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Identifying Novel Unc5b Interacting Partners to Understand Its Function in Cns White Matter

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Identifying Novel Unc5b Interacting Partners to Understand Its Function in Cns White Matter IDENTIFYING NOVEL UNC5B INTERACTING PARTNERS TO UNDERSTAND ITS FUNCTION IN CNS WHITE MATTER SAMUEL CLÉMOT-DUPONT February 2020 Integrated Program in Neuroscience Department of Neurology and Neurosurgery Montreal Neurological Institute Montreal, Quebec A Thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Master of Science © Samuel Clémot-DuPont 2020 Table of Contents ACKNOWLEDGEMENTS ........................................................................................................................... 3 ABSTRACT ................................................................................................................................................ 5 RÉSUMÉ ................................................................................................................................................... 6 AUTHOR CONTRIBUTIONS ...................................................................................................................... 7 INTRODUCTION ....................................................................................................................................... 8 GENERAL LITERATURE REVIEW ............................................................................................................. 10 The UNC5 family of proteins ............................................................................................................. 12 1.1. First Descriptions of the UNC5 family ............................................................................... 12 1.2. Structure of UNC5 proteins ............................................................................................... 13 1.3. Influence of UNC5s on cerebellar development ............................................................... 14 1.4. Implication of UNC5 proteins in axon guidance ............................................................... 15 1.5. Importance of UNC5B for vascularisation ......................................................................... 20 1.6. UNC5 expression and function in rodent CNS white matter ............................................ 23 MATERIALS AND METHODS .................................................................................................................. 27 RESULTS ................................................................................................................................................ 34 DISCUSSION ........................................................................................................................................... 56 Shorter internodes in 9-month-old UNC5B conditional knock out mice. ......................................... 57 UNC5B-BirA-Flag Flp-In T-REx HEK 293 cell line validation ............................................................... 60 Candidate UNC5B interacting proteins identified using the BioID assay ......................................... 62 DLG-1 and Scribble: ....................................................................................................................... 62 Band 4.1-like proteins: .................................................................................................................. 63 Tubulins and CNP: ......................................................................................................................... 63 Septins: .......................................................................................................................................... 64 Adducins: ....................................................................................................................................... 64 Junction plakoglobin: .................................................................................................................... 66 RAI14: ............................................................................................................................................ 67 Effect of netrin-1 in the UNC5B Bio ID assay .................................................................................... 68 UNC5B co-immunoprecipitations with tight junction proteins and DLG1 ....................................... 69 GENERAL DISCUSSION ........................................................................................................................... 71 SUMMARY AND CONCLUSION .............................................................................................................. 74 REFERENCES .......................................................................................................................................... 75 2 ACKNOWLEDGEMENTS I would like to start off by immensely thanking everyone mentioned in this section. Without your support, my master’s degree would have been much more difficult to accomplish. First and foremost, I would like to thank my supervisor and mentor, Dr. Tim Kennedy for guiding me through the hurdle that was my master’s degree. Especially the times I was a lost, stressed and frustrated individual. I am very lucky to have a supervisor with so much patience and tolerance but am extremely grateful that you enabled me to explore the world of neuroscience; a field that has always fascinated me but knew very little. I would also like to sincerely thank Ms. Kathleen Daigneault for basically holding my hand through essential steps needed to progress through my project. Without your help, I most likely would not have been able to reach the objectives I wanted to attain for this master’s project. I would additionally like to thank Lorne Taylor from the proteomics platform at the RI-MUHC for his guidance and step-by-step instructions for my data analysis. I would like to thank my committee members Dr. Jack Antel and Dr. Alyson Fournier, for grounding my research project and reminding me how I should prioritize my work in order to complete my degree in a sensible amount of time. I would also like to thank the Multiple Sclerosis Society of Canada for funding me and enabling me to conduct my research. I am extremely grateful to my colleagues and friends in the Kennedy lab. Omar for being my first mentor (and a great one at that) when I was first a lost puppy in the lab. You really helped relieve the initial stress of joining what I found was a big lab. Diane for being a great and patient mentor and friend, as well as Celina and Edwin for their constant support and great friendship, most of my lab techniques were also learned from you guys. Edwin for being able to tolerate me and being a fun and great roommate! I know you’ll do very well in 3 med-school! Jean-David (Jay Dee) for also being a great drinking buddy, friend and mentor. Even though we met later in my degree, we were able to form a good friendship very quickly. A big thank you to my friends back in Ottawa! I always looked forward to catching up with you guys on the rare occasions we got to. Most importantly, I would like to endlessly thank my dearest and best friend Fiona. You were instrumental to my master’s degree. You not only were a great roommate, but my master’s would have been much more difficult if you weren’t there constantly supporting me. I constantly depended on you and the memories we’ve formed the past years are unforgettable. I look forward to our future adventures together. I would like to thank my sister; your lively and unique personality always lightened my day when I would feel particularly gloomy. Viv, never change! Finally, to my parents. Your guidance, life lessons and endless encouragement made me the person that I am today. I hope I made you half as proud as I admire you and am proud of having such amazing parents. This one is for you! 4 ABSTRACT Netrin-1 and the netrin receptor deleted in colorectal cancer (DCC) have been demonstrated to functionally contribute to multiple stages in oligodendrocyte development and maturation in the central nervous system. Both are required for appropriate oligodendrocyte precursor cell (OPC) migration, as well as oligodendrocyte process extension, branching, and membrane elaboration. Another netrin receptor, UNC5B, is also highly expressed by OPCs and oligodendrocytes, but in contrast to netrin-1 and DCC, its functional contribution to oligodendrocyte maturation and myelination has not been studied. We have recently determined that UNC5B is enriched at the paranodal junctions that terminate the compact myelin internode. UNC5B null mice are embryonic lethal at ~E14. Thus, in order to study the contribution of UNC5B to myelination, we developed conditional knock-out (cKO) mice that selectively delete UNC5B from the oligodendrocyte lineage. Olig2 cre – UNC5Bflox/flox mice exhibit disruption of paranodal organization and an age dependent disruption of axonal subdomains. In this thesis, we further investigated the impact of UNC5B deletion on the length of myelin internodes, reporting shorter internodes in 9-month old UNC5B cKO mice compared to their wild type littermates. These findings reveal that UNC5B expression by mature myelinating oligodendrocytes is required to maintain and organize paranodal junctions. However, little is known regarding the proteins that interact with UNC5B and the signaling pathways associated with this protein. We therefore conducted a screen for UNC5B candidate interacting proteins that may function downstream of UNC5B using the biotin identification (BioID) proximity labeling assay. Our findings suggest previously unknown interaction of UNC5B with proteins associated with the cytoskeleton and with
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