4.2 Invoking Occam's razor: The BERP-deficient mouse as amodel for non-ataxic cerebellar dysfunction?

In discussing the phenotypic findings of the BERP-deficient mice, we will first take a

structure-function approach in this section, followed by a molecular approach in the next

section.

The neurological phenotype of the BERP-/- mice suggests the possibility of functional

abnormalities involving the hippocampus, amygdala, and cerebellum (Figure 24). This would

certainly make sense given that within the brain, BERP is highly expressed in these areas. If

we adopt the traditional view that the cerebellum is involved in motor coordination while the

hippocampus and amygdala (which form part of the limbic system) are involved in memory

formation and emotional responses, then the neurological phenotype of BERP-deficient mice

can mostly be linked to dysfunction these areas of the brain (Bear et al. 2001). The enhanced

fear/fear-driven learning and anxiety can quite easily be attributed to the

hippocampus/amygdala areas of the brain. Although the problem of understanding the true

nature of epileptogenesis is far from resolved, sclerosis (scarring) of the hippocampus is often

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associated with temporal lobe epilepsy (TLE), and patients with poorly controlled TLE often

benefit from surgical removal of temporal lobe structures including the hippocampus and

amygdala (Parrent and Lozano 2000; Blumcke et al. 2002). In addition, abnormalities of the

limbic system are also commonly thought to be involved in schizophrenia (Goldman and

Mitchell 2004). This leaves the cerebellum as the source of dysfunction for the impaired

performance of the BERP-deficient mice in the accelerating rotarod test. This interpretation

could leave us with the very reasonable assertion that BERP-deficient mice exhibit

phenotypic traits consistent with functional abnormalities of the hippocampus, amygdala,

and cerebellum.

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Figure 24. Neuroanatomical structures. Select anatomical structures within the human

brain. Please refer to text for details. Adapted from the American Health Assistance

Foundation (source: http://www.ahaf.org/alzheimers/about/understanding/anatomy-of-the-

brain.html).

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However, an alternative interpretation can also be made that ties all of these findings to the

cerebellum. According to long-standing tradition, the cerebellum is primarily thought to be

involved in movement, coordination and motor learning. However, in recent years, there is

mounting evidence that support an expanded role for the cerebellum. It is now implicated in

emotional/cognitive processes such as the formation of fear-memories, which were

traditionally thought to reside primarily within the limbic structures (Sacchetti et al. 2002).

Cerebellar abnormalities are present in neuropsychiatric disorders including anxiety,

schizophrenia and autism (Schutter and van Honk 2005; Andreasen and Pierson 2008).

Patients with epilepsy often have cerebellar atrophy; whether this is due to recurrent

seizures, longterm anticonvulsant therapy, or other factors is unclear. However, stimulation

of the cerebellum has the ability to stop seizure activity suggesting a role for the cerebellum in

modulating seizures (Velasco et al. 2005). So while many reported instances of cerebellar

dysfunction involve some degree of motor abnormality such as ataxia or tremor, the absence

of such findings does not exclude the possibility of cerebellum-based pathophysiology. For

example, there exists also the Cerebellar Cognitive Affective Syndrome (CCAS) that is

characterized by non-motor manifestations (Schmahmann and Sherman 1998) such as deficits

in executive function, cognition, language, and affect. This suggests that a cerebellar-centred

explanation for the observed phenotype in BERP-deficient mice is possible despite the lack

of an overt movement abnormality. In this way, BERP-deficient mice may represent a novel

model system for the study of non-ataxic cerebellar dysfunction.

To answer the question of whether the neurological phenotype seen in the BERP-deficient

mice can be attributed to cerebellar dysfunction (ie. whether the application of Occam's razor

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is truly appropriate in this case), one could generate a mutant mouse in which loss of BERP

can be anatomically and temporally controlled. This can be accomplished using the Cre/loxP

system in which a BERP conditional knockout mouse would be crossed with one that

expresses a Cre transgene under the transcriptional control of a cerebellum-specific gene

(Sandberg et al. 2000; Suzuki et al. 2004). The addition of an inducible element would

provide further specificity and power to such a system (Tsujita et al. 1999) in that BERP

deficiency could then be introduced in a mature animal. Depending on the results, further

refinement can then be achieved by examining the loss of BERP in other anatomical regions

such as the hippocampus or cortex, either on their own, or in combination with loss of

function in the cerebellum (Tsien et al. 1996; Guo et al. 2000).

4.3 What is the possible role of BERP in the CNS?

In attempting to define a possible molecular role for BERP in the CNS, several pieces of

evidence should be examined. BERP associates with myosin V via its c-terminal domain (El-

Husseini and Vincent 1999). Myosin V has been shown to be involved in the intracellular

trafficking of organelle and receptors (Provance and Mercer 1999; Wang et al. 2008). Both

BERP and myosin V are members of the CART complex, which is involved in the

constitutive recycling of transferrin receptors in HeLa cells (Yan et al. 2005). This raises the

possibility that the CART complex, and therefore BERP, may be involved in the intracellular

trafficking of other cell surface receptors that undergo constitutive endocytosis. Our results

from patch-clamp recordings show decreased mIPSC amplitude in brain slices derived from

BERP-/- compared to wild-type controls (Figure 20). Western blot analyses show decreased

surface expression of gamma2 subunit-containing GABAARs in cultured neurons derived

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from BERP-/- embryos compared to those from wild-type controls (Figures 21A and 21B).

Taken together, these data suggest BERP-/- mice have decreased GABAAR function in the

brain due to decreased numbers of GABAARs. In addtion, no statistically significant

difference was seen in GABAAR gamma2 subunit mRNA expression from the brains of

BERP-/- embryos compared with wild-type controls (Figure 21C), suggesting that the

decrease in GABAARs is likely a posttranscriptional phenomenon. Therefore, we

hypothesize that BERP is involved in the intracellular trafficking of GABAARs. If the

CART complex (or an analogous structure) exists in neurons and performs a similar function

to the one described for the transferrin receptor, then it would suggest that BERP is involved

in post-endocytic recycling of the GABAAR. BERP was identified as a possible

phosphoprotein in a large-scale analysis of proteins isolated from murine post-synaptic

densities (Trinidad et al. 2006). This suggests that in a phosphorylated state, BERP may

have a synaptic location. At this point however, without further studies examining the

effect(s) of BERP on GABAAR levels/distribution and possible physical interactions

between BERP and components of the GABAAR network, it is premature to try and

precisely place BERP into one of the five stages of the GABAAR lifecycle as described in

section 1.5.3.2.

4.4 Is BERP involved in neurite outgrowth?

Our results from cortical neuron cultures indicate that although the difference is small, neurite

outgrowth is enhanced in neurons derived from BERP-/- murine embryos (Figure 22). Van

Diepen et al. reported that deficiency (via knockdown by double stranded RNA) of L-TRIM,

the molluscan ortholog of mammalian TRIM2 and TRIM3, led to decreased neurite

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outgrowth in neurons cultured from the snail Lymnaea stagnalis (van Diepen et al. 2005).

This apparent discrepancy may represent differences in neuronal development between snails

and mice. In addition, in vitro knockdown of L-TRIM expression would likely elicit different

compensatory responses than a genetic deletion of TRIM3 present from the earliest stages of

development. In our studies, although neurite outgrowth was enhanced in ex vivo BERP-

deficient neuronal cultures, ultimately, the brains of the fully developed knockout animals

were morphologically indistinguishable from wild-type controls. This suggests that despite a

statistically significant difference in an ex vivo environment, the effect of BERP deficiency on

neurite outgrowth is not biologically significant in vivo. This again supports the importance

of considering biological compensation in the analysis of gene-deletion mutant animals.

4.5 Is BERP a functional homolog of D. melanogaster brat?

Brat is a D. melanogaster tumour suppressor that functions as a translational repressor of

hunchback (Sonoda and Wharton 2001) and as a negative regulator of ribosomal RNA

synthesis (Frank et al. 2002); it is also a negative regulator of self-renewal in Drosophila

neuroblasts (Betschinger et al. 2006). In addition, through the utility of an in vivo genetic

modifier screen, our laboratory determined that brat is a possible novel interacter of

Drosophila p53. Therefore, the identification of a brat mammalian homolog with similar

functions could provide exciting new opportunities to study abnormalities of the nervous

system. Brat belongs to a family of proteins that contain N-terminal zinc-finger and coiled

coil domains with a C-terminal NHL domain (Arama et al. 2000). The mammalian homologs

of brat are the three TRIM-NHL proteins: BERP/TRIM3, NARF/TRIM2, and

HT2A/TRIM32 (Reymond et al. 2001). Homologs can be either orthologs or paralogs (Fitch

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2000). Orthologs arise from speciation event(s) and likely have similar or analogous

functions. Paralogs arise from duplication event(s) and are less likely to have similar

functions to each other. Therefore, in the search of a mammalian homolog with similar

functions to brat, we employed the best reciprocal hit (BRH) strategy, which is commonly

used by biologists for identifying orthologs to study (Hulsen et al. 2006; Moreno-Hagelsieb

and Latimer 2008). In the BRH method, two cross-species proteins are considered orthologs

if they are each other's top hit using the National Center for Biotechnology Information's

(NCBI) basic local alignment search tool for proteins (BLASTP). In attempting to apply the

BLASTP BRH strategy to determine the mammalian brat ortholog, we encountered an

interesting situation. The top mammalian hit for brat was BERP/TRIM3 followed by

NARF/TRIM2. However, the top Drosophila hit for BERP/TRIM3 was a protein named

abba; brat was second. Therefore, brat does not have a BLASTP BRH partner in mammals.

In this scenario, we instead opted to use the best unidirectional BLASTP hit for brat and thus

chose BERP as the molecule upon which to focus our studies.

In our experiments on neural stem/progenitor cells isolated from BERP-deficient mice, no

effect of genotype on the self-renewal capacity, differentiation, and proliferation was seen

(Figure 23). Neural stem/progenitor cells from p53-deficient mice showed a dramatic

enhancement of self-renewal capacity which supports previously published reports (Meletis

et al. 2006). However, although neural stem/progenitors isolated from BERP-/-;p53-/- double

knockout mice showed a trend towards even greater increases in self-renewal and proliferative

capacity compared to those from p53-/- mice, the difference was not statistically significant

(Figure 23A and 23B). Published reports for NARF/TRIM2 indicate that it plays a role in

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neurodegeneration, possibly due to the accumulation of neurofilament light chain (Balastik et

al. 2008). A recently published report shows that HT2A/TRIM32, in a mammalian setting,

has very similar functions to brat in that it is able to inhibit cell proliferation, to undergo

asymmetric localization during mitosis, and to negatively regulate the self-renewal capacity of

murine neural stem/progenitor cells (Schwamborn et al. 2009). Therefore, although

experimental methodologies differed, based on the most current information to date, it

appears that of the three mammalian TRIM-NHL proteins, HT2A/TRIM32 has the most

analogous function to D. melanogaster brat and it is likely that these two molecules have an

orthologous relationship. In this context, it would appear that NARF/TRIM2 and

BERP/TRIM3 are likely paralogs of brat.

4.6 Why do knockout mice from the TRIM-NHL family notdevelop brain tumours?

Due to their homology to D. melanogaster brat (brain tumour), the mammalian TRIM-NHL

proteins continue to carry with them the expectation of tumourigenesis. Interestingly though,

none of the TRIM-NHL gene-deficient mice develop malignancies. This raises the possibility

of in vivo compensation with redundant molecules providing the "missing" tumour

suppressor functions. If this compensation is provided for by the other TRIM-NHL

proteins, then it is possible that simultaneous deficiency of more than one family member

may be required reveal a tumourigenic phenotype.

In addition, generated mice lacking a particular gene can have different phenotypes than their

human counterparts. A good example of this phenomenon is the p53 knockout mouse, which

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tends to develop lymphomas and sarcomas while in humans, p53-deficiencies tend to be

associated with carcinomas.

The timing of loss may also be important; an organism is more likely to be able to compensate

for early germline losses rather than later somatic losses that occur after "roles" and pathways

have already been determined. Such may be the case when comparing the lack of tumours in a

classical knockout mouse to LOH of a particular gene in human tumours. For example, BERP

(as TRIM3) was recently identified as the candidate tumour suppressor gene associated with

LOH of 11p15.5 found in 70 human malignant gliomas (Boulay et al. 2009); however, BERP-

deficient mice do not develop gliomas.

Lastly, in the generation of a classical knockout mouse, unless the size of the targeted locus is

small enough such that it can be entirely replaced/eliminated, then the possibility of residual

protein function should be considered. In keeping with standard and commonly employed

targeting strategies for creating null alleles, the BERP knockout mouse described in this thesis

was generated by replacing most of coding exons 2-5 (half the RING finger, all of the B-box,

coiled coil, and filamin/ABP280 domains) with a neomycin cassette (Figure 9A). In addition,

a stop codon was introduced at the start of exon 2 just after the splice acceptor site. Using a

mouse monoclonal antibody against the N-terminal aspect of BERP (BD Biosciences), the

82kDa band representing BERP was eliminated in the knockout mice (Figure 9C). As with

any gene-targeted mouse, there is always the possibility that any part of the gene not directly

replaced/eliminated by the targeting strategy could end up encoding partial proteins. In this

case, since the NHL domain was not replaced in the targeting strategy, the possibility of a

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partial protein with residual function can only be eliminated with an antibody capable of

detecting the C-terminus of BERP.

4.7 The p53/BERP /GABAAR relationship - A novel role for p53in the brain?

Riley et al. recently published a comprehensive overview of 129 known p53-regulated genes.

In order to be included in their analysis, a gene had to fulfill at least three of the following four

criteria: 1) "...the presence of a p53RE in the DNA close to or in the gene", 2) "...a

demonstration that the gene is either upregulated or down regulated at the RNA and protein

levels by the activated wild-type p53 protein (but not by the mutant protein)", 3) "...to clone

the p53RE from that gene, place it near a test gene, such as luciferase, and demonstrate that

the p53 protein can regulate the test gene", and 4) "...to use chromatin immunoprecipitation

with a p53-specific antibody to demonstrate the presence of the p53 protein on the RE site in

the DNA" (Riley et al. 2008). Our study of the human BERP genomic locus revealed the

presence of four putative p53RE (Figure 5B). One (BERP site B) is located upstream of exon

1 while the other three (BERP sites E, G, F) are located within intron 1 (Figure 5A). We

show that BERP expression is upregulated after 5FU treatment in the HCT116 cells that

contain wild-type p53 but not in isogenic cells that contain mutant p53 (Figure 7). In

addtion, we show upregulation of BERP expression in the brains of p53+/+ mice but not p53-

/- after PTZ injection (Figure 8). In a luciferase assay, BERP site E was able to support

transcription in the presence of wild-type but not mutant p53 (Figure 6B); a C → A point

mutation at position 4 rendered the site unresponsive to transcription regardless of p53 status

(Figure 6C). A chromatin immunoprecipitation assay using a p53-specific antibody (DO-1)

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shows that p53 can bind to BERP site E in HCT116 containing wild-type p53 but not in

HCT116 cells with mutant p53 (Figure 6A). These data fulfill the criteria set out by Riley et

al. and support the interpretation that BERP undergoes p53-dependent transcriptional

activation and is indeed a novel p53-regulated gene.

Since PTZ is a modulator of GABAAR function, its ability to upregulate BERP expression in

a p53-dependent manner suggests that: 1) PTZ is a novel activator of p53, 2) p53 is

downstream of GABAAR function, 3) BERP is downstream of p53. Also, based on the

evidence presented in Section 4.3 (“What is the possible role of BERP in the CNS?”), we

hypothesized that BERP is involved in receptor trafficking of the GABAAR. Taken

together, this suggests a feedback loop in which PTZ, acting as a stress to the neuroelectrical

homeostasis of the neuron, inhibits GABAAR function. This triggers the activation of p53

which in its capacity as a transcription factor, upregulates expression of its target gene BERP.

BERP then exerts its effects on the cell surface expression of GABAARs.

The existence of this new GABAAR - p53 - BERP pathway implies that p53 is involved in

the regulation of PTZ-induced effects on BERP. This suggests that p53, through BERP,

plays a novel role in the regulation of GABAAR signaling and seizure susceptibility in the

CNS. Further studies are required to more precisely elucidate the functions of p53 and BERP

in GABAAR signaling and their contributions to epileptogenesis.

4.8 PTZ – A novel therapy in the battle against cancer?

While approximately 50% of human cancers contain p53 mutations, this means that the other

50% does not. However, in the tumours that still retain wild-type p53, the function of p53 is

135

often abrogated due to signaling or regulation defects (Hainaut and Hollstein 2000).

Therefore, the recent demonstration that re-activation of p53 leads to tumour regression

(Ventura et al. 2007; Xue et al. 2007) underscores the importance of identifying compounds

capable of restoring p53 function in the roughly 50% of all human cancers that still retain

wild-type p53.

Triggers that activate the p53 pathway can be genotoxic (e.g. ionizing irradiation, ultraviolet

irradiation) or non-genotoxic (e.g. hypoxia, ribonucleotide depletion, microtubule disruption,

oncogene activation, replicative senescence). Genotoxic activators, including radiotherapy and

many chemotherapeutic drugs in current use, involve DNA damage, which can injure normal

tissues and may result in the development of secondary cancers. Therefore, non-genotoxic

compounds capable of activating p53 without the negative consequences associated DNA

damage would be therapeutically ideal.

PTZ, commonly used to generate seizures in animal models, is a compound that antagonizes

the functions of GABAARs (Huang et al. 2001) and does not cause neuronal apoptosis

(Planas et al. 1994; Valente et al. 2004; Koeller et al. 2008). Our results show that in

response to PTZ injection, BERP is upregulated in vivo in a p53-dependent manner (Figure

8). This suggests that PTZ is a novel activator of p53 transactivation functions. Since it does

not cause neuronal death, subconvulsive doses of this, or a pharmacologically similar

compound, may be effective in the treatment of human cancers and have the advantage of

being free from major side effects. Further studies are required to determine whether PTZ

should be classified as a genotoxic or non-genotoxic compound and whether it can activate a

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form of p53 with the appropriate posttranslational modifications that will lead to tumour

regression in vivo.

4.9 Closing Remarks

p53 is a central player in an organism's ability to respond to a variety of cellular stresses. Its

success as a tumour suppressor is contingent on its ability to preserve proliferative and

genomic stability in the face of many such insults. The identification of novel molecules

within the p53 network allow us the opportunity to expand our understanding of both known

and as yet unknown functions of this important transcription factor.

Prior to this work, there was no previously reported association between p53, BERP and

GABA A receptors. The findings in Chapter 2 support the interpretation that BERP is a

novel transcriptional target of p53 and that PTZ may be a novel activator of p53. The

findings in Chapter 3, through the generation and characterization of a BERP-deficient mouse

model, suggest that while BERP is dispensable for normal development, fertility, longevity,

tumour suppression, and adult neural stem cell renewal and differentiation, it may play a role

in fear learning and memory, seizure susceptibility, and the trafficking of GABA A receptors

in the brain. Taken together, this suggests a role for BERP in the modulation of GABA A

receptor-mediated seizure susceptibility as well as in p53 signaling. The convergence of these

two fields, through BERP, suggests that BERP may represent an important link to the

identification of a novel role for p53 in the regulation of "neuroelectrical homeostasis" in the

brain.

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