P75 Neurotrophin Receptor Function in Brain Development

p75 neurotrophin receptor function in brain development Sonja Meier BSc, MSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 Queensland Brain Institute Abstract Embryonic brain development is a complex process in which expression patterns of receptors and transcription factors control the generation of many different cell types from a common precursor, as well as their subsequent temporal and spatial distribution within different regions of the brain. Although these programs are tightly regulated to ensure formation of functional neuronal networks, the external cues that govern these processes are still largely unknown. The p75 neurotrophin receptor (p75NTR) has been identified as a key regulator in the development of a range of cell types, including neural progenitors of the peripheral nervous system. As a cell surface receptor, p75NTR can initiate direct environment-to-cell communication and coordinate important aspects of neurogenesis including survival, proliferation, specification, migration, and/or differentiation. However, the function of p75NTR in development of the central nervous system had not been studied comprehensively. The aim of the thesis is to elucidate the role of p75NTR in brain development and, more specifically, to investigate how neocortical progenitor fate is regulated by p75NTR using conditional p75NTR knockout mice. We found that p75NTR is most highly expressed during cortical development in post-mitotic neuronal cells, but that loss of p75NTR expression during embryogenesis in progenitor cells has widespread ramifications on the development of the neocortex and basal ganglia due to effects on progenitor populations. Specifically, p75NTR expression is required for the survival of neuron-specified intermediate progenitor cells (IPCs) and for the generation of appropriate numbers of pyramidal cortical neurons and parvalbumin (PV)-positive interneurons. Without p75NTR expression, a significant number of IPCs die prior to, or in the process of, undergoing neurogenic divisions, resulting in a depletion of the progenitor pool and subsequent reduction in neuronal production. Furthermore, loss of p75NTR expression in progenitors of the medial ganglionic eminences (MGE) reduces their ability to generate interneurons in culture and to differentiate into a PV-expressing subtype. In vivo, loss of p75NTR in the MGE selectively reduces the production of PV-expressing interneurons, presumably caused by reduced activity of the nuclear factor κB (NF-κB) pathway. These results demonstrate that p75NTR expression is required for normal cortical development by facilitating survival of cortical IPCs. i Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. ii Publications included in this thesis Meier, S, Alfonsi F, Kurniawan ND, Milne MR, Kasherman MA, Delogu A, Piper M, Coulson EJ, 2019. The p75 neurotrophin receptor is required for the survival of neuronal progenitors and normal formation of the basal forebrain, striatum, thalamus and neocortex. Development, doi: 10.1242/dev.181933 The weblink for the publication has been included in the thesis appendix. Data from this publication have been included in Chapters 3-5. Contributor Statement of contribution Sonja Meier (Candidate) Designed and performed the experiments (70%) Wrote the paper (70%) Fabienne Alfonsi Performed preliminary characterization and experiments (30%) Nyoman Kurniawan Carried out experiments, assisted with data analysis (10%) Michael Milne Carried out experiments (5%) Maria Kasherman Carried out experiments (5%) Alessio Delogu Carried out experiments (5%) Michael Piper (Co-supervisor) Wrote the paper (5%) Elizabeth Coulson (Primary supervisor) Designed experiments (10%) Wrote the paper (25%) Submitted manuscripts included in this thesis No manuscripts submitted for publication. iii Other publications during candidature Roig-Puiggros, S., et al., 2019. Construction and reconstruction of brain circuits: normal and pathological axon guidance. Journal of Neurochemistry, doi: 10.1111/jnc.14900 Boskovic, Z., et al., 2019. Regulation of cholinergic basal forebrain development, connectivity and function by neurotrophin receptors. Neuronal Signaling, 105(8- 9):871-903 Contributions by others to the thesis My supervisor Prof. Elizabeth Coulson, and co-supervisor A/Prof. Michael Piper contributed intellectually to this thesis through discussions, scientific guidance, and critical review of the project. Prof. Elizabeth Coulson also contributed intellectually to the conclusions discussed in this thesis, and critically reviewed the thesis draft. Parts of this thesis contain data previously published in: Meier, S, Alfonsi F, Kurniawan ND, Milne MR, Kasherman MA, Delogu A, Piper M, Coulson EJ, 2019. The p75 neurotrophin receptor is required for the survival of neuronal progenitors and normal formation of the basal forebrain, striatum, thalamus and neocortex. Development, doi: 10.1242/dev.181933 Overview of contribution by others (specific details are included in a paragraph on the page immediately preceding the respective chapters): Chapter 4: Fig. 4.4 A-C: Nissl staining and analysis performed by Fabienne Alfonsi (10%) Fig. 4.4 D-I: MRI scans performed by Nyoman Kurniawan (50%) Fig. 4.5A: Golgi Cox staining performed by Michael Milne (20%) Fig. 4.7: Experiment and analysis performed by Fabienne Alfonsi (100%) iv Chapter 5: Fig. 5.6 D-F: Staining and analysis performed by Lidia Madrid, under supervision of the thesis author Overall contribution of the thesis author to this thesis is estimated to be >80%. Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis. Research Involving Human or Animal Subjects All procedures were approved by the University of Queensland Anatomical Biosciences Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. This work in this thesis was part of the ethics approval numbers QBI/566/18, QBI/135/18/BREED, QBI/534/15, and QBI/084/15/NHMRC/ARC/BREED. The corresponding certificates have been included in the appendix of this thesis. v Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisor Prof. Elizabeth Coulson, who has been a constant source of support and inspiration throughout my PhD. Her wealth of knowledge, as well as her unwavering optimism and positive attitude, were critical to bringing this project to completion. Thank you, Lizzie, for teaching me to never, ever give up. My sincere thanks also go to my co-supervisor A/Prof. Michael Piper, who shared his knowledge of embryonic neurogenesis with me, and whose advice helped me turn a block of marble into a statue. Besides my supervisors, I would like to thank the members of my advisory team: Prof. Perry Bartlett, A/Prof. Tim Bredy, and Prof. Helen Cooper, for their input and encouragement throughout my PhD. I would like to thank the members of the Coulson and Piper Labs, past and present, for helpful discussions, ideas, and insights, and for providing a very positive and inclusive working environment. Special thanks go to Bree Rumballe, Dr. Lei Qian, Dr. Zoran Boskovic, and Michael Milne for sharing their scientific experience and technical know-how. I am extremely grateful to the QBI animal facility for establishing and maintaining mouse colonies, to the QBI microscopy team for their advice on imaging techniques and data analysis, and to Dr. Robert Sullivan for helpful discussions on sample preparation and sectioning methods. Special thanks go to Theodora Constantin for proofreading this document and for relentlessly chasing down typos and punctuation errors. Last but not least, I would like to thank my friends and family for always being there for me, even in the most difficult

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