Deficiency of Myelin Proteins During Brain Development in Fragile X Mice

Deficiency of Myelin Proteins during Brain Development in Fragile X Mice

David Lai Jiang

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto

Deficiency of Myelin Proteins during Brain Development in Fragile X Mice

David Jiang

Master of Science

Department of Pharmacology and Toxicology University of Toronto

2015 Abstract Fragile X syndrome (FXS) is the most common inherited form of mental impairment and

the leading genetic cause of autism. Our laboratory has previously reported that myelination is

delayed in the cerebellum of a mouse model of FXS. In my study, I assessed the spatial and

temporal aspects of this myelination defect and found that the defect occurs throughout the brain

in the early developing fragile X mouse. Additionally, I also examined the levels of epidermal

growth factor receptor (EGFR) and two of its ligands but found little to no deficiencies in the

fragile X brain. However, epidermal growth factor (EGF) levels in the plasma and thyroid of

adult fragile X mouse were found to be significantly reduced. Lastly, the fragile X mice were

treated with intranasal heparin binding EGF-like growth factor (HB-EGF) in an attempt to rescue

the myelination defect but the treatment did not promote early postnatal myelination.

Acknowledgements

First and foremost, I would like to acknowledge Dr. David R. Hampson for his continuous support and guidance in the past two years. I would also like to thank all of the current and past lab members for all the help and insights they have provided me with. Last but not least, I am very grateful to have Dr. Rebecca Laposa and Dr. Alexander Velumian shared with me their expertise during our committee meetings.

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Table of Contents

Abstract...... ii

Acknowledgments...... iii

Table of Contents...... iv

List of Tables...... vii

List of Figures...... viii

List of Appendices...... ix

List of Abbreviations...... x

1. Introduction...... 1

1.1 Fragile X Syndrome...... 2

1.11 Fragile X Syndrome and its Phenotypes...... 2

1.12 Functions of Fragile X Mental Retardation Protein...... 4

1.13 Mouse Model of Fragile X Syndrome...... 5

1.2 Myelination during Brain Development...... 6

1.21 Myelination and Oligodendrocyte Development...... 6

1.22 Myelin Basic Protein and 2',3'-Cyclic-nucleotide 3'-phosphodiesterase...... 9

1.23 Myelin-Axon Crosstalk...... 10

1.24 White Matter and Myelin Abnormalities in Autism and FXS...... 11

1.3 Role of Epidermal Growth Factor Receptor Signalling in Myelination...... 12

1.31 Epidermal Growth Factor Receptor and its Endogenous Ligands...... 12

1.32 EGFR Signaling Favors Glial Differentiation and Promotes

Myelinogenesis...... 14

1.4 The Functional Role of Thyroid Gland in Myelination...... 15

2. Hypotheses, Objective, and Rationales...... 17

2.1 Project 1 – Mapping the Myelination Defect in Fmr1 KO Mice...... 17

2.2 Project 2 – Investigation of the EGFR System in Fmr1 KO Mice...... 18

2.3 Project 3 – Intranasal Administration of HB-EGF...... 19

3. Methods...... 21

3.1 Animals...... 21

3.2 Western Blotting...... 21

3.3 EGF and thyroxine ELISA...... 23

3.4 Intranasal HB-EGF Administration...... 24

4. Results...... 26

4.1 MBP and CNPase expression in the Fmr1 KO and WT mouse brains...... 26

4.2 EGFR and pEGFR expression in the Fmr1 KO and WT mouse brains...... 31

4.3 EGF and HB-EGF levels in the WT and Fmr1 KO mouse brains...... 35

4.4 Analysis of EGF and thyroxine in the plasma and thyroid of adult Fmr1 KO and WT

mice...... 38

4.5 Effect of early-postnatal intranasal HB-EGF on MBP, CNPase, and NG2

Expression...... 40

5. Discussion...... 42

5.1 Delayed myelination in the early postnatal Fmr1 KO mouse brain...... 43

5.2 EGFR, EGF, and HB-EGF levels in the Fmr1 KO mouse brain...... 49

5.3 EGF deficiency in the plasma and thyroid of Fmr1 KO mouse...... 50

5.4 Effect of intranasal HB-EGF on early postnatal myelination...... 52

6. Conclusions...... 55

7. Future Directions...... 56

References...... 58

Appendices...... 76

List of Tables

Table 1. List of myelin protein and myelination-associated genes whose mRNA transcripts are

also FMRP substrates (Y) or not (N), as reported in Darnell et al., 2011, Ascano et al., 2012, and

Lucá et al., 2013...... 8

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List of Figures

Figure 1. Comparison of mouse versus human brain development...... 6

Figure 2. Representative western blots of MBP and CNPase expression in the mouse brain...... 27

Figure 3. MBP and CNPase expression profiles in the mouse brainstem and cerebellum...... 28

Figure 4. MBP and CNPase expression profiles in the mouse hippocampus and prefrontal cortex...... 30

Figure 5. Representative western blots of EGFR and pEGFR expression in the mouse brain.....32

Figure 6. EGFR and pEGFR expression in the early postnatal mouse brain...... 33

Figure 7. GAPDH-normalized EGFR expression in the PND 7 mouse cerebellum...... 34

Figure 8. EGF levels in the early postnatal cerebellum, hippocampus, and prefrontal cortex.....36

Figure 9. HB-EGF expression in the early postnatal mouse cerebellum...... 37

Figure 10. EGF and thyroxine (T4) levels in the adult mouse plasma and thyroid...... 39

Figure 11. Effect of intranasal HB-EGF treatment on the expression of myelin proteins and NG2

in the PND11 Fmr1 KO mouse brain...... 41

Figure 12. Effect of intranasal HB-EGF treatment on the expression of myelin proteins in the

PND11 WT mouse brain...... 42

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List of Appendices

Appendix 1. ELISA EGF measurements in the PND7 mouse brain...... 76

Appendix 2. ELISA EGF measurements in the PND13 mouse brain...... 76

Appendix 3. EGF ELISA measurements in the adult mouse plasma and thyroid...... 77

Appendix 4. Schematic of the dissection of mouse prefrontal cortex...... 78

Appendix 5. Representative EGF ELISA Standard curve...... 78

List of Abbreviations

CNPase: 2',3'-Cyclic-nucleotide 3'-phosphodiesterase

CNS: central nervous system

EGF: epidermal growth factor

EGFR: epidermal growth factor receptor

FMRP: fragile X mental retardation protein

FXS: fragile X syndrome

GAPDH: glyceraldehyde 3-phosphate-dehydrogenase

GDNF: glial cell line-derived neurotrophic factor

HB-EGF: herpin-binding EGF-like growth factor

KO: knock out

MBP: myelin basic protein

NG2: neural/glial antigen 2

NSC: neural precursor cell

OPC: oligodendrocyte precursor cell

PDGFR-α: platelet-derived growth factor receptor α

pEGFR: phospho-EGFR

PND: postnatal day

SEM: standard error of the mean

TGF- α: transforming growth factor α

UTR: untranslated region

WT: wild-type

1. Introduction

Fragile X Syndrome (FXS) is one of the most frequent genetic causes of mental retardation that affects millions of people worldwide. Ever since the discovery of the FMR1 gene mutations involved in this disease, extensive research has been performed FMR1 and its protein product fragile X mental retardation protein (FMRP). Yet, despite our best efforts, we still understand little about how the disease develops and no treatment has yet been shown to be effective. Interestingly, much of the work on FXS has focused on the deleterious effects of

FMR1 gene knockout on neurons and relatively little attention has been paid to the glial cells which constitute the majority of the cells in the brain.

Our laboratory has previously identified an early deficiency in myelination in the cerebellum of a mouse model of FXS (Pacey et al., 2013). This is an important finding because proper brain development requires precise timing and speed of the action potentials in an intricate network of neurons (i.e. spike-timing-dependent plasticity). Therefore, it is not difficult to see why it is crucial for myelination to occur at the right time and in the right places during the sensitive developmental period in which the neural connections are constantly being modified.

Although this work suggests that the oligodendrocytes are affected in FXS, the extent of the myelin deficiency in the whole fragile X brain and the factors that underlie this abnormality are still unknown. In an effort to better understand the myelination abnormalities in FXS, I utilized immunoblotting to examine the expression of myelin proteins in multiple regions and at multiple time points across the early developing fragile X brain. In addition to examining myelination, I also investigated the epidermal growth factor receptor (EGFR) signaling system in the fragile X mice due to its role in promoting oligodendrogenesis and myelination (Scafidi et al., 2014). For this, I measured the expression of EGFR and the levels of two of its ligands, epidermal growth

factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF), in fragile X mice. Lastly, in an attempt to rescue the early myelin deficiency in the fragile X mice, I examined the effect of

early postnatal intranasal administration of HB-EGF on myelination in fragile X and wild-type

(WT) mice.

1.1 Fragile X Syndrome

1.11 Fragile X Syndrome and its Phenotypes

Approximately 1-2% of the population is affected by mental retardation (Larson et al.,

2001), which is defined as “significant limitations in both intellectual functioning and adaptive behavior as expressed in conceptual, social and practical adaptive skills”. The underlying causes are both environmental and genetic with chromosome abnormalities being one of the genetic

causes. Mental retardation often involves the X-chromosome, explaining in part its higher

incidence in males compared to females (Lehrke, 1972). Amongst all the X-linked mental

retardation cases, FXS is the most prevalent disorder, accounting for approximately 20% of them

(Fishburn et al., 1983).

Human FXS is caused by the absence/deficiency of FMRP (Pieretti et al., 1991) as a result of transcriptional silencing of the FMR1 gene located on the X chromosome (Oberlé et al.,

1991). This is due to the expansion of CGG trinucleotide repeats in the 5’ untranslated region of the FMR1 gene (Oberlé et al., 1991). As a consequence, this CGG-rich region becomes hypermethylated and the gene is then silenced. There is a high degree of variation in the number of CGG repeats in the 5’ untranslated region of the FMR1 gene. In the normal population, this ranges from 5 to 54 repeats with an average of 30 repeat units (Fu et al., 1991). Those with the

CGG repeats ranging from 55 to 200 are termed premutation carriers. When the number of CGG

3 repeats exceeds 200, it is then characterized as a full mutation with abnormal methylation and the appearance of gap-like fragile sites on the active X chromosome (Gacy et al., 1995). FXS was one of the first diseases to be associated with trinucleotide repeat expansion, and to date at least

16 other neurological diseases have been found to be caused by this mechanism (Orr and Zoghbi,

2007).

Although not life threatening, FXS can severely impact the quality of life of affected individuals. For example, roughly 85% of males and 25–30% of females with a full FMR1 mutation have an IQ less than 70 (Loesch et al., 2004), with an average IQ of approximately 40 for males (Merenstein et al., 1996). Aside from intelligence, short-term memory for complex information, speech, and visuospatial skills are the most commonly affected abilities.

Interestingly, the severity of these deficits correlates with the deficiency of FMRP; individuals with only a partial decrease in FMRP levels are more likely to present a borderline normal or normal IQ without learning disabilities (Hagerman et al., 2006; Loesch et al., 2004). As the silencing of FMRP production in human FXS is not all or none, the lesser severity of these cases is likely due to a lower degree of FMR1 gene methylation which permits the production of at least some, albeit low, levels of FMRP. Since this disorder is X-linked, it is not surprising that females are generally much less affected. The majority of females with the full mutation have a normal or borderline normal IQ (Rousseau et al., 1994). However, most of the affected females still have psychological and behavioral deficits (Freund et al., 1993). These include social anxiety and avoidance, shyness, withdrawal, mood instability, irritability, depression, and hyperactivity. In additional to mental and behavioral disabilities, people with FXS also have abnormal physical characteristics such as macrocephaly, and macroorchidism (Lachiewicz and

Dawson, 1994). At the cellular level, FXS is characterized by the presence of abnormally long

and dense dendritic spines (Irwin et al., 2001), which is thought to be caused by abnormal spine

development or maturation (i.e. abnormal pruning).

1.12 Functions of Fragile X Mental Retardation Protein

It is clear that the deficiency of FMRP can lead to severe developmental abnormalities as

well as mental impairments. But what is the cellular function of FMRP? In the body, FMRP is

expressed in a variety of tissues (Khandjian et al., 1995). More importantly, it is found at high

levels in the brain and the testis (Devs et al., 1993), which correlates with the mental

impairments and macroorchidism phenotypes observed in FXS. As an RNA-binding protein,

FMRP acts as a translational regulator, being able to both suppress (Laggerbauer et al., 2001; Li

et al., 2001) and activate (Schütt et al., 2009; Ascano et al., 2012) mRNA translation.

Furthermore, the ability of FMRP to bind mRNAs led to the suggestion that it plays roles in

shuttling mRNA into and out of the nucleus, mRNA transport and localization in dendrites, and

synaptic protein synthesis in neurons (Antar et al., 2005; Dictenberg et al. 2008).

In the brain, FMRP is expressed in both neurons and glial cells (Pacey et al., 2007; Pacey

et al., 2013). While its expression in the neurons seems to be important for their spine

maturation (Comery et al., 1997) and synaptic plasticity (Huber et al., 2002), its role(s) in glial cells is still a relatively undeveloped area of research. Recent findings in astrocytes showed that

FMRP is important for regulating glutamate uptake (Higashimori et al., 2013). Its expression in the oligodendroglial lineage of cells seems to be restricted to the oligodendrocyte precursor cells

(OPCs) with a peak in expression during early development and gradually decreases with age

(Gholizadeh et al., 2015). This, combined with the observation of early developmental

myelination deficiency (Pacey et al., 2013), suggests a role of FMRP in the development and

maturation of OPCs.

1.13 Mouse Model of Fragile X Syndrome

The FMR1 gene was discovered in 1991. Shortly after its discovery, an Fmr1 knock out

(KO) mouse was produced in 1994 (The Dutch-Belgian Fragile X Consortium, 1994). Unlike the human FXS in which silencing of the FMR1 gene is due to expansion of the CGG repeats, the Fmr1 gene in this KO model was inactivated by a mutation within the exon 5 of the gene.

Nevertheless, this model was used study the pathology of the disease at the time as the KO mice displayed number of FXS phenotypes such as hyperactivity, learning deficits, and macroorchidism (enlarged testes). In addition, the increase in dendritic spine density and the abnormally long and immature spines observed in FXS patients are also observed in this mouse model (Irwin et al., 2001; Nimchinsky et al., 2001). Further study of the Fmr1 KO mouse showed that a form of protein synthesis-dependent synaptic plasticity termed long-term depression was enhanced in the hippocampus of the mice lacking FMRP (Huber et al., 2002).

This was consistent with a role of FMRP in translation repression and may also relate to the cognitive deficits observed in FXS patients since long-term depression is believed to be involved in memory and learning. It was shown more recently that synaptic functions were also affected in the amygdala of Fmr1 KO mice (Suvrathan et al, 2010). This might be related to the emotional pathologies of the disorder as the amygdala has a significant role in the formation of emotional memories (Zald, 2003). Together, these studies have further validated the mouse

Fmr1 KO model for gaining insights into FXS pathology.

1.2 Myelination during Brain Development

1.21 Myelination and Oligodendrocyte Development

Along with synaptogenesis and synaptic pruning, myelination is one of the defining features of early postnatal brain development. Myelination begins before birth at a very low level and accelerates in the postnatal period. In humans and rodents alike, it occurs rapidly during their early developmental stages of life (see Figure 1). It proceeds first in the hindbrain and moves rostrally (Bjelke and Seiger, 1989) in response to a functional demand with the areas important to early nursing developing first, followed by the other motor and sensory regions and finally the learning areas (Downes and Mullins, 2014).

Oligodendrocytes are glia cells that produce myelin, which is essential for saltatory conduction of action potentials in the central nervous system (CNS). An oligodendrocyte can extend many processes, each of which contacts and repeatedly wraps itself around a segment

Figure 1. Comparison of mouse versus human brain development.

axon to form myelin. On the same axon, different oligodendrocytes produce myelin segments that are adjacent to each other. The number of processes that form myelin from a single

oligodendrocyte can vary greatly between different CNS locations and between different species,

from 40 in the rat optic nerve (Peters et al., 1991) to 1 in the cat spinal cord (Bunge et al, 1961).

Due to this heterogeneity, oligodendrocytes were classified into 4 types: type I that form short,

thin myelin sheaths around 15-30 small diameter axons, type IV that form long, thick myelin

sheaths around 1-3 large diameter axon(s), and type II and III which are intermediary (Butt et al.,

1995).

Oligodendrocytes go through several stages of development before maturing to myelin-

forming cells. Rodent oligodendrogial lineage of cells has been studied in detail using the

expression of specific proteins as markers of the different stages of oligodendrocyte development

(i.e. mature oligodendrocyte marker adenomatous polyposis coli and the OPC marker NG2).

Interestingly, oligodendrocytes are also the cell population in the central nervous system with the

highest turnover, and as such, the adult CNS contains oligodendrocyte precursor cells at all

stages of maturation (Dawson et al., 2003).

Mature oligodendrocytes arise from OPCs, which are characterized by their expression of

platelet derived growth factor receptor alpha (PDGFR-α) (Pringle et al., 1992) and neural/glial antigen 2 (NG2) proteoglycans (Stallcup and Beasley, 1987). Unlike mature oligodendrocytes,

OPCs are both proliferative and motile cells. The process of OPC maturation begins with an initial contact, which induces a number of molecular rearrangements in the future myelinating cell, including the association of the tyrosine-kinase fyn with lipid-rich membrane microdomains

(Czopka et al., 2013; Krämer-Albers and White, 2011), the suppression of RhoA activity (Baer et al. 2009), and the local enrichment of phosphoinositides in the glial membrane (Goebbels et al.

2010; Snaidero et al. 2014). Moreover, directed mRNA transport (i.e. myelin basic protein mRNA) and local protein translation is activated (Muller et al., 2013; Wake et al., 2011). Since

myelin basic protein (MBP) mRNA is a substrate of FMRP (see Table 1), one can postulate that

the absence of FMRP in OPCs could have detrimental consequences on their maturation.

Table 1. List of myelin protein and myelination‐associated genes whose mRNA transcripts are also FMRP substrates ( Y) or not (N), as reported in Darnell et al., 2011, Ascano et al., 2012, and Lucá et al., 2013. Gene Name Darnell Ascano Ascano Ascano Ascano Luca Symbol et al., et al., et al., et al., et al., et al., 2011 2012 2a 2012 2b 2012 2c 2012 2d 2013 Myelin Proteins MBP Myelin basic protein Y Y Y N N CNP 2’,3’‐cyclic nucleotide 3’‐ Y Y Y N Y phosphodiesterase PLP1 Proteolipid protein 1 Y N N N N MAG Myelin‐associated N N N N N glycoprotein MOG Myelin oligodendrocyte N N N N N glycoprotein MPZ Myelin protein zero N N N N N PMP22 Peripheral myelin protein 22 N N N N Y OMG Oligodendrocyte myelin N N N N N glycoprotein Oligodendrocyte Precursor Cell Development Olig1 Oligodendrocyte transcription N N N N N factor 1 Olig2 Oligodendrocyte transcription N N Y N Y factor 2 Sox10 SRY‐box containing gene 10 N N N N N Fyn Fyn proto‐oncogene Y Y Y Y Y Thra Thyroid hormone receptor Y N Y N Y alpha EGFR and its Ligands EGFR Epidermal growth factor N Y N N Y Y receptor EGF Epidermal growth factor N N N N N Hbegf Heparin‐binding epidermal N N Y N Y growth factor like factor

1.22 Myelin Basic Protein and 2',3'-Cyclic-nucleotide 3'-phosphodiesterase

Oligodendrocyte maturation is accompanied by the expression of certain myelin proteins

such as MBP, which is one of the first to be expressed at detectable quantities (Verity and

Campagnoni, 1988). MBP comprises a major part of the cytosolic protein of compact myelin

and is present both in the central and peripheral nervous systems. Moreover, it is actually a

family of isoforms of different molecular masses that are generated by alternative splicing of the

MBP gene. The molecular masses of these major isoforms are 21.5, 20.2, 18.5, and 17.2 kDa in

humans and 21.5, 18.5, 17, and 14 kDa in mice (Campagnoni and Macklin, 1988). However,

these isoforms are not all expressed at the same levels during different stages of life. In the adult,

the predominant isoforms are the 18.5 and 17.2 kDa isoforms in humans and 18.5 and 14 kDa

isoforms in mice, constituting approximately 95% of the MBPs (Staugaitis et al., 1990). On the

other hand, the 17 and 21.5 kDa isoforms in mice, and the 20.2 and 21.5 kDa isoforms in humans

appear early during development and are mainly expressed during myelinogenesis. Interestingly,

they are also re-expressed in chronic lesions of multiple sclerosis with their expression

correlating with re-myelination (Capello et al., 1997). These early-expressed MBP isoforms are

also actively transported from the cytoplasm to the nucleus, which suggests a regulatory role for

them in myelination (Pedraza et al., 1997).

A well-established function of MBP is its participation in the formation of compact myelin. During initial myelin compaction, the intracellular surface of the oligodendroglial membrane is resistant to forming tight appositions due to the presence of highly negatively charged phospholipids. However, as its name indicates, MBP is a basic protein, and the negatively charged membrane attracts basic proteins (Harauz et al., 2009). Thus, MBP neutralizes the membrane phospholipids and pulls two bilayers together (Aggarwal et al., 2013),

which forms the major dense line and allows the myelin to grow in thickness (Min et al., 2009).

Without MBP, oligodendrocytes cannot form compact myelin, as observed in the shiverer mice

(Roach et al., 1985). Interestingly, peripheral myelination can proceed without MBP due to the

compensation by basic intracellular domain of myelin protein zero (Martini et al., 1995).

Aside from MBP, 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) is another

protein that is specifically expressed in myelin, where it is localised in the cytoplasm of non-

compact myelin (Trapp et al., 1988). Rather than serving as a marker for mature myelin

(although it is also expressed in mature myelin), the appearance of CNPase seems to be one of

the earliest events of myelination (Braun et al., 1988), and it is thought to play a role in

oligodendrocyte development (Gravel et al., 1996). The enzymatic activity of CNPase allows it

to hydrolyzes artificial substrates, 2’,3’-cyclicnucleotides into their 2’-derivatives. However, the

biological importance of its enzymatic function is obscure (Kursula, 2008), especially since

2’,3’-nucleotides have not been detected in the brain (Vogel and Thompson, 1988). Interestingly,

the catalytic function of CNPase has been suggested to be related to that of RNase A (Sakamoto

et al., 2005). Furthermore, the N-terminus sequence of CNPase has been suggested to be

structurally homologous to the N-terminus kinase domain of an RNA repair enzyme T4 poly

nucleotide kinase (Wang et al., 2002). Therefore, it is possible that the function of CNPase is

related to RNA metabolism.

1.23 Myelin-Axon Crosstalk

The relationship between the oligodendrocyte and the neuron goes beyond the facilitation

of axonal conductance; extremely complex interactions can occur between neurons and

oligodendrocytes at every stage of myelination and consequently underlie myelin defects. Injury

to the oligodendrocyte can lead to axonal degeneration (Pohl et al., 2011) and vice versa. Thus,

after maturation, the integrity and survival of the myelinated axons depend on oligodendrocyte

support. One of the ways oligodendrocytes can affect axonal functions is the direct transfer of materials, such as mRNAs and proteins through extracellular vesicles that are triggered by

neurotransmitter release from the axons (Frühbeis et al., 2013). In addition, oligodendrocytes

also have the capacity to support axons through glial cell line-derived neurotrophic factor

(GDNF) (Wilkins et al., 2003) and connexins (Altevogt et al., 2002) which may allow for the

passage of small metabolites. In addition to signalling, there is also a metabolic exchange

between the oligodendrocyte and the axon. Essentially, the oligodendrocyte supplies the pyruvate or lactate to the neurons to meet their energy demands and the neuron in return supplies the oligodendrocyte with its metabolite N-acetylaspartate for the biosynthesis of myelin lipids

(Nave and Werner, 2014). Overall, the intimate relationship between the oligodendrocyte and the neuron highlight the two as an inseparable functional unit and challenges the traditional view of neurons being the principal cells of the brain.

1.24 White Matter and Myelin Abnormalities in Autism and FXS

The importance of myelin is probably best exemplified in the MBP-deficient shiverer

mouse and in the human multiple sclerosis condition. Aside from multiple sclerosis, myelination

abnormalities have also been associated with other brain diseases such as autism. Magnetic

resonance imaging studies on young autistic children between the ages of 0.5-3.3 years have

revealed a first accelerated growth of white matter, seen in the fractional anisotropy (indicator of

white matter integrity) measurements (Ben Bashat et al., 2007; Wolff et al., 2012), that seems to

then slow down after the age of 1 (Wolff et al., 2012). In agreement with these findings, another

study that examined autistic children between the ages of 6-13 years saw widespread reductions

in white matter fractional anisotropy values in the autistic group compared to the control group

(Barnea-Goraly et al., 2010). Furthermore, the fact that the same reduction was also seen in the

unaffected siblings of the autistic children in the same study suggests an underlying genetic basis

for the aberration.

Unlike autism, myelination in FXS has not been studied extensively. However, an early

study offered preliminary evidence of white matter alternations in FXS (Barnea-Goraly et al.,

2003). Similar to autism, the study observed decreased fractional anisotropy values in the brains

of 13-22 year-old FXS females compared to the controls. Using a mouse model of FXS, our lab

has recently discovered that myelination in the cerebellum of early postnatal Fmr1 KO mice is

delayed (Pacey et al., 2013). At postnatal day (PND) 7 and PND 15, cerebellar expression of

both MBP and CNPase is significantly lower in the Fmr1 KO than in the WT mice. Moreover,

electron microscopy of cerebellar axons revealed that the PND 7 (but not PND 15) FXS

cerebellum contained fewer myelinated axons and thinner myelin sheaths than the WT

counterpart. The fact that these changes in the early postnatal Fmr1 KO mouse cerebellum were

accompanied by a decreased number of OPCs suggests that impaired maturation or function of

OPCs underlie the delayed myelination. Most importantly, it appears from studies on both

autism and FXS that proper early developmental myelination is crucial to the proper

development and functioning of the brain later on in life.

1.3 Role of Epidermal Growth Factor Receptor Signalling in

Myelination

1.31 Epidermal Growth Factor Receptor and its Endogenous Ligands

Epidermal growth factor receptor (EGFR, also known as ErbB1) belongs to the ErbB

family of receptor tyrosine kinases which also include HER2/neu (ErbB2), HER3 (ErbB3), and

HER4 (ErbB4) (Schlessinger, 2002). Upon ligand binding, EGFR forms homo- or heterodimers and its intrinsic kinase domain become activated, resulting in autophosphorylation of specific

tyrosine residues in the cytoplasmic tail (Schlessinger, 2002). There are seven known

endogenous ligands of EGFR: epidermal growth factor (EGF), heparin-binding EGF-like growth

factor (HB-EGF), transforming growth factor α, amphiregulin, betacellulin, epiregulin, and

epigen (Harris et al., 2003). However, in this study, I focused solely on EGF and HB-EGF.

EGF is a small protein of 53 amino acids with a molecular weight of approximately 6

kDa (Carpenter and Cohen, 1979). EGF is derived from proteolytic cleavage of its

transmembrane precursor (prepro-EGF) of approximately 1200 amino acids in length (Carpenter

and Cohen, 1979). However, the metalloproteinases(s) involved in the cleavage of prepro-EGF

remain to be identified. In terms of its biological source, EGF is first discovered in the rodent

submaxillary gland, the most abundant source of EGF. Since then, it has also been found in

other tissues, namely the kidneys, the thyroid gland, and the brain (at low levels). In addition,

EGF is detectable in the blood. Although platelets seem to be the main source of blood EGF

(Oka and Orth, 1983) the submaxillary gland can also secret EGF into the blood and contribute

to its non-platelet EGF content (Kurachi and Oka, 1985; Tebar et al., 2000).

HB-EGF is another ligand EGFR. Its precursor, proHB-EGF, is an 206 amino acid,

plasma membrane-anchored protein that undergoes metalloproteinase-dependent processing to

an 86 amino acid secreted protein (Higashiyama et al., 1992; Sahin et al., 2004). Interestingly,

the human, but not murine proHB-EGF also serves as the receptor for diphtheria toxin

(Mitamura et al., 1995). Like EGF, HB-EGF is also found in a variety of tissues, but most

importantly it is also found in the brain and at a much higher level (approximately 100x) than

EGF (Piao et al., 2005). Therefore, it is not surprising that HB-EGF has been proposed as the

major EGFR ligand in the brain (Oyagi and Hara, 2012).

1.32 EGFR Signaling Favors Glial Differentiation and Promotes

Myelinogenesis

The most well-known function of EGFR is probably its mitogenic effects to increase cell proliferation and survival. Due to this, the vast majority of the studies on EGFR signalling are focused in the area of cancer research. However, in my M.Sc. research I focused on the role of

EGFR signaling in myelination.

EGFR, EGF, and HB-EGF are expressed in the brain (Aguirre et al., 2004; Piao et al.,

2005) and both EGFR and HB-EGF expression have been documented in the oligodendroglial

lineage of cells (Aguirre et al., 2004; Nakagawa et al., 1998) where they exert positive effects on

the generation of OPCs and myelinogensis. For instance, through EGFR signalling, EGF has been shown to increase myelination of brain cell aggregates in vitro with an increase in MBP and

CNPase expression (Almazan et al., 1985). This growth factor likely also exerts protective effects on oligodendrocytes as its expression increases in white matter after chronic hypoxia

(Scafidi et al., 2014) and can also promote oligodendrocyte process formation after injury in vitro (Knapp and Adams, 2004). Furthermore, HB-EGF administration immediately following chronic hypoxia was able to promote oligodendrocyte regeneration in the white matter and rescue behavioral deficits (Scafidi et al., 2014). Interestingly, enhancing EGFR expression in the oligodendrocytes itself was able to facilitate oligodendrogenesis and myelin repair (Aguirre et al.,

2007; Scafidi et al., 2014). As the oligodendroglial lineage of cells are first derived from neural

precursor cells (NSCs) in the subventricular zone, it is no surprise that EGFR signaling favors

the glial differentiation of NSCs (Galvez-Contreras et al., 2013; Romero-Grimaldi et al., 2011;

Sun et al., 2011).

1.4 The Functional Role of Thyroid Gland in Myelination

One of the many functions of the thyroid gland is the production of the hormone

calcitonin by thyroid C cells to regulate calcium levels in the body. In addition to its roles in

calcium homeostasis, the thyroid gland also possesses follicular cells that produce thyroid

hormones which are essential to brain maturation and function. This is apparent in thyroid

disorders. For example, hypothyroidism has been associated with affective disorders,

polyneuropathy, and loss of cognitive functions (Joffe and Sokolov, 1994; Jaggy and Oliver,

1994; Ganguli et al., 1996). Interestingly, maternal hypothyroidism has also been linked to

autism spectrum such as that children whose mothers were hypothyroid during pregnancy have

an increased risk of having autism spectrum disorders (Román et al., 2013; Andersen et al.,

2014).

With respect to myelination, thyroid hormone has been shown to directly regulate MBP

expression (Farsetti et al., 1991). Furthermore, hypothyroidism has been found to reduce MBP

expression in the perinatal brain (Ibarrola and Rodríguez-Peña, 1997) and cause delayed and

hypomyelination (Balázs et al., 1969; Malone et al., 1975; Noguchi and Sugisaki, 1984) whereas

hyperthyroidism seems to accelerate myelination (Adamo et al., 1990). This is probably due to

the effect of thyroid hormone in increasing OPC generation (Calza et al., 2002) and its action on

the OPC thyroid hormone receptors to promote their differentiation (Billon et al., 2002). In

addition, the thyroid gland is also known as one of the major sources EGF in the body (Dagogo-

Jack, 1992) and the follicular cells express both EGF and its receptor EGFR (van der Laan et al.,

1995). Therefore, EGF and other EGFR ligands might be able to indirectly influence

myelination in the brain through their actions on the thyroid and its production of thyroid

hormones.

2. Hypotheses, Objectives, and Rationale

2.1 Project 1 – Mapping the Myelination Defect in Fmr1 KO Mice

Hypotheses

1) Deficiencies in myelin proteins MBP and CNPase occur in the brainstem, cerebellum,

hippocampus, and prefrontal cortex of the brain in Fmr1 KO mice during the early postnatal

period at PND7 and PND15, but not in adult mice.

2) Based on the caudal to rostral progression of myelination, I also hypothesize that the

MBP and CNPase deficiencies in the Fmr1 KO brain will be observed first in the brainstem and

cerebellum before the hippocampus and the prefrontal cortex.

Objective

The objective was to conduct a thorough analysis of myelination in the cerebellum,

brainstem, hippocampus, and prefrontal cortex of PND 7, 13, and adult Fmr1 KO mice in

comparison to their WT controls. To examine myelination, I measured the overall expression of

MBP and CNPase in each of the four brain regions in these mice using western blotting.

Rationale

The rationale behind the extension of the delayed myelination defect to the brainstem,

hippocampus, and prefrontal cortex is based on 2 postulations: 1) global loss of FMRP would

affect all oligodendrocytes in the brain, not just those in the cerebellum as seen in Pacey et al.,

2013 and 2) myelination of the brain is a directional process that begins in the brainstem and

proceeds rostrally. Therefore, a delay in myelination in regions that are myelinated earlier (i.e.

brainstem and cerebellum) could stall the rostral movement and delay the timing in regions that

are myelinated later (i.e. hippocampus and prefrontal cortex). Moreover, due to the rostral

progression of myelination, this deficit should appear first in the rostral areas of the brain that are

the first to be myelinated. By the same logic, myelination in these rostral areas should also be

the first to mature and therefore the first to catch up to WT levels (seen previously in the cerebellum in Pacey et al., 2013).

2.2 Project 2 – Investigation of the EGFR System in Fmr1 KO

Mice

Hypothesis

The levels of EGFR, phospho-EGFR (pEGFR), EGF, and HB-EGF in the cerebellum,

hippocampus, and prefrontal cortex are lower in Fmr1 KO mice than those of the age-matched

WT mice at PND7 and PND15.

Objectives

1) The first objective was to examine the levels of expression of EGFR and pEGFR in the

cerebellum, hippocampus, and prefrontal cortex of PND 7, 13 WT and Fmr1 KO mice using quantitative western blotting. It was important to examine both EGFR and its active form pEGFR because the latter is the activated form of the receptor that would exerts its effects on myelination.

2) The second objective was to measure the level of EGF and HB-EGF in the cerebellum, hippocampus, and prefrontal cortex of PND 7 and 13 WT and Fmr1 KO mice.

3) The third objective was the measure the level of EGF in the plasma, kidney, and

thyroid gland of adult WT and Fmr1 KO mice.

Rationale

EGFR signaling is known to play a role in the proliferation and development of

oligodendrocytes. For example, administration of an EGFR ligand has been shown to be able to

significantly boost OPC production and induce remyelination in the white matter (Scafidi et al.,

2014). However, the rationale to tackle the EGFR system not only stems from its role in

myelination, but also from the fact that both EGFR and HB-EGF mRNAs have been shown to

interact with FMRP (Table 1). Although studies on autism have found decreased blood EGF

levels in autistic individuals (Suziki et al., 2007; Onore et al., 2012; Russo, 2013), whether there

is a dysregulation of the EGFR system in FXS has yet to be explored. Together, the findings

above suggest that the EGFR system could be abnormal in FXS and the abnormality could be a

contributing factor to the myelin deficit seen in FXS.

2.3 Project 3 – Intranasal Administration of HB-EGF

Hypothesis

Intranasal HB-EGF administration during the early postnatal period (PND3-6) restores

the MBP and CNPase deficiencies in Fmr1 KO mice later at PND 11.

Objective

The object in this second phase of the EGFR project was to examine and compare the

expression of MBP, CNPase, and NG2 expression in the brainstem, cerebellum, and prefrontal

cortex between the Fmr1 KO-saline, the Fmr1 KO-HB-EGF, and the WT mice. The comparison

between the WT and the Fmr1 KO-HB-EGF mice allowed us to determine the effect of HB-EGF on levels of MBP and CNPase in Fmr1 KO mice in relation to WT levels.

Rationale

It is clear that myelination is deficient throughout the developing fragile X brain.

Although it is still unknown whether this early myelin deficiency contributes to any of the behavioral phenotypes seen in the adult fragile X mice, considering the importance of proper myelination during the early developmental period, especially to ensure normal neuronal firing

and development of the neural circuitry, it is tempting to connect the myelin deficits to the

behavioral deficits. Therefore, in order to investigate this, the first rational step is to correct the

myelin deficit. As recent evidence suggests, intranasal administration of HB-EGF to neonatal

mice promoted oligodendrogenesis and myelin repair after chronic hypoxia (Scafidi et al., 2014).

Therefore, we hope to be able to boost up myelin production in the early postnatal Fmr1 KO mice that are myelin-deficient by using intranasal HB-EGF treatment.

3. Methods

3.1 Animals

All animal experiments were carried out in accordance with the guidelines set by the

Canadian Council on Animal Care and were approved by the University of Toronto Animal Care

Committee. All WT and Fmr1 KO mice used were of the C57/BL6 background. Fmr1 KO mice

(backcrossed > 10 generations on the C57BL/6 background) were generously provided by Dr.

William Greenough, University of Illinois, and bred at the University of Toronto. All Fmr1 KO mice were the offspring of homozygous Fmr1 KO parents.

3.2 Western Blotting

All samples of mouse brainstem, cerebellum, hippocampus, and prefrontal cortex were

dissected on ice (see appendix 4 for the dissection of prefrontal cortex), frozen on dry ice, and

stored at -80°C. To prepare for western blotting, the tissues were first homogenized in ice-cold

lysis buffer (50 mM Tris-HCl, 1% SDS, 1x protease inhibitor cocktail [Roche], and 1x

phosphatase inhibitor cocktail 2 [Sigma-Aldrich] pH 7.4) at the following volume: weight of wet

tissue (mg) / 30 * 100 mL to yield a protein concentration of approximately 3 µg/mL. Protein

quantification was performed using the QuantiPro BCA assay kit (Sigma-Aldrich). The protein

concentrations were measured in duplicates using the DU 730 UV/VIS scanning

spectrophotometer (Beckman Coulter, Mississauga, ON) at 562 nm absorbance. Tissue

homogenates were prepared for SDS-PAGE in 4x sample buffer (8% SDS, 250 mM Tris, 40%

glycerol, pH 6.8) with 100 mM of dithiothereitol, and 1 µL of bromophenol blue. The samples

were then sonicated for 10 seconds with a MicrosonTM ultrasonic cell disruptor (Heat Systems

Ultrasonics, Farmingdale, NY) after which they were heated to 95°C for 3 minutes and then

cooled down to room temperature before loading polyacrylamide gel.

Equal amounts of total protein were loaded in each lane of the stacking gel and separated

by electrophoresis in 8%, 10%, or 12% polyacrylamide gels, depending on the molecular weight

of the target protein. The amount of the total protein loaded (5–30 µg) depended on the

abundance of the target protein. A total of 6-8 samples and a pre-stained protein standard (Bio-

Rad) were loaded in each gel every time. After electrophoresis, the gels and the nitrocellulose membranes were soaked in transfer buffer (48 mM Tris, 34 mM glycine, 1.5 mM SDS, pH 9.2) for 15 minutes. The proteins were then transferred to a nitrocellulose membrane using either the

Bio-Rad Trans-Blot semi-dry transfer system for 1 hour at 20V or the wet transfer system for 1 hour at 300 mA at 4°C, and then blocked with 5% skim-milk (BioShop) in the wash buffer (100 mM Tris, 15 mM NaCl, 0.05% Tween-20, pH 7.6) at room temperature for 1 hour. The membranes were washed 3 x 15 minutes with wash buffer and incubated with primary antibodies

(diluted in wash buffer) overnight at 4°C.

The following primary antibodies used: rat anti-MBP (1:500 – 1:1000, Millipore), mouse anti-CNPase (1:1500, Millipore), mouse anti-glyceraldehyde 3-phosphate-dehydrogenase

(GAPDH) (1:80 000, Sigma-Aldrich), rabbit anti-EGFR (1:200, Santa Cruz), rabbit anti-pEGFR

(1:500, Novus), rabbit anti-beta-actin (1:5000, Abcam) and rabbit anti-HB-EGF (1:200, Santa

Cruz). The next day, the membranes were washed 3 times 15 minutes each with wash buffer and incubated for 2 hours at room temperature with secondary antibodies (diluted in wash buffer with

3% skim-milk). The following horseradish peroxidase-conjugated secondary antibodies were used: goat anti-mouse (1:4000, Jackson ImmunoResearch), donkey anti-rat (1:4000, Jackson

ImmunoResearch), and goat anti-rabbit (1:4000, Jackson ImmunoResearch). After secondary

antibody incubation, the membranes were washed 3 x 15 minutes again in wash buffer before

immunoreactivity was detected by incubating the membranes in SuperSignal West Pico

Chemiluminescence substrate (Fisher Scientific, Pittsburgh, PA) for 5 minutes.

For quantification, the signal intensity was measured using the Alpha Innotech

FluorChem® Chemiluminescent Imaging System. The membranes were exposed for 1-10

minutes, depending on signal intensity, before immunolabeling density was analyzed using

AlphaEaseF image analysis software by measuring the integrated density value per area of the target protein and GAPDH or beta-actin bands. The intensity of the antibody bands was then calculated as a ratio to GAPDH or beta-actin (for EGFR and pEGFR) intensity. The protein expression levels for KO mice are reported as percentages of the WT expression ± standard error of the mean (SEM) with WT expression adjusted to 100%. The data from WT and Fmr1 KO mice were compared using the two-tailed unpaired Student’s t-test assuming either equal or unequal variances depending on whether the variances were statistically different.

3.3 EGF and thyroxine ELISA

All samples of mouse cerebellum, hippocampus, and prefrontal cortex (see Appendix 4

for the dissection of prefrontal cortex) were dissected on ice, frozen on dry ice, and stored at -

80°C. To obtain the plasma, blood was collected via cardiac puncture and then immediately

transferred to heparin-coated Eppendorf tubes and spun in a microfuge at 20000×force of gravity

for 20 minutes. The supernatant was then collected as the plasma and stored at -80°C until analysis. To prepare the tissues for ELISA, they were first homogenized in ice-cold PBS with 1x protease inhibitor cocktail (Roche) at a volume of 200 uL (for all samples) and subsequently sonicated for 10 seconds with a MicrosonTM ultrasonic cell disruptor (Heat Systems Ultrasonics,

Farmingdale, NY). The tissue homogenates were then spun in a microfuge at 20000×force of

gravity for 2 minutes and the supernatants were then used for protein quantification and ELISA.

Protein quantification was performed using the QuantiPro BCA assay kit (Sigma-Aldrich). The protein concentrations were measured in duplicates using the DU 730 UV/VIS scanning spectrophotometer (Beckman Coulter, Mississauga, ON) at 562 nm absorbance. EGF and thyroxine concentrations in the samples were determined using the Mouse EGF Quantikine

ELISA Kit (R&D Systems) and the Mouse/Rat Thyroxine (T4) ELISA kit (Sigma-Aldrich). All

reagents were supplied by the kits and all experimental procedures were followed as outlined in

their separate manuals. Standard curves were constructed for the calculation of EGF

concentrations from raw absorbance values (see Appendix 5 for a representative EGF ELISA

standard curve). Samples were assayed in duplicate with 50µL of the sample supernatant used

for each duplicate. The EGF detection range of for this kit was 1.64-500 pg/mL of the protein.

The absorbance readings of the samples were measured with a microplate reader (VICTOR3

1420, PerkinElmer precisely®) that was generously provided by Dr. Carolyn Cummings at the

Leslie Dan Faculty of Pharmacy, University of Toronto.

3.4 Intranasal HB-EGF Administration

From postnatal day (PND) 3 to 6, Fmr1 KO mice received 100 ng per g of body weight of recombinant human HB-EGF (hHB-EGF) (R&D Systems) in saline twice daily between the hours of 9:00 -10:00 and 17:00 - 18:00. For each treated Fmr1 KO mouse litter, half of the pups received hHB-EGF and the other half received saline as control. The experimenter was blinded to the identity of the solutions to be administered throughout the entire experiment and one of the two groups of mice was tail-clipped in order for them to be identified. A 20 µL pipette

(Pipetman®) attached with a 0.5-10 µL tip (Axygen) was used to deliver the solution in 2-4µL

droplets to either naris with the animal in the ventral-side-up position. After delivery of the

entire volume, the animal was then kept in the same position for 1-2 minutes ensure absorption.

The pups were kept separate from their mother until every pup in the litter was treated. The pups

were dissected on PND 11.

4. Results

4.1 MBP and CNPase expression in the Fmr1 KO and WT mouse

brains

The overall expression of two myelin markers MBP and CNPase in the cerebellum,

brainstem, hippocampus, and prefrontal cortex of the mice was assessed by western blotting.

Representative western blots of MBP and CNPase expression in each of the brain regions at

three developmental time points (PND 7, PND 13, and 2 month-old adult) were collectively

shown in Figure 2. Western blots of PND 7 hippocampus and prefrontal cortex were omitted

due to undetectable MBP and CNPase expression. For quantification, all densitometric values of

MBP and CNPase were normalized to those of GAPDH and then expressed as a percentage of

the average WT value (set to 100%). For MBP, the 17 kDa and the 18.5 kDa bands were

quantified together since they were not well separated in the gels.

At PND 7 there was significantly lower expression of MBP (all isoforms) and CNPase in

the Fmr1 KO brainstem and cerebellum compared to their WT counterparts (Figure 3). Fmr1

KO brainstem MBP expression was, ~60% lower (average of the 4 isoforms) while CNPase was

~25% lower than that of the WT brainstem (Figure 3A). Following a similar trend, MBP and

CNPase expression in the cerebellum were ~50% and ~25% lower in the Fmr1 KO mice when

compared to the WT mice (Figure 3A). Interestingly, at PND 13, MBP (all isoforms) and

CNPase expression were not different between the Fmr1 KO and WT mouse brainstem (Figure

3A) and cerebellum (Figure 3B). In the adult 2 month-old mice, MBP (all isoforms) and CNPase

expression in the brainstem (Figure 3A) and cerebellum (Figure 3B) were also not different

between the Fmr1 KO and WT mice.

A Brainstem B Cerebellum

PND 7

PND 13

Adult

C Hippocampus D Prefrontal Cortex

PND 13 Undetectable MBP

Adult

Figure 2. Representative western blots of MBP and CNPase expression in the mouse brain. A collection of western blots showing the expression of 21.5 kDa, 17/18.5 kDa, and 14 kDa MBP isoforms and CNPase, along with the loading control protein GAPDH in the A) brainstem, B) cerebellum, C)

hippocampus, and D) prefrontal cortex of PND 7, PND 13, and adult mice. Both MBP and CNPase were below the limit of detection in the PND 7 prefrontal cortex and hippocampus and therefore the blots were not shown.

A. Brainstem WT fmr1 KO WT KO WT KO WT KO 150 n = 7 n = 8 n = 12 n = 12 n = 8 n = 7 P = 0.0006 P = 0.002 P = 0.0002 P = 0.0003 100 *

50 * * *

Expression (% of WT) 0

21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase

kDa kDa kDa kDa kDa kDa

PND7 PND13 Adult

B. Cerebellum WT fmr1 KO

WT KO WT KO WT KO 200 n = 7 n = 8 n = 12 n = 12 n = 8 n = 7

150 P = 0.0009 P = 0.011 P = 0.0018 P = 0.018 100 * * * 50 *

Expression (% of WT) 0 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase kDa kDa kDa kDa kDa kDa PND7 PND13 Adult

Figure 3. MBP and CNPase expression profiles in the mouse brainstem and cerebellum. Summary of the quantitative western blotting results of MBP (21.5, 17/18.5, and 14 kDa isoforms) and CNPase expression in A) the brainstem and B) the cerebellum of PND 7, PND 13, and adult mice. MBP and CNPase expression was normalized with GAPDH expression and all normalized values were expressed as a percentage of the average WT value (adjusted to 100%). *indicates statistical significance compared to the respective WT group (P < 0.05, two-tailed unpaired t-test).

Figure 4 provides a summary of MBP and CNPase expression in the hippocampus and

prefrontal cortex of the Fmr1 KO and WT mice. Note that analysis was not performed on PND

7 prefrontal cortex because MBP expression in this brain region was below the limit of detection

at PND 13. Unlike the PND 7 brainstem and cerebellum, MBP and CNPase expression were

undetectable in the PND 7 hippocampus in both Fmr1 KO and WT mice (Figure 4A). In the

PND 13 hippocampus, MBP (all isoforms) expression was on average ~55% lower and CNPase

only a trend of ~50% lower in the Fmr1 KO mice when compared to the WT mice (Figure 4A).

In the PND 13 prefrontal cortex, MBP was undetectable and CNPase was ~30% lower in the

Fmr1 KO mice relative to the WT mice (Figure 4B). In the adult hippocampus, MBP and

CNPase expression were not different between the Fmr1 KO and WT mice (Figure 4A). In the adult prefrontal cortex however, the expression of 21.5 kDa and 14 kDa MBP isoforms were lower in the Fmr1 KO mice, albeit not statistically significantly different than the WT mice, whereas the 17/18.5 kDa MBP isoforms and CNPase were statistically significantly lower by

~30% in the Fmr1 KO mice (p < 0.05) compared to the WT mice (Figure 4B).

In summary, the analysis of myelin proteins in the Fmr1 KO mice showed significantly lower levels of MBP and CNPase in all of the 4 brain regions analyzed, albeit at different ages with the deficit appearing earlier in the brainstem and the cerebellum than in the hippocampus and prefrontal cortex. Moreover, in the adult Fmr1 KO prefrontal cortex, some MBP isoforms remained reduced.

A. Hippocampus WT fmr1 KO WT KO WT KO WT KO 150 n = 4 n = 4 n = 8 n = 8 n = 8 n = 7 P=0.022 P=0.013 P=0.017 P=0.07 100 * * Undetected 50 *

Expression (% of WT) 0 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase kDa kDa kDa kDa kDa kDa

PND7 PND13 Adult

B. Prefrontal Cortex WT fmr1 KO WT KO WT KO n = 8 n = 8 n = 11 n = 12 P=0.0025 P=0.0021 P=0.0042 100 * * * Undetected 50

0 Expression (% of WT) 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 14 kDa CNPase kDa kDa kDa kDa

PND13 Adult

Figure 4. MBP and CNPase expression profiles in the mouse hippocampus and prefrontal cortex. Summary of the quantitative western blotting results of MBP (21.5, 17/18.5, and 14 kDa isoforms) and Delete empty space CNPase expression in A) the hippocampus and B) the prefrontal cortex of PND 7, PND 13, and adult mice. MBP and CNPase expression was normalized with GAPDH expression and all normalized values were expressed as a percentage of the average WT value (adjusted to 100%). *indicates statistical significance compared to the respective WT group (P < 0.05, two-tailed unpaired t-test).

4.2 EGFR and pEGFR expression in the Fmr1 KO and WT mouse

brains

Brain region-specific expression of EGFR and pEGFR during the early postnatal period

at PND 7 and PND 13 were assessed by western blotting. For quantification purposes, all

densitometric values of EGFR and pEGFR were normalized to those of β-actin and then

expressed as a percentage of the average WT value (set to 100%). Representative western blots

of EGFR and pEGFR expression in the cerebellum, hippocampus, and prefrontal cortex at PND

7 and PND 13 are shown in Figure 5. Note that the expression of pEGFR was below the limit of detection in the PND 13 cerebellum and the blot was therefore omitted from Figure 5.

As shown in Figure 6A, EGFR and pEGFR expression in the PND 7 cerebellum and

EGFR expression in the PND 13 cerebellum were not different between the Fmr1 KO and WT

mice. In the PND 7 hippocampus, EGFR and pEGFR expression also did not differ between the

Fmr1 KO and WT mice (Figure 6A). However, in the PND 13 hippocampus, EGFR expression

is ~25% lower in the Fmr1 KO mice compared to the WT mice while pEGFR did not differ

between the 2 genotypes (Figure 6B). In the PND 7 prefrontal cortex, EGFR expression was not

different between the Fmr1 KO and WT mice while pEGFR expression was ~30% lower in the

Fmr1 KO compared to their WT controls (Figure 6C). At PND 13, the prefrontal cortex

expression of both EGFR and pEGFR were not different between the two genotypes (Figure 6C).

A. Cerebellum

EGFR pEGFR

PND 7

PND 13 Undetectable pEGFR

B. Hippocampus EGFR pEGFR

PND 7

PND 13

C. Prefrontal Cortex

EGFR pEGFR

PND 7

PND 13

Figure 5. Representative western blots of EGFR and pEGFR expression in the mouse brain. A collection of western blots showing the expression of EGFR and pEGFR, along with the loading control protein β-actin in the A) cerebellum, B) hippocampus, C) prefrontal cortex of PND 7 and PND 13 mice.

WT fmr1 KO A. Cerebellum WT KO WT KO WT KO WT KO n = 4 n = 4 n = 4 n = 4 n = 12 n = 12 n = 4 n = 4

100

50 Undetected

0 Expression (% of WT) EGFR pEGFR EGFR pEGFR

PND7 PND13

WT fmr1 KO B. Hippocampus WT KO WT KO WT KO WT KO n = 8 n = 8 n = 8 n = 8 n = 12 n = 12 n = 8 n = 8 P<0.0001 100 *

0 Expression (% of WT) EGFR pEGFR EGFR pEGFR PND7 PND13

WT fmr1 KO C. Prefrontal Cortex WT KO WT KO WT KO WT KO n = 8 n = 8 n = 8 n = 8 n = 8 n = 8 n = 8 n = 8 P=0.014 100 * 50

0 Expression (% of WT) EGFRpEGFREGFRpEGFR PND7 PND13

Figure 6. EGFR and pEGFR expression in the early postnatal mouse brain. Summary of the quantitative western blotting results of EGFR and phospho-EGFR (pEGFR) expression in A) the cerebellum B) the hippocampus, and C) the prefrontal cortex of PND 7, and PND 13 mice. Analysis of PND 7 prefrontal cortex was omitted. MBP and CNPase expression was normalized with beta-actin expression and all normalized values were expressed as a percentage of the average WT value (adjusted to 100%). *indicates statistical significance compared to the respective WT group (P < 0.05, two-tailed unpaired t-test).

Due to the fact that the mRNA of the loading control protein β-actin interacts with FMRP

(Darnell et al., 2011; Ascano et al., 2012) and therefore the possibility that the absence of FMRP in the Fmr1 KO mice could have altered β-actin expression along with the values of EGFR and pEGFR that were normalized to it, additional EGFR western blots were performed on the PND 7 cerebellum using GAPDH (not listed in Darnell et al., 2011 and Ascano et al., 2012) as the loading control protein. As shown in Figure 7B, the PND 7 cerebellar expression of EGFR normalized to GAPDH was also not different between the Fmr1 KO and WT mice. Therefore, using β-actin as a loading control produced comparable results as those from using GAPDH instead.

To summarize the EGFR results, although EGFR expression in the brain was mostly similar in the Fmr1 KO and WT mice during early development, there was a reduction in EGFR in the PND13 hippocampus and in the pEGFR expression in the PND7 prefrontal cortex in the

KO mice. PND 7 cerebellum A

B n = 8 n = 8

Figure 7. GAPDH-normalized EGFR expression in the PND 7 mouse cerebellum. A) Representative western blot of the EGFR and GAPDH expression in the PND 7 cerebellum. B) A summary of the GAPDH-normalized EGFR expression in the PND 7 cerebellum. EGFR expression in the Fmr1 KO group was expressed as a percentage of WT EGFR expression (adjusted to 100%). Two-tailed, unpaired t-test was used as the

statistical test. 35

4.3 EGF and HB-EGF levels in the WT and Fmr1 KO mouse

brains

In the mouse brain, EGFR signaling depends on both expression of the receptor and the

levels of its ligands. Two of the endogenous EGFR ligands, EGF and HB-EGF were

investigated via ELISA and western blotting respectively. A summary of the EGF ELISA results

in the PND 7 and 13 mouse brains is presented in Figure 8 and the data of each individual PND 7

and PND 13 mouse listed in Appendix 1 and 2 respectively. In the PND 7 and PND 13

cerebellum (Figure 8A), hippocampus (Figure 8B), and prefrontal cortex (Figure 8C), the levels

of EGF were not statistically different between the Fmr1 KO mice and the WT mice. However,

the average EGF levels in the Fmr1 KO cerebellum showed a decreasing trend in both PND 7

and PND 13 mice (Figure 8A). This trend was also seen in the PND 13 hippocampus (Figure

8B). However, the PND 7 hippocampus had the opposite trend, with EGF levels being higher in

the Fmr1 KO mice than in the WT mice (Figure 8B). In the PND 7 and PND 13 prefrontal

cortex, both Fmr1 KO and WT mice had similar levels of EGF (Figure 8C).

Representative western blots as well as a summary graph of HB-EGF expression in the

PND 7 and PND 13 cerebellum are shown in Figure 9. Overall there was no difference in HB-

EGF expression between the Fmr1 KO and WT cerebellum at both PND 7 and PND 13 (Figure

9B). Unfortunately, the expression of HB-EGF in the other brain regions (i.e. PND 7

hippocampus) was below the level of detection in both Fmr1 KO and WT mice (western blot not

shown).

A. Cerebellum WT fmr1 KO WT KO WT KO 2.5 n = 8 n = 8 n = 8 n = 8

2 P = 0.19 P = 0.28 1.5

0.5

EGF (pg/mg protein) 0 PND7 PND13

B. Hippocampus

WT fmr1 KO

WT KO WT KO 1.5 n = 8 n = 8 n = 8 n = 8

P = 0.27 1 P = 0.34

0.5

EGF (pg/mg protein) 0

PND7 PND13

C. Prefrontal Cortex WT fmr1 KO

1 WT KO WT KO n = 8 n = 8 n = 4 n = 4

0.5

EGF (pg/mg protein) 0 PND7 PND13

Figure 8. EGF levels in the early postnatal cerebellum, hippocampus, and prefrontal cortex. Summary of the EGF ELISA results in A) the cerebellum B) the hippocampus, and C) the prefrontal cortex of PND 7, and PND 13 mice. All EGF concentrations were normalized with the total protein concentrations in the same samples. P > 0.05 for all comparisons (two-tailed, unpaired t-test).

A Cerebellum

PND 7

PND 13

B WT fmr1 KO WT KO WT KO 150 n = 8 n = 8 n = 4 n = 4

100

HB‐EGF Expression (% of WT) 0 PND 7 PND 13

Figure 9. HB-EGF expression in the early postnatal mouse cerebellum. A) Representative western blots of HB-EGF and GAPDH expression in the PND 7 and PND 13 cerebellum. B) A summary of the HB-EGF expression in the PND 7 and PND 13 cerebellum. HB-EGF expression in the Fmr1 KO group was expressed as a percentage of WT HB-EGF expression (adjusted to 100%). Two-tailed, unpaired t- test was used as the statistical test.

4.4 Analysis of EGF and thyroxine in the plasma and thyroid of

adult Fmr1 KO and WT mice

In addition to the brain, I also measured EGF levels in the plasma and thyroid gland of

adult 2-month-old Fmr1 KO and WT mice. Figure 10A and 10B shows a summary of the EGF

ELISA results in the plasma and thyroid respectively with the data of each individual adult

mouse provided in Appendix 3. It should be noted here that the both the thyroid and the plasma

EGF levels were quite variable from mouse to mouse and therefore Grubb’s test was performed

on the data (alpha = 0.05) to remove outliers. Overall, Fmr1 KO mice showed significantly

lower levels of EGF in both the plasma and thyroid. The average EGF levels in the KO mice

were roughly 15% and 30% of the WT levels in the plasma and the thyroid respectively (Figure

10A and 10B).

Since a lower EGF content in the thyroid could be due to an altered thyroid function, I

subsequently measured the plasma thyroxine levels in the adult WT and Fmr1 KO mice with

ELSIA. As shown in Figure 10C, the plasma thyroxine level was not different between the WT

and the Fmr1 KO mice.

To summarize the analysis of EGFR ligands, EGF and HB-EGF levels in the early

developing Fmr1 KO mouse brain were not statistically different from the WT levels despite the

large differences between the mean WT and KO values seen in the cerebellum and the

hippocampus. In the adult mouse plasma and thyroid however, Fmr1 KO mice showed

significantly lower EGF levels than their WT counterpart and this was not accompanied by any

detectable differences in the plasma thyroxine level.

A. Plasma

P = 0.027

B. Thyroid

P = 0.0067

C. Plasma T4 N= 8 8 N= 8 6

2 Plasma T4 (µg/dL) 0 Adult WT Adult KO

Figure 10. EGF and thyroxine (T4) levels in the adult mouse plasma and thyroid. Summary of the

EGF ELISA results in A) the plasma, and B) thyroid of adult (2 month-old) mice. C) Summarizes the

thyroxine (T4) ELISA results in the plasma of adult mice. Grubbs’ Test was used to determine the statistical outlier (alpha = 0.05) that were excluded from this figure. The left panel shows scatter-plots of all the sample values that were included in the summary histograms in the right panel. All EGF concentrations in the thyroid were normalized with the total protein concentrations in the same samples. * indicates statistical significance compared to the WT group (P < 0.05, two-tailed, unpaired t-test).

4.5 Effect of early-postnatal intranasal HB-EGF on MBP, CNPase,

and NG2 expression

In an attempt to boost myelination during the early developmental period, Fmr1 KO mice

were treated with intranasal HB-EGF to increase EGFR signaling in their developing brains.

After treatment from PND3 to 6, the mice were sacrificed on PND11 for the analysis of myelin

proteins. Figure 11 presents a summary of the expression of MBP, CNPase, as well as NG2 in the brainstem, cerebellum, and prefrontal cortex after the intranasal treatment. In the Fmr1 KO

mouse brainstem, intranasal HB-EGF significantly reduced the expression of the 14kDa MBP by

roughly 30% (Figure 11B) while in the brainstem there were only trends toward a decrease in the

expression of 21.5kDa MBP and CNPase (Figure 11B). The expression of MBP and CNPase

were undetectable in the PND11 Fmr1 KO prefrontal cortex by western blotting (results not shown).

Since EGFR signaling is known to contribute to the generation of NG2+ OPCs, I also examined the expression of NG2 the brainstem, cerebellum, and prefrontal cortex of these mice.

However, HB-EGF treatment did not significantly alter NG2 expression in these brain regions at

PND11 (Figure 11D).

We also investigated the effects of intranasal HB-EGF treatment in the WT C57/BL6 mouse as described above in the Fmr1 mouse. The brainstem and cerebellum were analyzed in saline vs. HB-EGF treated WT mice. Surprisingly, in the brainstem HB-EGF treatment induced a decrease in myelin protein expression, while in the cerebellum the HB-EGF treated mice showed higher levels of MBP and no change in CNPase (Figure 12 A and B). However, in all cases the effects were not significant.

A. 41 Brainstem Cerebellum

Fmr1 KO‐Saline Fmr1 KO‐HB‐EGF B. N = 11 N = 10 140 120 P = 0.01 P = 0.089 P = 0.078 100 80 * 60 40 20 of Saline) 0 21.5 17/18.5 14 kDa CNPase 21.5 17/18.5 CNPase kDa kDa kDa kDa

Expression of Myelin Proteins (% Brainstem Cerebellum C. Brainstem Cerebellum

Prefrontal Cortex

Fmr1 KO‐saline Fm1 KO‐HB‐EGF

D. 140 N=8 N=7 N=7 N=7 N=8 N=7 120 100 80 60 40 20 0

NG2 Expression (% of Saline) Brain stem Cerebellum Prefrontal Cortex

Figure 11. Effect of intranasal HB-EGF treatment on the expression of myelin proteins and NG2 in the PND11 Fmr1 KO mouse brain. A) Representative western blots of MBP and CNPase expression and C) of NG2 expression in the Fmr1 KO mouse brain after HB-EGF treatment. B) Summary of the MBP and CNPase expression profiles in the PND11 Fmr1 KO brainstem and cerebellum after HB-EGF treatment. D) Summary of NG2 expression in the brainstem, cerebellum, and prefrontal cortex after HB-EGF treatment. MBP, CNPase, and NG2 expression was normalized with GAPDH expression and all normalized values were expressed as a percentage of the average saline value (adjusted to 100%). *indicates statistical significance compared to the res pective saline group (P < 0.05, two-tailed unpaired t-test). 42

A. Brainstem

WT‐Saline WT‐HB‐EGF B. N = 8 N = 7 600 500 400 300

Saline) 200 100 0 Protein Expression (% of 14 kDa 17/18.5 21.5 kDa CNPase kDa C. Cerebellum

WT‐Saline WT‐HB‐EGF D. N = 8 N = 7 140 120 100 80 60

Saline) 40 20 0 Protein Expression (% of 14 kDa 17/18.5 21.5 kDa CNPase kDa Figure 12. Effect of intranasal HB-EGF treatment on the expression of myelin proteins in the PND11 WT mouse brain. A) and C) Representative western blots of MBP and CNPase expression in the WT mouse brainstem and cerebellum, respectively, after HB-EGF treatment. B) and D) Summaries of the MBP and CNPase expression profiles in the PND1WT brainstem and cerebellum, respectively, after HB-EGF treatment. MBP and CNPase expression were normalized with GAPDH expression and all normalized values were expressed as a percentage of the average saline value (adjusted to 100%). P > 0.05 for all comparisons (two-tailed unpaired t- test).

5. Discussion

5.1 Delayed myelination in the early postnatal Fmr1 KO mouse

brain

As the flow of information within the brain is limited by the rate of action potentials,

processes such as myelination have been developed to optimize the transmission of action

potentials along axons. Myelination achieves this in two ways. First, by wrapping the axons

with myelin, the axons are insulated to reduce the leaking of current out of axons and thus to

increase the distance along the axon that a local current can flow passively. Second, myelin

changes the propagation of action potentials from a continuous wave of depolarization to

salutatory conduction. More specifically, the speed of action potentials is greatly increased

because with myelin, an action potential that is generated at a node of Ranvier elicits a current

that flows passively down the axon until it reaches the next node of Ranvier and elicits another

action potential there. Essentially, wrapping the axon in segments of myelin allows the action

potential to “jump” down the gaps in between the segments. Therefore, it is not hard to see that

the thickness of myelin, which determines the distance of passive current flow, would also

determine the maximum possible distance between nodes of Ranvier and ultimately the speed of

action potentials.

On this note, our lab has previously demonstrated that myelination is deficient in the

early postnatal Fmr1 KO mouse cerebellum (Pacey et al., 2013). Pacey et al., 2013 reported

reductions in MBP and CNPase expression in the cerebellum of the Fmr1 KO mice at PND7 and

at PND 15 that later normalized to WT levels by PND 30. That study has also demonstrated

reductions in both the thickness of myelin and the number of myelinated axons in the same

region at PND7 but not at PND15. Additionally, these deficits were accompanied by a deficit in

both NG2 expression and the number of NG2+/PDGFRα+ OPCs that were proposed to be the

underlying cause of the myelin deficiencies.

In order to expand our findings on these abnormalities in the Fmr1 KO mice, my project

therefore aimed to not only confirm our previous findings in the cerebellum, but also to extend

the analysis of myelination into several other brain regions in order to create a spatial-temporal

profile of the myelin deficiency in the Fmr1 KO mouse. This is an important first step to take

before attempting to restore the myelination in Fmr1 KO mouse to WT levels.

As expected, we found that the myelination is deficient in the cerebellum of the Fmr1 KO mice at PND 7 (Figure 3B), as also reported in Pacey et al., 2013. However, whereas Pacey et al., 2013 reported a significant reduction in MBP at PND15, my results showed lower MBP in

Fmr1 mice, albeit not significant, in the PND13 Fmr1 KO mouse cerebellum. Although this could be explained by a difference in the sample sizes used in the two studies, Pacey et al., 2013 also reported that electron microscopy of the PND7 and PND15 cerebellar axons showed reduced myelin thickness and number of myelinated axons in the Fmr1 KO mice only at PND7 and not PND15. This rather supports my finding that myelination in the Fmr1 KO cerebellum has largely (but perhaps not completely) caught up to WT levels at PND13.

In addition to the cerebellum, I also analyzed myelination in the brainstem, hippocampus, and prefrontal cortex. Similar to the cerebellum, myelin proteins in the Fmr1 KO mouse brainstem were deficient at PND7 but not at PND13 or in adults (Figure 3A). The connection between the brainstem and FXS is not obvious at first glance because FXS is most prominently

understood as mental retardation while the brainstem is well known for its roles in controlling

unconscious functions such as digestion and respiration rather than higher order cognitive

functions. However, roughly 25% of the children with FXS also develop seizures that are

eventually resolved later in childhood (Hagerman and Stafstrom, 2009). This is interesting

because the brainstem is known to be involved in the generation of some types of seizures (Chiba

et al., 2005; Kohsaka et al., 1999), and moreover, brainstem demyelination has been proposed to

induce seizures (Libenson et al., 1994). Furthermore, the exclusively early timing of the

myelination deficiency is consistent with the early occurrence of seizures in fragile X children.

In contrast to the cerebellum and brainstem, the hippocampus and prefrontal cortex are

located more rostrally and are myelinated later during the postnatal period. In the hippocampus, the myelin proteins were undetectable by western blotting on PND7 in both WT and Fmr1 mice and deficient in Fmr1 KO mice at PND13 but not in 2 month-old (young adult) mice (Figure 4A).

This suggests that the myelin deficiency in Fmr1 KO mice occurs only when myelination is in the early stage and when it is highly active. Since myelination begins first in the brainstem and the cerebellum, the myelin deficiency is first seen in these regions at PND7 prior to the hippocampus at PND13. Although this deficiency in the hippocampus is not seen in 2 month-old

Fmr1 KO mice, the “restoration” of myelination in this region probably occurred earlier (around

PND25) when the speed of myelination plateaus as it approaches adult levels (Meier et al., 2004).

The prefrontal cortex is the last brain region to be myelinated and in humans the process is not complete until early adulthood (Sowell et al., 1999). Therefore, it is not surprising that

MBP, a marker for mature myelin, remained undetectable in the prefrontal cortex at PND13 in both the WT and the Fmr1 KO mice (Figure 4B). In contrast to MBP, CNPase is one of the earliest myelin proteins to be synthesized by developing oligodendrocytes and its expression precedes that of MBP (Baumann and Pham-Dinh, 2001). CNPase expression was detected and found to be lower in the PND13 Fmr1 KO mouse prefrontal cortex, foreshadowing the MBP

deficiency that had yet to be observed. Interestingly, the two myelin proteins were still deficient

in the 2 month-old adult Fmr1 KO mouse prefrontal cortex (Figure 4B). More recent evidence

has found that in the human prefrontal cortex, myelination extends beyond late adolescence

(Miller et al., 2012) and synaptic pruning likely continues until the age of 30 (Petanjek et al.,

2011). Furthermore, another study found that 2 month-old mouse prefrontal cortex myelination

is plastic and can be altered by social isolation (Liu et al., 2012). In summary, the finding that

myelin is deficient in the Fmr1 KO prefrontal cortex in young adults is a potentially important

finding because it could have direct implications for the behavioral abnormalities observed in the

Fmr1 KO mice that are linked to prefrontal cortex. For example, people with FXS often suffer

from deficits in attention, inhibitory control, and cognitive flexibility, all of which have been

linked to the prefrontal cortex (Reiss and Hall, 2007; Chudasama and Robbins, 2006).

A reoccurring observation with the myelin deficit is that myelin protein levels are, with

the exception of the prefrontal cortex, eventually restored to WT levels with increasing age.

However, even a temporary myelin deficit could still have long-lasting impact on neuronal

development. Spike-timing-dependent-plasticity is one mechanism through which this can

happen. It is the process through which the strengths of neuronal connections are adjusted based

on the relative timing of a neuron’s input and output action potentials. Since the timing of

neuronal firing is intimately tied to myelination, a delay in myelination during the early

developmental period could drastically alter spike-timing-dependent-plasticity during this period

and leave repercussions that persist through adulthood. For example, this could alter the

development of sensory neurons and formation of cortical maps, which have been proposed to be

heavily dependent on spike-timing-dependent-plasticity (Larsen et al., 2010; Song and Abbott,

2001).

In support of this notion that early postnatal myelination is crucial to the development of

the brain, delayed myelination have been characterized in children with developmental delays, suggesting that myelination is intimately linked to brain maturation (Pujol et al., 2004).

Additionally, there is also evidence that even a temporary deficit in early myelination can alter the behavioral outcomes of affected rodents. For example, maternal immune activation with polyinosinic-polycytidilic acid and direct hippocampal lysophosphatidylcholine injections were both able to induce hypomyelination in the hippocampus of affected juvenile animals that later reverted to normal levels in adult animals (Makinodan1 et al., 2008; Makinodan2 et al., 2008).

However, even with normal myelination, the adults exhibited behavioral abnormalities such as

deficits in prepulse inhibition, methamphetamine-induced hyperactivity and anxiety-related

behaviors (Makinodan2 et al., 2008). This suggests that perturbations to early myelination can

produce lasting effects and affect behavior later in life.

What are the mechanisms underlying the myelin deficiency and then the recovery with

age? We know that both OPCs and mature oligodendrocytes express FMRP (Pacey et al., 2013),

and that FMRP expression in the oligodendroglial lineage of cells gradually decreases as the

mice matures (Gholizadeh et al., 2015). Since FMRP has been show to interact with mRNAs of

MBP and CNPase (Table 1), an obvious explanation to account for the MBP and CNPase

deficiency in Fmr1 KO mice would be that FMRP normally acts as a translational activator of

both proteins and therefore, without FMRP, their levels are lower in the Fmr1 KO mice.

Although this is unlikely since FMRP have already been shown to be a translational repressor of

MBP (Wang et al., 2004; Giampetruzzi et al., 2013), it does not rule out the possibility of FMRP

being involved in the transport of MBP and CNPase mRNAs to the oligodendrocyte processes

where they are needed.

Table 1 contains a list of genes whose proteins are either found in myelin or are linked to

myelination. Interestingly, out of the first 6 myelin proteins listed in Table 1 (MBP to

oligodendrocyte myelin glycoprotein [OMG]), MBP and CNPase are the only 2 that have been

listed as FMRP substrates by both Darnell et al., 2011 and Ascano et al., 2012. This further

justifies the rationale to focus on examining MBP and CNPase in the Fmr1 KO mice. In

addition to the myelin proteins, proteins that are involved in the development and maturation of

OPCs, such as oligodendrocyte transcription factors 1 and 2, SRY-box containing gene 10, fyn

proto-oncogene, and thyroid are also listed in Table 1. Oligodendrocyte transcription factors 1 and 2 are well known to be required for the differentiation of oligodendrocyte progenitors in to mature oligodendrocytes (Meijer et al., 2012). SRY-box containing gene 10 and fyn proto- oncogene control the terminal differentiation of OPCs and the initiation of myelination (Stolt et al., 2002; Yamauchi et al., 2012). On the other hand, thyroid hormone receptor alpha functions in early OPC development by triggering the arrest of OPC proliferation and acting in Purkinje neurons and astrocytes to create an environment that is favorable of OPC differentiation (Picou et al., 2012). Therefore, the fact that oligodendrocyte transcription factor 2, fyn proto-oncogene, and thyroid hormone receptor alpha were listed as FMRP substrates (Table 1) raises the possibility that the differentiation and maturation of OPCs in the Fmr1 KO mice could have been impaired by altered levels of these 3 proteins.

Theoretically, there are two more possibilities that could explain the myelin deficit, the first being a delayed onset of myelination, and the second being a slower rate of myelination.

Eventually, as myelination in the WT mice reaches completion, it slows down and eventually stops so that the delayed myelination in the Fmr1 KO mice catches up to that of the WT mice.

Although the current results cannot directly revolve between the two possibilities, the previous

study proposed that it was caused by a reduced number of OPCs (Pacey et al., 2013), making the

“slower rate of myelination due to less OPCs” possibly a more likely explanation.

5.2 EGFR, EGF, and HB-EGF levels in the Fmr1 KO mouse brain

Our interest in studying the EGFR and its ligands in the Fmr1 KO mouse stemmed from the role of EGFR signaling in the production and development of oligodendrocytes (Aguirre et al., 2007). I investigated the expression of EGFR/pEGFR, EGF, and HB-EGF in parallel with

MBP and CNPase in the cerebellum, hippocampus, and prefrontal cortex using western blotting for EGFR/pEGFR and HB-EGF and ELISA for EGF (due to its small molecular size). As shown in Figure 6, the overall expression of EGFR and pEGFR were not very different between the

Fmr1 KO and WT mice in the three brain regions at PND7 and 13. This was somewhat

surprising considering EGFR mRNA has been shown to be a substrate of FMRP (Table 1). Only

EGFR expression in the PND13 hippocampus and pEGFR expression in the PND7 prefrontal were found to be lower in the Fmr1 KO mice compare to the WT mice. However, the differences were moderate (~20%) and appeared to be sporadic. Therefore, further investigation is required before any conclusions can be drawn.

Admittedly, although not shown in the results, the anti-EGFR antibody also recognized

several other bands in the EGFR western blots whose identities remained unknown. This,

together with the lack of use of a negative EGFR control, limits the validity of the results since

the analysis of those EGFR western blots assumed that the presumed EGFR bands were visible

due to the presence of EGFR. In order to address this problem, future studies should include

tissues samples from EGFR KO mice as negative controls. It could be difficult, however, to

obtain such samples since EGFR KO mice have low viability that varies depending on the strain

of the mice (Sibilia and Wagner, 1995). Luckily, homozygous EGFR KO mice of the C57BL/6

background have been shown to be able to survive for up to 8 days after birth (Miettinen et al.,

1995), making it possible to collect their samples postnatally.

In addition to EGFR, EGF levels were also measured and no significant differences were

seen between the Fmr1 KO mice and WT mice in all three brain regions (Figure 8). It should be

mentioned that the high variability in the EGF measurements could be due to the fact that the

EGF level in the brain is normally very low (100 times lower than HB-EGF), and that the EGF

measurements were mostly bordering the limit of detection of the ELISA assay. Along with

EGF, analysis of HB-EGF expression in the PND7 and PND13 cerebellum did not reveal any

differences between the Fmr1 KO and the WT mice (Figure 9) despite being listed in Ascano et

al., 2012 as an FMRP substrate (Table 1). Thus, I did not pursue to analyze HB-EGF in the other

brain regions.

5.3 EGF deficiency in the plasma and thyroid of Fmr1 KO mouse

The plasma and the thyroid gland are known to be two of the sources of EGF in the body

(although not the only two). Analysis of EGF in the plasma and the thyroid gland of adult mice showed significantly lower EGF levels in the Fmr1 KO mouse plasma and thyroid. As lower levels of plasma/serum EGF have also been reported in children with autism spectrum disorder disorders (range of disorders that share a considerable overlap of symptoms with FXS) (Suzuki et al., 2007; Onore et al., 2012; Russo, 2013), decreased EGF levels might be implicated in the

pathophysiology of fragile X syndrome. This is further supported by the fact that EGF not only plays an important role in myelination, but also regulates neuronal growth, proliferation,

differentiation, and migration. Since EGF can readily cross the blood-brain barrier, measuring

the plasma EGF might be a good way to indirectly measure the brain EGF content as its level in

the brain might be too low to provide accurate readings with direct ELISA measurements.

In the plasma, platelets are the main source of EGF (Oka and Orth, 1983; Ben-Ezra et al.,

1990), albeit not the only source (Lev-Ran et al., 1990). Upon activation, the platelets release

EGF into the blood along with a number of other factors and proteins. Therefore, the number of

platelets contained in the mouse plasma samples could have been a source of variability in the

plasma EGF measurements. In terms of the plasma platelet number, the plasma samples that

were collected in this study were most likely platelet-poor-plasma as the 20 min centrifugation

time at 2000×g would have eliminated some or even most of the platelets (Sultan, 2010); the

generation of platelet-free-plasma requires a centrifugation force of at least 10,000×g.

Interestingly, FMRP is also expressed in the platelets where it is postulated to play a role in RNA

metabolism (Lauzière et al., 2012). However, whether this has any direct consequences on

platelet EGF expression in fragile X syndrome remains to be investigated.

In addition to the plasma, the thyroid EGF level was also found to be significantly lower

in the adult Fmr1 KO mice than the WT controls (Figure 10). Additionally, the thyroid EGF

levels were found to be much higher than that of the plasma and the brain. In the thyroid, EGF

and EGFR expression is found in the thyroid hormone-synthesizing follicular cells but not in the

calcitonin-synthesizing C cells (van der Laan et al., 1995). Interestingly, FMRP is also

expressed in the thyroid (Hinds et al., 1993) although its cell type-specific distribution is still

unclear. Nevertheless, the absence of FMRP in the thyroid of the Fmr1 KO mice could have

contributed to the lower levels of EGF in the gland.

As a mitogen, EGF is known to stimulate cell proliferation. It also inhibits thyroid-

specific functions (ie. iodide uptake) (Asmis et al., 1995) and therefore primarily plays a role in

cellular maintenance and growth in the thyroid gland. Since it has been shown that thyroxine

increases thyroid EGF levels (Ozawa et al., 1991), altered thyroid EGF levels could be indicative

of altered thyroxine levels and therefore altered thyroid function. To investigate this possibility,

I measured plasma thyroxine levels in the 2 month-old adult Fmr1 KO and WT mice. However,

I found no difference in the plasma thyroxine levels between the two groups (Figure 10C). In

fact, our finding in the Fmr1 KO mice is consistent with another study that examined thyroid

function in human males with FXS and found no difference in thyroxine levels between their

fragile X subjects and controls (Bregman and Leckman, 1990). To shed light on the possible

explanations of the decreased EGF levels in the adult Fmr1 KO thyroid, another study found that

the level of dietary iodine had a positive correlation with the level of EGF in the thyroid

(Dagogo-Jack, 1994). They reported that changing the dietary iodine content had a direct effect

on the thyroid EGF level while thyroid function remain the same. As EGF is known to inhibit

thyroid iodide uptake, low levels of the thyroid EGF would favor iodide uptake in conditions

where dietary iodine is low. Since the level of dietary iodine in my study would have been

similar between the Fmr1 KO and WT mice, this opens up the possibility of deficiencies in

iodine absorption or iodide uptake in the Fmr1 KO mice.

5.4 Effect of intranasal HB-EGF on early postnatal myelination

In an effort to restore the myelin deficiency during the early postnatal period, I

administered HB-EGF intranasally to Fmr1 KO mice from PND3 to PND6. Although the

treatment conditions used in this study were similar (ie. same treatment scheme and dosage, but

different starting age and endpoint) to another study that reported significant effects of HB-EGF

in rescuing myelin deficiency (Scafidi et al., 2014), my results did not show an increase in the

expression of MBP and CNPase in cerebellum and brainstem of Fmr1 KO mice after HB-EGF treatment (Figure 11B). In fact, the opposite was observed where the 14 kDa MBP isoform expression was decreased in the brainstem of the HB-EGF-treated Fmr1 KO mice. In order to investigate this further I also examined NG2 expression (marker for OPCs) in HB-EGF-treated

Fmr1 KO mice as multiple studies have shown that EGFR signaling induces an increase in the number of NG2+ OPCs (Aguirre et al., 2007; Ivkovic et al., 2008; Scafidi et al., 2014). However,

I found (using western blotting) that there was no overall increase of NG2 protein expression in the cerebellum, brainstem, and the prefrontal cortex of HB-EGF-treated Fmr1 KO mice (Figure

11D). In order rule out the possibility that the effect of HB-EGF treatment or the lack thereof is genotype-specific, I also treated WT mice with intranasal HB-EGF by using the same treatment scheme. However, the treated WT mice also showed a trend towards decreasing expression of

MBP and CNPase in the brainstem while in the cerebellum CNPase expression showed no change and MBP expression was elevated but not significant due to high variability (Figure 12).

It is tempting to conclude that the intranasal administration of HB-EGF simply failed to deliver sufficient amount of the growth factor into the mouse brains as I did not monitor whether

HB-EGF entered the brains. However, it should be noted that the rationale for using the intranasal route for HB-EGF delivery is that it allows the molecules to bypass the blood-brain barrier and enter rapidly from the nasal cavity into the brain predominantly through the olfactory nerve to the olfactory bulb, and through the trigeminal nerve to the brainstem (Dhuria et al.,

2010). A study investigating intranasally administered radiolabelled IGF, a molecule with comparable size to HB-EGF, reported rapid entry of IGF into the brain as soon as 30 minutes

after administration (Thorne et al., 2004).

A possible explanation for the lack of a positive effect of HB-EGF treatment is suggested

by a study that saw that constitutive EGFR signalling in oligodendrocytes actually inhibited the

final differentiation of OPCs (Ivkovic et al., 2008). In the Scafidi et al., 2014 study that

investigated a mouse model of hypoxia to mimic the neonatal brain injury in infants born very

preterm, HB-EGF treatment was initiated on PND11 as opposed to PND3 in my study. Perhaps

an overactive EGFR signaling induced by the exogenous HB-EGF in the Fmr1 KO mouse brain during a period where myelination is still in its early stages has greater negative consequences on the process compared to later time points in brain development. Furthermore, in Scafidi et al.,

2014, although HB-EGF treatment increased MBP expression in the white matter compared to the saline-treated controls, it only did so in the state of hypoxia-induced injury and not in the normoxia condition. Myelin repair and developmental myelination may therefore be two fundamentally different processes such as that HB-EGF might not induce developmental myelination the same way (or at all) as it induces myelin repair. Despite the lack of a positive effect of HB-EGF on MBP expression, the most puzzling observation was the lack of its effect on NG2 expression. A possible explanation could be that the Scafidic et al., 2014 study only examined the white matter and quantified the number of NG2+ cells rather than total NG2 protein expression. Therefore, it could be that the effect of HB-EGF on NG2 expression was

localized to only the white matter and the changes in the total protein expression of NG2 were

too small to be accurately detected by western blotting. Therefore, future studies should examine

NG2 with an immunohistochemical approach and focus only on the white matter.

6. Conclusions

The ultimate goal of my M.Sc. thesis research was to better understand the myelination

defect in the Fmr1 KO mice. This requires answers to the questions of where, when, and how

this defect occurs in mice lacking the FMRP. The “where” and the “when” were addressed in the first project where I examined the expression of two important myelin proteins in multiple brain regions and at multiple time points. We concluded that the myelination deficit occurs globally throughout the entire Fmr1 KO brain and only transiently during the window of early myelination. As a result, its pattern of appearance parallels the caudal to rostral direction of

myelin progression. Although this defect is mostly rectified in the adult brain (except for the

prefrontal cortex), its occurrence during the period of early brain development suggests that it

could still produce lasting neuronal impairments.

Project two of my thesis was an attempt to answer the “how” question. Since EGFR

signaling is closely tied to a number of important biological processes in the brain, such as

myelination, we wondered whether the absence of FMRP in the Fmr1 KO mice could have

perturbed the EGFR system which could be a mechanism underlying the myelination defect.

However, my results showed little to no impairments in the expression of EGFR and its ligands

EGF and HB-EGF in the Fmr1 KO brains and suggested that this was not the case. Interestingly however, I did detect a decrease in the level of EGF in the adult Fmr1 KO mouse plasma and thyroid which suggests the possibility of thyroid dysfunction. However, thyroxine levels in the plasma were not altered in the Fmr1 KO mice, indicating that the deleterious effects of thyroid

EGF deficiency, if any, lie elsewhere.

In my third project I treated the Fmr1 KO mice with intranasal HB-EGF in an attempt to restore the myelin deficiency. However, intranasal HB-EGF treatment did not show a positive

effect on the expression of the myelin proteins in the early developing Fmr1 KO mice. Although

NG2 expression was also not increased by HB-EGF treatment as I expected, the changes could

have been too small to be adequately quantified by western blotting. Therefore further

investigation using more sensitive techniques, such as immunohistochemistry, are required.

7. Future Directions

As the mechanism(s) behind the myelination defect remains to be elucidated, it would be

interesting to see whether the defect was solely due to the loss FMRP in the oligodendroglial

lineage of cells, or perhaps was also due to the loss of FMRP in the neurons and astrocytes that

also interact with oligodendrocytes. To investigate this, one could inject viral vectors with a

construct that includes FMRP and an oligodendrocyte-, neuron-, or astrocyte-specific promotor

into the Fmr1 KO mice to selectively restore FMRP in these three populations of the cells

separately and see whether the myelination defect could be rescued. However, this approach

might not be the best considering that the myelination defect occurs early in development and

therefore there may not be enough time for the defect to be rescued. An alternative approach

would be to selectively knock out Fmr1 in the oligodendrocytes, the neurons, or the astrocytes

and examine the consequences on myelination.

Furthermore, in order to validate the myelination defect as a truly important pathological

feature of FXS, a link between the myelination defect and the Fmr1 KO behavioral deficits must

be established. This requires increasing and decreasing the early developmental myelination in

the Fmr1 KO mice by compounds and small molecules and then evaluating the effect of the

changes in myelination on a range of fragile X behavioral tests. Examples of such tests would

include audiogenic seizure induction, marble burying test, motor activity, elevated plus maze,

and prepulse inhibition. Whereas the myelination-promoting effect of HB-EGF may be injury-

dependent, glatiramer acetate can promote mylinogenesis and oligodendrogenesis in vivo in the

developing nervous system under non-pathological conditions (From et al., 2014). Glatiramer

acetate was developed as a drug to treat multiple sclerosis. The drug is composed of random

polymers of four amino acids (glutamic acid, lysine, alanine, and tyrosine) that are found in MBP

and is thought to treat multiple sclerosis by suppressing the immune response (Ziemssen and

Schrempf, 2007) and by exerting neurotrophic effects via increasing the expression of growth

factors such as insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor

(BDNF) (From et al., 2014). Therefore, it would be interesting to determine whether glatiramer

acetate could restore the myelin deficiency in the Fmr1 KO mice and whether this

pharmacological treatment could correct the abnormal behaviors in these KO mice.

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Appendices

Appendix 1. ELISA EGF measurements in the PND7 mouse brain. Genotype Mouse ID Sex Cerebellum Hippocampus Prefrontal Cortex (pg/mg protein) (pg/mg protein) (pg/mg protein) WT W19N‐1 M 5.650149 0.642549 0.594116 W19N‐2 F 1.181282 0.7218 0.773159 W19N‐3 F 1.527227 1.010362 0.768078 W19N‐4 M 1.676746 0.556592 1.889785 W28N‐1 M 1.95627 0.167012 0.149326 W28N‐2 M 0.373552 0.070135 0 W28N‐3 M 0.232967 0.120741 0 W28N‐4 M 0.203437 0.058481 0 Frm1 KO K19N‐1 M 0.682064 0.689961 1.177059 K19N‐2 F 1.182381 0.907506 0.526598 K19N‐3 F 1.603122 0.898578 0.753456 K19N‐4 F 1.033596 1.062183 0.829068 K25N‐1 M 0.202573 0.039231 0.035295 K25N‐2 F 0.155278 3.017348 0.24574 K25N‐3 M 0.210997 0.065588 0.060627 K25N‐4 F 0.223261 0.110691 0.014145

Appendix 2. ELISA EGF measurements in the PND13 mouse brain. Genotype Mouse Sex Cerebellum Hippocampus Prefrontal Cortex ID (pg/µgprotein) (pg/µgprotein) (pg/µgprotein) WT W14N‐1 F 0.8318 0.0873 W14N‐2 M 0 0.3878 W14N‐4 F 0.1898 0.1825 W14N‐5 F 0.1376 2.6112 W20N‐1 M 0.288371 0.385269 0.302801 W20N‐2 F 0.1825 0.374563 0.614619 W20N‐3 M 0.183069 0.456013 0.607636 W20N‐4 F 0.577197 0.615314 0.471934 Fmr1 KO K13N‐1 M 0 0.2671 K13N‐2 M 0 0.2083 K13N‐3 M 0.1103 0.0433 K13N‐4 M 0.0339 0.0725 K16N‐1 M 0.205548 0.373012 0.324603 K16N‐2 M 0.317102 0.450326 0.396558 K16N‐3 F 0.336842 0.734267 0.647396 K16N‐4 M 0.400797 0.614255 0.386742

Appendix 3. EGF ELISA measurements in the adult mouse plasma and thyroid. Genotype Mouse ID Sex Plasma (pg/mL) Thyroid (pg/mg protein) WT 37359 F 0 62.72 37358 F 0 19.32 37360 M 98.55* (outlier) 3739.31* (outlier) 37361 M 4.25 196.18 39620 M 11.15 389.46 39619 M 6.63 112.86 39642 M 31.03 558.04 39643 M 11.89 559.02 W31N‐1 F 37.54 122.58 W31N‐2 F 7.98 125.27 W31N‐3 F 2.76 107.76 W31N‐4 F 4.20 231.24 W33N‐1 F 1.15 90.56 W33N‐2 F 17.46 37.93 W33N‐3 F 3.41 117.14 W33N‐4 M 71.14 391.52 Fmr1 KO 37380 F 0 14.96 37381 F 0 16.25 37382 F 0 14.17 37384 M 4.63 53.85 39633 F 0.74 109.47 39634 F 0.61 168.03 39632 F 3.62 56.71 39654 M 5.45 386.62* (outlier) K31N‐1 F 0.82 17.04 K31N‐2 F 1.08 94.53 K31N‐3 F 1.34 126.52 K31N‐4 F 3.13 13.87 K32N‐1 F 0.65 98.22 K32N‐2 F 0.74 48.31

K32N‐3 F 0.96 18.41 K32N‐4 F 3.83 21.77

K33N‐1 M 1.13 68.99 K33N‐2 M 4.72 46.62

K33N‐3 M 4.95 57.19 *Grubbs’ Test (alpha = 0.05) was used to determine statistical outliers.

Appendix 4. Schematic of the dissection of mouse prefrontal cortex.

A. B.

Appendix 5. Representative EGF ELISA Standard curve. A) A standard curve of absorbance value versus EGF concentration. B) In order to linearize the standard curve, the log of absorbance was plotted against the log of EGF concentration. The equation of the line of best fit of the linearized standard curve was then used to calculate EGF concentrations from raw absorbance measurements