Deficiency in Mbd2 Is Sufficient to Cause Behavioral Impairments in Mice

DEFICIENCY IN MBD2 IS SUFFICIENT TO CAUSE BEHAVIORAL IMPAIRMENTS IN MICE

Lidiya Zavalishina

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto

DEFICIENCY IN MBD2 IS SUFFICIENT TO CAUSE

BEHAVIORAL IMPAIRMENTS IN MICE

Master of Science Graduate Department of Physiology University of Toronto 2010

ABSTRACT

Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor

molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain

function and behavior remains largely unexamined. To begin elucidating whether MBD2

influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared

their hippocampal electroencephalographic activity during exploration, and performed protein

and mRNA expression assessments. The results indicate that mutant mice display a heightened

anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-

type mice. However, these behavioral differences were not paralleled by neurophysiological

impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2

mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was

significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the

hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce

behavioral impairments in mice without introducing gross deficits in hippocampal

neurophysiology.

ACKNOWLEDGEMENTS

I am deeply thankful to many great people who have guided me through this science journey, helped me answer my never ending questions, and supported me. One person who inspired and taught me the most is my supervisor, Dr. James Eubanks. I was extremely fortunate to have Dr.

Eubanks as my mentor. Since the moment we met Dr. Eubanks never ceased to teach me how to

really think about science. His scientific curiosity, intelligence and “contagious” passion for

science served as my main source of inspiration throughout this project. Thank you Dr. Eubanks!

I am also grateful to my caring and brilliant committee members, Dr. Liang Zhang, Dr. Sheena

Josselyn and Dr. Mac Burnham, for their support and guidance. Their insightful comments and

words of encouragement not only had an impact on my project but also on my life. I would like

to thank the members of my lab, Richard Logan, Guanming Zhang, Andreea Popescu, Elena

Sidorova, Ewelina Maliszewska-Cyna, Jennifer D’Cruz, and Carl Fisher for teaching me new

methods and techniques and for making my time in the lab pleasant and exciting. I would also

like to thank the members of Dr. Liang Zhang’s lab. Chiping Wu, Joe Hayek and Liang Zhang

have provided help and support with electrophysiology related experiments that quite truthfully,

scared me at first. Thank you for knowing when to provide me with an insightful advice or a box

of chocolate.

I would also like to thank the Ontario Rett Association for their sincere interest in our work and

for giving me the privilege to meet the girls and their families. These beautiful girls were my main source of inspiration throughout this project. I dedicate my work to them.

I am thankful to my family who has always wholeheartedly supported me in everything that I do.

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TABLE OF CONTENT

Title Page...... I

Abstract...... II

Acknowledgements...... III

Table of Content...... IV

List of Figures……...... IX

List of Abbreviations...... XI

1. Introduction...... 1

1.1. Epigenetics and DNA methylation……………………………………..……………..….1

1.1.1 Overview of Epigenetics………………………………………………….……...... 1

1.1.2. DNA methylation-mediated transcriptional repression……………….….………..2

1.2. Example of an epigenetic regulator: MeCP2 and Rett Syndrome (RTT)……..….……....4

1.3. Autism Spectrum Disorders (ASD) and Epigenetics…………………………...….……..8

1.3.1. Overview of Autism Spectrum Disorders…………………………………………8

1.3.2. Causes of Autism Spectrum Disorders……………………………….……………9

1.3.3. Mouse models of Autism Spectrum Disorders……………………..…………….10

1.3.4. ASD, MBD proteins and Electroencephalographic abnormalities…….………....10

1.4. Methyl-CpG-binding proteins …………………………………..………………………11

1.4.1.Methyl-CpG-binding domain protein 1 (MBD1)…………………………....……15

1.4.2. Methyl-CpG-binding domain protein 3(MBD3)……...…………………...……..16

1.4.3. Methyl-CpG-binding domain protein 4 (MBD4)…………………………...……16

1.4.4. Kaiso protein…………………………………………………………………..…17

1.5. Methyl-CpG-binding domain protein 2 (MBD2)…………………………...…..……....17

1.5.1.MBD2 and maternal behavior……………………………………………….……18

1.5.2. MBD2 and immune system………………...…………………………………….19

1.5.3.The role of MBD2 in embryogenesis and postmititic tissues…………………….19

1.5.4. MBD2 as a demethylase enzyme and its role in tumor suppression……………..20

1.6. Hypothesis and the goals of my project…………………………………………………22

2. Materials and Methods...... 23

2.1. Animal subjects...... 23

2.1.1. Generation of Mbd2 (-/-) mice...... 23

2.1.2. Genotyping...... 26

2.1.3. Statistical Analysis…………..…………………………………………...... …..……...…27

2.2. Behavioral Assessments...... 28

2.2.1. Motor Function Assessments ...... 28

2.2.1.1. Open Field Apparatus ………………………………………………………….28

2.2.1.2. Coordination and Balance Function Rotarod Test …………………………….29

2.2.2. Socialization Assays………………………………………………………….……….32

2.2.2.1. Socialization Test………………………………………………………………32

2.2.2.2. Nesting Test…………………………………………………………………….33

2.2.3. Novel Object Recognition Task……………………………………………….....……34

2.2.4. Anxiety-like Behavioral Assessments………………………………………………...35

2.2.4.1. Light and Dark Preference Test……………………………………………...…35

2.2.4.2. Elevated Plus Maze Assessment……………………………………………….36

2.3. Electroencephalographic (EEG) Assessments…………………………………………..39

2.3.1. Electrode Implantation…………………………………………………..……….39

2.3.2. EEG Recordings………………………………………..……………….………..39

2.3.3. EEG Data Analysis………………………………………………….….………...40

2.4. Western Blot ………...…………..………………………………………………...……..41

2.4.1. Tissue harvesting …………………………………………………………………41

2.4.2. Preparation of protein samples for western blots………………………………….41

2.4.3. Protein concentration measurement for western blots…………………………….….41

2.4.4. Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis ……………….42

2.4.5. Densitometric analysis…………………………………………………………….43

2.5. Quantitative -Real -Time PCR……………………………………………….………..…44

2.5.1. Samples preparation and specific primers used……………………...…..……….44

2.5.2. PCR running conditions and data analysis……………………………….………..44

3. Results ………………………...... 46

3.1. Mbd2-deficient animals display behavioral alteration………………………………………46

3.1.1. Mbd2-deficient animals display impaired motor performance………………..…..46

3.1.1.1. Impaired Activity Distribution...... 46

3.1.1.2. Impaired Mobility Distribution………………………..…………………….46

3.1.1.3. Mbd2 null mice show enhanced motor coordination performance…….……53

3.1.2 Mbd2- deficient mice show impairments in social interactions……………….….56

3.1.2.1. Mbd2 null mice spent less time socially interacting…………….….…...... 56

3.1.2.2. Mbd2 null mice initiated fewer social interactions………………….…....…56

3.1.2.3. Mbd2 null mice displayed more Social Avoidance Behavior……….…...….59

3.1.3. Mbd2 null animals display deficits in nest-building behavior………………..….64

3.1.3.1. Diminished interaction with the neslet……………………………………...64

3.1.3.2. Impaired nest quality………………………………………………………...64

3.1.4. Mbd2- null mice display increased anxiety-like behavior………..…………..…..72

3.1.4.1. Increased anxiety-like behaviors during Light Dark Preference Test………72

3.1.4.1.1. No differences in Light and Dark chamber preference…………….….72

3.1.4.1.2. Impaired Risk Assessment behaviors…………...…….……………....72

3.1.4.2. Increased anxiety during Elevated Plus Maze testing……………………….77

3.1.5. Mbd2- deficient mice show deficits in object recognition…………..….....…….82

3.2. Electroencephalographic (EEG) Assessments…………………………………….………...85

3.2.1. Mbd2-deficient mice display normal theta waveform during exploration…….…85

3.3. Assessment of MeCP2, MBD1, MBD2, MBD3 and GR mRNA expression levels in Mbd2 null neural tissues………………………………………………...……94

3.3.1. MeCP2 mRNA levels are elevated in the cortex of Mbd2-null mice……….…..94

3.3.2. Reduced GR mRNA expression in neural Mbd2-null tissues…………....……...94

3.4. Assessment of MeCP2 protein expression levels in the cortex, hippocampus and cerebellum of Mbd2 null and wild-type mice samples………….………………………………….99 4. Discussion ………..………...... 107

4.1. MBD2-deficient mice display behavioral alterations...... 108

4.1.1. Mutant mice show locomotion alterations…………………………………………108

4.1.1.1. Mbd2 null mice exhibit motor impairments...... 108

4.1.1.2. Mbd2 null mice show enhanced coordination ability...... 109

4. 1. 2. Socialization behaviors are deficient in Mbd2 null mice…………...………….…111

4.1.2.1. Nesting behavior is impaired in Mbd2 null mice……………….………..111

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4.1.2.2. Impairments in sociability and preference for social novelty……...…….112

4.1.3. MBD2-deficient mice display elevated anxiety-like behaviors……………...…….114

4.1.4. Mbd2 null mice exhibit recognition memory impairments………….....…………..115

4.2. Preservation of basic Electroencephalographic (EEG) activity in Mbd2 null mice…….....116

4.3. The mRNA and protein expression of candidate systems in Mbd2 null neural tissues…....117

4.3.1. Increased MeCP2 mRNA expression levels in Mbd2 null derived cortical tissues..118

4.3.2. MeCP2 Protein expression alterations in Mbd2 null derived tissues…..……….....118

4.4. Proposed models for observed behavioral alterations……………………………………..120

4.4.1. Could alterations in Glucocorticoid Receptor expression account for impaired behavior of Mbd2 null mice? ………………………..………………….121

4.4.2. Could MeCP2 changes in Mbd2 null neural tissues account for observed behavioral deficits?...... 124

4.5. Future Directions and Potential Clinical Implications……………………………………128

4.5.1. MBD2 is a potential candidate for studying ASD………………………………128

4.5.2. MBD2 ablation as a potential cancer treatment……………………….…………129

References…………………………………………….…………………………….…...……..130

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LIST OF FIGURES

Introduction

Figure 1: Function and structure of MeCP2 and MeCP1…………………..……………………..6

Figure 2: Structure of MBD proteins and Kaiso……………………..…………………………..13

Materials and Methods

Figure 3: Generation of Mbd2 (- /+) ……………………………...………………………….….24

Figure 4: Pictures of behavioural assays used in this study……………………………………...30

Figure 5: Diagram of the Elevated Plus Maze…………………………………………………...37

Results

Figure 6: Activity Distribution of Mbd2 null and their age-matched wild-type control mice assessed during a 60 minute Open Field test……..…………...………...47

Figure 7: Rearing behavior displayed by mutant and wild-type mice…………………………...49

Figure 8: Mobility Distribution expressed as total mobility, fast mobility and slow mobility in wild-type and Mbd2 null mice……………………………….…………..………….51

Figure 9: Motor coordination performance on the rotating rotarod……………………………...54

Figure 10: Amount of time wild-type and Mbd2 null mice spent interacting with another mouse during the two trials of the Social Interaction test………………………….…..57

Figure 11: Number of interactions wild-type and Mbd2 null mice initiated during the two trials of the Social Interaction test……………………………………………………60

Figure 12: Social Avoidance Behavior displayed by Mbd2 null and wild-type age matched control mice during the two trials of the Social Interaction Assay…………62 . Figure 13: Nesting behaviors performed by wild-type and Mbd2 null mice…………...…….….66

Figure 14: Pictures of the nests 24 hours after a new neslet was introduced to the cage…….….68

Figure 15: The resultant nests made by wild-type and Mbd2 null mice…………………………70

Figure 16: Time spent in the Light and Dark Compartments………………………………...….73

Figure 17: Risk Assessment behaviors performed during Light and Dark behavioral test….…..75

Figure 18: Total amount of time wild-type and mutant mice spent in the Middle Platform, Open Arm, and the Closed Arm of the Elevated Plus Maze……...…………...…….78

Figure 19: Time spent in the distal area expressed as a fraction of total time in the Closed arms of the Elevated Plus Maze……………………………….………...…...80

Figure 20: Novel Object Recognition Test…………………………………………………..…..83

Figure 21: Hippocampal theta rhythm in wild-type mice during home cage exploration…………………………………………………………...………….86

Figure 22: Hippocampal theta rhythm in Mbd2-deficient mice during home cage exploration……………………………………………………………………....88

Figure 23: Sample of a Power Spectrum analysis of theta rhythm for wild-type and Mbd2 null mice (8-13 months)………..……………………………………………..90

Figure 24: Peak theta frequency observed during exploration in wild-type and Mbd2 null mice (8-13 months)…………...... 92

Figure 25: mRNA expression levels of MeCP2, MBD1, MBD2, and MBD3 in the cortex, hippocampus and striatum in Mbd2 null and wild-type mice……………...…96

Figure 26: mRNA expression levels of Glucocorticoid Receptor (GR) in the cortex, hippocampus and striatum relative to Hprt expression……………………………….98

Figure 27: MeCP2 protein expression in the hippocampus of wild-type and Mbd2 null mice...101

Figure 28: MeCP2 protein expression in the cortex of wild-type and Mbd2 null mice…….…..103

Figure 29: MeCP2 protein expression in the cerebellum of wild-type and Mbd2 null mice…...105

Discussion

Figure 30: Proposed mechanisms of MBD2 transcriptionally regulating GR and MeCP2…….126

List of Abbreviations A: Adenine ASD: Autism Spectrum Disorder C: Cytosine cDNA: complementary DNA CGIs: CpG islands CORT: cotricosterone CREB1: CAMP responsive element binding protein 1 Ct: cycle threshold C-terminus: Carboxy-terminus CxxC: cysteine-rich regions zink-finger motif DI: Discrimination Index DNA: Deoxyribonucleic acid DNMT1: DNA methyltransferase 1 DNMT3A: DNA methyltransferase 3A DNMT3B: DNA methyltransferase 3B EB: Elution buffer EEG: Electroencephalography Fmr1: a gene encoding for a protein called fragile X mental retardation protein in mouse GAPDH: Glyceraldehyde-3-phosphate dehydrogenase G: glycine residue HDAC: Histone deacetylase HPA: hypothalamic-pituitary-adrenal axis Hprt: Hypoxanthine-guanine phosphoribosyltransferase Htr2c: serotonin 2c receptor IRSF: International Rett Syndrome Foundation Il4: interleukin-4 MBD: Methyl-CpG binding domain MBD1: Methyl-CpG-binding domain protein 1 MBD2: Methyl-CpG-binding domain protein 2 MBD3: Methyl-CpG-binding domain protein 3 MBD4: Methyl-CpG-binding domain protein 4 MeCP1: Methyl CpG binding protein 1 MeCP2: Methyl CpG binding protein 2 MECP2: gene encoding methyl CpG binding protein 2 in humans Mecp2: gene encoding methyl CpG binding protein 2 in mouse mg: Milligram mM: Millimolar mRNA: Messenger ribonucleic acid N-terminus: Amino terminus NGF1-A: nerve growth factor -inducible protein-A NMD: Nonsense-mediated mRNA decay NLGNs: neuroligins NPC: No Primer Control NRXNs: neurexins

NR3C1: neuron-specific glucocorticoid receptor promoter NTC: No Template Control NuRD: Nucleosome remodelling and histone deacetylase activity complex OD: Optical densitometry R: arginine residue RbAp46: retinoblastoma suppressor (Rb)-associated protein 46 RbAp48: retinoblastoma suppressor (Rb)-associated protein 48 PBS: Phosphate buffered saline PCR : Polymerase chain reaction PTC: Premature termination codon RT-PCR: Real time polymerase chain reaction RTT: Rett Syndrome Q: Quadrant SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sin3A: SIN3 homolog A, transcriptional regulator siRNA: small interfering RNA T: thymine TCA: total time in the closed arms TOA: total time in the open arms TMA: total time in the middle arms TRD: Transcriptional repressor domain TRIS: Tris(hydroxymethyl)aminomethane Ube 3A: ubiquitin protein ligase 3A μg: microgram V: volt WT: Wild-type Zf: zinc finger

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Chapter 1

Introduction

1.1. Epigenetics and DNA methylation

1.1.1. Overview of Epigenetics

Epigenetics is a term used to describe the process of functionally relevant genome

modifications that do not include changes in the nucleotide sequence directly (Zhang et al.,

2010). Shown to result in gene expression changes, the concept of epigenetics is now widely

investigated. A predominant epigenetic mechanism is DNA methylation. In the mammalian

genome it is accomplished by covalent modification at the fifth carbon (C5) on the cytosine

residues in CpG dinucleotides. These CpG dinucleotides are not evenly distributed across the

human genome, and are often concentrated in dense pockets called CpG islands (CGIs). Most

CpG sites are methylated at a frequency of 60% - 90% (Bird et al., 1980), while CpG islands,

which are found in promoters of highly expressed genes are not methylated (Cross and Bird,

1995). DNA methylation is accomplished and maintained by three DNA methyltransferases

which are DNMT1, DNMT3A and DNMT3B. DNMT1 is the most abundant methyltransferase in somatic cells and has a high preference for hemimethylated DNA ( Robertson et al., 1999). As

a result, it is also known as a maintenance methyltransferase as it transfers patterns of

methylation to a newly synthesized strand after DNA replication (Robertson et al., 2000).

The importance of proper methylation became appreciated when mice lacking DNMT1

displayed abnormal development and died prematurely (Li et al., 1992). In addition, mice

lacking DNMT1 and DNMT3, specifically in the forebrain postnatal postmitotic neurons

displayed abnormalities in cell size, neural plasticity and showed defects in learning and memory

(Feng et al., 2010). DNMT3A and DNMT3B are de novo methyltransferases that are responsible

for establishing global cytosine methylation patterns at unmethylated DNA cites during early

embryogenesis. Usually methylation patterns are stable and heritable in all somatic and

differentiated cells and are maintained by DNMT1. However during cell’s developmental stages,

the process of reprogramming (dynamic processes of methylation and demethylation) take place

with the help of these de novo methyltransferases (Wolf et al, 2001), (Kim et al., 2009).

1.1.2. DNA methylation-mediated transcriptional repression

The primary function of DNA methylation is to repress transcription. This is done with

the help of specific transcriptional repressors that recognize methylated CpG sites, recruit other

factors and turn off transcription. This function of methylated dependent transcription was first

shown when methylation of CpG sites on the gene promoter resulted in the lack of transcription

of that gene. In contrast, non-methylated promoter sites on the genes were associated with

transcriptional activation (Bestor and Tycko, 1996). A few years after, the importance of DNA methylation became even more appreciated. Walsh et al. showed that DNA methylation plays a critical role in proper mouse embryogenesis by transcriptionally repressing the function of

repetitive promoters (Walsh et al., 1998). More recent studies have verified this finding and have

shown that DNA methylation is implicated in the regulation of proper mouse development. It

does so by silencing tissue specific genes and repetitive DNA elements in fibroblast cultures

(Jackson-Grusby et al., 2001). In 1989 the first protein with methyl-binding activity, now known

as methyl – CpG - binding protein 1 (MeCP1), was identified (Meehan et al., 1989). It is a large 2

protein complex, 400-800 kDa in size and plays a role in the methylation-mediated repression of transcription both in vitro and in vivo. It requires at least 13 methylated CpGs for proper

transcriptional repression and its function was reported to correlate with methylation density

(Boyes et al., 1991).This complex is made of methyl-CpG-binding domain 2 (MBD2), together with histone deacetylases 1, 2 (HDAC1) and (HDAC2), RbAp46, RbAp48 (Ng et al., 1999) (Fig.

1C). Repression of gene transcription occurs when a protein containing a methyl-CpG-binding

domain (MBD) binds to methylated CpG nucleotides on that gene (Hendrich and Bird, 1998).

The proposed mechanism by which DNA methylation leads to transcriptional repression was

suggested to involve direct interference with the transcription factors binding to DNA and direct blockage of the sites needed for transcription. Specifically, it was shown that some transcription factors failed to bind to DNA when their target sequences were methylated (Comb and

Goodman, 1990). However, another indirect theory of methylation dependent repression was

proposed. It attempted to explain the observations that DNA methylation is capable of

repressing transcription at some distance (Ben-Hattar et al., 1989). In contrast to the direct

theory, it was proposed that a methyl- CpG-binding protein specifically binds to methylated

DNA and recruits transcriptional co-repressors, known as histone deacetylases (HDACs).

HDACs remove the acetyl groups from the histones, thereby revealing the positive charges on the proteins that then lead to a tight binding between the positive charges on the proteins and negatively charged DNA, finally resulting in gene transcriptional repression. Even though the debates about which theory of methylation dependent transcription better explains the process

are still ongoing, it is clear that DNA methylation associates with transcriptional silencing of the

methylated gene (Zupkovitz et al., 2006).

1.2. Example of an epigenetic regulator: MeCP2 and Rett Syndrome (RTT)

The importance of epigenetic factors became appreciated when mutations in MeCP2

were shown to cause to Rett Syndrome. RTT is a severely debilitating autism-spectrum condition

that affects mostly girls with the prevalence of 1 in 10,000 cases worldwide. It is characterized

by the presence of repetitive hand wringing or clasping movements, a loss of motor abilities, lack of coordination, ataxia, and occurrence of seizures that vary in severity (Hagberg et al., 2000).

Rett girls exhibit many common autistic features such as hypersensitivity and increased anxiety in novel situations, indifference to surrounding environment, and loss of social interaction abilities. MeCP2 is located on the X chromosome, and as a result of X chromosome inactivation in females, Rett syndrome girls possess mosaic gene expression and a highly varied phenotype

(Zoghbi et al., 1990). In contrast, MeCP2 mutations on the X chromosome in males usually lead

to little phenotypic variation and embryonic lethality in a majority of cases.

Mutations of MeCP2 are also associated with other neurobehavioral abnormalities,

ranging from mild learning disabilities to autism, X-linked mental retardation, and infantile

encephalopathy (Moretti et al., 2006). MeCP2 is a highly abundant chromosomal nuclear protein

that is detected in most tissues, but has the highest prevalence in postmitotic neurons

(Shahbazian et al., 2002). It is believed that it plays an important function in neuronal

maturation, dendritic arborization and maintenance (Francke et al., 2006). It has been estimated

that about 85% of all Rett syndrome cases are due to MECP2 missense, nonsense, and frameshift

mutations, as well as deletions of several nucleotides and even whole exons (Ravn et al.,2005),

(Amir et al., 1999). Up to date over 300 mutations have been reported within MECP2 gene

(Christodoulou et al., 2003). It is therefore not surprising that resulting phenotypic differences

seen in Rett syndrome patients are so diverse. While many of these mutations have been found

throughout the MECP2 gene, they tend to occur disproportionally often in domains of functional

importance. MeCP2 contains two essential domains: methyl binding domain (MBD) which binds

methylated CpG dinucleotides, and a transcriptional repression domain (TRD) (Nan et al., 1998).

It is now known that MeCP2 protein modulates gene expression through chromatin

remodeling, acting both as an activator or an inhibitor of gene activity. Initially, the primary role

of MeCP2 protein was believed to repress transcription by binding to methylated CpG dinucleotides and recruiting co-repressor and chromatin remodeling proteins. Specifically, TRD domain of MeCP2 was shown to associate with co-repressor SIN3A and recruit histone

deacetylases (HDACs) (Nan et al., 1998) (Fig. 1 A). However, recently it was proposed that

MeCP2 also acts as transcriptional activator and associates with CREB1 (Chahrour et al., 2008)

(Fig. 1 B).

Figure 1: Function and structure of MeCP2 and MeCP1.

A) MeCP2 acts as a transcriptional repressor. It binds methylated DNA and recruits Sin3A

co-repressor and histone deacetylases (HDACs), resulting in deacetylation of histones.

Consequently, chromatin becomes compact and the process of transcription ceases.

B) MeCP2 also acts as a transcriptional activator by associating with CREB1 transcriptional

factor.

C) MBD2 acts as a part of the MeCP1 complex. MBD2 like MeCP2 associates with histone

deacetylases (HDACs) and thereby acts as a transcriptional repressor.

(Figure 1 -modified from IRSF database, Australia - http://mecp2.chw.edu.au/mecp2)

Figure 1

1.3. Autism Spectrum Disorders (ASD) and Epigenetics

Recently, the involvement of epigenetic regulatory mechanisms in the pathogenesis of

autism and autism spectrum disorders has been suggested. An extensive study investigated the

involvement of MBD family members in the etiology of ASD in a large clinical population.

Cukier at al. found over 46 alterations in MBD-containing members in 190 tested autistic individuals. As a result the investigators concluded that the entire MBD family plays a role in the etiology of autism (Cukier et al., 2009).

1.3.1. Overview of Autism Spectrum Disorders

Autism is a complex, behaviorally defined disorder that affects young children worldwide

(Muhle et al., 2004). Autism has an incidence of approximately one in every 1,000 individuals in the general population and has a skewed male to female ratio of about 4:1 (Fombonne et al.,

2002). Impairments in the three main behavioral parameters characterize this syndrome: 1) social interaction 2) language, communication, and imaginative play and 3) range of interests and activities (Muhle et al., 2004). Specifically, children with autism display impairments in social interaction, difficulty in communication as well as a severely restricted scope of interests.

Autism is a condition that is grouped under a broad class of Autism Spectrum Disorders (ASDs).

ASDs include other related developmental and cognitive disabilities such as childhood disintegrative disorders, Rett syndrome, Asperger syndrome and altogether affect approximately

1 in 150 children. Similarly to autism, these conditions are characterized by impairments in reciprocal social interaction, communication as well as restricted and stereotyped patterns of interests and activities (Kumar et al., 2009).

1.3.2. Causes of Autism Spectrum Disorders

Despite many efforts to elucidate causes of ASDs, they still remain poorly understood. It

is speculated that a complex interplay between genetic predispositions and many environmental

factors contribute to these disorders. A high concordance rates of autism in monozygotic twins

(60–91%) and dizygotic twins (0–6%) point to a strong genetic component in development and

heritability of the disorder (Folstein et al., 2001). However, only a few studies have identified

specific genes involved in autism etiology (Ritvo et al., 1985), (Bailey et al., 1995), (Greenberg et al., 2001). Recently, mutations in the family of transsynaptic adhesion molecules, known as

neurologins (NLGNs) and neurexins (NRXNs) have been implicated in autism. They are

synaptic cell-adhesion molecules that connect presynaptic and postsynaptic neurons at synapses,

mediate signaling across the synapse, and play a critical role in synaptic maturation. Primarily

mutations in NLGN3 and NLGN4 were found in patients with ASD and were associated with

some autism-like features in mice. The precise mechanism of action is still unclear, but a few theories have been proposed. One potential mechanism proposes impairment in the transport of mutated neurologins to the cell surface, leading to reduced induction of synaptic formation.

(Chubykin et al., 2005).

1.3.3. Mouse models of Autism Spectrum Disorders

Up to date a few mouse models have been developed that recapitulate some of the

cardinal features of autism and autism spectrum disorders. Most of these represent monogenic

aberrations and include the loss of function of Fmr1, Mecp2 and ubiquitin protein ligase 3A

(Ube3A). In addition, as previously mentioned, mouse models with NRXNs and NLGNs mutations are also used to elucidate autism etiology. Interestingly, female mutant mice lacking one form of neurexin 1α, were viable and fertile but displayed impaired maternal behavior.

Specifically, mice were characterized by maternal indifference towards their pups that was

independent of pup genotype (Geppert et al., 1998). However, other mouse models have also

been designed to study the effects of environmental factors exclusively. For example, these

include early exposure of mice to risk factors such as valproic acid, inflammatory agents or

maternal stress, all of which are believed to associate with autism (Moy S., 2008). Other studies

have emphasized the need to incorporate environmental influences and genetic predisposition in

investigating the etiology of autism. It was proposed that epigenetic regulation, DNA methylation specifically, serves as a link between environmental exposures and genetics (Zhao et al., 2003). This theory is particularly feasible, considering the discovery of a fundamental epigenetic role of MeCP2 in Rett syndrome etiology.

1.3.4. ASD, MBD proteins and Electroencephalographic (EEG) abnormalities

In addition to social interaction abnormalities, 75 % of ASD patients are also diagnosed with mental retardation, 10-30 % of patients experience epileptic seizures and 20-50% display 10

electroencephalographic abnormalities (Gabis et al., 2005).Routine awake EEG analyses

revealed epileptoform abnormalities in adolescents and young adults with autism (Rossi et al.,

2000). Specifically, about 10 % of children diagnosed with autism were shown to have

paroxysmal EEG pattern not always accompanied by clinical seizures. In addition, electrical

status epilepticus was seen in slow wave sleep in autistic children (Rapin et al., 1995). However,

the rates of epilepsy in general population is from 1 to 4 % (Sander and Shorvon, 1987). The

incidence of seizures in patients with autism usually peaks at around 12-18 months of age,

followed by a slow increase throughout childhood. The second peak in incidence takes place in

adolescence (Volkmar et al., 1998). Interestingly, many types and severity levels of seizures

were reported in children with autism. However, another study identified disproportionate

number of cases with partial seizures (Rossi et al., 2000). Recently EEG analysis of MeCP2-

deficient mice revealed abnormal spontaneous rhythmic EEG discharges of 6–9 Hz in

somatosensory cortex during awake immobile states. In addition, mutant mice displayed a theta

rhythm in the hippocampus characterized by a significantly reduced peak frequency (D’Cruz et

al., 2010). Collectively, all of these findings suggest that abnormal EEG patterns frequently

occur in autism spectrum disorders and warrant further investigation.

1.4. Methyl-CpG-Binding Proteins

In addition to MeCP2, now four other MBD-containing proteins have been described.

These are MBD1, MBD2, MBD3 and MBD4 (Fig. 2). The MBD sequence between all five

members is well conserved with 45%-75% overall amino acid identity. Additionally, Kaiso

protein is also involved in transcriptional repression but does not contain a classical MBD

domain. (Filion et al., 2006).While Methyl-CpG binding proteins display homology within their

MBD domains, the transcriptional repression domains (TRD) in MeCP2, MBD1 and MBD2 are

non-homologous and are important for interaction with various co-repressor complexes

(Hendrich and Bird, 1998). MBD proteins are ubiquitously expressed in all somatic tissues,

although at varying levels. However with the exception of RTT mice and Mbd3 knockout mice,

animals lacking other MBD protein members display a relatively mild phenotype. Considering

that MBD proteins were shown to simultaneously bind to the same promoter regions, many

speculations exist of the potential functional redundancy between MBD family members

(Matarazzo et al., 2007). This hypothesis is supported by the observation that several MBD

proteins were detected at the same methylated promoter in human cancer cell lines. However,

other studies in primary human lung fibroblasts have not observed several MBD proteins occupy

methylated sites. Intriguingly, MeCP2 was unable to colonize methylated sites vacated by MBD2

after it was depleted from lung fibroblast cells. Surprisingly, upon MeCP2 removal, MBD2 was

shown to migrate into about half of the binding sites normally occupied by MeCP2 (Klose et al.,

2005).These data suggest that MeCP2 has a binding preference that is distinct from that of

MBD2 and that MeCP2 is reluctant to replace MBD2. This preference could be explained by the recent finding that MeCP2 requires a run of four or more A/T bases adjacent to a methylated

CpG in order to efficiently bind the methylated sequence. However, MBD2 does not seem to

show such a preference and instead binds to single methylated CCGG sites regardless of flanking

sequence (Klose et al., 2005).

Figure 2: Structure of MBD proteins and Kaiso.

Methyl-CpG binding proteins display homology within their MBD domains. The transcriptional repression domains (TRD) in MeCP2, MBD1 and MBD2 are non-homologous and are important for interaction with various co-repressor complexes. MBD1 is able to bind unmethylated DNA with the help of third cysteine-rich regions zink-finger motif (CxxC). MBD2 has a unique stretch of glycine and arginine (GR) residues. In addition MBD2 is unique in that its MBD and TRD are situated right next to each other. MBD3 has no equivalent MBD domain and is therefore unable to bind methylated DNA. MBD4 contains a glycosylase domain that is used for DNA repair.

Kaiso is not a member of MBD family, but it binds methylated DNA through its triple zinc finger (zf) domains.

Figure modified from the review paper. (Bogdanovic et al, 2009)

Figure 2

MeCP2 MBD TRD

MBD1 MBD CxxC CxxC CxxC TRD

MBD2 GR MBD TRD

MBD MBD3

MBD4 MBD glycosylase

Kaiso POZ/BTB ZF ZF ZF

1.4.1. Methyl-CpG-binding domain protein 1 (MBD1)

MBD1 is the largest protein of the MBD family. It is a unique protein that is capable of

repressing transcription by binding to both methylated and unmethylated promoters. It associates

with unmethylated DNA through a unique cysteine - rich motif (CXXC) that other MBD proteins

do not possess (Fujita et al., 2000). Recently it was shown that MBD1 acts as an epigenetic

regulator in mouse development. It plays a critical role in the regulation of several autism-related behaviors such as social interaction, anxiety, depression, and sensorimotor gating in mice. Mice lacking Mbd1 are otherwise grossly normal and live nearly normal life spans without any motor

deficits. Due to complexity and diversity of these behavioral impairments, it is believed that

MBD1 somehow regulates a whole network of pathways underlying these abnormalities. The precise mechanism of MBD1 action is still unknown, but it is speculated that MBD1 transcriptionally modulates some of the key components involved in proper mouse development.

Specifically, it was established that MBD1 acts as a key regulator for serotonin 2c receptor

(Htr2c) by binding to its promoter. As a result, mice lacking Mbd1 were found to have increased

RNA and protein levels of serotonin receptors. This study confirmed a previously hypothesized

link between serotonin and autism and emphasized the importance of epigenetic regulators in

mammalian brain development and proper cognitive functioning. MBD1’s extensive expression

profile in neural progenitors and neurons also suggests of the importance of MBD1. While the

presice mechanism of action still remains unclear, it became evident that Mbd1 plays a role on

hippocampal neurogenesis, synaptic plasticity and learning in mice (Allan et al., 2008).

1.4.2 .Methyl-CpG-binding domain protein 3(MBD3)

MBD3 is very similar to MBD2 over most of its length with 71 % overall amino acid

identity (Hendrich et al., 1998). Despite this sequence similarity, mammalian MBD3 does not

directly bind to methylated DNA but requires additional factors. This is due to sequence

variations at two amino acid residues on MBD3 that are not present in other MBD family

members. One of the amino acids substitutions is from a tyrosine to phenylalanine. As a result of

the substitution, a hydrogen bond between amino acid and methylated DNA is disrupted.

Consequently, direct binding between DNA and protein factors is prevented (Ohki et al., 2001).

Like the majority of MBD containing proteins, MBD3 is believed to play a role in transcriptional

repression and does so by associating with HDAC1 and HDAC2 that function to modify

chromatin. MBD3 acts as a component of the nucleosome remodeling and histone deacetylation

(NuRD) co-repressor complex. NuRD is a co-repressor molecule that is recruited to DNA by

other repressor proteins. NuRD is important for proper embryonic development, as mice lacking

it are not viable. (Wade et al., 1999). Additionally, loss of MBD3 in mice was also associated

with embryonic lethality. Thus, both NuRD and MBD3 are important for proper development in

mice (Hendrich et al., 2001).

1.4.3. Methyl-CpG-binding domain protein 4 (MBD4)

MBD4 is a mismatch repair protein. It is the only member of MBD family that is not

involved in transcriptional repression. Methylation of the cytosine base is inherently mutagenic

as it spontaneously deaminates to form thymine and results in T-G base pair mutation (Bestor et

al., 1996). MBD4 preferentially binds to T-G mismatches and repairs them (Hendrich et al.,

1999). MBD4 has a glycosylase domain that removes T when it is mismatched with G base pair

without cleaving the DNA strand (Hendrich et al., 2003). However, recent evidence suggests that

MBD4 may also act as a transcriptional repressor (Kondo et al., 2005).

1.4.4. Kaiso protein

Kaiso is a protein that uses zinc finger (zf) domain to bind both methylated and non-

methylated DNA. It represses transcription but lacks the classical MBD motif (Prokhortchouk et

al., 2001), (Filion et al., 2006). In addition, Kaiso requires at least two symmetrical methylated

sites for proper binding to DNA, while MBD family members need only one methylated site

(Hendrich et al., 1998).

1.5. Methyl-CpG-binding domain protein 2 (MBD2)

MBD2 is a component of the methyl-CpG binding protein 1 (MeCP1) complex and is

necessary for normal MeCP1 formation. MeCP1 was the first methyl-CpG binding complex that

was described and shown to represses transcription (Bird et al., 1999). Interestingly, the release

of MBD2/MeCP1 from the nuclei can be accomplished by low salt, suggesting that the binding

of this complex to DNA is not very strong. Indeed, it was shown that the formation of the stable

complex with DNA requires densely methylated DNA (Ballestar et al., 2003). Like the other

members of MBD family, MBD2 associates with histone deacetylases (HDACs) and is involved

in transcriptional repression (Feng et al. 2001). Two variants of MBD2 protein exist:MBD2a and

MBD2b. MBD2b has an N-terminal truncation with translation starting at the beginning of

MBD domain, thus making MBD2b smaller of the two variants (31 KDa). While the size of

MBD2a is 43 KDa. MBD2 and MBD3 are more closely related to each other than to the other

MBD proteins having 75% similarity (Hendrich and Bird, 1998). Up to date the expression of

MBD2 in the brain has only been done qualitatively. MBD2 is well expressed in the hippocampal and cerebellar brain regions in the adult. However, quantitative assessments of

MBD2 expression in the brain remain to be performed (Jung et al., 2002). Because of such high degree of protein similarity, Hendrich et al., in 1999 decided to investigate if these two proteins

interact genetically. They generated Mbd2 (-/-) and Mbd3 (-/-) mice, crossed them and observed reduced viability in Mbd3 (+/-) with absence of Mbd2. Therefore, they concluded that Mbd2 and

Mbd3 interact. In addition, other in vitro studies showed that MBD2 recruits NuRD and MBD3 as a part of the NuRD complex to methylated DNA (Hendrich and Bird, 1998).This raised the possibility that MBD2 and MBD3 also interact biochemically (Matarazzo et al., 2007). However, up to date the hypothesis of biochemical interaction between them still remains to be investigated. Another study reported that MBD2, in addition to MBD3 binds to NuRD but formed mutually exclusive Mi-2/NuRD chromatin remodelling complexes (Le Guezennec et al.,

2006).

1.5.1.MBD2 and maternal behavior

Hendrich et al. in 1999 were also the first to describe the phenotype of Mbd2- deficient

mice. MBD2 null mice were reported as viable, fertile and having a normal life span. The only

abnormality that Mbd2 null mice displayed was their impaired maternal behavior. The average litter size from Mbd2 (-/-) parents was half the size of Mbd2 (-/+) mice. In addition, the average

size of pups from null females was significatly lower. Intersetingly, this finding was not related

to the genotype of the father, but instead was explained exclusively by the genotype of the

mother. Therefore, Hendrich et al. concluded that Mbd2 deficiency in females results in

abnormal nurturing behavior (Hendrich et al., 1999).

1.5.2. MBD2 and immune system

In addition to the role MBD2 plays in transcriptional silencing of methylated promoters

and regulation of maternal behavior (Hendrich et al., 2001), a new function of MBD2 was

recently described. Kersh et al. reported how MBD2 aids in the differentiation of naïve CD8 T

cells into effector and memory cells. Specifically, MBD2 acts as an intracellular regulator of T

cells differentiation and helps to prevent viral reinfection (Kersh et al., 2006). Normally, MBD2

acts as a transcriptional repressor and silences Il4 gene that encodes interleukin-4 in naïve T

helper cells. However, because IL4 is present in absence of MBD2, the levels of Il4 are

abnormally high in differentiating type 2 T helper cells. Even though the precise mechanism of

MBD2 action is still unclear, the importance of MBD2 in normal immune response development

is becoming appreciated.

1.5.3.The role of MBD2 in embryogenesis and postmititic tissues

Many studies have looked at the involvement of methyl-CpG-binding proteins in

embryogenesis. Specifically, two members of MBD family (MeCP2 and MBD2) and Kaiso

proteins were investigated. It was discovered that in mammals, MBD proteins are not essential

for embryogenesis as no loss of neurorogenesis was detected in neural stem cell cultures lacking

MBD2, MeCP2 and Kaiso. Interestingly, Caballero et al. reported an initial defect in neuronal

specification when all three genes were missing, followed by a self-reversible outcome.

Caballero and his group concluded that the function of MBD proteins becomes important

postnatally, probably to fine tune expression of postmitotic tissues (Caballero et al., 2009). In

addition, they tested if MBD2, Kaiso and MeCP2 play a redundant role in proper mouse

development and created a triple null mouse: Mbd2 (-/-), Kaiso Zbtb (-/-) and MeCP2 null. The

resultant phenotype was characterized by previously observed Rett like symptoms, without

worsening of the phenotype. However, the investigators themselves addressed an inherent

problem with their study as they did not perform tests to assess any subtle behavioural alterations

that could result from a triple null. The additional tests would have been especially valuable

considering that Mbd2 (-/-), MeCP2 (-/-) and Kaiso- deficient mice had a significantly reduced

life span. The observation that MBD2 and MeCP2 deficient mice as well as triple knockout

mice died at a younger age compared to MeCP2 null mice suggests that MBD2 and Kaiso

somehow compensate for the loss of MeCP2 (Caballero et al., 2009). Collectively, it is clear that

MBD2 plays an important role in proper develoment and that further analysis of Mbd2 (-/-) mice

is warranted.

1.5.4. MBD2 as a demethylase enzyme and its role in tumor suppression

Hypermethylation of tumor suppression gene promoters and consequently transcriptional

silencing of these genes, have long been implicated in cancer development and progression

(Ballestar et al., 2003). As a result of this, MBD family members have been investigated in the

field of cancer. Yet, MBD2 is the member that has been most extensively studied. The results of

adenoma studies showed that MBD2-deficient mice are resistant to developing intestinal cancers

(Sansom et al., 2003). Specifically, Sansom et al. showed that Mbd2 not only inhibits the

development of intestinal adenomas in the tumor-prone Apc Min/ + mouse, but also shows Mbd2

dosage is crucial for the observed tumor resistant effect. When Mbd2-deficient mice were

crossed on to an Apc Min/ + background, mice with the complete Mbd2 deletion survived

significantly longer than control animals, while Mbd2 -/+ showed an intermediate survival. In

addition, the number of tumors as well as their size were the lowest in Mbd2 null animals

(Sansom et al., 2003). Furthermore, ablating MBD2 showed inhibited tumor growth in the lungs,

breast, prostate and colorectal cancers (Slack et al, 2002), (Campbell et al., 2004), (Pakneshan et

al., 2005) (Shukeir et al., 2006). Precisely how MBD2 supresses tumorigenesis remains to be

investigated. In addition to facilitating transcriptional repression, MBD2 is also speculated to act

as a DNA 5-methylcytosine demethylase (Bhattacharya et al., 1999). Based on this hypothesis it is then feasible that MBD2 removes methyl groups from the tumor suppressor genes, and thereby interferes with methyl associated transcriptional repression. MBD2 acts as a demethylase by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine followed by the release of the oxidized methyl group (Hamm et al., 2008). However, this finding is controversial as the global methylation levels were not reported to change in MBD2 null mice. Yet, in support for MBD2 demethylation role, the researchers proposed to assess DNA methylation at single site resolution rather than determining a global methylation pattern. These experiments remain to be conducted and up to date, the debate over its demethylase activity continues (Bhattacharya et al., 1999).

Intriguingly, after witnessing the dramatic reductions in cancer development as a result of MBD2 ablation, many cancer studies have proposed MBD2 as a new target of anticancer drug development. However, because the role of MBD2 in brain functioning has yet to be studied, it is imperative to assess its function in the brain before therapeutically ablating MBD2.

1.6. Hypothesis and the goals of my project

Considering that MBD2 serves an important part of the MeCP1 complex, I hypothesize

that the loss of MBD2 is sufficient to elicit behavioural impairments in mice. The role of

epigenetic regulators in proper mammalian development and functioning is becoming

increasingly clear. The results of the previous investigations demonstrate the critical roles

MECP2 and MBD1 proteins have in proper neural development. Mice lacking Mecp2 and Mbd1

were shown to have behavioural alterations that resemble an autism-like phenotype. Therefore it

can be speculated that the loss of MBD2 could also affect behavioural responses of animals to

anxiety producing tests and even contribute to socialization behaviour impairments. Even

though Mbd2 is well expressed in neural stem cells and neurons, its biological function

specifically in the brain, has not yet been extensively studied (Caballero et al., 2009). Hence, my

project was specifically aimed at investigating the following:

- Characterize the behavioural phenotype of Mbd2 null mice. Specifically, the aim of the

project was to assess their general locomotion skills, anxiety-like behavioural responses

during anxiety assessment tests, socialization behaviours, and preference for novelty.

- Study electroencephalographic recordings of wild-type and MBD2-deficient mice during

the active exploration behavioural state.

- Assess MeCP2 protein level expression in the cortex, cerebellum and hippocampus of

wild-type and Mbd2- deficient mice

- Assess mRNA expression profiles of MBD1, MBD2, MBD3, and MeCP2 expression

levels in wild-type and Mbd2-deficient mice in neural tissues.

Chapter 2

Materials and Methods

2.1. Animal subjects

2.1.1. Generation of Mbd2 (-/-) mice

Mbd2 null mice were generated and generously provided by Adrian Bird (Edinborough,

Scotland) and obtained directly from Dr. Jane Roskams (University of British Columbia). Mice

lacking MBD2 are viable, fertile and develop without any gross deficits and have a seemingly

normal life span. Additionally, no observed differences in weights of MBD2-deficient mice were

detected (data not shown).The exon 2 of the Mbd2 gene was replaced by the promotorless βgeo

cassette as described by Hendrich et al. (Hendrich et al., 2001) (Fig. 3). As a result of this

genetic manipulation, transcription of Mbd2 was terminated at the stop site located in the βgeo cassette, producing a truncated Mbd2 transcript. Specifically, the transcription was initiated but was only continued through exon 1 and intron 1. The translation of the truncated Mbd2 produced

183 amino acids N-terminus but stopped in the middle of methyl-CpG-binding domain (MBD)

(Hendrich et al., 2001). As a result of this stop codon disruption, nonscence mediated mRNA decay (NMD) is induced. Consequently, mRNA with a premature stop codon is degraded and the synthesis of truncated proteins is prevented (Amrani et al., 2006).

All procedures were conducted in accordance with the guidelines established by the

Canadian Council on Animal Care and all experimentation was reviewed and approved by local

Animal Care Committees prior to the onset of the study.

Figure 3: Generation of Mbd2 (- /+)

To generate Mbd2 (- /+) mice, the exon 2 of the Mbd2 gene was replaced by the

promotorless βgeo cassette as described by Hendrich et al. (Hendrich et al., 2001). This resulted

in the termination of Mbd2 at the stop site located in the βgeo cassette, producing a truncated

Mbd2 transcript. The transcription was initiated but was only continued through exon 1 and

intron 1. The translation of the truncated Mbd2 produced 183 amino acids N-terminus but

stopped in the middle of methyl-CpG-binding domain (MBD) (Hendrich et al., 2001).

Figure 3

Mbd2 MBD2

1 23

X Β geo

Mbd2 (‐/+) Β geo truncated MBD2

1 23

2.1.2. Genotyping

For subject genotype analysis, tails of mice were biopsied and digested with 20 mg/ml

Proteinase K in a lysis solution for 6 hours in water bath set at 55oC. This was followed by the

addition of ethanol. Then, the samples were placed in binding columns, centrifuged at 6,500 x g

for 1 minute and washed twice with 500 ul of Wash Solution. The samples were once again

centrifuged at 6,500 x g for 1minute. Genomic DNA was then eluted from the binding column

with Elution Solution. The samples were stored at -20oC and used as templates for the

Polymerase Chain Reaction (PCR). Master Mix contained PCR buffer as well as 4 different

primers for detecting Mbd2 wild-type or Mbd2 deficient allele. The wild-type Mbd2 allele was

detected using primer P 61 5’-ACG CTG GCC TAG TGT CGT GC -3’ in conjunction with a

wild-type allele P49 5’-AAGAACAAGCAGAGACTCCG-3’. Mutant Mbd2 allele was detected

using primer P62 5’TTGTGGTTGTGCTCAGTTC-3’ in conjunction with another null allele

EnP1 5’-TCCGCAAACTCCTATTTCTG-3’. A single band of 243 base pairs indicated a wild-

type subject, while a single band of 150 base pairs indicated a null subject. When both bands

were present, the subject was heterozygous. Genomic DNA from wild-type mice was used as a

positive control to detect the wild-type allele. DNA from MeCP2 null mouse as well as samples

without any genomic DNA (Master Mix only) were used as negative controls. PCR amplification

was conducted on an MJ Research Thermocycler using a program of 35 cycles of denaturation at

94oC for 1 minute, annealing at 55oC for 30 seconds, and extending at 72oC for 30 seconds. After

the reaction was complete 5 µl of 6x loading buffer dye was added to the samples. The samples

were run and products were visualized on ethidium bromide-stained 2 % agarose gel.Mbd2 (-/-)

mice were generated by crossing heterozygous mice for Mbd2 (-/+) with other heterozygous

mice. All the experiments for this study were performed using Mbd2 (-/-) mice.

2.1.3. Statistical Analysis

Statistical analysis for the results of socialization behaviors, open field locomotion

assessments and anxiety-like behaviors were performed using a two-way ANOVA (genotype and

age) with Bonferroni post hoc correction using GraphPad Prism Software 5. The results of the

rotating rotorod were analyzed by a non-paired, two-tailed Student’s t-test in Microsoft Excel

software, followed by Bonferroni post hoc correction. The results of Novel Object Recognition

task were analyzed with two-way ANOVA with repeated measures with Bonferroni post hoc

correction using GraphPad Prism Software 5. The analysis of the western blots, real time PCR

results and EEG recordings were performed using non-paired, two-tailed Student’s t-test in

Microsoft Excel software. The probability of less than 0.05 was considered significant.

2.2 Behavioural Assessments

Pictures of the assays used in this study are depicted in Figure 4.

2.2.1. Motor Function Assessments

2.2.1.1. Open Field Apparatus

Motor activity was assessed during the light cycle between the hours of 8: 00 am and

14:00pm to minimize the effects of circadian rhythms. An automated movement detection

system (AM1053 activity monitors; Linton Instrumentation, UK) was used to measure motor

behavior in the open-field arena. Mice were individually placed in a plexiglass box (20 cm × 30

cm) surrounded by a frame (45 cm × 25 cm). Inside that apparatus is an array of 24 infrared

beams that form a grid across two levels and detect motion. When the mouse moves in the box,

the beam is broken and an activity count, dependent on the type of movement, is registered.

Different activities can be detected with this apparatus depending on the location of the mouse in

the apparatus as well as the level and the speed of beam breaks. For example, slow activity

counts were registered if successive beam breaks are separated by > 200 ms, while fast activity

was recorded when the interval was < 200 ms. In addition, behavioral parameters such as total

activity, locomotion and exploratory behaviors as total and center field rearing (forepaws

elevated from the floor) were obtained from the assessment. Many other additional parameters

were the percentage of time spent walking, rearing or immobile; the number of fast and slow

locomotive mobility counts; the number of total rearing events; the number of fast and slow

rearing events in the entire field and the number of total rearing events in the center region of the

open field. Motor behavior was recorded during a 60 minute test session for each mouse and

odors were controlled by wiping the interior of the box with 70 % ethanol solution after each test

(Jugloff et al., 2008).Open field tests were performed for two age cohorts of Mbd2 null and

control mice (3-5 months old) and (8-13 months old). The number of subjects for the test was

the following: young group (wild-type n = 42, Mbd2 null n = 35), older group (wild-type n = 70,

Mbd2 null n = 40).

2.2.1.2 Coordination and Balance Function Rotarod Test

To determine the ability to perform well-coordinated locomotor activity, mice were

examined with an automated movement detection system as previously described (Jugloff et al.,

2008). Animals were placed on a slowly revolving (3 rpm) rod that was set to accelerate until the final speed of 35 rpm during 10 min trial time. Latency to fall was recorded automatically by laser beam break in three daily trials, performed at 60-min intervals. In total, mice were subjected to four consecutive days of testing. Subjects were excluded if they did not actively

balance on the rotating rod and instead, rotated in a circle and went around the rod three

consecutive times. For the rotarod behavioral test, overall motor performance was expressed as

the mean time spent on the rod before falling, as well as the final speed in rpm before falling off

the rod. Rotarod tests were performed for two separate age cohorts of Mbd2 null and control

mice (3-5 months old) and (8-13 months old). The number of subjects for each group was the

following: young group (wild-type n = 29, Mbd2 null n = 21), older group (wild-type n = 15,

Mbd2 null n = 29).

Figure 4: Pictures of behavioural assays used in this study.

A) Open Field Apparatus

B) Rotarod Test

C) Socialization Test

D) Nest Building Assay

E) Novel Object Recognition Test

F) Light and Dark Preference Test

G) Elevated Plus Maze

Figure 4

A) B)

C) D)

Delay

2.2.2. Socialization Behaviour Assays

2.2.2.1. Social Interaction Test

Social interaction was assessed based on a published paradigm with modifications

(Crawley, 1999, 2004). For the assessment of mouse preference for social novelty as well as behavioural reactivity of mice to novel or co-housed partners, the following sets of tests were conducted. Three days prior to the test, the experimental animals were individually habituated to

the testing conditions consisting of the clear box (44 cm × 22 cm × 29 cm) with a wire tent

placed at the center (15 cm × 12.5 cm) for 30 minutes daily. The wire tent permitted visual,

auditory, olfactory and some tactile contact between the experimental mouse and the partner, all

of which are required for the subject to detect social cues emitted by the partner mouse in the

wire cage and initiate social interaction. Eighteen hours prior to the experiment a partner C57

BL/6 mouse that was age, gender and weight matched was placed in the wire tent for 15 minutes.

That was done to ensure that the partner mouse was habituated to the wire tent and displayed low

levels of activity during the test. In addition, locating the partner mouse inside the wire tent

ensured that all the social approaches were initiated only by the subject mouse while also

preventing the aggressive fighting. The time the subject animal spent near the wire tent sniffing

the wire, as well as the number of approaches to the wire cage with a partner were recorded.

These behaviours were assessed by the observer blinded to genotype and analysed to indicate the

level of social interest displayed by the test mouse. Before the experiment, the subject mouse was placed in the wire tent for 10 minutes for the final habituation procedure. The social interaction test was performed two consecutive times; the experimental mouse was exposed to their co-housed partner (at least 3 weeks of co-housing) and then exposed to a novel mouse for

10 minutes. The stranger mice were age, gender and weight matched C57BL/6 mice that had no

prior physical contact with the test subject. In addition, avoidance behaviour was recorded. This

was assessed when the subject mouse climbed on top of the wire cage and avoided interactions

with the partner mouse. The amount of time as well as the number of avoidance climbs were

recorded and analyzed. The wire tent was cleaned with 70 % ethanol and allowed to completely

air dry after each new interaction to eliminate all odors.

Social interaction tests were performed for two separate age cohorts of Mbd2 null and

control mice (3-5 months old) and (8-13 months old). The number of subjects for the social

interaction tests was the following: young group (wild-type n = 10, Mbd2 null n = 5), older group

(wild-type n = 14, Mbd2 null n = 19).

2.2.2.2. Nest Building Behaviour Test

Nest building behaviour in mice was assessed with a paradigm previously described

(Samaco et al., 2008 and Moretti et al., 2005). Mice were single-housed and given a new nesting

material, a 2.5 cm × 2.5 cm piece of pressed cotton. The amount of time and the number of

interactions that animal initiated with the neslet during the first 30 minutes of exposure were

recorded. Also, 24 hours after the initial introduction of the nesting material, the height and the

width of the resultant nest were recorded. In addition, pictures of the nests were taken .The

assessments were done for two age cohorts of wild-type and Mbd2 null mice (3-5 months old),

(8-13 months old) and the number of subjects was the following: young group (wild-type n = 11,

Mbd2 null n = 7), older group (wild-type n = 19, Mbd2 null n = 18).

2.2.3. Novel Object Recognition Test

Mbd2 null and wild-type mice were tested for the object recognition memory in a clear

plastic box (44 x 22 x 29 cm).The procedure was slightly modified from the paradigm previously

described (Vaucher et al., 2002). Each animal was exposed to three objects that differed in shape, color, and texture. The objects were selected from a larger pool of objects and were similar and attractive enough for mice to spend approximately equal time exploring them. Mice were habituated to the testing environment for 15 minutes for seven days. On the test day, two objects were placed at the two ends of the plastic box, equal distance from the imaginary center line.

Mice were allowed to explore the two objects for 10 minutes and exploratory activity (i.e., time spent in object-directed exploration) was recorded. After a delay of 5 minutes, mice were re- exposed for 5 minutes to the original object and a novel object. Time spent exploring each object was recorded. The same procedure was repeated for 5 minutes after a 1 hour delay period. Object exploration was considered to be any behavior that involved touching the object with the forepaws, nose or sniffing at the object within a distance of 1.5 cm. Number of interactions that mice initiated with the objects, as well as the duration of these interactions was recorded. In addition, discrimination index (DI) was also calculated for each mouse as the time spent exploring new object “tN” over the total time spent exploring new “tN” and familiar object “tF”

DI = tN/(tF + tN) (Niewiadomska et al., 2006), (Maroun and Akirav, 2009). After each exposure,

the objects and the cage were wiped with 70% ethanol to eliminate all odor cues. The

assessments were done for one age cohort of older wild-type and Mbd2 null mice (8-13 months old, wild-type n = 16, Mbd2 null n = 10).

2.2.4. Anxiety-like Behavioral Assessments

2.2.4.1. Light and Dark Preference Test

The following behavioural paradigm was used to assess anxiety- related responses in

mice, and was based on a well-known procedure as previously described (Bouwknecht et al.,

2002). On the test day, mice were transferred from the colony room to the test room and were

left undisturbed for at least 45 minutes to acclimate to new settings (Bouwknecht et al., 2002).

All tests were performed between 08:00 am and 14: 00. Mice were placed in a clear plastic box

(44 cm × 22 cm × 29 cm) unequally divided in two chambers of which one is dark (1/3 of total surface) and the other area (2/3) is illuminated. A small opening at the centre of the dividing wall

(5 cm × 5 cm) allowed mice to freely move between the light and dark chambers. Each test lasted 5 minutes and started by placing a mouse in the middle of the light chamber facing the opening to the dark chamber. The behavior of the mice was recorder and later scored by an investigator blinded to genotype. Specifically, the amount of time spent in the illuminated and dark compartments were recorded as well as the number of transitions between the chambers.

For the transition, all four paws had to cross the line that separated the light and the dark compartments. In addition, risk assessment behavior was also calculated. It was recorded when mice did not perform a full four paws transition between compartments, but only stretched their heads from the dark to the lit compartments. The number of these inquisitive behaviors as well as their latency was used as a measure of risk assessment behavior. The following behavioral assays were performed for two age cohorts of Mbd2 null and wild-type mice (3-5 months old) and (8-13 months old). The number of subjects for each group was the following: young group (wild-type n

= 20, Mbd2 null n = 29), older group (wild-type n = 47, Mbd2 null n = 30).

2.2.4.2. Elevated Plus Maze

The apparatus consisted of a plus-maze with two lit open arms (30 cm × 5 cm) and two closed arms (30 cm × 5cm × 19 cm).The arms radiated from a central platform (5× 5 cm) and

were raised at the height of 50 cm above the floor. At the start of the 5 minute test session, a

mouse was placed on the central platform, facing an open arm, while its behavior videotaped for

5 minutes. An investigator was blinded to subjects’ genotype. The mouse was assigned the

central platform location whenever its two paws were on it. Similarly, when mice had its all four

paws in one of the arms, it was assigned the corresponding compartment. Each arm was divided

into 2 areas, proximal and distal. Proximal area is immediately attached to the central platform

and distal is the area away from the platform. Parameters recorded were the amount of time spent

on the proximal and distal areas of the open arm, proximal and distal areas of the closed arm, total time in the open arms (TOA), total time in the closed arms (TCA) as well as total time spent

in the middle area (TMA) (Clement et al., 2007) (Fig. 5). The assay was performed for two age

cohorts of Mbd2 null and control mice (3-5 months old) and (8-13 months old). The number of

subjects was the following: young group (wild-type n = 12, Mbd2 null n = 30), older group

(wild-type n = 32, Mbd2 null n = 29).

Figure 5: Diagram of the Elevated Plus Maze.

The elevated plus maze is made of 4 arms, 2 of which are closed and 2 that are open. The mouse

is placed in the middle area and is allowed to explore the arms for 5 minutes. Proximal area is the

closest one to the middle platform, while distal area is away from the platform. Parameters

recorded were the amount of time spent on the distal and proximal areas of the open arm, distal

and proximal areas closed arm, total time in the open arms (TOA), total time in the closed arms

(TCA) as well as total time spent in the middle area (TMA).

Figure 5

Closed Arm

Distal area

Proximal area

Distal area Proximal area Open Arm Middle Proximal area Distal area Open Arm Platform

Proximal area

Distal area

Closed Arm

2.3. EEG Assessments

2.3.1. Electrode Implantation

Electrode implantation for Mbd2 null mice and their corresponding wild-type control

group was carried out according to the procedures previously outlined (Wu et al., 2008). In

summary, wild-type mice and Mbd2-deficient mice were deeply anaesthetized. Then polyimide-

insulated stainless steel wire electrodes with outside diameter of 125 μm were inserted in the

hippocampal CA1 (Bregma, −2.3 mm; lateral, 1.7 mm; depth,2.0 mm) and contralateral somatosensory cortex (Bregma, −0.8 mm; lateral, 1.8 mm; depth, 1.5 mm). In addition, a reference electrode was positioned near the cortical electrode (Bregma, −3.8 mm; lateral, 1.8 mm; depth, 1.5 mm). Mbd2 null and their aged matched control mice were implanted between 8 and 12 months of age.

2.3.2. EEG Recordings

Animals were allowed to recover from the surgery for at least seven days before EEG

recordings were taken. All the recordings were obtained while the animals were in their natural

behavioral states of wakefulness during the morning hours (exploration that was specifically

assessed during sniffing behavior). Two extracellular amplifiers with extended head-stages

(Model-300, AM Systems Inc., Carlsborg, WA, USA) were used to obtain EEG recordings. The

head stage was connected to the electrode caps of the mice using soft wires and female pins and

was located approximately 10 cm above their home cage. EEG signals were recorded in a

frequency band of 0.01–1000 Hz, amplified 1000 times, and then digitized (Digidata 1300, Axon

Instruments, CA, USA). The data were then sampled (at 60 kHz), stored, and analyzed using

Pclamp software (version 9, Molecular Devices, Axon Instruments). Recording sessions varied 39

from 45 min to 3 h to allow for the observation of all the behavioral states. Individual subjects were recorded a minimum of three times, over a period of 4–6 weeks, with at least 3 days in between the recordings from the same subject.

2.3.3. EEG data analysis

Hippocampal theta patterns were identified as a sinusoidal waveform in the 6–10 Hz

range. For a consistent theta waveform throughout testing, only one corresponding exploratory

behavior of inquisitive sniffing was used. To calculate the dominant theta frequency during

exploratory sniffing, the epochs of 3-s period were obtained and bandpass filtered (0.5–200 Hz).

In addition power spectral plots (with 50% window overlap and frequency resolution of 0.41 Hz) were generated for each recording. To determine the peak theta frequency of each epoch, each one was analyzed with the spectral plot. However, to determine the corresponding theta peak frequency per animal, a minimum of 25 epochs from different recording sessions were averaged.

EEG recordings with less than 25 epochs of theta rhythms were excluded from the study (Wu et

al., 2008, D’Cruz et al., 2010).

2.4. Western Blots

2.4.1. Tissue harvesting

After electrophysiology and behavioral assessments were carried out on mutant mice and

their age matched controls, mice were sacrificed by cervical dislocation. Cortex, cerebellum, striatum and hippocampal regions from older mice (8-13 months of age) were dissected and stored at – 80oC for further processing for protein and RNA samples.

2.4.2. Preparation of protein samples for western blots

Specific brain parts (cortex, cerebellum, hippocampus and striatum) were slowly thawed

on ice. Lysis buffer was added to the samples and contained 50 mM Tris (pH8.0), 1% NP40,

150mM NaCl, 1 mM PMSF (Bishop, Cat # PMS123), 2mM Na3VO4,1 tablet of Protease

inhibitor cocktail tablet, 1 ug/ml of Aprotinin (Bioshop, Cat # APR600), Leupeptin (Bioshop,

Cat # LEU001).The samples that were then homogenized with a needle and a syringe on ice.

Different volumes of lysis buffer were added to samples: 500 ug to the cortex and cerebellum,

200 ug to striatum and hippocampus. After brain samples were thoroughly homogenized, they

were centrifuged for 15 min at 1000xg at 4oC. The supernatant was removed and stored at -80 o C

until further use.

2.4.3. Protein concentration measurement for western blots

The protein concentrations of individual samples were determined using the Bradford

protein assay (Invitrogen, Carlsbad CA, BioRad, Cat # 500 - 0006) and absorbance values were

measured at 595 nm wavelength using the spectrophotometer (Beckman model DU640). The

recorded values were normalized to the blank reading of a lysis buffer. Five standard protein

concentrations were determined and plotted to generate a standard curve, which was then used as

a reference to determine protein concentration of the samples. Three replicates for each sample

were used and then averaged to calculate the final absorbance value for each sample.

2.4.4. Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Samples were prepared for gel electrophoresis by addition of loading buffer (50 mM

Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% Beta-mercaptoethanol, 12.5 mM EDTA and

0.02% bromophenol blue) and heating to 95oC for five minutes to denature the proteins. Ten ug

of protein samples were loaded on each gel and resolved by electrophoresis on a 5% acrylamide

stacking gel and 12.5% resolving acrylamide gel in Tris-glycine Laemlli running buffer (25 mM

Tris, 192 mM glycine and 0.1% SDS) at a constant voltage of 100 V for 2 hours. The proteins

were then transferred to a nitrocellulose membrane overnight in the solution of transfer buffer

(25 mM Tris, 192 mM glycine, 20% methanol) at a constant voltage of 23V at 4oC. After the

transfer, the membranes were prehybridized for two hours at room temperature with 5% non-fat dry milk solution dissolved in TBST washing buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween

20). This blocking buffer was used to reduce non-specific protein binding. The membranes were then incubated overnight with a chicken anti-human MeCP2 C-terminus antibody diluted to 1:

30,000 in a blocking solution at 4oC (a gift from Dr. Janine LaSalle, University of California,

Davis). Following primary incubation, the blots were washed extensively in TBST washing buffer, and then incubated for two hours with secondary horseradish peroxidase-conjugated anti chicken antibody diluted in blocking solution to 1: 10,000. Specific immunoreactivity was visualized using enhanced chemiluminescence (GE Healthcare, Amersham ECL Western

Blotting Detection Reagents) and molecular weights were determined with pre-stained markers.

To control for the amount of loaded protein for each well, the membranes were stripped and

incubated with GAPDH primary antibody overnight. Specifically, the membranes were

incubated at 55o C for 30 minutes in a solution of stripping buffer (10% SDS, 1 M Tris pH 6.7,

Beta-mercaptoethanol). The membranes were then washed for 10 min in TBST three times and

placed in a blocking solution for 2 hours at room temperature. Following blocking, the blots

were re-probed overnight with GAPDH primary antibody at 4 o C (1: 10,000). Once again, the

blots were extensively washed in TBST and placed for 2 hours at room temperature in a blocking

solution with secondary anti- mouse antibody (1: 10,000). Following TBST washes, the

chemiluminescence solution was applied as previously mentioned and membranes were exposed

and results analyzed.

2.4.5. Densitometric analysis

Densitometric analysis was done on all Mbd2 null and wild-type protein samples to

assess MeCP2 and GAPDH protein expression levels. The relative optical densities (RODs) were

measured for each sample on the immunoblots using MCID Elite 6.0 program. Three to five

background readings were taken from areas between each sample line and average background

assessment was calculated for every blot. This background value was then subtracted from the

ROD sample value. The ROD – background value for MeCP2 was divided by the corresponding

GAPDH value for normalization calculations. These normalized values were analyzed for

statistical significance with Student’s t-test, plotted, and expressed as percent changes from

control subjects.

2.5. Quantitative real - time – PCR

2.5.1. Samples preparation and specific primers used

Real-time PCR was performed based on procedures described previously (Zhao et al.,

2001). Mbd2 null and wild-type mice were sacrificed and followed by dissections of cortex,

hippocampus, striatum and cerebellum. Total RNA was extracted with RNeasy Mini Kit

(Qiagen, # 74104) following manufacture’s guidelines. RNA concentrations were determined by a Nanodrop TM 1000. To generate cDNA for each sample, one µg of total RNA was used in reverse transcription reaction (RT) with the Superscript II First Strand Synthesis System

(Invitrogen, #18064-014). An inventoried Taqman probe and primer mixtures (Applied

Biosystems) were used in the PCR reaction for MECP2 (Mm00465017_m1), MBD1

(Mm00522100_m1), MBD2 (Mm00521967_m1), MBD3 (Mm00488961_m1), Glucocorticoid

Receptor (Mm00433832_m1), and HPRT1 (Mm03024075_m1). The MBD2 primer used spans the exon 3 and exon 4 of the mbd2 gene but is preserved in the targeted knockout. The

TaqMan® Master Mix Reagent Kit (Applied Biosystems, # 4304437) was used to amplify and quantify each transcript of interest in 10 µl reactions and contained 20 ng of cDNA (0.76 μl of

cDNA, 0.9 μl of yellow dye, and 2.34 μl of distilled water) and 6 μl of primer mix (5 μl of

Master Mix, 0.5 μl of primer and 0.5 μl of blue dye).

2.5.2. PCR running conditions and data analysis

The following quantitative- PCR Universal TaqMan PCR conditions were used: initial

step at 50 °C for 2 min, followed by 95° C for 10 min, then 95° C for 15 sec and finally at 60°C

for 1 min and repeat of 40 cycles. The reaction was run in 96-well optical plates and the

expression of MECP2, MBD1, MBD2, MBD3, Glucocorticoid Receptor and housekeeping gene

Hypoxanthine-guanine phosphoribosyltransferase 1(HPRT1) was assessed by 7900HT Fast Real-

Time PCR System (Applied Biosystems). All the samples were loaded on the same plate in

quadruplicates and run simultaneously to avoid inter-plate variability. In addition, to verify

specific amplification, negative control samples were loaded and ran on the same plate. No

Primer Control (NPC) contained 4 μl of cDNA mix and 6 μl of distilled water without a primer

mix. The other No Template Controls (NTC) were made of 6 μl primer mix and 4 μl of water

only. The results were recorded as a cycle threshold (Ct) values, analyzed by Sequence Detection

System (SDS) Software 2.2. and averaged from the values obtained from each reaction. Gene

expression profiles for MeCP2, MBD1, MBD2, MBD3 and Glucocorticoid Receptor were

normalized to the corresponding HPRT1 levels for each sample.

Chapter 3

Results

3.1. Mbd2-deficient animals display behavioral alteration

3.1.1. Impaired motor performance

3.1.1.1. Impaired Activity Distribution

Even thought mutant mice do not exhibit any obvious signs of motor impairments, we

wanted to investigate whether Mbd2 null mice display more subtle locomotion deficits.

Therefore, I assessed the performance of young mice (3-5 months) and older mice (8-13 months)

in the open field apparatus. This test measures the levels of the overall motor performance in

mice during a 60 minute test period. Our results revealed that Mbd2- deficient mice displayed a

lower level of overall activity compared to wild-type mice (Fig. 6 A). Additionally, mutant mice

in both age groups spent significantly less time walking and considerably less time in the mobile

state (Fig. 6 B, C). The extent of rearing, expressed as the percentage of time was significantly

reduced in both age groups of mutant mice compared to age-matched wild-type animals (Fig.

7A). Also the degree of center rearing (beam breaks per hour) was significantly lower in mutant

mice form both age groups, compared to control animals (Fig.7 B). Male and female mice were

equally represented in the following assay and no significant gender – associated alterations in

performance were found.

3.1.1.2. Impaired Mobility Distribution

Older Mbd2 null mice were found to display alterations in fast and slow mobility

compared to wild-type mice. Specifically, mutant mice had significantly fewer beam break

counts per hour during both slow and fast mobility (Fig.8 A, B).

Figure 6: Activity Distribution of Mbd2 null and their age-matched wild-type control mice

assessed during a 60 minute Open Field test.

A) The overall activity of wild-type (n = 42 young group, n = 70 older group) and Mbd2 null

(n = 35 young group, n = 40 older group) mice expressed as beam breaks per hour.

Younger mutant mice (3-5 months) on average displayed a similar level of overall

activity relative to control younger mice. However older Mbd2 null mice displayed

significantly fewer beam breaks per hour as a measure of overall activity compared to

age-matched wild-type mice. *P < 0.05 compared with wild-type, two-way ANOVA

(genotype and age) with Bonferroni post hoc correction.

B) The extent of mobility in wild-type (n = 42 young group, n = 70 older group) and Mbd2

null (n = 35 young group, n = 40 older group) was measured during a one hour Open

Field test. It was expressed as the percentage of total test time.Mbd2-deficient mice from

both age groups spent significantly less time in the mobile state. *P < 0.05 compared with

wild-type, two-way ANOVA (genotype and age) with Bonferroni post hoc correction.

D) The duration of walking was assessed in wild-type (n = 42 young group, n = 70 older

group) and Mbd2 null (n = 35 young group, n = 40 older group) mice during a one hour

Open Field test. It was expressed as the percentage of total test time.Mbd2-deficient

mice from both age groups spent significantly less time walking. *P < 0.05 compared

with wild-type, two-way ANOVA (genotype and age) with Bonferroni post hoc

correction.

Figure 6 Activity

A) 15000 * WT MBD2 Null

10000

5000

Beam BreaksBeam Per Hour 0 3-5 months 8-13 months

B) Percentage of time mobile

30 * * WT

25 MBD2 Null

5 Percentage (%) of time (%) of Percentage 0 3-5 months 8-13 months

Percentage of time walking 30 * * WT 25 MBD2 Null

rcentage of time (%) time of rcentage 5 48 Pe 0 3-5 months 8-13 months Figure 7: Rearing behavior displayed by mutant and wild-type mice.

A) Rearing in wild-type (n = 42 young group, n = 70 older group) and Mbd2 null (n = 35

young group, n = 40 older group) mice was measured during a one hour Open Field test

and expressed as the percentage of total test time.Mbd2-deficient mice from both age

groups spent significantly less time rearing. *P < 0.05 compared with wild-type, two-way

ANOVA (genotype and age) with Bonferroni post hoc correction.

B) Center rearing behavior of wild-type (n = 42 young group, n = 70 older group) and Mbd2

null (n = 35 young group, n = 40 older group) measured during a one hour Open Field

test and expressed as beam break per hour. Mutant mice in both age cohorts displayed

significantly fewer beam break per hour compared to age-matched wild-type mice. *P <

0.05 compared with wild-type, two-way ANOVA (genotype and age) with Bonferroni

post hoc correction.

Figure 7

A) Percentage of time rearing

* 25 * WT

20 MBD2 Null

Percentage (%) time of 0 3-5 months 8-13 months

B) Center Rearing

* 300 * WT

Null 200

100

BreaksBeam Per Hour 0 3-5 months 8-13 months

Figure 8: Mobility Distribution expressed as total mobility, fast mobility and slow mobility for young (3-5 months) and older (8-13 months) wild-type and Mbd2-null mice.

A) Total mobility in wild-type (n = 42 young group, n = 70 older group) and Mbd2-null

(n = 35 young group, n = 40 older group) mice was assessed as the total number of beam

breaks in one hour. Younger Mbd2 null mice did not differ significantly with respect to

their total mobility. However, older mutant mice displayed significant reductions in total

mobility. *P < 0.05 compared with wild-type, two-way ANOVA (genotype and age) with

Bonferroni post hoc correction.

B) Fast mobility in wild-type (n = 42 young group, n = 70 older group) and Mbd2-null

(n = 35 young group, n = 40 older group) mice was assessed as the total number of beam

breaks in one hour. Mutant mice from the older age cohort displayed significant

reductions in fast mobility. *P < 0.05 compared with wild-type, two-way ANOVA

(genotype and age) with Bonferroni post hoc correction.

C) Slow mobility in wild-type (n = 42 young group, n = 70 older group) and Mbd2-null

(n = 35 young group, n = 40 older group) mice was assessed as the total number of beam

breaks in one hour. Mutant mice from the older age cohort displayed significant

reductions in slow mobility. *P < 0.05 compared with wild-type, two-way ANOVA

(genotype and age) with Bonferroni post hoc correction.

Figure 8 Total Mobility * 5000 A) WT 4000 MBD2 Null

3000

2000

1000

Beam BreaksBeam PerHour 0 3-5 months 8-13 months

Fast Mobility

4000 * WT B) 3500 MBD2 Null 3000

2500 2000 1500

1000 500 Beam BreaksBeam Per Hour 0 3-5 months 8-13 months

Slow Mobility

* 1500 C) WT 1250 MBD2 Null

1000

750

500 Breaks Per Hour 52 250

Beam 0 3-5 months 8-13 months 3.1.1.3. Mbd2 null mice show enhanced motor coordination performance during the rotarod test

To further test motor coordination, animals were subjected to the rotarod assay for three

trials a day for four consecutive days. The mean latency to fall for each day was recorded. Mbd2-

deficient young mice spent similar amounts of time on the rotating rod compared to wild-type

mice, except for the time duration on the last trial during the last day. During the last trial Mbd2

null mice stayed significantly longer on the rotarod (Fig. 9 A). However, Mbd2 null mice in the

older age group on average performed better at this behavioral task compared to wild-type

littermate controls. In particular, Mbd2 null mice spent significantly more time on the rotating

rod on the last trial of the third day and the last day of training during trials 10 and 12 (Fig. 9 B).

These data collectively demonstrate that Mbd2 null mice are capable of motor learning and have

good motor coordination skills. Additionally, male and female mice were equally represented in

the following assay and no significant gender – associated alterations in performance were

found.

Figure 9: Motor coordination performance on the rotating rotarod.

A) Average time wild-type (n = 29) and mutant mice (n = 21) spent on the rotarod before

falling for a cohort of young mice (3-5 months). Mbd2 null mice spent significantly more

time on the rotating rotarod during the last third trial on day 4. *P < 0.05 compared with

wild-type, Student’s two tailed t-test, followed by Bonferroni post hoc correction.

B) Average time wild-type (n = 15) and mutant mice (n = 29) spent on the rotarod before

falling for a cohort of older mice (8-13 months). Mutant mice spent significantly more

time on the rotating rod before losing their balance during the third day third trial and

during the last training day on two trials. *P < 0.05 compared with wild-type, Student’s

two tailed t-test, followed by Bonferroni post hoc correction.

Figure 9

A) Time Spent on the Rotorod Before Falling (3-5 months of age)

500 WT * 450 MBD2 Null

lling(sec) 400

350 re f 300

250 200 150

Seconds befoSeconds a 100 Day 1 Day 2 Day 3 Day 4

B) Time Spent on the Rotorod Before Falling (8-13 months of age)

500 * * WT 450 MBD2 Null 400 * 350 300

250 200 150 100 (sec) Seconds before falling Day 1 Day 2 Day 3 Day 4 55

3.1.2. Socialization Behavioral Impairments

3.1.2.1. Mbd2- deficient mice show impairments in social interactions without physical contact

The test of social interaction was used to assess behavioral reactivity of test mice to

familiar and novel mice. The assay consisted of two social interaction trials and was based on the

paradigm previously described (Crawley, 2004).During the first trial, a test mouse interacted

with a previously familiar co-housed mouse. The second trial involved the introduction of a

novel mouse to the test cage. The number and duration of interactions initiated by the test mouse were recorded and analyzed for each trial.

Control and Mbd2- null mice in the young age cohort (3-5 months) spent on average the

same amount of time interacting with a familiar mouse during trial 1 (Fig. 10 A). In contrast,

older Mbd2 (-/-) mice spent on average 51% less time in direct contact with a previously co-

housed mouse during trial 1 compared to their age- matched control group (Fig. 10 A).

During the second trial no significant differences in the duration of interactions between

young mutant and wild-type mice were observed. Both young groups displayed an increased

interest in a novel mouse. Young wild-type mice spent on average twice the amount of time with

a novel mouse compared to the time with a co-housed mouse from trial 1. Similarly Mbd2- null

mice spent on average 1.8 more time with a mouse during trial 2 (Fig. 10 B). However, mutant

mice in the older group were less social and spent significantly less time interacting with a novel

mouse compared to control mice (Fig. 10 B). On average, older Mbd2-deficient mice spent 44 %

less time with a novel mouse compared to age matched wild-types (Fig. 10 B). Additionally,

male and female mice were equally represented in the following assay and no significant gender

– associated alterations in performance were found.

Figure 10: Amount of time wild-type and Mbd2 null mice spent interacting with another

mouse during the two trials of the Social Interaction Test.

A) The amount of time wild-type (n = 10 younger group, n =14 older group) and Mbd2 null

(n = 5 younger group, n = 19 older group) mice spent interacting with a Co-housed

Familiar mouse during Trial 1 of the Social Interaction assay. No significant differences

in the amount of interaction time were observed between wild-type and Mbd2-null mice

in the young age group. However, older mutant mice spent significantly less time

compared to older wild-type mice interacting with their co-housed mouse during Trial 1.

*P < 0.05 compared with wild-type, two-way ANOVA (genotype and age) with

Bonferroni post hoc correction.

B) The amount of time older wild-type (n = 10 younger group, n =14 older group) and Mbd2

null (n = 5 younger group, n = 19 older group) mice spent interacting with a Novel mouse

during Trial 2 of the Social Interaction assay. Younger mutant and wild-type mice spent

similar amount of time exploring the novel mouse. However, significant differences in

the amount of interaction time were observed between older wild-type and Mbd2-null

mice. *P < 0.05 compared with wild-type, two-way ANOVA (genotype and age) with

Bonferroni post hoc correction.

Figure 10

A) Time Spent With a Co-Housed Mouse During Trial 1

140 WT 120 MBD2 Null 100

80 * 60 40 20 Amount of time (sec) time of Amount 0 3-5 months 8-13 months

B) Time Spent W ith a Novel Mouse During Trial 2

140 WT

120 MBD2 Null 100 * 80 60

Amount of time (sec) time of Amount 20 0 3-5 months 8-13 months

3.1.2.2. Mbd2-null mice initiated fewer social interactions

An additional parameter of social activity was the number of interactions initiated by the

test mouse. Younger and older Mbd2- null mice initiated roughly the same number of social

interactions as their age-matched wild-type mice during the first trial (Fig. 11 A). During the

second trial, younger Mbd2 – deficient mice displayed a similar level of interest in a novel

mouse compared to age-matched wild-type mice (Fig. 11 B). However, when older mutant mice

were exposed to a novel mouse, they initiated significantly fewer socialization seeking behaviors

(Fig. 11 B).

3.1.2.3. Mbd2-null mice displayed more of Social Avoidance Behavior

The duration of social avoidance behavior was assessed as the time mice spent on the top

of the wire tent, away from the partner mouse. Younger Mbd2-deficient mice avoided social

interactions during both trials for approximately the same time as their age-matched control mice

(Fig.12 A, Fig. 12 B). However, mutant mice from the older age cohort (8-13 months) displayed

significantly more time avoiding the novel mouse during trial 2 compared to wild-type mice

(Fig. 12 B). Yet, this avoidance behavior did not differ significantly between older wild-type and

Mbd2-null mice during the first trial, suggesting that older mutant mice avoided the interactions with a novel mouse only (Fig.12 A). Overall these data indicate that older Mbd2-deficient mice

have impairments in social interactions.

Figure 11: Number of interactions wild-type and Mbd2 null mice initiated during the two trials of the Social Interaction test.

A) The number of times wild-type (n = 10 younger group, n =14 older group) and Mbd2 null

(n = 5 younger group, n = 19 older group) mice spent interacting with their Co-housed

Familiar partner mouse during Trial 1. No significant differences in the social interaction

interest were found.

B) The number of times wild-type (n = 10 younger group, n =14 older group) and Mbd2 null

(n = 5 younger group, n = 19 older group) mice spent interacting with their novel partner

mouse during Trial 2. Older MBD2-deficient mice initiated significantly fewer

interactions compared to older wild-type mice. *P < 0.05 compared with wild-type, two-

way ANOVA (genotype and age) with Bonferroni post hoc correction.

Figure 11

A) Number of Interactions with a Co-housed Mouse During Trial1

60 WT 50 MBD2 Null

Number of interactionsNumber of (n) 0 3-5 months 8-13 months

B) Number of Interactions with a Novel Mouse During Trial 2

60 WT 50 MBD2 Null * 40

Number interactions of (n) 0 3-5 months 8-13 months

Figure 12: Social Avoidance Behavior displayed by Mbd2 null and wild-type age matched control mice during the two trials of the Social Interaction Assay.

A) Amount of time wild-type (n = 10 younger group, n = 14 older group) and Mbd2 null

(n = 5 younger group, n = 19 older group) mice spent avoiding social interactions during

Trial 1 of the Social Interaction test. No statistically significant changes between the behavior

of wild-type and mutant mice were observed.

B) Amount of time wild-type (n = 10 younger group, n =14 older group) and Mbd2 null

(n = 5 younger group, n = 19 older group) mice spent avoiding social interactions during

Trial 2 of the Social Interaction test. Older mutant mice spent significantly more time

avoiding social interactions with a stranger mouse compared to age-matched wild-type mice.

*P < 0.05 compared with wild-type, two-way ANOVA (genotype and age) with Bonferroni

post hoc correction.

Figure 12

A) Time Spent on Avoidance Behavior During Trial 1

300 WT 250 MBD2 Null

200

150

100

50 Amount of time (sec) time of Amount

0 3-5 months 8-13 months

B) Time Spent on Avoidance Behavior During Trial 2

300 WT MBD2 Null 250

200

150

100

50 Amount of time (sec) time of Amount

0 3-5 months 8-13 months

3.1.3. Mbd2 null animals display deficits in nest-building behavior

3.1.3.1. Diminished interaction with the neslet

Nest building behavior is a commonly used assay for measuring home cage activity

related to social behavior (Moretti et al., 2005). Considering previous reports of impaired

maternal behavior in MBD2 - deficient mice, learning about nest building behavior becomes

particularly informative. We tested this skill in two separate age cohorts of mice: younger (3-5 months of age) and older (8-13 months of age). Male and female mice were equally represented

in the following assay and no significant gender – associated alterations in performance were

found. A new neslet was introduced to a mouse cage and the behavior of mice was recorded for

30 minutes. Twenty four hours later the quality of the final nest was assessed. Wild-type mice

from the young age group (3-5 months) approached the new neslet on average 1.6 times more

than mutant mice of the same age (Fig. 13 A). However, younger Mbd2 null mice spent on

average similar amount of time as wild –type mice tearing and actively manipulating nesting

material (Fig. 13 B). MBD2-deficient mice from the older cohort (8-13 months) displayed

impaired nest building behavior. They spent significantly less time interacting with the nesting

material and approached the neslet significantly fewer times (Fig. 13 A, B).

3.1.3.2. Impaired nest quality

The final quality and height of the resultant nest were assessed 24 hours after the introduction of the cotton neslet. As illustrated in Fig. 14, the resultant nests made by wild-type and Mbd2-null mice significantly differed in quality. In the younger age group 83.3% (10/12) of wild-type mice had a completely processed and chewed neslet, resulting in a well fluffed nest.

Similarly, 81% (17/21) of control mice from the older group chewed the cotton neslet

completely. However, only 28.5% (2/7) of Mbd2- null younger mice and 21.7% (5/23) of Mbd2-

null older mice had nest of similar quality. The height of the resultant nest was also significantly

different between wild-type and MBD2-deficient mice. The height of the nests made by younger

and older mutant mice was on average 43% and 44% smaller compared to age-matched control

groups, respectively (Fig. 15).

Also the appearance of the nests differed between the groups. As previously noted by

Moretti et al., nests made by the wild-type mice had a cocoon- like shape (Moretti et al., 2005).

In the wild-type older group 71.4% (17/21) of mice built a cocoon-like nest while 66% (8/12) of

younger control mice produced a nest of similar quality (Fig.16). Conversely, the nests of mutant

mice did not resemble a cocoon and were less organized and much wider in the parameter.

Specifically, only 14.3% (1/7) of nests made by younger mice and 13.04% (3/23) of resultant

nests by older mice had a similar cocoon-like appearance (Fig. 16).

Figure 13: Nesting behaviors performed by wild-type and Mbd2-null mice.

A) Number of interactions wild-type (n = 11 younger group, n =19 older group) and

Mbd2 null (n = 7 younger group, n = 18 older group) mice initiated with a new neslet

during the first 30 minutes of the test. The behavioral assay was performed on two age

cohorts of mice: (3-5 months) and (8-13 months). Significant differences were

observed between the number of interactions initiated by wild-type and Mbd2-null

mice from both age cohorts. Mutant mice displayed significantly fewer interactions

compared to age-matched wild-type mice. *P < 0.05 compared with wild-type, two-

way ANOVA (genotype and age) with Bonferroni post hoc correction.

B) The amount of time wild-type (n = 11 younger group, n =19 older group) and Mbd2

null (n = 7 younger group, n = 18 older group) mice spent interacting with a neslet

during the first 30 minutes of the test. Significant differences in time were observed

between wild-type and Mbd2-null mice from both age cohorts. *P < 0.05 compared

with wild-type, two-way ANOVA (genotype and age) with Bonferroni post hoc

correction.

Figure 13

50 * WT * 40 MBD2 Null

Number interactions of (n) 0 3-5 months 8-13 months

200 * WT * MBD2 Null 150

100

Amount of time (sec) time of Amount

0 3-5 months 8-13 months

Figure 14: Pictures of the nests 24 hours after a new neslet was introduced to the cage.

A) A representative example of the nests made by wild type mice (n = 11) from the young

group (3-5 months).

B) A representative example of the nests made by wild type mice (n = 19) from the older

group (8-13 months).

C) A representative example of nests made by Mbd2-null mice (n = 7) from the young group

(3-5 months).

D) A representative example of nests made by Mbd2-null mice (n = 18) from the older group

(8-13 months).

Figure 14

A) B)

C) D)

Figure 15: The resultant nests made by wild-type and Mbd2 null mice.

A) The height of the resultant nest measured in cm, 24 hours after the new neslet was

placed in a cage. Significant differences were observed between the nest height in wild-

type and Mbd2 null mice from both age cohorts. *P < 0.05 compared with wild-type,

two-way ANOVA (genotype and age) with Bonferroni post hoc correction.

B) Number of nests with a cocoon like shape expressed as a percentage of total number of

nests. These nets were made by wild-type and Mbd2 null young (3-5 months) and older

(8-13 months) mice.

Figure 15

A) *

4.0 * WT 3.5 MBD2 Null 3.0

2.5 2.0

1.5 1.0

0.5

The height of the heightThe of nest (cm) 0.0 3-5 months 8-13 months

B) Nests with a cocoon-like shape

20 (%) Percentage

0 young WT young Null older WT older Null 71

3.1.4 Mbd2 - null mice display increased anxiety behavior

3.1.4.1 Mbd2 - null mice display increased anxiety during Light Dark Preference Test

3.1.4.1.1. Mbd2-null mice show no differences in Light and Dark chamber preference

To determine whether Mbd2- null mice experience elevated anxiety levels, I assessed

their performance in the light-dark preference test (Crawley, 1999). Briefly, mice were placed in

a chamber unequally divided into dark and illuminated compartments. The amount of time mice

spent in each compartment as well as the number of transitions between them was recorded.

Mbd2 null mice spent on average the same amount of time in the Light and Dark compartments

as their age-matched wild-type control mice (Fig. 16 A, B). Male and female mice were equally

represented in the following assay and no significant gender – associated alterations in

performance were found.

3.1.4.1.2 .Impairments in Risk assessment behaviors

In addition, we also assessed the number of risk assessment behaviors as the measure of

anxiety. Younger mutant mice displayed similar number of risk assessment behaviors as wild-

type mice (Fig. 17 A). However, we found that older Mbd2 null mice displayed significantly

fewer risk assessment behaviors (Fig. 17 A). Another parameter of anxiety behavior was the

amount of time risk assessing expressed as a percent fraction of total time spent in the dark

chamber. Older Mbd2-deficient mice spent significantly lower percentage of time engaged in

risk assessing behavior relative to wild-type mice (Fig. 17 B). Once again, these behavioral

alterations were not seen in younger mutant mice (Fig. 17 B). Taken together, it is clear that

Mbd2-null mice from the older age cohort are more anxious and less inquisitive than their age- matched control mice.

Figure 16: Time spent in the Light and Dark Compartments.

A) The amount of time wild-type (n = 20 younger group, n = 47 older group) and Mbd2 null

(n = 29 younger group, n = 30 older group) mice spent in the Dark compartment of the

Light Dark Apparatus. Mutant mice spent on average similar amount of time as wild-type

mice in the Dark compartment of the apparatus.

B) The amount of time wild-type (n = 20 younger group, n = 47 older group) and Mbd2 null

(n = 29 younger group, n = 30 older group) time spent in the Light compartment of the

Light Dark Apparatus. Mutant mice spent on average similar amount of time as wild-type

mice in the Light compartment of the apparatus.

Figure 16

Dark Compartment A)

300 WT MBD2 Null

200

100

ount of time (sec)ount time of

0 3-5 months 8-13 months

Light Compartment

200 WT ) Legend sec 150

100

Amount of time ( time of Amount

0 3-5 months 8-13 months 74

Figure 17: Risk Assessment behaviors performed during Light and Dark behavioral test.

A) Number of risk assessment behaviors performed by wild-type (n = 20 younger group,

n = 47 older group) and Mbd2 null (n = 29 younger group, n = 30 older group) mice

during a five minute Light Dark behavioral assay. No differences in behavior were

observed between young wild-type and mutant mice. However, a significant difference

was found in the risk assessment behavior of older mutant mice. *P < 0.05 compared

with wild-type, two-way ANOVA (genotype and age) with Bonferroni post hoc

correction.

B) The duration of each risk assessment behavior expressed as a percent fraction of total

time spent in the dark chamber. A significant difference between Mbd2-null and wild-

type mice was found at 8-13 months of age period. *P < 0.05 compared with wild-type,

two-way ANOVA (genotype and age) with Bonferroni post hoc correction.

Figure 17

* 20 WT hav MBD2 Null 15

sse 5

0 3-5 months 8-13 months

risk aNumber of be ssment iors (n)

20 * WT MBD2 Null 15

0 3-5 months 8-13 months

Percentage risk time of (%) assessing

3.1.4.2. Mbd2-/- mice displayed increased anxiety during Elevated Plus Maze testing

Elevated Plus Maze test was also used to assess anxiety-like behaviors in mice. To

summarize, mice were placed in the middle platform of the apparatus during a five minute

testing session. The amount of time mice spent in the Open arms (proximal and distal areas),

Closed arms (proximal and distal areas) and the middle platform was recorded. I found that

Mbd2-/- mice spent on average the same amount of time as control mice in the open arms, closed

arms and the middle platform in a young age group (Fig. 18 A, B, C). Yet older Mbd2 null mice

spent significantly more time in the closed arms of the apparatus and significantly less time in

the middle platform compared to older wild-type mice (Fig.18 A,B,C). In addition, further

thorough data analysis revealed subjects’ preference for different areas of the test. Specifically,

older Mbd2 null mice spent significantly more time in the distal area of the closed arm compared

to their age-matched controls (Fig. 19 A).This suggests of greater anxiety experienced by mutant

mice and their preference for a safe location (far end of the closed arms).However young mice

did not show such preference. Specifically, there were no significant changes in the amount of

time spent in the distal area of the closed arm, expressed as the percentage of total time in the

closed arm (Fig. 19 A).Additionally, mutant mice did not show a preference for the distal area of

the open arms (Fig. 19 B).Collectively, these data indicate that Mbd2 null mice display a

phenotype consistent with heightened anxiety at an older age. Male and female mice were

equally represented in the following assay and no significant gender – associated alterations in

performance were found.

Figure 18: Total amount of time wild-type and mutant mice spent in the Middle Platform,

Open Arm, and the Closed Arm of the Elevated Plus Maze.

A) Total amount of time wild-type (n = 12 younger group, n = 32 older group) and Mbd2

null (n = 30 younger group, n = 29 older group) mice spent in the Middle Platform of the

Elevated Plus Maze during a five minute test period. No significant differences in the

platform preference were displayed by younger mutant mice. However, older Mbd2 null

mice spent significantly more time in the Middle platform compared to older wild-type

mice. *P < 0.05 compared with wild-type, two-way ANOVA (genotype and age) with

Bonferroni post hoc correction.

B) Total amount of time wild-type (n = 12 younger group, n = 32 older group) and Mbd2

null (n = 30 younger group, n = 29 older group) mice spent in the Open Arms of the

Elevated Plus Maze during a five minute test period. No statistically significant

differences in the Open Arm preference were found.

C) Total amount of time wild-type (n = 12 younger group, n = 32 older group) and Mbd2

null (n = 30 younger group, n = 29 older group) mice spent in the Closed Arms of the

Elevated Plus Maze during a five minute test period. Older Mbd2 null mice spent

significantly more time in the Closed Arms of the Elevated Plus Maze test. *P < 0.05

compared with wild-type, two-way ANOVA (genotype and age) with Bonferroni post

hoc correction.

Figure 18 Time in the Middle Platform A) * 100 WT 80 MBD2 Null

20 Amount of time (sec) time of Amount

0 3-5 months 8-13 months

Time in the Open Arms 100 B) WT 80 MBD2 Null

(sec) time of Amount 0 3-5 months 8-13 months

Time in the Closed Arms * C) 250 WT

200 MBD2 Null

150

100

50 mount of time (sec) time of mount 79 A

0 3-5 months 8-13 months Figure 19: Time spent in the distal area expressed as a fraction of total time in the Closed arms of the Elevated Plus Maze.

A) Percentage of time wild-type (n = 12 younger group, n = 32 older group) and Mbd2

null (n = 30 younger group, n = 29 older group) mice spent in the distal area of the

Closed arm as a fraction of total time spent in the Closed arms. Older Mbd2 deficient

mice spent significantly more time in distal arm of the Closed arms compared to age-

matched wild-type mice. *P < 0.05 compared with wild-type, two-way ANOVA

(genotype and age) with Bonferroni post hoc correction.

B) Percentage of time spent in the distal area of the Open arm as a fraction of total time

spent in the Open arms. Mutant and wild-type mice spent on average the same

amount of time in the distal area of the open arms.

Figure 19

Percentage of Time in the Closed Arm Distal Area/ Total Time in the Closed Arm A)

80 WT * MBD2 Null 60

Percentage (%) time of 0 3-5 months 8-13 months

B) Percentage of Time in the Open Arm Distal Area/Total Time in the Open Arm

40 WT

MBD2 Null 30

Percentage of time (%) time of Percentage 0 3-5 months 8-13 months

3.1.5. Mbd2- deficient mice show deficits in object recognition task

To assess whether Mbd2 null mice display deficits in object recognition and memory, we

assessed their performance in the novel object recognition task. Briefly, mice were introduced to two objects that they explored for 10 minutes that were then designated as old or familial objects.

After a five minute delay, mice were given an old object and a novel object that they had no

previous contact with. We assessed the duration of time mice spent exploring the new and the

old objects during the first trial. The second trial took place after a one hour long delay. Only one

older cohort of mice (8-13 months) was tested for this novel object recognition ability. Also male and female mice were equally represented in the following assay and no significant gender –

associated alterations in performance were found. We found that mutant mice exhibited a

reduced level of interest towards a novel object compared to wild-type mice after a five minute delay period (Fig. 20 B). Specifically, Mbd2 null mice spent less time exploring the novel object during the first trial (Fig. 20 B). Interestingly, mutant mice spent similar amount of time as wild- type mice exploring the old object (Fig. 20 A). In addition to time duration, a novelty discrimination index (DIN) was also used to measure the degree of interest for the novel toy

(Niewiadomska et al., 2006). DIN was calculated as: DIN = tN/ (tF + tN), where tN = exploration

time of the novel object and tF = mean exploration time of the familiar objects. Intriguingly,

statistically significant changes in DIN values for trial 1 and trial 2 were found between wild-type

and mutant mice (Fig. 20 C).

Figure 20: Novel Object Recognition Test

A) The amount of time (sec) wild-type (n = 16) and Mbd2 null (n = 10) mice spent

interacting with an Old Object during the two trials of the novel object recognition test.

No statistically significant changes in time duration were found between wild-type and

Mbd2 null mice.

B) The amount of time (sec) wild-type (n = 16) and Mbd2 null (n = 10) mice spent

interacting with a Novel Object during the two trials of the novel object recognition test.

Mbd2-deficient mice spent significantly less time interacting with a Novel Object during

the first trial. *P < 0.05 compared with wild-type, two-way ANOVA with repeated

measures with Bonferroni post hoc correction.

C) Discrimination Index (DI) values calculated during two trials. Statistically significant

changes were found for DI values for trial 1 and trial 2. *P < 0.05 compared with wild-

type, two-way ANOVA with repeated measures with Bonferroni post hoc correction.

Figure 20 Amount of time spent exploring the Old Object A) 20 WT MBD2 Null 15

(sec) time of Amount 0 trial 1 trial 2

B) Amount of time spent exploring the Novel Object

* 40 WT MBD2 Null 30

10 Amount of time (sec)time of Amount 0 Trial 1 Trial 2

C) Discrimination Index Values

* 0.8 * WT

MBD2 Null 0.6

0.4

0.2

crim

Dis0.0 ination Index (DI) 84 trial 1 trial 2

3.2. Electroencephalographic (EEG) Assessments

3.2.1. Mbd2-deficient mice display normal theta waveform during exploration

I tested whether hippocampal theta wave activity is present in Mbd2 null mice during

cage exploration behaviors. As the animals were exploring their environment (sniffing), clear theta waveform was observed in the hippocampus in both wild-type (Fig. 21 A, B) and mutant mice (Fig. 22 A, B). In addition, spectral analysis of all the theta waveforms during a recording session revealed no significant differences in the peak theta frequency between wild-type and

Mbd2-null mice (Fig. 23 A, B). The average theta frequency for the 9 to 13 month-old wild-type mice (n = 7) was 8.47 Hz, while the average frequency for the age-matched Mbd2 null mice

(n = 5) was 8.42 Hz (Fig. 24 A).Thus, these data indicate that Mbd2 null hippocampus is able to generate theta activity with the theta peak frequency similar to wild-type hippocampus.

Additionally, during EEG theta rhythm characterization, no obvious abnormal neuronal activity or epileptoform – like discharges were seen in the recordings of mutant mice. However a more rigorous analysis is required to confirm a total EEG preservation.

Figure 21: Hippocampal theta rhythm in wild-type mice during home cage exploration.

A) Representative EEG tracings of CA1 hippocampal region and somatosensory cortex in a

wild-type mouse. The top segment shows the theta rhythm recorded from the hippocampus while the bottom trace depicts the corresponding low amplitude cortical activity.

B) Trace from A) but with shown a greater resolution.

Figure 21

hippocampus

1 mV

2 s

cortex

hippocampus

1 mV 0.5 s

cortex

Figure 22: Hippocampal theta rhythm in Mbd2-deficient mice during home cage

exploration.

A) Representative EEG tracings of CA1 hippocampal region and somatosensory cortex in a

Mbd2 null mouse. The top segment shows the theta rhythm recorded from the

hippocampus while the bottom trace depicts the corresponding low amplitude cortical

activity.

B) Trace from A) but shown with a greater resolution.

Figure 22

A) hippocampus

1 mV

2 s

cortex

B) hippocampus

1 mV 0.5 s

cortex 89

Figure 23: Sample of a Power Spectrum analysis of theta rhythm for wild-type and Mbd2

null mice (8-13 months).

A) A representative sample of the power spectrum analysis of a theta waveform recorded

from a wild-type animal. This spectrum contains 30 theta segments observed during

exploration and sniffing of the home cage environment. All of these segments are

averaged, giving a peak theta frequency for each animal. In the following example, the

peak theta frequency is 8.5 Hz.

B) A representative sample of the power spectrum analysis of a theta waveform from a

Mbd2 null animal. This spectrum contains 30 theta segments recorded during exploration

and sniffing of the home cage environment. These segments are averaged, producing a

peak theta frequency for each animal. In the following example, the peak theta frequency

is 9 Hz.

Figure 23 Power Spectrum F:\09211003_reduced_1.abf A) 0.004

0.003

0.002

Amplitude (mV² / Hz) / (mV² Amplitude

0.001

-10 -5 0 5 10 15 20 25 30 35 40 45 50 Frequency (Hz)

Power Spectrum B) F:\MBD2 recordings\08211000_reduced_1.abf

0.06

0.04

Amplitude (mV² / Hz) / (mV² Amplitude

0.02

-5 0 5 10 15 20 25 30 35 40 45 Frequency (Hz)

Figure 24: Peak theta frequency observed during exploration in wild-type and MBD2 null

mice (8-13 months).

A) No significant difference in the frequency of dominant theta frequency observed during

exploratory behavior was found between wild-type and Mbd2-deficient mice. Student’s

unpaired t-test, (P< 0.05).

Figure 24

10 9 8 7

6 5 4 3 2

Peak theta frequency (Hz) 0 WT MBD2 null

3.3. Assessment of MeCP2, MBD1, MBD2, MBD3 and Glucocorticoid receptor mRNA

expression levels in Mbd2 null neural tissues.

3.3.1. MeCP2 mRNA levels are elevated in the cortex of Mbd2 null mice

Although Mbd2 null mice model has been used extensively, up to date no studies have

examined mRNA profiles of the other MBD transcripts in neural tissues of these mice.

Therefore, one of the aims of my study was to determine whether mRNA levels of MeCP2,

MBD1 and MBD3 in the brain change as a result of MBD2 deficiency. I performed a quantitative real-time PCR analysis in Mbd2 null and wild-type cortical, hipppocampal and striatum tissues.

The mRNA MeCP2 expression relative to Hypoxanthine-guanine

phosphoribosyltransferase (Hprt) was significantly higher in Mbd2 null cortical tissues compared

to MeCP2 expression in age-matched control samples (Fig. 25 A). However, MeCP2 mRNA

expression in the hippocampus and striatum did not differ significantly between wild type and

mutant samples (Fig. 25 B, C). As expected, MBD2 mRNA levels were significantly lower in all

three brain regions in Mbd2 null mice compared to wild-type tissues.

3.3.2. Reduced GR mRNA expression in neural Mbd2 null tissues

A heightened anxiety-like behavioral phenotype observed in Mbd2 null mice prompted

the assessment of Glucocorticoid Receptor levels in neural tissues of these mice. Specifically,

Glucocorticoid Receptor expression was assessed in the cortex, hippocampus and striatum

samples of older Mbd2 null mice (8-13 months) and their age-matched control cohort. The

levels of Glucocorticoid Receptor mRNA (relative to Hprt) were significantly lower in the

hippocampus and cortex of Mbd2 null mice (Fig. 26 A). Similarly, Glucocorticoid Receptor

striatum mRNA levels were also lower in Mbd2 null samples, but the differences were not

statistically significant (Fig. 26 A).

Figure 25: mRNA expression levels of MeCP2, MBD1, MBD2, and MBD3 in the cortex, hippocampus and striatum of aged mice (8-13 months) in Mbd2 null and wild-type mice

A) mRNA expression levels of MeCP2, MBD1, MBD2 and MBD3 relative to Hprt in the

cortex of Mbd2 null and wild-type mice. MeCP2 mRNA expression was significantly

higher in the cortex of Mbd2 null animals. As expected, MBD2 mRNA expression was

significantly lower in the cortex of Mbd2-deficient mice. *P < 0.05 compared with

wild-type, non-paired, two-tailed Student’s t-test.

B) mRNA expression levels of MeCP2, MBD1, MBD2 and MBD3 relative to Hprt in the

hippocampus of Mbd2 null and wild-type mice. As expected, MBD2 mRNA expression

was significantly lower in the hippocampus of Mbd2-deficient mice. *P < 0.05

compared with wild-type, non-paired, two-tailed Student’s t-test.

C) mRNA expression levels of MeCP2, MBD1, MBD2 and MBD3 relative to Hprt in the

striatum of Mbd2 null and wild-type mice. As expected, MBD2 mRNA expression was

significantly lower in the striatum of Mbd2-deficient mice. *P < 0.05 compared with

wild-type, non-paired, two-tailed Student’s t-test.

Figure 25

mRNA expression in the cortex A) 3.5 3.2 WT 2.9 MBD2 Null 2.6 2.3 * 2.0 1.7 * 1.4 1.1 0.8 0.5 0.2 mRNAlevels relative to Hprt MeCP2 MBD1 MBD2 MBD3

mRNA expression in the hippocampus B)

3.4 WT 3.1 2.8 MBD2 Null 2.5 2.2 * 1.9 1.6 1.3 1.0 0.7 0.4 0.1 mRNA levels relative to levels Hprt mRNA MeCP2 MBD1 MBD2 MBD3

mRNA expression in the striatum

3.5 C) WT 3.0 MBD2 Null 2.5 *

2.0 1.5

1.0 levels relative to Hprt relative levels 0.5 97 0.0 mRNA MeCP2 MBD1 MBD2 MBD3 Figure 26: mRNA expression levels of Glucocorticoid Receptor in the cortex, hippocampus

and striatum of older mice (8-13 months) relative to Hprt expression.

A) Glucocorticoid Receptor mRNA level expressed relative to Hprt in three brain

regions. The mRNA values are expressed relative to Hprt levels. mRNA levels were

significantly lower in the cortex and hippocampus of Mbd2-null animals. *P < 0.05

compared with wild-type, non-paired, two-tailed Student’s t-test.

Figure 26

A) mRNA Glucocorticoid Receptor expression

1.50 * * WT 1.25 MBD2 Null

1.00

0.75

vel 0.50

0.25

0.00 mRNA le mRNA to Hprt relative s Cortex Hippocampus Striatum

3.4. Assessment of MeCP2 protein expression levels in the cortex, hippocampus and cerebellum of Mbd2 null and wild-type mice samples.

Seeing significantly elevated MeCP2 mRNA levels in the cortex of Mbd2 null mice prompted us to further assess MeCP2 expression profile in mutant mice (8-13 months of age).

We speculated that MeCP2 protein expression would parallel mRNA expression and show higher levels in the cortex of Mbd2 null animals. However, this was not observed. Instead, the levels of MeCP2 were significantly lower in Mbd2 null derived hippocampal and cortical tissues

(Fig.27, Fig. 28). Additionally, protein MeCP2 levels were significantly elevated compared to

Hprt expression in the cerebellum of mutant mice (Fig. 29).

100

Figure 27: MeCP2 protein expression in the hippocampus of wild-type and Mbd2 null mice

A) Representative western blot of wild-type and Mbd2 null hippocampal samples (8-13

months of age). Anti-human MeCP2 antibody raised in chicken was used to detect the C-

terminus of MeCP2 protein. Positive control was obtained from MeCP2 mouse brain

nuclear extracts, while negative control was derived from Mecp2 null neural tissue.

B) Following initial hybridization, the blots were stripped and then re-probed with an

antibody against GAPDH that served as a loading control. The histogram shows the

densitometric data from 5 wild type and Mbd2 null hippocampal samples, normalized to

GAPDH levels. The OD ratio averaged from Mbd2- deficient samples was significantly

lower compared to OD ratio from the control samples. *P < 0.05 compared with wild-

type, non-paired, two-tailed Student’s t-test.

101

Figure 27

A) - + wt wt wtwt null nullnull null

72 kDa MeCP2 Protein

55 kDa

wt wt wtwt null nullnull null

GAPDH Protein 37 kDa

B) MeCP2 protein expression in hippocampus

* 1.0

0.8

0.6

0.4

0.2

0.0 WT MBD2 Null OD ratio (normalized to GAPDH)

102

Figure 28: MeCP2 protein expression in the cortex of wild-type and Mbd2 null mice

A) Representative western blot of wild-type and Mbd2 null cortical samples (8-13 months of

age). Anti-human MeCP2 antibody raised in chicken was used to detect the C-terminus of

MeCP2 protein. Positive control was obtained from MeCP2 mouse brain nuclear extracts,

while negative control was derived from Mecp2 null neural tissue.

B) Following initial hybridization, the blots were stripped and then re-probed with an

antibody against GAPDH that served as a loading control. The histogram shows the

densitometric data from 5 wild type and Mbd2 null cortical samples, normalized to

GAPDH levels. The OD ratio averaged from Mbd2- deficient samples was significantly

lower compared to OD ratio from the control samples. *P < 0.05 compared with wild-

type, non-paired, two-tailed Student’s t-test.

103

Figure 28

- + wtwt wt wt null null null null MeCP2 Protein 72 kDa

55 kDa

wt wt wt wt null null null null

GAPDH Protein 37 kDa

B) MeCP2 protein expression in the cortex

* 0.5

0.4

0.3

0.2

0.1

0.0 WT MBD2 Null OD ratio (normalized to GAPDH) to (normalized ratio OD

104

Figure 29: MeCP2 protein expression in the cerebellum of wild-type and Mbd2 null mice.

A) Representative western blot of wild-type and Mbd2 null cerebellum samples (8-13

months of age). Anti-human MeCP2 antibody raised in chicken was used to detect the C-

terminus of MeCP2 protein. Positive control was obtained from MeCP2 mouse brain

nuclear extracts, while negative control was derived from Mecp2 null neural tissue.

B) Following initial hybridization, the blots were stripped and then re-probed with an

antibody against GAPDH that served as a loading control. The histogram shows the

densitometric data from 5 wild type and Mbd2 null cerebellum samples, normalized to

GAPDH levels. The OD ratio averaged from Mbd2- deficient samples was significantly

higher compared to OD ratio from the control samples. *P < 0.05 compared with wild-

type, non-paired, two-tailed Student’s t-test.

105

Figure 29

A) + - wtwt wt wt null null null null

MeCP2 Protein 72 kDa

55 kDa

wt wt wtwt null nullnull null

GAPDH Protein 37 kDa

B) MeCP2 protein expression in cerebellum

* 2.5

PDH) A 2.0

1.5

1.0

0.5

0.0 WT MBD2 Null ODratio (normalized toG

106

Chapter 4

Discussion

The significance of epigenetic regulation in proper brain development and functioning is becoming increasingly clear. Recently, mice lacking MBD-containing proteins were shown to

have serious behavioral impairments and EEG alterations. So far, very little is known about

MBD2, another member of MBD protein family. Besides having a seemingly normal

appearance, viability and fertility, Mbd2 null mice were reported to display impaired maternal

behavior. Until now however, the role of MBD2 in the brain has not been described. The aim of

this study was to assess whether loss of MBD2 leads to behavioral and neurophysiological

impairments in mice. Three principal findings have emerged from this study. First, Mbd2 null

mice display an array of behavioral alterations. Second, basic neurophysiological properties are

preserved in Mbd2-deficient mice. Third, the analysis of candidate genes revealed alterations in both mRNA and protein expression levels in Mbd2 null neural tissues. Taken together, these results suggest that MBD2 plays an important role in proper functioning and that lack of MBD2

contributes to an autism-like behavioral phenotype in mice

107

4.1. Mbd2-deficient mice display behavioral alterations

4.1.1. Mutant mice show locomotion alterations

4.1.1.1 Mbd2 null mice exhibit motor impairments

The open field arena test allowed us to determine general motor activity of Mbd2 null and

wild-type mice. This test also accounts for the location of the mouse in the cage as well as the

speed of movements, thereby producing an array of motor assessment parameters (Jugloff et al.,

2008). The mice were separated based on age in two groups : young mice (3-5 months) and

older group (8-13 months). By doing so we wanted to assess whether any motor parameters

change as mice get older. Consequenly, the behavior of Mbd2 null mice was compared to the

behavior of age- matched control mice. We observed significant motor alterations in both young

and older groups of Mbd2 null mice. Mutant mice from both age cohorts spent less time rearing,

mobile and walking.Older Mbd2 null mice displayed reduced overall activity, while young mice

did not. In addition, only older mutant mice showed overall reduced fast and slow mobility.

Fast mobility was recorded when the interval between beam breaks was greater than 200 ms.

Therefore, these data suggest that mutant mice from both age cohorts performed fewer fast

movements compared to control mice. In contrast, slow mobility, was measured when the

interval between beam breaks was less than 200 ms. These impairments suggest that as mutant

mice age, their slow movements and fast movements become affected. These findings are also

consistent with their general decrease in total mobility.

It has been previously reported that motor testing in an open field apparatus serves as an anxiety promoting stimulus for rodents. Even though rodents naturally tend to explore a novel environment, open fields are aversive and counter normal behavioral responses. Therefore, open

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field apparatus also serves as a measure of anxiety-like behaviors. Anxiety is assessed by the extent to which mice avoid the center of the open field test (Clément Y., 2007).Our results revealed that Mbd2 null mice from both age cohorts displayed significantly less center rearing that is indicative of a greater experienced anxiety.Collectively, these results indicate that MBD2 plays a role in proper motor performance in mice and also suggest of the elevated anxiety-like behaviors in mutant mice associated with MBD2 deficiency.

4.1.1.2 Mbd2 null mice show enhanced coordination ability

We then assessed whether coordinated movement and balance would also be altered in

Mbd2 null mice using the accelerating rotarod test. This assay tests the ability of an animal to balance on a rotating rod as the speed of the rotation increases. As a result, it becomes more difficult for mice to keep their balance and mice tend to fall down from the rod (Carter et al..,

2001). Surprisingly, Mbd2 null mice showed an enhanced motor coordination performance during rotarod testing. They spent significantly more time on the rotating rod before losing their balance during most of the trials. This elevated coordination ability was especially pronounced in older Mbd2 null mice, suggesting that this is an age-associated phenomenon. Since wild-type and mutant animals showed similar rates of rotarod learning throughout the trial sessions, we believe that the observed differences in performance were mostly due to innate enhanced coordination ability of Mbd2 null mice.

This result was unexpected and currently we cannot identify the mechanism to explain this observed enhanced performance of mutant mice. At this point we can only speculate of potential reasons and propose two possible explanations. First, it is feasible that some compensatory changes occurred in the cerebellum associated with MBD2 loss. Interestingly, we

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identified increased levels of MeCP2 protein in the cerebellum of Mbd2 null mice. We speculate

that this MeCP2 upregulation in the cerebellum could somehow lead to such changes in

behavior. Considering that cerebellum is one of the brain regions primarily involved in balance

and locomotion, we speculate that MeCP2 upreguation in that particuar brain region could

enhance cerebellar control of balance and locomotion in Mbd2 null mice (Bastian, 2004).

Previously we also saw that over - expression of MeCP2 in the brain is enough to induce a

noticeable change in behavioral phenotype (Collins et al., 2004). Therefore, it is feasible that

over - expression of MeCP2 in the cerebellum leads to observed enhanced coordination in Mbd2

null mice. However, since cerebellum is only one of the brain regions involved in balance and

coordination, additional experiments will be required to delineate whether cerebellum is the main

candidate responsible for this enhanced coordination. One of the behavioral tests used to assess

balance that is associated mainly with the cerebellum is a Dowel Assay (Opal et al., 2004).

Implementing this assay to test fine balance skills in MBD2 could be a worthwhile task.

Alternatively, we propose another potential explanation for enhanced motor coordination

and balance behavior in mutant mice. Previously in our lab we observed abnormally high mRNA

levels of CCL21 in cortical and hippocampal brain regions of Mbd2 null-derived tissues

(unpublished data). CCL21 is a chemokine that is associated with generation and maintenance of pain after neuronal damage (Detlof et al., 2009). Additionally, high levels of CCL21 mRNa were noted after neuronal damage (Eiko et al., 2005). It is feasible that falling from the rotarod is a more painful experience for Mbd2 null mice because of their increased pain sensitivity. As a result of this higher degree of pain associated with falling, they stay longer on the rotating rod.

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4. 1. 2. Socialization behaviors are deficient in Mbd2 null mice

4.1.2.1. Nesting behavior is impaired in Mbd2 null mice

Nest building behavior, as a parameter of home cage activity, reveals important

information about socialization in mice (Samaco et al., 2008), (Moretti et al., 2005). Nests serve

many central functions for mice such as shelter, conservation of body heat, and were even

reported to indicate fitness levels in mice (Bult and Lynch, 1997). In addition, the degree of nest

utilization also serves as an indication of socialization with respect to nurturing behavior as

parents retrieve their pups to the nest and share it with them (Moretti et al., 2005). Thus, nesting

behavior can serve as an important parameter of an animal phenotype characterization. Our

results showed that young and older mutant mice spent less time interacting with the neslet

during first 30 minutes compared to age-matched control animals. Additionally, the number of

approaches that Mbd2 null mice initiated towards the nesting material were also significantly fewer. Furthermore, the resultant nests made by mutant mice from both age cohorts were significantly smaller in height and were less sophisticated compared to the nests made by wild- type mice. Previously, reduced interaction with the nesting material was suggested to indicate

lack of interest in nest building and hint to socialization impairments in mice (Moretti et al.,

2005). Collectively these data indicate that Mbd2 null mice show impairments in nest building.

Only two previous studies have assessed nest building activity in a strain of mice

lacking another MBD factor. Intrestigly, when Samaco et al. examined the nesting behavior in

MeCP2-deficient mice, they reported similar nest building impairments. In their study Samaco et

al. also observed that only a small percentage of these mice shred the new nesting material, and

built nests of comparable quality to those of control mice (Samaco et al., 2008, Moretti et al.,

2005). Our study is the first to assess nesting behavior in Mbd2 null mice. 111

Current findings also complement the previous results on impaired maternal behavior in

Mbd2 null mice. Hendrich et al. reported reduced average size of pups as well as lower litter

numbers born to Mbd2 null mothers. Building a proper nest and utilizing it serves many

important functions for mice. Previously, nest buiding behavior has been positively correlated

with litter number and pups’ body weight (Bult and Lynch, 1997). In addition, highest nesting

quality was associated with the highest ratios of litter survival. In light of this, it is feasible that

poor survival litter outcomes could result from nest building impairments rather than maternal

behavior exclusively. Taken together, our results complement previous reports on impaired

maternal behavior of MBD- deficient mice and offer new insight into related nesting behaviors.

4.1.2.2. Mbd2-deficient mice display impairments in sociability and preference for social novelty

Many rodents, including mice, live in strong social communities in the wild, seek contact

and display social interest towards other rodents (Nadler et al., 2004). Likewise, mice living in

captivity display a similar need for socialization (Van Loo et al., 2004). Considering that

socialization plays such a prominent role in normal murine functioning, many methods for the

evaluation of social behavior have been described. Therefore, one of the principal goals of this

study was to characterize a socialization profile of Mbd2 null mice. To observe and quantify

social interactions, mice were exposed to their co-housed mouse followed by a socialization

session with a novel mouse. At a younger age, Mbd2-deficient mice displayed similar levels of

interest towards their co-housed partner as wild-type mice. However, older Mbd2 null mice

displayed a social approach deficit. Specifically, they initiated fewer interactions with their

partner mouse that were also shorter in duration. This is suggestive of an age-associated reduction in sociability observed in Mbd2-null mice group exclusively. Present findings parallel

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social behavior phenotype of MeCP2-deficinet mice, as previously reported by Moretti et al.

They showed that mutant mice spent less time near the partition that housed an unfamiliar

mouse, a behavior indicative of a social interaction impairment (Moretti et al., 2005).

In addition to investigating sociability in mice, present study also assessed their

preference for social novelty. When animals were exposed to a novel mouse during a second

trial, young mice spent similar amount of time interacting with it as wild-type mice. However,

older mice showed a diminished interest in social interactions. Surprisingly, rather than directing

their social interest towards a new mouse, older Mbd2 null mice spent more time avoiding this

interaction. What is even more intriguing is that older mutant animals showed no avoidance

behavior towards their partners during the first trial. It suggests that, in contrast to wild-type

mice, older mutant mice do not show a preference for novel social interaction, but rather avoid it.

Another recent study assessed socialization behaviors in Mbd1-null mice. Interestingly,

these animals were also shown to exhibit reduced interest in social interaction. In a social

preference test, contrary to wild-type mice, Mbd1-null animals spent more time interacting with a novel toy rather than a novel mouse (Allan et al., 2008).This observation prompted authors to

speculate of the important role MBD -containing proteins may play in the regulation of social

behavior. The results of our study further support this hypothesis. MBD2 is now a third member

of methyl binding domain protein family to play a role in socialization behavior. The precise

mechanism still remains to be investigated, but the importance of epigenetic regulation becomes

more evident.

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4.1.3. MBD2-deficient mice display elevated anxiety-like behaviors

Having seen reduced centre rearing activity of Mbd2 null mice during the open field test,

prompted us to assess the anxiety levels of these animals more thoroughly. Generally, mice are

inquisitive creatures that often engage in explorative behaviors of new environments. However, they are also cautious of brightly lit areas as well as wide open spaces and display anxiety-like behaviors when subjected to these aversive conditions. Light and dark preference box as well as elevated plus maze are the two most commonly used paradigms used to assess anxiety-like behaviors in mice (Crawley, 1999). The time spent in the dark box, and the number of risk assessment behaviors between chambers, are the two commonly implemented measurements of anxiety-related behavior. It is now know that anxious animals spend more time in the dark box and perform fewer risk assessments compared to less anxious mice (Clement et al., 1998).

Surprisingly, the results of our study revealed that Mbd2 null mice from both age cohorts do not display a preference for the dark chamber. Yet, older mice demonstrated an anxiety- related impairment. Specifically, older mutant mice were shown to be less inquisitive and displayed reduced risk assessment behavior compared to control mice of similar age.

The findings of heightened anxiety were also observed through the elevated plus maze

testing. Elevated plus maze tests anxiety -like behavior induced by the unconditioned aversion to

heights and open spaces (Pellow et al., 1985).Even though mice prefer to spent more time in the

enclosed arms of the maze, they still like to explore novel environments and often visit the open

arms. Despite their inquisitive tendency to explore unfamiliar spaces, exposure to the open arms

of the maze is still a stressful and anxiety-provoking situation. This was demonstrated by

elevated plasma corticosterone concentrations in animals confined to the open arms as compared

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with those confined to the closed arms (Hennessy et al, 1979). Younger mutant mice showed

similar location preferences as control animals. However, older Mbd2 null mice preferred to spend more time in the closed arms of the maze, suggesting of higher stress levels compared to age-matched wild-type mice. Additionally, older MBD2-deficient mice preferred the distal area of the closed arms. Spending more time in the distal area of the closed arms- being the most secured location of the apparatus, is also consistent with higher anxiety levels. Previous studies have assessed the order of arm preference and found that mice chose closed arms to central to open arms, indicating an inclination to spend more time in the secured sections of the maze

(Espejo et al., 1997). Therefore, the observation that older mutant mice spent less time in the central middle platform and more time in the closed arms, once again point to higher experienced anxiety levels and increased preference for the safety of the closed compartment. Taken together, the results of these two assays indicate that Mbd2 null mice display enhanced anxiety levels at an older age.

Interestingly, similar anxiety-like behaviors were observed in other MBD–deficient

mouse models. For example, mice lacking MBD1 showed preference for the dark compartment of the light dark preference test and spent more time in the closed arms of the elevated plus maze test (Allan et al., 2008). Additionally, MeCP2 null mice are also known to display comparable

heightened anxiety-like behaviors (Stearnsa et al., 2007). Hence, the results of the present study

further illustrate the central role of MBD- containing proteins in the regulation of anxiety.

4.1.4. Mbd2 null mice exhibit novel object recognition impairments

To determine whether mutant mice display deficits in object recognition we assessed

their preference for a novel object after a 5 minute delay, and after a one hour long delay. Mutant

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mice exhibited reduced levels of interest towards a novel object compared to wild type mice after a 5 minute delay period, suggesting that their object recognition memory was impaired.

However, their preference for a novel object was not affected after a one hour delay. What is even more intriguing is Mbd2 null mice showed a significant impairment in object recognition after 5 minute delay and one hour delay, when measured through the Discrimination Index assessment. Previously, poor performance on this task was linked to hippocampal dysfunction

(Broadbent et al., 2004). However, it seems that they are either unable to discrimiate between novel and familiar objects, or that Mbd2 null mice simply do not have an innate preference for novelty that control mice display. Further detailed memory assessments need to conducted to deleinate the causes of observed behavioral alterations.

4.2. Preservation of basic Electroencephalographic (EEG) activity in Mbd2 null mice

Having identified an array of behavioral deficits in Mbd2-deficient mice, I examined cortical and hippocampal EEG activity in these mutant animals. I wanted to determine if these mice display any neurophysiological alterations, and if present, whether they would correlate with the observed behavioral impairments. Hippocampal theta patterns were observed and recorded when mice engaged in exploratory sniffing behaviors. For each recording I assessed a dominant peak frequency and compared it to the frequency obtained from the traces of control mice. No differences in theta peak frequencies were observed in mutant and wild type mice. Our results show that no gross EEG alterations such as abnormal discharges or seizures are seen in

Mbd2 null mice. Yet, the preservation of the hippocampal theta activity is not consistent with the abnormal object recognition behavior seen in Mbd2 null mice. EEG results hint to the

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hippocampal preservation associated with MBD2 deficiency, while impaired object recognition

suggest hippocampal dysfunction.

This lack of gross EEG alterations in Mbd2 null mice is different from the significant

alterations associated with MeCP2 deficiency in mice. Recently the results from our lab showed the presence of abnormal spontaneous, rhythmic EEG discharges in the somatosensory cortex of

MeCP2-deficient mice. In addition these mice also have a reduced peak theta frequency (D’Cruz et al., 2010). It would be interesting to perform similar EEG assessments for mice lacking MBD1 to determine if removing MBD motif is implicated in neurophysiological alterations.

4.3. The mRNA and protein expression of candidate systems in Mbd2 null neural tissues

4.3.1. Increased MeCP2 mRNA expression levels in Mbd2 null derived cortical tissues

Finally, after seeing behavioral alterations and EEG activity preservation in Mbd2 null

mice, we assessed mRNA and protein expression levels of potential candidate genes.

Specifically, we wanted to determine whether expression of MBD family members will change

as a result of MBD2 deficiency in neural tissues. MBD protein family consists of five members:

MBD1, MBD2, MBD3, MBD4 and MeCP2. All of these proteins contain a methyl binding domain (MBD) sequence with 45%-75% overall amino acid identity and except for MBD3 bind methylated DNA (Hendrich and Bird, 1998). Additionally, with the exception of MBD4, they

also contain a transcriptional repression domain (TRD) and function to repress transcription. Due

to these similarities, a few studies have suggested a genetic interaction between these members

and even speculated of a functional redundancy between MBD family members (Hendrich and

Bird), (Franckem et al., 2006). Interestingly, we observed a significantly higher expression of

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MeCP2 mRNA in Mbd2 null cortical tissues compared to age-matched control samples. These

data suggest of a transcriptional upregulation of MeCP2, potentially to compensate for the loss of

MBD2. However, no similar significant increase in MeCP2 mRNA expression was observed in

hippocampal or striatal samples. This could be due to the high degree of variability observed in

the samples. Therefore, before attributing this change in expression to MeCP2 upregulation in

the cortical tissues exclusively, the sample size needs to be increased.

Intriguingly, MBD2 mRNA expression was the highest in wild-type samples in all the

hippocampal and striatum samples. However, the expression of MBD1 and MBD3 was more

elevated in the cortex and striatum of wild-type mice. This robust MBD2 expression probably

serves an important functional role in these brain regions. This speculation is consistent with an

array of behavioral impairments that we observed in Mbd2 null mice. Yet, the precise role and

the mechanism of action of MBD2 in proper neural functioning remain to be investigated. As an

epigenetic regulator, MBD2 was shown to act as a transcriptional repressor and a demethylation

enzyme, yet its demethylation function is still not well accepted. Yet, which loss of function

leads to the observed behavioral deficits remains unknown.

4.3.2. MeCP2 Protein expression alterations in MBD2 null derived tissues

Seeing alterations in mRNA MeCP2 levels prompted us to study MeCP2 protein

expression. We wanted to determine whether MeCP2 protein levels would parallel the observed elevated mRNA MeCP2 expression. Western blot analysis revealed that, surprisingly, protein

levels of MeCP2 were significantly reduced in the cortex and the hippocampus of Mbd2 null

tissues. However, as mentioned above, real – time - PCR analysis showed that MeCP2 mRNA in the cortex was increased by almost fourfold and no significant change was observed in the

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hippocampal samples. In addition, MeCP2 protein expression was significantly elevated in the

cerebellum samples in MBD2 null derived tissues. Taken together, the data suggest that the levels of MeCP2 are altered in Mbd2 null neural tissues and that mRNA and protein expression levels are not parallel. This finding suggests of a possible postranscriptioanal modification of

MeCP2 RNA that could happen through polyadenylation. Interestingly and somewhat

reassuringly, another study also saw similar disoconnect between MeCP2 mRNA and protein

levels. Shahbazian et al. looked at the expression profiles of MeCP2 mRNA and protein in both

human and mice derived tissues and found a similar lack of correlation between the two

(Shahbazian et al., 2002). They conluded that the levels of MeCP2 transcripts are

postranscriptioanlly controlled and are tissue specific. According to their study, MeCP2 protein

is particularly abundant in the brain, spleen and lung. It would be interesting to see if these

tissues will also show altered MeCP2 levels in MBD2 null mice.

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4.4. Proposed models for observed behavioral alterations

4.4.1. Could alterations in Glucocorticoid Receptor expression account for impaired behavior of

Mbd2 null mice?

While many systems contribute to elevated anxiety levels, one particularly attractive

system is Glucocorticoid Receptor system. Many previous findings have linked the involvement

of hypothalamic-pituitary-adrenal (HPA) axis with stress responsiveness and altered

glucocorticoid receptor expression. Glucocorticoids belong to the class of stress hormones shown

to regulate anxiety (Wei et al.., 2004). Elevated anxiety levels displayed by Mbd2 null mice led

us to suspect of the role MBD2 could have in the regulation of HPA axis. To test whether mutant

mice display alterations in HPA axis, and show differences in the GR expression, we assessed

their mRNA glucocorticoid receptor levels with a quantitative real-time-PCR assay. We expected

to see lower levels of Glucocorticoid Receptor, as a previous study reported decreased

Glucocorticoid Receptor mRNA in the hippocampus associated with high doses of cotricosterone

circulating in the blood (CORT) and higher anxiety levels (Herman and Spencer, 1998).As expected, we found reduced Glucocorticoid Receptor mRNA levels in the hippocampus and cortex of Mbd2 null derived brain regions. Low Glucocorticoid Receptor expression in the hippocampus is especially significant, since hippocampus is the area that is strongly impilcated

in glucocorticoid negative-feedback regulation (Jacobson and Sapolsky, 1991) Therefore,

reduced Glucocorticoid Receptor mRNA levels in Mbd2 null mice suggest of an increased

anxiety that is most likely associated with elevated CORT. It would be interesting to confirm

this by directly assessing the levels of CORT in the blood of these mice.

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The results of another recent study provide additional support for our speculations of GR

misregulation associated with MBD2 deficiency. In their study McGill et al. explained elevated

anxiety-like behaviors in MeCP2 null mice also through disregulation of the HPA axis. The

researchers noted that MeCP2 null mice displayed higher levels of glucocortiocid release

following mouse restrain as well as increased expression of genes regulated by glucocorticoids

(McGill et al., 2006). They also found that nornally MeCP2 binds to Crh promoter and

suppresses its transcription. This is an important finding that emphasizes the role of epigenetic

regulation in a stress response. It is feasible that other MBD containing epigenetic factors also

play a similar role.

Additionally, many studies have looked at the relationship between maternal care and

stress levels in pups, regulated through the HPA axis. Maternal behavior was proposed to

“program” HPA responses to stress in the offspring. This is particularly interesting, considering

previous reports of impaired maternal behavior in Mbd2 null mice. Liu at el. found that maternal behavior alters the development of hypothalamic pituitary adrenal axis (HPA) response to stress in pups. Specifically, pups that received more licking and grooming from their mothers during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocorticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone mRNA(Liu et al., 1997).

A mechansim was propoed that attempted to explain these observations. It was shown

that high degree of licking and grooming rearing behaviors lead to increased serotonergic tone in

the hippocampus of pups. Consequently, this activated cAMP and cAMP-dependent protein

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kinase-A (PKA) that led to CREB phosphorylation and finally drove the expression of the nerve

growth factor -inducible protein-A (NGF1-A). NGF1-A is a transcription factor that binds to the

first exon of GR. Upon binding of NGF1-A to its site, the expression of GR is increased.

However, this explanation only addresses short term changes in stress levels,

associated with the immediate effects of serotonergic (5- HT) tone. Yet rats displayed elevated

levels of anxiety throughout life. A new mechanism was therefore needed that would explain

life- long changes in stress response, long after matrenal care -driven activation of NGF1-A.

This new theory uses the concecpt of eipgenetics to explain this intriguing relationship. As it turns out, frequent licking and grooming by rat mothers is associated with glucocorticoid receptor gene demethylation early on in life, which leads to marked expression of GR.

Specifically, it was shown that NGFI-A binding sequence on the promoter of GR is undergoing many alternating methylation demethylation modifications. The sequence is unmethylated right

before birth but then becomes methylated during the the first day after birth. However, during

the next few weeks one key site in the sequence becomes unmethylated in the pups of high

licking and grooming mothers.Therefore, these pups show high levels of GR expression and

consequently show lower anxiety levels (Sapolsky et al., 2004).

The identity of the demethylase protein that removes methyl groups from NGF1 site

during the first few weeks of life is still unknown. However, considering that MBD2 was

reported to act as a demethylase, it is feasible that MBD2 could be that demethylase protein.

Before making such assumptions, it is worth emphasizing that the demethylase function of

MBD2 is still controversial and not well accepted in the scientific community. Nevertheless,

unpublished data by Weaver et al. show that MBD2 protein binds to the promoter of GR at the

NGF1-A binding site. They speculate that it acts as a demethylase and removes methylation form

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the GR site, thereby leading to increased GR transcription. However, to act as a demethylating enzyme MBD2 requires the binding of NGF1-A to its site on GR. It is yet unknown how NGF1-

A recruits MBD2 to GR sites (Weaver et al., 2009). What is clear is that MBD2 either directly

or indirectly leads to decreased GR mRNA expression (Fig. 30).

This proposed mechanism is consistent with our observed behavioral results. When

MBD2 is missing, NGF1A sequence on GR is no longer demethylated. Due to this constant

presence of methylation, the transcription of the GR gene is repressed and as a result we see

lower levels of GR mRNA expression. Therefore, the elevated anxiety-like behaviors seen in

mutant mice could be explained with HPA axis misregulatoin, associated with MBD2 deficiency

(Weaver et al., 2009).

In addition, recent human studies identified a relationship between childhood abuse,

decreased GR mRNA levels and increased cytosine methylation of a neuron-specific

glucocorticoid receptor promoter (NR3C1). These findings emphasize a significant function of

epigenetic programming in glucocorticoid receptor expression and the importance of proper

demethylation (McGowan et al., 2009). However, the identity of the demethylating enzyme has

yet to be determined. Considering recent findings by Weaver et al. it is feasible that MBD2 could

serve a similar function in human GR regulation (Weaver et al., 2009).

Seeing reduced mRNA GR levels help to explain elevated anxiety-like behaviors.

Consequently, heightened anxiety observed in Mbd2 null mice could also explain impaired

animal performance as seen from the other behavioral assessments. Precisely, we speculate that

increased anxiety-like phenotype, associated with reduced GR levels, could partially explain

their impaired socialization behaviors as well as some of the observed motor impairments in

mutant animals. It was previously proposed that increased anxiety during a social encounter with

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an adult mouse affects social interactions initiated by a test mouse (Nadler et al., 2004).

Considering that higher anxiety levels are so prominent in older Mbd2-deficient mice, it is conceivable that anxiety could reduce socialization in older mice. Similarly, elevated anxiety levels could also affect observed motor performance in mice due to higher occurrences of freezing behaviors, as previously described (Clement et al., 1998). However, it is feasible that mutant mice display socialization impairments that are independent of their elevated anxiety phenotype. Similarly, their motor impairments could also arise from an unrelated to elevated anxiety levels problem.

4.4.2. Could MeCP2 changes in Mbd2-null neural tissues account for observed behavioral deficits?

Alterations in MeCP2 levels could provide a clue for explaining the behavioral impairments seen in Mbd2 null mice. Previously published data suggest that over-expression of

MeCP2 by at least a two-fold leads to a severe progressive neurological phenotype in mice.

Additionally, it is becoming appreciated that MeCP2 expression levels need to be maintained within a narrow range before serious behavioral impairments develop (Collins et al., 2004).

Therefore, we hypothesize that MeCP2 alterations seen in Mbd2 null neural tissues are associated with an impaired behavioral phenotype seen in these mice. We also speculate that

MBD2 acts as a transcriptional repressor of MeCP2 and in the absence of MBD2, the transcription of MeCP2 is no longer repressed (Fig.30). Interestingly methylated sequences have been identified on MeCP2, implying that MeCP2 could be regulated by another MBD-containing protein (Nagarajan et al., 2008). Though consistent with the observed elevated MeCP2 mRNA expression, further development of this hypothesis requires additional investigation. First and 124

foremost, we need to determine whether MBD2 and MeCP2 actually interact. This can be verified with a Chromatin immunoprecipitation (ChIP).

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Figure 30: Proposed mechanisms of MBD2 transcriptionally regulating GR and MeCP2.

A) A mechanism of GR gene expression epigenetically regulated by MBD2. We speculate that

MBD2 interacts with GR promoter and either directly or indirectly leads to GR

transcription. MBD2 was proposed to act as demethylating enzyme that removes

methylation from NGF1A binding site on GR exon 1, allowing NGF1-A to bind to its site

and thereby facilitates GR transcription.

(The figure is modified from Ian Weaver, 2009).

B) A mechanism of MeCP2 transcriptional repression facilitated by MBD2. We speculate that

MBD2 binds to methylated sites of MeCP2 promoter and thereby leads to transcriptional

repression of MeCP2. However, in the absence of MBD2, the expression levels of MeCP2

mRNA are high. This proposed mechanism is consistent with our observed results.

Specifically, mRNA MeCP2 levels were elevated in the cortex of Mbd2- deficient tissues.

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Figure 30

A) NGF1‐A MBD2 GR transcription

GR exon 1 promoter

Low GR transcription NGF1‐A

GR exon 1 promoter

MBD2 Transcriptional repression CH3 of MeCP2 CpG MeCP2 promoter

CH3 CpG Transcription of MeCP2

MeCP2 promoter 127

4.5. Future Directions and Potential Clinical Implications

4.5.1. MBD2 is a potential candidate for studying ASD

Collectively, the behavioral impairments displayed by Mbd2 null mice suggest of an

autism-like phenotype. Reduced sociability is one of the principal features of autism and ASDs.

Seeing socialization impairments in mice suggests of the potential role MBD2 could have in

autism etiology. Interestingly, in addition to impaired social kills, one other cardinal feature of

ASDs is preference for sameness and a routine (Kanner, 1943). Since our results indicate that

mutant mice avoid interactions with a novel mouse but do not show such behavior towards a

familiar mouse, we can speculate that they prefer sameness rather than novelty. This is consistent

with an autism spectrum phenotype. Additionally, it is also worth noting that anxiety,

specifically associated with novelty, is a commonly occurring comorbidity of autism (Piven et

al., 1997), (Zoghbi et al., 1990). Interestingly, we saw that Mbd2 null mice show elevated

anxiety-like behaviors when animals are subjected to new environments. Therefore, taken

together the results of this study suggest that Mbd2 – deficient older mice display autism- like

behavioral features. However, it is worthwhile to note that younger MBD2 null mice did not display this autism-like behavioral phenotype. Considering that autism spectrum disorders usually begin in infancy and are diagnosed by the third year of life, suggest that MBD2 might not be involved in the classic form of ASD. Similairly, it is feasible that other MBD factors might be able to compensate for MBD2 loss at a younger age and thus explain lack of behavioral abnormalities in young animals. However, considering that presently only a few mouse model of autism exist, and that older Mbd2 null mice recapitulate many cardinal features of autism, this study could help to establish Mbd2-deficient mice as a new model of ASDs. In support if this

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proposed new model of ASD, MBD members were already implicated in autism and ASDs in a

large patient study (Cukier et al., 2009). However this study did not separate between different

MBD members and in the future it would be interesting to assess whether patients with ASD

have a higher rate of MBD2 mutations.

4.5.2. MBD2 ablation as potential cancer treatment

Additionally, there is a great deal of interest in MBD2 in the filed of cancer research.

Many cancer studies reported that deficiency of Mbd2 suppresses tumorigenesis (Sansom et al.,

2003) (Slack et al., 2002), (Campbell et al., 2004), (Pakneshan et al., 2005) (Shukeir et al.,

2006). As a result, it was proposed that MBD2 ablation could serve as a new therapy for

anticancer drug development. At the time of these studies no detailed assessment of MBD2

deficient animals was performed. It was only known that removal of MBD2 in mice did not

result in any gross abnormalities. These mutant mice had a normal appearance, viability and

fertility. The only known impairment associated with Mbd2 removal was abnormal maternal

behavior. However, the results of my study challenge this assumption and reveal that MBD2

deletion in mice results in serious behavioral impairments. Specifically, older Mbd2 null mice show deficits in locomotion, socialization impairments and elevated anxiety-like behaviors. It is also now clear that removal of MBD2 leads to mRNA changes of other MBD-containing members, and that MeCP2 protein levels also become affected. Taken together, we have shown the importance of MBD2 in the proper brain functioning. Therefore, if cancer researchers were to pursue the development of an anti -cancer drug that ablates MBD2, the drug needs to be designed that would not cross a blood brain barrier and thus spare its function in neural tissues.

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