Deficiency of Myelin Proteins during Brain Development in Fragile X Mice by 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 © Copyright by David Lai Jiang 2015 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. ii 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. iii 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 iv 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 v 7. Future Directions......................................................................................................................56 References......................................................................................................................................58 Appendices.....................................................................................................................................76 vi 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 vii 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 viii 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