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:

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 of times. Without your love and continued support I would not be where I am today.

vi Financial support

Sonja Meier is supported by an International Postgraduate Research Scholarship, a UQ Centennial Scholarship, and a QBI Top-up scholarship. Prof. Elizabeth Coulson receives support from the Clem Jones Centre for Ageing Dementia Research and the Australian Research Council (ARC LP110100403). We acknowledge the support of the Queensland NMR Network and the National Imaging Facility (a National Collaborative Research Infrastructure Strategy capability) for the operation of a 16.4T MRI at the Centre for Advanced Imaging, the University of Queensland. Optical imaging using wide-field fluorescence microscopes and slide scanners, and image analysis using Neurolucida were performed at the Queensland Brain Institute's Advanced Microscopy Facility, generously funded through ARC LIEF grant LE130100074.

Keywords p75 neurotrophin receptor, cortical development, parvalbumin interneurons, neuronal progenitor survival

vii Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060103 Cell Development, Proliferation and Death, 20% ANZSRC code: 110903 Central Nervous System, 50% ANZSRC code: 110902 Cellular Nervous System, 30%

Fields of Research (FoR) Classification

ANZSRC code: 1109 Neurosciences, 90% ANZSRC code: 0601 Biochemistry and Cell Biology, 10%

viii Dedication

This thesis is dedicated to my mother

Mirella Meier

and to the loving memory of my father

Paul Meier (1953 – 2017)

Contents

List of figures ...... 1 List of abbreviations ...... 3 Chapter 1: Introduction ...... 9 1.1 Embryonic neurogenesis ...... 9 1.1.1 Early patterning of the developing brain and origin of telencephalic neurons ...... 9 1.1.2 Cellular and molecular mechanisms underlying proliferation and differentiation ...... 10 1.2 Neurotrophin signaling in central nervous system development ...... 13 1.2.1 Neurotrophin receptors ...... 13 1.2.2 p75NTR and Trk receptors in survival and migration of neuronal cells ...... 14 1.2.3 p75NTR in differentiation, cell cycle regulation, and lineage progression ...... 15 1.3 Research aims ...... 18 Chapter 2: Materials and methods ...... 21 2.1 Mice...... 21 2.2 Preparation and administration of tamoxifen ...... 23 2.3 Preparation and administration of BrdU ...... 23 2.4 Tissue preparation ...... 23 2.5 Nissl staining ...... 24 2.6 Immunohistochemistry ...... 24 2.7 Golgi Cox staining ...... 26 2.8 Behavioral testing ...... 27 2.9 Magnetic resonance imaging ...... 27 2.10 Culturing of MGE progenitors and interneuron differentiation ...... 28 2.11 Immunocytochemistry ...... 29 2.12 Western blot analysis ...... 30 2.13 Microscopy ...... 33 2.14 Statistical analysis ...... 33 Chapter 3: Spatiotemporal mapping of p75 neurotrophin receptor expression in the developing mouse telencephalon ...... 35 3.1 Introduction...... 35 3.1.1 Chapter summary ...... 35 3.1.2 p75NTR expression in neurons ...... 35 3.1.3 p75NTR expression in progenitors ...... 36 3.1.4 p75NTR expression in other cell types ...... 37 3.2 Results ...... 39 3.2.1 The p75 neurotrophin receptor is expressed transiently in newly born neurons during brain development ...... 39 3.2.2 p75NTR is expressed in a subpopulation of intermediate progenitors ...... 41 3.2.3 p75NTR is expressed in a subpopulation of neurogenic progenitors ...... 43

3.2.4 p75NTR is expressed in tangentially migrating neurons ...... 45 3.3 Discussion ...... 47 Chapter 4: Cell-autonomous and non cell-autonomous effects of p75NTR signaling regulate normal formation of the neocortex and basal forebrain ...... 51 4.1 Introduction...... 51 4.1.1 Chapter overview ...... 51 4.1.2 Knockout strategy and conditional p75NTR knockout mouse strains ...... 51 4.1.3 Chapter aims and hypothesis ...... 53 4.2 Results ...... 54 4.2.1 p75NTR deletion in conditional knockout mouse strains is efficient and specific ...... 54 4.2.2 Nestin-Cre p75in/in mice have decreased brain volume with a disproportionate reduction of the neocortex and basal ganglia ...... 57 4.2.3 p75NTR is required for the development of cortical interneurons and upper-layer pyramidal neurons ...... 63 4.2.4 p75NTR is required for the development of neurons of the ventral telencephalic lineage . 67 4.2.5 Heterozygous knockout of p75NTR in MGE derived progenitors reduces cortical layer thickness and the number of PV interneurons ...... 69 4.2.6 Heterozygous knockout of p75NTR in Nkx2.1-positive progenitors leads to a reduced number of cholinergic basal forebrain neurons but not cortical SST interneurons ...... 73 4.3 Discussion ...... 76 4.4 Conclusion...... 80 Chapter 5: p75NTR function in progenitor survival and neuronal differentiation ...... 83 5.1 Introduction...... 83 5.1.1 Chapter overview ...... 83 5.1.2 Cell survival and cell death ...... 83 5.1.3 Cortical neuron lineage markers in dorsal and ventral progenitor zones ...... 84 5.1.4 Chapter aims and hypothesis ...... 86 5.2 Results ...... 87 5.2.1 p75NTR is required for the survival of cortical neuron progenitors and production of later born neurons ...... 87 5.2.2 Effect of embryonic p75NTR expression loss on survival and production of different subpopulations of cortical neurons ...... 93 5.3 Discussion ...... 96 5.4 Conclusion...... 100 Chapter 6: p75NTR induces NF-κB phosphorylation and regulates the survival and differentiation of intermediate progenitor cells ...... 103 6.1 Introduction...... 103 6.1.1 Chapter overview ...... 103 6.1.2 p75NTR in cell survival ...... 103 6.1.3 p75NTR in cell cycle regulation ...... 104 6.1.4 Chapter aims and hypothesis ...... 105 6.2 Results ...... 106 6.2.1 Intrinsic p75NTR expression is required for survival of interneuron progenitors ...... 106

6.2.2 p75NTR is required for the survival of neurogenic intermediate progenitor cells in the medial ganglionic eminence ...... 109 6.2.3 Heterozygous knockout of p75NTR decreases phosphorylation of p65 S536 and expression of the cell cycle regulatory protein p27 in MGE progenitors ...... 116 6.3 Discussion ...... 118 6.4 Conclusion...... 121 Chapter 7: Thesis summary and conclusions ...... 125 7.1 Significance ...... 125 7.2 Summary of findings and conclusions ...... 126 7.3 Additional thoughts ...... 129 Bibliography ...... 130 Appendix ...... 142

List of figures

Chapter 1

Figure 1.1 Progenitor hierarchy and relationships during corticogenesis……… 11

Chapter 3

Figure 3.1 Relative p75NTR protein abundance in different cell types of the mouse CNS measured with label-free fractionation mass spectrometry……………………………………………………………… 38 Figure 3.2 Spatiotemporal mapping of p75NTR expression in the developing telencephalon…………………………………………………………….. 40 Figure 3.3 p75NTR expression in progenitors………………………………………. 42 Figure 3.4 A subset of neurogenic progenitors at the border of the subventricular zone express p75NTR…………………………………… 44 Figure 3.5 p75NTR is expressed in dorsolaterally projecting fiber tracks and in tangentially migrating neurons in the cortical intermediate and marginal zone…………………………………………………………….. 46

Chapter 4

Figure 4.1 Qualitative assessment of p75NTR expression loss in three conditional knockout strains…………………………………………….. 55 Figure 4.2 Nestin-Cre (NesCre) p75in/in mice show reduced growth and are born in Mendelian ratios………………………………………………………. 59 Figure 4.3 Basic behavioural analysis of adult Nestin-Cre (NesCre) p75in/in mice 60 Figure 4.4 Reduced brain volume in Nestin-Cre (NesCre) p75in/in mice………… 62 Figure 4.5 Golgi silver stain showing changed neuronal morphology in Nestin- Cre (NesCre) p75in/in mice………………………………………………. 65 Figure 4.6 Immunohistochemical analysis of cortical layering in Nestin-Cre (NesCre) p75in/in mice……………………………………………………. 66 Figure 4.7 Nestin-Cre (NesCre) p75in/in mice have reduced numbers of interneurons and cholinergic neurons…………………………………. 68 Figure 4.8 Image of control (p75wt/fl) and knockout (Nkx2.1-iCre p75wt/in) mice.... 69 Figure 4.9 Deep layer thickness and number of PV interneurons is decreased in Nkx2.1-iCre p75wt/in mice……………………………………………... 71 Figure 4.10 Nkx2.1-iCre p75wt/in mice have reduced numbers of cholinergic neurons and PV interneurons…………………………………………... 74

1 Chapter 5

Figure 5.1 Sequential marker expression in the dorsal neuronal lineage……….. 85 Figure 5.2 Increased cleaved caspase 3 activation in the telencephalon of Nestin-Cre (NesCre) p75in/in mice………………………………………. 89 Figure 5.3 Reduced number of proliferative cells and newborn neurons in Nestin-Cre (NesCre) p75in/in cortices…………………………………... 90 Figure 5.4 Reduced size of the developing telencephalon in Nestin-Cre (NesCre) p75in/in mice……………………………………………………. 92 Figure 5.5 Apoptosis in embryonic Emx1-iCre p75in/in and Nkx2.1-iCre p75wt/in mice correlating with reduced MGE size in Nkx2.1-iCre p75wt/in mice. 94 Figure 5.6 Loss of p75NTR expression in ventral and dorsal progenitors does not affect the number of neocortical intermediate progenitor cells and newly born neurons……………………………………………………… 95

Chapter 6

Figure 6.1 MGE-derived GABAergic neurons can be cultured in vitro...... 107 Figure 6.2 Heterozygous deletion of p75NTR reduces the ability of interneurons to survive in vitro…………………………………………………………. 108 Figure 6.3 Apoptotic cells in Nkx2.1-iCre p75wt/in mice display the morphological features of proliferating progenitors……………………………………. 110 Figure 6.4 Enlarged multinucleic mCherry-positive cells in Nkx2.1-iCre p75wt/in mice undergo cell death…………………………………………………. 111 Figure 6.5 The majority of apoptotic cells in Nkx2.1-iCre p75wt/in mice are neurogenic progenitors………………………………………………….. 113 Figure 6.6 Preliminary data indicating increased apoptosis upon p75NTR expression loss in ventral IPCs…………………………………………. 115 Figure 6.7 Heterozygous knockout of p75NTR in MGE progenitors decreases phosphorylation of p65 and lowers p27 expression, but does not affect Erk1/2 phosphorylation………………………………………...... 117

Chapter 7

Figure 7.1 Combination hypothesis of intrinsic and extrinsic factors influencing cortical development of p75NTR knockout mice………………………. 127

2 List of abbreviations

3D Three-dimensional AEBSF 4-benzenesulfonyl fluoride hydrochloride ANOVA Analysis of variance AraC Cytosine arabinoside hydrochloride Ascl1 Achaete-scute family BHLH transcription factor 1 BDNF Brain-derived neurotropic factor Bex1 Brain-expressed X-linked protein 1 bFGF Basic fibroblast growth factor BrdU 5-Bromo-2'-deoxyuridine BSA Bovine serum albumin CAI Centre for Advanced Imaging Cdc42 Cell division control protein 42 homolog CGE Caudal ganglionic eminences ChAT Choline acetyltransferase CNS Central nervous system CP Cortical plate CPu Caudate putamen CR Calretinin CSF Cerebrospinal fluid Ctip2 COUP-TF-interacting protein 2 DAPI 2-(4-amidinophenyl)-1H -indole-6-carboxamidine DCX Doublecortin DIV Day in vitro Dll1 Delta-like protein 1 Dlx2 Distal-less homeobox 2 DMSO Dimethyl sulfoxide DTT Dithiothreitol E Embryonic day EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGL External granule layer Emx1 Empty spiracles homeobox 1 ErbB2, 4 Erythroblastic oncogene B 2, 4

3 FA Fractional anisotropy FGF2 Fibroblast growth factor 2 FLASH Fast low angle shot GABA Gamma aminobutyric acid GAD67 Glutamic acid decarboxylase 67 GAPDH Glyceraldehyde 3-phosphate dehydrogenase GCPs Granule cell progenitors GFP Green fluorescent protein GLAST Glutamate aspartate transporter 1 GnRH Gonadotropin-releasing hormone-1 Gsh2 Genetic-screened homeobox 2 GTP Guanosine triphosphate HBSS Hanks' balanced salt solution HDB Horizontal diagonal band of Broca HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hes1,5 Hairy and enhancer of split-1, -5 HRP Horse radish peroxidase ICD Intracellular domain iCre Codon-improved cre recombinase IGF-1 Insulin-like growth factor-1 IKK IκB kinase IPC Intermediate progenitor cell IZ Intermediate zone IκBα Inhibitor of nuclear factor κB, alpha JNK c-Jun N-terminal kinase LC MS/MS Liquid chromatography mass spectrometry LFQ Label-free quantification LGE Lateral ganglionic eminences LINGO-1 Leucine rich repeat and Immunoglobin-like domain-containing protein 1 LTD Long term depression LTP Long term potentiation MAGE Melanoma antigen gene MAPK Mitogen-activated protein kinase Mash1 Mammalian achaete scute homolog-1

4 MGE Medial ganglionic eminences MOPS 3-(N-morpholino) propanesulfonic acid MRI Magnetic resonance imaging mRNA Messenger RNA MS Medial septum MZ Marginal zone NA Signal average NE Neuroepithelial cells NesCre Nestin-Cre NF-κB Nuclear factor κB NGF Nerve growth factor Ngn2 Neurogenin-2 Nkx2.1 NK-2 homeobox 1 NPY Neuropeptide Y NRAGE Neurotrophin receptor-interacting MAGE homolog Nrg1 Neuregulin 1 NRIF Neurotrophin receptor interacting factor NT-3, -4 Neurotrophin-3, -4 P Postnatal day p27/Kip1/CDKN1B Cyclin-dependent kinase inhibitor 1B Par Polarity complex Pax6 Paired box protein 6 PB Phosphate buffer PBS Phosphate buffered saline PBS-T Phosphate buffered saline-Tween-20 Pen-Strep Penicillin/Streptomycin PFA Paraformaldehyde PI3K Phosphoinositide 3-kinase PLL Poly-L-lysine PMSF Phenylmethylsulfonyl fluoride POA Preoptic area PSPB Pallial-subpallial boundary PVDF Polyvinlidene difluoride Ras Rapidly accelerated fibrosarcoma RGP Radial glial progenitor

5 RIP2 Receptor interacting protein-2 Rpm Rotations per minute SC-1 Schwann cell factor 1 SDS Sodium dodecyl sulfate SEM Standard error of the mean SHH Sonic hedgehog SorCS2 Sortilin related VPS10 domain containing receptor 2 SOV Sodium orthovanadate SST Somatostatin SVZ Subventricular zone T3 Triiodothyronine T4 Tetraiodothyronine Tbr1, 2 T-Box brain transcription factor 1, 2 TBS Tris buffered saline TBS-T Tris buffered saline-Tween-20 TE Echo time TNF Tumour necrosis factor TR Repetition time Trk Tropomyosin receptor kinase V Ventricle VDB Vertical diagonal band of Broca VZ Ventricular zone Wnt Wingless

Chapter 1: Introduction

1.1 Embryonic neurogenesis

1.1.1 Early patterning of the developing brain and origin of telencephalic neurons

The murine gestation period lasts 19-21 days, and embryonic neurogenesis, defined as the production of neurons from pluripotent stem cells, starts from embryonic day (E) 10 and continues until birth (Dehay and Kennedy, 2007). By E12, the developing telencephalon can be divided into two major progenitor zones, the dorsal pallium and ventral subpallium. While the pallium gives rise to cortical glutaminergic projection neurons, the subpallium produces the majority of gamma aminobutyric acid (GABA)-ergic cortical interneurons, olfactory neurons, and neurons of the basal ganglia and amygdala (Flames et al., 2007; Molyneaux et al., 2007; Silberberg et al., 2016; Yun et al., 2001). The distinct progenitor zones of the pallium and subpallium are formed by mutually exclusive expression of sets of transcription factors and regulatory gradients of secreted morphogens, such as wingless type (Wnt) and Sonic hedgehog (Shh), which define the dorso-ventral axis of the neural tube (Ulloa and Marti, 2010). In the pallium, the neuronal lineage is defined by the expression of transcription factors including paired box 6 (Pax6), and empty spiracles homeobox 1 (Emx1), and subsequently T-box brain transcription factor 2 (Tbr2), neurogenin-2 (Ngn2), and Tbr1 (Carney et al., 2009). Conversely, expression of Genetic-screened homeobox 2 (Gsh2) in the subpallium controls the expression of neuronal-lineage transcription factors achaete- scute family BHLH transcription factor 1 (Ascl1) (originally named Mash1 for mammalian achaete scute homolog-1), distal-less homeobox 2 (Dlx2) and NK-2 homeobox 1 (Nkx2.1) (Puelles et al., 2000). The gradient in gene expression between Pax6 and Gsh2 defines the pallial-subpallial boundary (PSPB) (Carney et al., 2009). The subpallium can be further subdivided into the medial, lateral, and caudal ganglionic eminences (MGE, LGE, CGE), the preoptic area (POA), and septum (Puelles et al., 2000). Throughout the course of neurogenesis, neurons are produced in the highly proliferative zones lining the ventricles, the ventricular zone (VZ) and subventricular zone (SVZ). Newly born neurons migrate from their origin to their target destination following a path of molecular and cellular cues. Cortical projection neurons are produced dorsally and follow a radial migration pattern, forming the six cortical layers in an inverse sequential order (Molyneaux et al., 2007). This is achieved using a scaffold of radial glial progenitor (RGP) cell processes that initially span the whole width of the early cortex (Hatten, 1990). In comparison, approximately 70% of cortical inhibitory interneurons are born in the MGE and undergo a

9 more complex migration pattern, traversing tangentially into the cortex by crossing the PSPB to their final destination within the cortical layers (Marin and Rubenstein, 2001). The MGE also produces interneurons for the striatum and cholinergic neurons for the basal forebrain, while the LGE mainly produces interneurons for the olfactory system (Marin et al., 2000; Stenman et al., 2003). Thus, subpallial progenitor zones produce a range of neuronal subtypes, the timing of differentiation of which vary widely. There are few studies that explore the extrinsic and intrinsic molecular cues that govern the generation of different cell types from a certain progenitor zone. For example, it is still largely unknown what drives interneuron subtype specification, and whether interneurons are fate-restricted from birth or acquire their specific characteristics in response to environmental cues. Therefore, although proliferation, differentiation and migration are universal principles of neurogenesis, regulatory mechanisms underlying these processes may be different depending on the place of origin and timing of birth of specific types of neurons. The following sections outlines common molecular and cellular mechanisms that control neurogenesis, and highlights signaling pathways that are candidates for subtype specific regulation of development.

1.1.2 Cellular and molecular mechanisms underlying proliferation and differentiation

The first steps in neurogenesis are progenitor expansion and subsequent differentiation of neuronal progenitor cells into immature neurons, which then migrate to their target region to integrate into neuronal networks. At the onset of neurogenesis, neuroepithelial cells (NE) that form the neural tube give rise to more fate-restricted RGPs, the stem cells from which most neurons and glial cells in the brain are derived (Molyneaux et al., 2007). From E11 to about E13.5, cortical RGPs produce, by asymmetric division, one identical daughter cell and an immature neuron which migrates out of the proliferation zone towards its neuronal target network. However, from E14 onwards, neurogenesis switches from a one-stage to a two- stage production of neurons, where an intermediate progenitor cell (IPC) rather than a neuron is generated from the RPG. The IPC can undergo a few rounds of proliferative divisions but the final cell cycle of an IPC is a neurogenic division to form two neurons (Fig. 1.1A; Molyneaux et al., 2007).

10 A B

Figure 1.1: Progenitor hierarchy and relationships during corticogenesis. A) Neuroepithelial cells (NE) undergo proliferative (symmetrical) divisions during early embryonic stages (

At the core of this differentiation process is the production of two different cell types from a common precursor, which can happen in two ways. Firstly, a polarized neural progenitor cell can produce two identical cells if the cleavage plane is oriented such that both daughter cells receive a similar proportion of the polarised cytoplasm and membrane of the parent cell (Fig. 1.1B). However, if the mitotic cleavage plane is oriented such that molecules at the apical side are distributed into one cell and the basal constituents into the other, intrinsically different daughter cells are generated (Fig. 1.1B; Gotz and Huttner, 2005). Alternatively, extrinsic asymmetric division can occur if the daughter cells are initially identical, but one receives fate-determining signals not received by the other. On a molecular level, several prerequisites are required for a cell to switch from symmetric to intrinsic asymmetric cell division mode. These include cell polarity, cleavage plane orientation and distribution of cell-fate determining proteins within the daughter cells (Fig. 1.1B; Kosodo et al., 2004). Furthermore, in order for a cell to differentiate into a neuron, exit of the cell cycle is a fundamental step, requiring the induction of a new gene expression program and acquisition of a more specialized phenotype (Galderisi et al., 2003). In agreement with this, expression of the anti-proliferative gene TIS21 has been shown to selectively mark cells that are about to undergo the final neurogenic divisions (Iacopetti et

11 al., 1999), which last longer than progenitor-generating cell cycles. Moreover, cell cycle arrest has been shown to be sufficient to force cells from a proliferation to a differentiation program, leading to a depletion of the progenitor pool and premature maturation of neurons (Caviness et al., 2003; Knoepfler et al., 2002). Conversely, failure to arrest the cell cycle leads to excessive proliferation and a delay in the production of mature neurons (Caviness et al., 2003; Lee et al., 1994a). Together, these studies suggest that cell cycle regulation is a major factor controlling the overall number of neurons in the cortex. RGP identity in the developing cortex is maintained by Neuregulin 1 (Nrg1), which signals through its receptors erythroblastic oncogene B 2 (ErbB2) and ErbB4, and by the mitogens fibroblast growth factor 2 (FGF2) and insulin-like growth factor-1 (IGF-1) that promote a proliferative state and inhibit neurogenesis (Mairet-Coello et al., 2009; Schmid et al., 2003; Vaccarino et al., 1999). Asymmetric division of RGPs on the other hand has been shown to depend on the assembly of the Polarity complex (Par), including Par3, Par6, and the small guanosine triphosphate (GTP)ase cell division control protein 42 homolog (Cdc42). In the daughter cell that retains a larger amount of Par3, Notch signaling is increased, resulting in this cell maintaining its RGP identity, while the second daughter cell downregulates Notch signaling and becomes an IPC (Bultje et al., 2009). Other studies have confirmed that Notch signaling can be used to distinguish RGPs from IPCs (Mizutani et al., 2007), and that timing of Notch ligand Delta-like 1 (Dll1) expression correlates with that of Ngn2 and Ascl1, two transcriptional regulators of neurogenesis in the pallium and subpallium, respectively (Castro et al., 2006). Although decreased Notch signaling is important for the acquisition of an IPC identity, other extrinsic signaling pathways have been shown to regulate the cell cycle and promote neuronal differentiation. They are, however, more complex, and it is unclear to what extent they are redundant or complementary to each other. One example is the Wnt/beta-catenin pathway that promotes either proliferation or neuronal differentiation depending on the cellular context of its expression (Munji et al., 2011). Similarly, neurotrophic signaling can regulate specific aspects of neurogenesis in certain progenitor populations, and activation of the p75 neurotrophin receptor (p75NTR) in particular can induce cell cycle exit and neural differentiation (Young et al., 2007). However, p75NTR signaling is largely context dependent, and its role in neuronal lineage progression in the developing central nervous system (CNS) is not well defined. Due to its flexibility, the neurotrophic system is a candidate for fine-tuning the timing of development via cell cycle regulation. The following section summarizes the contribution of neurotrophic signaling, especially through p75NTR, on aspects of neurogenesis that have been identified in different progenitor populations of the CNS.

1.2 Neurotrophin signaling in central nervous system development

1.2.1 Neurotrophin receptors

Neurotrophins can activate two major types of receptors, tropomyosin receptor kinases (Trk), and p75NTR. Trk A, B, and C are specific receptors for their respective type of neurotrophins, with nerve growth factor (NGF) activating TrkA, brain-derived neurotropic factor (BDNF) and Neurotrophin-4 (NT-4) activating TrkB, and NT-3 activating TrkC. Neurotrophin binding to Trk receptors initiates dimerization and transphosphorylation of the intracellular kinase domains. This can lead to the induction of three main signaling pathways: (1) the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (Ras/MAPK) pathway, which promotes neuronal differentiation and neurite outgrowth, (2) activation of phosphoinositide 3-kinase (PI3K)/Akt, which promotes neuronal survival, and (3) protein kinase C-regulated pathways, associated with Trk function in promoting synaptic plasticity (reviewed in Reichardt, 2006). p75NTR is a member of the tumour necrosis factor (TNF) receptor superfamily and can bind to all four types of mature neurotrophins, albeit with lower affinity than their specific Trk receptors. p75NTR can also bind to the unprocessed pro-forms of neurotrophins and can associate with Trk A, B and C, leucine rich repeat and Immunoglobin-like domain-containing protein 1 (LINGO-1) and the Nogo receptor, as well as the death receptor sortilin (Meabon et al., 2015; Mi et al., 2004; Underwood and Coulson, 2008). Depending on the presence or absence of co-receptors, neurotrophin binding to p75NTR can lead to activation of the nuclear factor κB (NF-κB) pathway and cell survival (Hamanoue et al., 1999; Mattson and Meffert, 2006), activation of apoptotic c-Jun N-terminal kinase (JNK) pathway and caspase cascades (Friedman, 2000; Kenchappa et al., 2006; Linggi et al., 2005; Salehi et al., 2002), or modulation of RhoA activity and regulation of axonal outgrowth (Yamashita et al., 1999). Whereas the survival promoting effects of p75NTR are mainly attributed to NF-κB activation and the enhancement of Trk signaling via the Ras/MAPK and PI3K/Akt pathways (Jin et al., 2008; Nakamura et al., 1996), p75NTR -mediated death signaling is more complex, with death pathways activated by various means including pro-neurotrophin binding and association with sortilin (Skeldal et al., 2012). However, it has been shown that binding of neurotrophins to p75NTR can lead to different functional outcomes depending on the cellular context and timing of expression (Barrett and Bartlett, 1994), and that p75NTR can alter ligand affinity of Trk co-receptors (Brennan et al., 1999; Matusica et al., 2013). In addition, proteolytic cleavage by α- and γ-secretases has been emphasized as a key element of p75NTR -induced

13 signaling, adding yet another aspect to the multiplicity of p75NTR actions (Coulson et al., 2000; Kenchappa et al., 2006; Matusica et al., 2013; Skeldal et al., 2011). Unlike Trk receptors, the p75NTR intracellular domain (ICD) lacks catalytic activity to initiate signaling. However, it has been suggested that ligand binding induces conformational rearrangement of the p75NTR receptor dimer, exposing binding sites for intracellular adaptor molecules and leading to Trk-independent induction of downstream signaling (Charalampopoulos et al., 2012). Indeed, the p75NTR ICD has the ability to bind a large range of intracellular regulatory adaptor proteins (Barker and Salehi, 2002; Di Zazzo et al., 2013; Pincheira et al., 2009; Vilar et al., 2006). Expression of p75NTR has been shown to be transient and restricted to specific populations of cells and is downregulated after development in most tissues. Expression is maintained in the cholinergic neurons of the basal forebrain where it is thought to regulate plasticity of the cholinergic system throughout adulthood (Boskovic et al., 2019), in postnatal and adult embryonic stem cells residing in the rat SVZ (Young et al., 2007), in sensory and motor neurons of the peripheral nervous system, and in cerebellar granule neurons (Courtney et al., 1997; Jacobs and Miller, 1999). More recently, low levels of p75NTR messenger RNA (mRNA) have been shown to be co-expressed with parvalbumin (PV) in cortical interneurons of adult mice (Baho et al., 2018). During nervous system development, p75NTR is expressed in the stem cells of the neuronal crest precursor population prior to the onset of neurogenesis (Stemple and Anderson, 1992). In contrast, during neurogenesis in the developing mouse telencephalon, expression is highest in post-mitotic cells where it co-localizes with the neuronal marker beta III tubulin (Meier et al. 2019, chapter 2). This indicates a shift in p75NTR expression during neurogenesis, and upregulation in IPCs and immature neurons make p75NTR an interesting candidate for regulation of IPC differentiation.

1.2.2 p75NTR and Trk receptors in survival and migration of neuronal cells

Historically, most studies exploring neurotrophic signaling during nervous system development have focussed on the peripheral nervous system, where limited amounts of neurotrophins secreted by a target region controls the extent of innervation. Trk receptors have been shown to promote survival of newly born neurons in this context (reviewed here (Reichardt, 2006) and here (Huang and Reichardt, 2001)). This has been established in studies where deletion of NGF, BDNF, or their respective Trk receptors led to a substantial loss of sensory and sympathetic ganglia (Crowley et al., 1994; Jones et al., 1994; Smeyne et al., 1994). In the CNS however, Trk function has been found to be more complex, with loss of Trk function not always leading to apoptosis.

14 One example to illustrate this are cortical subplate neurons, which are generated early during cortical development, and are positive for both TrkB and p75NTR (Ghosh and Shatz, 1993). This transient population of neurons is located beneath the developing cortical plate (CP) and is important for the development of thalamocortical connections, forming a substrate for the establishment of these tracks and then undergoing programmed cell death postnatally (Allendoerfer and Shatz, 1994; Ghosh and Shatz, 1993; Naegele et al., 1991). Studies in cultured rat subplate neurons have shown that p75NTR promotes the survival of these cells in response to BDNF and NT-3 via the sphingomyelinase/ceramide pathway, and that this effect is independent of Trk receptor signaling (DeFreitas et al., 2001). Furthermore, TrkB and TrkC are expressed in proliferating progenitors in the VZ/SVZ of the cortex and ablation of one or both of these receptors has been shown to reduce proliferation and neuronal migration but without inducing apoptosis in vivo (Bartkowska et al., 2007). TrkB and TrkC, but not TrkA and BDNF, are also expressed in interneuron progenitors in the GE (Gorba and Wahle, 1999; Klein et al., 1990). Knockout of TrkB in the MGE also leads to reduced migration of cortical interneurons without affecting their survival (Polleux et al., 2002). TrkB is mainly expressed in the leading edge of migrating interneurons but not in the cell soma, suggesting that TrkB fulfils a specific function in regulation of migration of these neurons (Polleux et al., 2002). In contrast, ablation of TrkB in the LGE, where striatal neurons and interneurons of the olfactory bulb arise from, has been shown to induce apoptosis of these progenitors in the VZ/SVZ and result in a reduced size of the striatum (Baydyuk et al., 2011). However, no similar studies have been conducted to investigate the effect of p75NTR signaling in cortical neuronal progenitors. In this thesis, we show that p75NTR promotes the survival of IPCs, and that loss of p75NTR signaling in this context reduces the progenitor pool and subsequent production of upper-layer cortical projection neurons and PV-expressing interneurons.

1.2.3 p75NTR in differentiation, cell cycle regulation, and lineage progression

In addition to its role in migration and survival, p75NTR has been shown to regulate aspects of neuronal lineage progression. It is well established that cell cycle arrest is closely linked to differentiation in neuronal cells and that regulation of cell cycle proteins itself is sufficient to induce genetic differentiation programs. All neurotrophins have been implicated in proliferation and cell cycle regulation, and there is evidence that p75NTR activation can also play a role in this process.

In vitro studies have proposed a mechanism of p75NTR-mediated cell cycle regulation via

15 interaction with intracellular binding partners. One p75NTR-interacting factor that has been implicated in controlling neurotrophin-induced cell cycle arrest is brain-expressed X-linked protein 1 (Bex1). In p75NTR-positive PC12 cells, Bex1 expression oscillates during the cell cycle and NGF or BDNF facilitates Bex1-p75NTR interaction, most likely in a Trk-independent fashion (Vilar et al., 2006). Bex1 is rapidly phosphorylated by Akt, which prevents its degradation. It has been shown that Bex1 competes with receptor interacting protein-2 (RIP2) for the p75NTR binding site, with the latter being required for the activation of the NF- κB pathway and neuronal differentiation in response to NGF stimulation in PC12 rat pheochromocytoma cells. Therefore, Bex1 interaction with p75NTR promotes cell cycle progression by preventing NF-κB signaling and the resultant differentiation. Other p75NTR interacting factors involved in cell cycle regulation are neurotrophin receptor interacting factor (NRIF)1 and NRIF2, which have been shown to decrease proliferation and increase cell cycle arrest (Benzel et al., 2001; Casademunt et al., 1999), the zinc finger protein Schwann cell factor 1 (SC-1; Chittka and Chao, 1999), and the neurotrophin receptor- interacting melanoma antigen gene MAGE homolog NRAGE (Salehi et al., 2000).

A role for p75NTR in the regulation of neuronal differentiation has also been shown in vivo. During postnatal development, p75NTR is expressed by cerebellar granule cell progenitors (GCPs) in the external granule layer (EGL) (Courtney et al., 1997). It has been shown that TrkB is expressed in the developing cerebellum and that its activation by BDNF regulates survival and migration of GCPs (Zhou et al., 2007). A recent study revealed that p75NTR is expressed in the EGL where it regulates the cell cycle exit of neuronal progenitors (Zanin et al., 2016). The p75NTR expression pattern differs from that of TrkB, suggesting that these receptors fulfil distinct roles in cerebellar development. Cells in the EGL of p75NTR-deficient mice continue proliferating for an increased time span, and increased levels in cyclin E1 suggest a delayed cell cycle exit. Interestingly, the p75NTR-induced cell cycle exit in GCPs was shown to be mediated by binding of pro-NT-3 and association with the sortilin co- receptor SorCS2 (Zanin et al., 2016). Although association of p75NTR with sortilin receptor and binding to pro-NTs has been shown to induce cell death in most cells, pro-NT-3 has been shown to interfere with the SHH pathway needed to induce proliferation in GCPs, further highlighting the context-dependent outcomes of p75NTR activation.

It is worth mentioning that inappropriate cell cycle regulation by p75NTR can also lead to the induction of cell death as a secondary effect, independently from the classical apoptotic pathway. Cell cycle re-entry in newly born neurons has been shown to occur naturally to trigger tetraploidy, such as retinal ganglion cells and cortical projection neurons (Lopez-

16 Sanchez and Frade, 2013; Morillo et al., 2010). This has been shown to be dependent on expression of p75NTR and also occurs in disease conditions such as Alzheimer’s disease (Frade and Lopez-Sanchez, 2010). Cell-cycle re-entry mediated by p75NTR has been shown to take place in response to NGF binding in developing retinal ganglionic cells (Morillo et al., 2012). Using chick retinal ganglionic cells, the cited study provides evidence that p75NTR activation leads to activation of p38MAPK and subsequent phosphorylation of the transcription factor E2F4, which in turn mimics the signal that initiates the cell cycle and triggers re-entry-associated apoptosis. This mechanism is likely independent of TrkA, as a previous study has shown that antibodies against p75NTR, but not TrkA, can prevent cell cycle re-entry (Frade, 2000).

In addition to cell cycle regulation, non cell-autonomous p75NTR has been shown to be required for the acquisition of a GABAergic phenotype of interneurons in the basal forebrain in response to BDNF and NGF treatment (Lin et al., 2007). The fact that p75NTR expression in cholinergic basal forebrain neurons can induce gene expression in GABAergic neurons that do not express the receptor provides evidence that neurotrophic signaling can affect cell fate and differentiation in an indirect manner.

Signaling outcome of p75NTR activation varies widely across different neuronal subpopulations. Presence or absence of co-receptors, ligand availability, and regulated proteolytic cleavage can affect how p75NTR controls neuronal function (Matusica et al., 2013; Vicario et al., 2015)). The variety of signaling outcomes of p75NTR activation underpin the requirement of in vivo studies to determine which of its functions are physiologically relevant in a specific context and cell type. p75NTR -/- mice have been used to study its function, however; complete p75NTR knockout models have the limitation of combination of direct (such as cell-intrinsic effects on neuronal differentiation) and indirect (effects not specific for neuronal progenitors) phenotypes accumulating during development with subtle or transient phenotypes may be missed over effects that are either more pronounced or that have been compensated for. Therefore, to study the complex process of embryonic brain development, conditional p75NTR knockout mouse strains provide a more powerful tool that allows investigating the pathways regulated by p75NTR in specific cell populations at a given time during neurogenesis.

17 1.3 Research aims

The aim of this project was to determine the function of p75NTR in neurogenesis during brain development, particularly during the generation of cortical pyramidal neurons and interneurons, by using conditional p75NTR knockout mouse strains. This was first addressed by analysing the phenotype of Nestin-Cre p75in/in mice, in which p75NTR expression was lost in all neuronal progenitors prior to the start of neurogenesis. Subsequently, mice with more restricted spatial and temporal gene deletion were used to answer specific questions regarding the cell-autonomous effects of p75NTR on the observed phenotypes.

a) Which cortical progenitor cells express p75NTR and what role does p75NTR play in their fate?

b) Is the effect of the loss of p75NTR in dorsal and ventral cortical progenitors the same?

c) What are the molecular and cellular mechanisms by which p75NTR regulates the normal development of cortical progenitors?

Chapter 2: Materials and methods

2.1 Mice

All procedures were approved by the University of Queensland Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Animals were housed in the Queensland Brain Institute (QBI) animal facility in groups of 3-5 animals in individually ventilated cages, maintained on a 12 h light/dark cycle (lights on at 7:00 a.m.), with food and water provided ad libitum. Contact Sentinels are the primary tool used for monitoring the health of the colonies held. Each sentinel tested will represent up to 4 strains on a rack (max. 100 cages). Cages are only changed in HEPA filtered change stations. All feed, bedding, nesting material, boxes, feed hoppers, and water bottles are autoclaved before transferred into the rooms. Mice are housed in Optimice IVC caging, with double HEPA filters and built-in ventilation. Food and water is available ad libitum, and materials are provided for nesting and enrichment. Reverse osmotic water is used for all rodent drinking water. Breeding colonies are maintained in a pathogen-free environment; for experimental procedures, animals were transferred to a Standard Barrier level for access by researchers. Both male and female animals were used for all experiments (2-6 months old for adult animals, embryonic stages as specified in the results sections). Genotyping was performed by The University of Queensland Australian Equine Genetics Research Centre (AEGRC). p75NTR floxed mice (p75fl/fl) The p75NTR floxed strain harbors a transgenic construct containing a floxed exon 1 of p75NTR (NGFR/TNF16) as well as an inverted mCherry reporter gene and stop codon. Upon cre recombinase expression, the construct is inverted and p75NTR expression switched for mCherry (p75in/in; Boskovic et al., 2014).

Nestin-Cre transgenic mice The Nestin-Cre strain used in this thesis have been described previously (Tronche et al., 1999). These mice express cre recombinase under the control of the nestin promoter. Nestin is expressed from approximately E10.5 onwards in all neuronal progenitors (Dubois et al., 2006). By crossing Nestin-Cre mice to the p75fl/fl strain, a homozygous conditional knockout of p75NTR in all cells of the nervous system was achieved.

21 Nkx2.1-iCre transgenic mice The Nkx2.1-iCre transgenic mice were obtained from obtained from William D Richardson and Nicoletta Kessaris (University College London; animal code #G178) and have been described previously (Kessaris et al., 2006). These mice harbour a codon-improved cre (iCre) recombinase controlled by the Nkx2.1 promoter. By crossing these mice to our p75fl/fl strain, a heterozygous conditional deletion of p75NTR in all progenitors of the MGE from E10.5 onwards was achieved. Heterozygous p75NTR knockout animals (Nkx2.1-iCre+/-; p75wt/in) were infertile and therefore no homozygous animals were generated.

Emx1-iCre transgenic mice The Emx1-iCre transgenic mice were obtained from William D Richardson and Nicoletta Kessaris (University College London; animal code #H151) and have been described previously (Kessaris et al., 2006). These mice harbour an iCre recombinase controlled by the Emx1 promoter. By crossing Emx1-iCre mice to the p75fl/fl strain, a homozygous conditional knockout of p75NTR in dorsal progenitors was achieved.

Tis21-GFP knock-in; p75NTR floxed mice Tis21-GFP mice harbour a GFP reporter gene under the control of the Tis21 promoter (Haubensak et al., 2004). In these mice, GFP is expressed in all progenitors that are about to undergo neurogenic divisions. Tis21-GFP mice were sourced from a colony in the QBI animal facility and crossed onto our p75fl/fl strain to obtain animals homozygous for both alleles (Tis21-GFP+/+; p75fl/fl). These mice were used as breeders in timed mating experiments with Nkx2.1-iCre+/- mice to obtain experimental Tis21-GFP+/-; Nkx2.1-iCre+/-; p75wt/in knockout and control Tis21-GFP+/-; Nkx2.1-iCre-/-; p75wt/fl wild type embryos.

Ascl1-CreERT2; tdTomato knock-in transgenic mice Ascl1-CreERT2 mice (Jax Mice Database strain Ascl1tm1(Cre/ERT2)Jejo/J; stock No. 012882) crossed to a CAGfloxStop-tdTomato strain (JAX Mice Database strain B6.Cg- Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; stock no. 007914), were kindly provided by Dr. Dhanisha Jhaveri. In these mice, Cre-mediated recombination in Ascl1-positive cells can be induced by tamoxifen administration. We crossed these mice to our p75NTR floxed strain to obtain animals in which p75NTR knockout as well as constitutive expression of the tdTomato marker gene could be induced at different timepoints in ventral progenitors.

22 2.2 Preparation and administration of tamoxifen

Tamoxifen (Sigma #T5648) was sterilized by mixing with 100% ethanol (10% of the final volume), and was dissolved in corn oil (Sigma) at 37°C and protected from light to produce a stock solution of 25 mg/ml. The solution was prepared freshly on the day of injection. Pregnant dams were injected intraperitoneally at E11.5 (48 h prior to embryo collection) with a single dose of 75 mg/kg tamoxifen. Mice were housed separately after tamoxifen administration to prevent cross-contamination.

2.3 Preparation and administration of BrdU

5-Bromo-2'-deoxyuridine (BrdU, Sigma) was dissolved in phosphate buffered saline (PBS) with 1% dimethyl sulfoxide (DMSO). Pregnant dams were injected intraperitoneally either 1 h or 24 h before embryo collection with 50 mg/kg BrdU solution.

2.4 Tissue preparation

The day of vaginal plug was considered E0.5, and birth was considered as postnatal day 0 (P0). For embryos younger than E16.5, whole embryo heads were drop-fixed overnight in 4% paraformaldehyde (PFA) in PBS before processing for histological analysis. The brains of E14.5 and older embryos were dissected from the skull post drop-fixation. E16.5 embryos were transcardially perfused with 4% PFA, after which whole heads were post-fixed overnight, and the brains dissected from the skull. Adult mice were deeply anesthetized with sodium pentobarbitone (VIRBAC) and perfused with 4% PFA through the left ventricle of the heart. Brains were removed from surrounding tissues and post-fixed in 4% PFA. For cryosectioning, fixed samples were cryo-protected overnight by immersion in 20% sucrose in PBS, and embryonic coronal tissue sections (30 µm) were collected on Superfrost Plus slides. Staining of adult tissues (P60 and older) was performed on 40 µm coronal sections. For free-floating vibratome sections, the tissue was embedded in 4% agarose in PBS before sectioning.

23 2.5 Nissl staining

40 µm floating sections were mounted on Superfrost Plus slides and allowed to dry, dehydrated in 100% ethanol, after which they were defatted for 15 min in xylene. The sections were rehydrated in 95% and 70% ethanol and H2O, then stained in 0.1% cresyl violet acetate solution for 10 min. The stained sections were rinsed in H2O and destained with 70% ethanol until the desired intensity was reached, at which point the reaction was stopped by immersing slides into 100% ethanol followed by xylene. The sections were then mounted in DePex mounting medium (VWR International).

2.6 Immunohistochemistry

For immunofluorescence labeling without antigen retrieval, tissue sections were incubated for 1 h in blocking buffer (5% horse serum, 0.1% Triton-X 100 in PBS). The sections were then incubated overnight at room temperature (~24°C) in primary antibody diluted in blocking buffer. They were subsequently washed in PBS containing 0.1% Triton-X 100 at room temperature before being incubated with the appropriate fluorescent secondary antibody (1/1000; Jackson Immunoresearch Laboratories) for 2 h at room temperature and protected from light. Cell nuclei were stained with 2-(4-amidinophenyl)-1H-indole-6- carboxamidine (DAPI; 1/2000, Sigma). Sections were mounted on Superfrost slides and coverslipped with Dako fluorescence mounting medium. For immunofluorescence labeling with antigen retrieval, tissue sections were mounted onto Superfrost slides and allowed to dry completely before incubating in 10 µM sodium citrate buffer at 85°C for 30 min. The slides were then washed in PBS and the subsequent blocking, antibody incubation, and mounting steps were performed as described above. Bright field immunohistochemistry was performed as described previously (Hamlin et al., 2013). Briefly, sections were washed in 0.1 M phosphate buffer (PB), pH 7.4, and then incubated in 3% H2O2, 50% ethanol to inactivate endogenous peroxidase. The sections were subsequently washed in 0.1 M PB and incubated for 1 h at room temperature in blocking solution (PB, 10% horse serum, 0.1% Triton-X 100). Primary antibodies were diluted in blocking buffer before being applied onto the sections for overnight incubation at room temperature. The sections were then washed at room temperature in PBS containing 0.1%Triton-X 100 before incubation with biotinylated secondary antibody and ABC reagents (Vector Elite Kit, Vector Laboratories; 6 µl/ml avidin and 6 µl/ml biotin). Black immunoreactivity was revealed by a nickel-intensified diaminobenzidine reaction. Sections

24 were then washed in PB and gradually dehydrated in ethanol and xylene before being mounted in DePex mounting medium (VWR International).

Primary antibodies Primary antibodies included goat anti-p75NTR (extracellular domain; immunofluorescence 1/200; immunohistochemistry 1/1000; R&D Systems AF1157), rabbit anti-Ds Red (mCherry and tdTomato) (1/500; Clontech 632496), goat anti-tdTomato (1/1000, Origene AB8181- 200), mouse anti-PV (1/1000; Millipore MAB1572), rabbit anti-calretinin (1/2000; Swant 7699/3H), rabbit anti-activated caspase 3 (1/500; Cell Signaling 9661L), and mouse anti-β3 tubulin (TUJ1; 1/2000; Promega G712A), used without antigen retrieval (note: for embryonic tissue, the anti-p75NTR antibody was found to work best on vibratome sections using non- frozen tissue). In addition, rat anti-COUP-TF-interacting protein 2 (Ctip2) (1/500; Abcam ab18465), rabbit anti-Tbr1 (1/500; Abcam ab31940), rat anti-EOMES (Tbr2) (1/500; Life Technologies 53-4875-80), rat anti-somatostatin (SST) (1/1000; Milipore MAB354), rabbit anti-neuropeptide Y (NPY) (1/2000; Immunostar 22940), mouse anti-Ki67 (1/500; BD Biosciences 556003), mouse anti-Mash1 (Ascl1) (1/200, BD Biosciences 556604), and rat anti-BrdU (1/500; Serotec MCA2060) were used following antibody retrieval.

Imaging Low resolution images and images of whole adult sections for cell counts were acquired on a Zeiss Axio Imager Epifluorescence Microscope with a fully motorized X-Y-Z stage, and an upright fluorescence slide scanner (Metafer VSlide Scanner by MetaSystems using Zeiss Axio Imager Z2) with a 20x air objective. Fluorescent filter sets for DAPI, FITC (530/350 nm), Cy3 (620/660 nm), and Cy5 (633/647 nm) were used. High resolution images and images for cell counts in embryonic tissue sections were acquired with a Nikon Plan Apo Lambda 40x/1.15 NA water objective and a Plan Apo Lambda 60x/1.4 NA oil-immersion objective on a spinning disk confocal microscope (Diskovery; Andor Technology, UK) built around a Nikon Ti-E body (Nikon Corporation, Japan) and equipped with two Zyla 4.2 sCMOS cameras (Andor Technology), 405nm, 488nm, 561nm, 640nm lasers, and controlled by Nikon NIS software.A tile scan of the region of interest was performed and z-stacks were collected across the depth of the tissue (0.5 µm step size).

Quantification For quantification of immunohistochemical stainings, a minimum of 3 mice were used for each genotype. To ensure that the same cutting angle was applied between experimental

25 groups, embryonic coronal sections were matched using 4 points of reference: choroid plexus/cortical hem and POA/MGE for dorsal and ventral, and the LGE for lateral orientation, based on the Allen Developing Mouse Brain Reference Atlas (www.developingmouse.brain- map.org). Cell counts, cell size, and volume and thickness measurements were performed using Imaris 7.2.3 software (Bitplane) and ImageJ. Quantification of the number of cortical interneurons in the adult brain was performed in a defined area of the cortex (400 µm width, 40 µm depth) spanning the pial-white matter extent of the cortex. Cell density was calculated as the number of cells in regions of interest of equal size that covered layers V and VI.

2.7 Golgi Cox staining

Mice were perfused transcardially with 0.04% PFA in NaCl, and their brains were removed. The brains were incubated in Golgi-Cox solution overnight, with the Golgi-Cox solution being replaced the next day, and again after one week. After two weeks, the brains were placed in 30% sucrose in NaCl for 3 days and subsequently sectioned into 150 µm thick slices using a vibratome. Tissue sections were mounted onto gelatin-coated Superfrost Plus slides and dried at 4˚C for 24 h, followed by another 24 h at room temperature. To visualize the staining, sections were washed sequentially in dH2O for 1 min, in 30% ammonia for 10 min, and in fresh ammonia (30%) for 10 min. Following a dH2O wash, the sections were incubated for 5 min in 5% thiosulphate, followed by another 5 min in fresh thiosulphate (5%), before being washed for 3 min in each of 30% and 70% ethanol, and twice for 5 min in 100% ethanol, and >1 h in xylene.

Imaging Z-stacks and tile scans of individual neurons were acquired using the brightfield settings on a Zeiss Axio Imager Epifluorescence Microscope with a fully motorized X-Y-Z stage, using a 40x water immersion objective.

Quantification Neurolucida Neuron Tracing Software (MBF Bioscience) was used to measure neurite length and soma size in Golgi Cox samples, and to perform Sholl and complexity analyses. Neurite complexity was calculated as described previously (Pillai et al., 2012). Neurons were selected for tracing according to their location (layer V in the somatosensory cortex), position in the section (soma roughly in the center, no dendrites cut off) and minimal overlap with other neurons.

2.8 Behavioral testing

Male and female six-month old Nestin-Cre p75in/in and p75fl/fl mice were used for all behavioral tests. Animals were adjusted to the experimental facilities for at least 30 min before commencement of behavioural tests.

Rotarod and grip strength motor function testing The mice were placed on a rotating rod and the time until they fell off was measured. The initial rotarod speed was set to 4 rotations per minute (rpm) and was gradually increased (acceleration rate of 1 rpm/sec) until a maximum speed of 40rpm. The mice were left on the rotating bar for a maximum of 120 sec. Fore- and hind limb grip strength was tested by pulling mice evenly across a metal bar attached to a grip strength meter (Harvard apparatus). 3 measurements were taken per animal and limb pair then averaged.

Elevated plus maze Each mouse was placed in an elevated plus maze apparatus (Pellow et al., 1985) for 5 min under bright light conditions (400 lux) with the total time spent in the open and closed arms being measured using EthoVision XT tracking software (Noldus).

Novel arm recognition test Mice were placed in a Y maze with one arm of the maze closed off (left and right were alternated randomly between animals) for 10 min, as described previously (Qian et al., 2018). Novel arm recognition was tested 24 h after training by placing the mice back into the Y maze with all arms open for 5 min at 150 lux, and time spent in each arm was measured using the EthoVision XT tracking software (Noldus).

2.9 Magnetic resonance imaging

3-month old mice were perfused transcardially with 4% PFA (as described in section 2.4) and whole heads were post-fixed overnight. Brains were removed from the skull the next day and immersed in 0.1 M PBS with 0.2% gadopentetate dimeglumine (Magnevist, Bayer) for four days. A small animal magnetic resonance imaging (MRI) system (16.4T vertical bore; Bruker Biospin; ParaVision v6.01) with a 15 mm linear SAW coil (M2M Imaging) and a

27 Micro2.5 imaging gradient was used. Three-dimensional (3D) diffusion-weighted imaging spin-echo sequences were acquired with echo time (TE) = 23 ms, repetition time (TR) = 200 ms, signal average (NA) = 1, diffusion pulse/mixing times (δ/Δ) = 2.5/12 ms, spectral bandwidth = 50 kHz, 30 direction diffusion-encoding with b-value = 5000 s/mm2, 2 b=0 images (without diffusion-weighting), and field of view (FOV) = 19.6 x 11.4 x 8.4 mm with matrix = 196 x 114 x 84 to produce images at 0.1 mm isotropic resolution. The acquisition time was 17 h. A 3D gradient echo Fast Low Angle Shot (FLASH) sequence was acquired with TR/TE = 50/12 ms, 30 degrees flip angle, NA = 1, the same spectral bandwidth and FOV as the DWI sequence but with matrix = 654 x 380 x 280 to produce images at 0.03 mm isotropic resolution; the acquisition time was 40 min.

Volumetric analysis was performed using an adult C57/BL6 MRI brain atlas developed in- house at the Centre for Advanced Imaging (CAI) at the University of Queensland (Liu et al., 2016a; Ullmann et al., 2014), and the Brookhaven C57/BL6 ex vivo brain MRI atlas (Ma et al., 2005). These atlases were registered into the FLASH images using FMRIB Software Library linear and non-linear registration (FLIRT and FNIRT) (Jenkinson et al., 2012). Measurements were extracted using ITK-SNAP (http://www.itksnap.org; Yushkevich et al., 2006)). The volume of the basal forebrain was measured by drawing a region of interest on the diffusion-weighted fractional anisotropy (FA) image using the mouse brain atlas by Franklin and Paxinos (2007) as a guide and as described previously (Kerbler et al., 2013).

2.10 Culturing of MGE progenitors and interneuron differentiation

Dissections of MGEs MGE progenitors were obtained by dissecting MGEs of E13.5 heterozygous p75NTR knockout (Nkx2.1-iCre+/-; p75wt/in; tdTomato+/-) and control (Nkx2.1-iCre-/-; p75wt/fl; tdTomato+/-) embryos as previously described (Franchi et al., 2017). Briefly, embryos were kept on ice and the heads were separated for dissections. The skull and the dorsal part of the telencephalon were removed to expose the ganglionic eminences. The inner part of the heart-shaped structure (as seen from above) was excised with 4 vertical and one horizontal cut. Dissections were performed bilaterally and the MGEs collected in an Eppendorf tube containing 0.5 ml HBSS/HEPES Pen-Strep (Hanks' Balanced Salt solution (HBSS; Life Technologies) containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma), 100 units/ml of penicillin, and 100 µg/ml streptomycin (Pen-Strep; Life Technologies)).

Cell seeding and culture maintenance MGE progenitors were cultured in 24-well plates on circular Menzel coverslips (15 mm diameter; pre-treated with 1 M hydrochloric acid overnight at 60°C) for immunocytochemical analysis, or in 6-well plates without coverslips for Western blot analyses. Cell culture surfaces were coated with 200 µg/ml poly-L-lysine (PLL; Sigma) overnight at 4°C. The next day, the culture surface was washed 3 x with PBS and coated for 2 h at 37°C with 20 µg/ml laminin (Life Technologies), washed with PBS and placed in maintenance medium (neurobasal medium (Life Technologies) with 2% (v/v) B-27 supplement (Life Technologies), 1% (v/v) GlutaMAX (Life Technologies), and 1% (v/v) antibiotic-antimycotic (Life

Technologies)) at left to equilibrate at 37°C and 5% CO2 until cell seeding. To disperse the MGE tissue and obtain single cell suspensions, 100 µg/ml DNAse I (Sigma) and 0.05% trypsin (ethylenediaminetetraacetic acid (EDTA)-free, Life Technologies) final concentration were added to each tube and incubated for 10 min at 37°C. The trypsin reaction was stopped by adding 850 µl warm plating medium (maintenance medium with 10% fetal bovine serum (Sigma)) and the tissue was sedimented by centrifuging for 5 min at 100 x g. The supernatant was removed, and the tissue resuspended and mechanically dissociated in 180 µl of warm maintenance medium by pipetting up and down. Viable cell counts were determined via trypan blue staining and manual counting in a hemocytometer. 50’000 cells per well were seeded in 24-well plates, and 200’000 cells per well in 6-well plates, respectively, and the cells were allowed to attach overnight at 37°C and 5% CO2. The next day (day in vitro (DIV) 1), 50% fresh maintenance medium containing 150 ng/ml BDNF (PeproTech; 50 ng/ml final concentration) was added to each well to stimulate neuronal differentiation. On DIV 4, 2 μM cytosine arabinoside hydrochloride (AraC; Sigma) was added to the media to prevent expansion of glial cells. 50% of the media was changed every 72 hours.

2.11 Immunocytochemistry

On DIV 15, interneuron cultures were fixed and processed for immunocytochemical staining. The medium was aspirated and cells washed once with cold PBS. Fixation was performed by adding 0.5 ml of freshly prepared 4% PFA in PBS (pH range 7.2 – 7.4) and incubating for 15 min at room temperature. The fixed cells were washed 3 x with PBS and stored at 4°C in PBS. For immunofluorescent labeling, cells were incubated for 1 h in blocking buffer (5% horse serum, 0.1% Triton-X 100 in PBS). The coverslips were then removed and incubated

29 cell-side down in 30 µl of primary antibody diluted in blocking buffer overnight at 4°C in a humidified chamber. They were subsequently washed in PBS containing 0.1% Triton-X 100 at room temperature before being incubated with the appropriate fluorescent secondary antibody (1/1000; Jackson Immunoresearch Laboratories) for 2 h at room temperature and protected from light. Cell nuclei were stained with DAPI (1/2000, Sigma). Coverslips were mounted on Superfrost slides with Dako mouting medium and the edges were sealed with clear nail polish.

Primary antibodies Primary antibodies included rabbit anti-Ds Red (tdTomato) (1/500; Clontech 632496), mouse anti-glutamic acid decarboxylase 67 (GAD67) (1/1000; Millipore MAB5406), and mouse anti- βIII tubulin (TUJ1; 1/1000; Promega G712A).

Imaging Coverslips were imaged on a Zeiss Axio Imager Epifluorescence Microscope with a fully motorized X-Y-Z stage. 3 tile scans of 5x5 tiles were acquired using a 20x air objective. Exposure times for all channels were kept constant between all samples of the same experiment.

2.12 Western blot analysis

Protein extraction from tissue culture To prepare samples from interneuron cultures, on DIV 15, the cells were washed once with cold PBS and scraped with 250 µl lysis buffer per well (10 mM Tris-base, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.8) containing freshly added sodium orthovanadate (SOV, 1 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), and Complete Inhibitor Cocktail (1%, Roche). The scraped cells were transferred to 1.5 ml Eppendorf tubes and incubated on ice for 15 min. Lysates were then centrifuged at 15,000 x g (top speed) for 30 min at 4°C and the supernatants transferred to a fresh tube. The total protein concentration of each sample was determined with a Pierce BCA protein assay kit (Thermo Scientific). Samples were stored at -80°C until analysis.

Protein extraction from embryonic tissue The MGEs of embryos from Nkx2.1-iCre x p75fl/fl timed matings were dissected at E13.5 as described in section 2.10 and snap frozen on dry ice. To prepare samples from embryonic

30 tissue, the MGEs of 3 embryos (6 MGEs total) of each genotype (Nkx2.1-iCre+/-; p75wt/in for experimental, and Nkx2.1-iCre-/-; p75wt/fl for control embryos) were pooled to obtain enough protein for 2 Western blot analyses. If less than 3 embryos per genotype / litter were obtained, they were combined with embryos from other litters of the same genotype (see figure legends for details). 45 µl of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Triton X- 100, 1% sodium deoxycholate, 1 mM EDTA, 1 mM SOV, 1 mM PMSF, 1 mM 4- benzenesulfonyl fluoride hydrochloride (AEBSF), 1% Complete Inhibitor Cocktail (Roche) and 1% PhosSTOP Phosphatase Inhibitor Cocktail (Roche), pH 7.4) was added to each sample and the tissue was triturated by pipetting up and down with a p200 pipette. The homogenized tissue was incubated on ice for 15 min and then centrifuged at 15,000 x g (top speed) for 30 min at 4°C. The supernatants were transferred to a fresh tube and immediately prepared for Western blot analysis.

Gel electrophoresis and transfer Protein from the soluble fraction were used in all analyses. The lysates were denatured by adding 1 part NuPAGE LDS sample buffer (Invitrogen, 4x concentrated) with 0.1 M dithiothreitol (DTT) to 3 parts of sample (v/v) and heating at 90°C for 10 min. Equal amounts of sample (20 µg of protein for interneuron culture lysates, 3 MGEs for embryonic tissue) were loaded in each lane on a NuPAGE Novex 4-12% Bis-Tris Protein Gel (Life Technologies). Gels were run at 100 V for 10 min and further 65 min at 130 V in NuPAGE 3- (N-morpholino) propanesulfonic acid-sodium dodecyl sulfate (MOPS-SDS) running buffer in a Novex Gel System Mini chamber (Life Technologies). Subsequently, the gels were blotted on a methanol-activated polyvinylidene difluoride membrane (PVDF) Immobilon-FL membrane (Millipore) via wet-tank electro-transfer for 90 min at 90 V in NuPAGE Transfer Buffer (Life Technologies) and 20% methanol. The membranes were blocked in 5% bovine serum albumin (BSA) in tris buffered saline (TBS) (for MGE lysates) or PBS (for cell culture lysates) with 0.1% Tween-20 (TBS-T / PBS-T) for 1 hour at room temperature and then incubated overnight at 4°C in primary antibody diluted in their respective blocking buffer. They were subsequently washed 3 x in TBS-T / PBS-T before being incubated with the appropriate fluorescent secondary antibody (1/1000; Jackson Immunoresearch Laboratories, for cell culture samples) for 2 h at room temperature, or appropriate horse radish peroxidase (HRP) coupled secondary antibody (1/2000, Cell Signaling, for MGE samples) for 1 h at room temperature and protected from light. Membranes probed with HRP-coupled secondary antibodies were washed 3 x in TBS-T and once in TBS before incubating with SuperSignal West Femto Substrate (Pierce) for 5 min and protected from

31 light. All membranes were imaged using the Odyssey Fc Dual-Mode Imaging System (Li- Cor Biosciences).

Primary antibodies Primary antibodies included mouse anti-GAD67 (1/1000; Millipore MAB5406), mouse anti- βIII tubulin (TUJ1; 1/1000; Promega G712A), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1/4000; Abcam ab9484), rabbit anti-NF-κB (D14E12) (1/1000; Cell Signaling 8242), rabbit anti-phospho-NF-κB (Ser536, 93H1) (1/1000; Cell Signaling 3033), inhibitor of NF-κB alpha (IκBα) (L35A5 amino terminal antigen) (1/1000; Cell Signaling 4814), mouse anti-p27 (Kip1, Cyclin-Dependent Kinase Inhibitor 1B (CDKN1B), clone 57/Kip1/p27) (1/1000; BD Biosciences 610242), rabbit anti-Erk1/2 (MAPK p44/42) (1/1000; Cell Signaling 9102), and rabbit anti-phospho-Erk1/2 (Thr202/Tyr204, phospho MAPK p44/42) (1/1000; Cell Signaling 9101S).

Stripping and re-probing of PVDF membranes Membranes were stripped and re-probed when detection of proteins in the same membrane was required (e.g. for the loading control GAPDH). To remove the antibodies, the membranes were incubated in stripping buffer (62 mM Tris-base, 2% SDS, 7% 2- mercaptoethanol, pH 6.7) for 30 min at 50°C under constant mixing in a Hybaid oven. The membrane were then washed 3 x in TBS-T / PBS-T. Blocking and primary antibody incubation were then repeated as described above.

Quantification Western blot images were quantified using the Image Studio Lite software (Li-Cor Biosciences, version 5.2). Analysis was performed using the ‘Rectangle’ function to quantify the band signal, and the the ‘Median Background Method’, a local background subtraction algorithm. For analyses of MGE samples the band intensity of each protein was normalized to GAPDH to correct for smaller MGE size and lower amount of total protein loaded for p75NTR knockout embryos. To calculate the level of phosphorylation, the signal of total protein was taken as 100% and the percentage of the phosphorylated form calculated (e.g. the signal of phospho-Erk1/2 was measured against the signal of pan-Erk1/2).

32 2.13 Microscopy

All imaging was performed at the Queensland Brain Institute Advanced Imaging Facility.

2.14 Statistical analysis

Graphs were created using Graph Pad Prism version 7.0. Unless stated otherwise, results are displayed as mean ± standard error of the mean (SEM) and statistical testing was performed using a Student’s t-test, one or two-way analysis of variance (ANOVA), or Chi- squared test as specified in the figure legends. Normal distribution of the data was assessed with Graph Pad Prism software using the Shapiro-Wilk normality test.

Chapter 3: Spatiotemporal mapping of p75 neurotrophin receptor expression in the developing mouse telencephalon

3.1 Introduction

3.1.1 Chapter summary

Previously, most major studies investigating p75NTR expression and function focused on the peripheral nervous system. To our knowledge, no comprehensive spatiotemporal mapping of this receptor has been reported for the developing brain. In this section, we investigate the time point at which p75NTR expression is first detectable, as well as how its expression pattern changes throughout the process of embryonic neurogenesis in the mouse telencephalon. Furthermore, we explore which cell types express p75NTR in order to gain a better understanding of the function p75NTR plays in the developing brain.

3.1.2 p75NTR expression in neurons

During development, the role of p75NTR has primarily been characterized as mediating the survival or developmental programmed cell death of newly born neurons in response to neurotrophin binding. This has been best described in sympathetic neurons of the peripheral nervous system, where p75NTR regulates the extent of target innervation by controlling neuronal number (Lee et al., 1994b). In the adult nervous system however, expression of p75NTR is normally limited to a restricted number of neuronal subtypes. The best studied p75NTR-positive adult neuronal population are the cholinergic basal forebrain neurons, in which high expression of this receptor is maintained throughout life. p75NTR knockout studies using a cre recombinase expressed under the control of the choline acetyltransferase (ChAT) promoter have shown that p75NTR signaling regulates several aspects of basal forebrain development. Deletion of the p75NTR gene increased the cell size and number of cholinergic basal forebrain neurons, as well as the extent of cholinergic innervation into the cortex (Boskovic et al., 2014). Furthermore, these developmental changes in the cholinergic axonal wiring and synaptic connectivity were accompanied by alterations in spatial idiothetic navigation and fear extinction paradigms, suggesting that developmental loss of p75NTR can have a long lasting impact on brain function (Boskovic et al., 2014; Boskovic et al., 2018). Other studies have identified additional neuronal subtypes that retain a low level of p75NTR expression. One study has shown that p75NTR modulates hippocampal glutamatergic synapses and mediates long term depression (LTD; Lu et al., 2005) in combination with the

35 sortilin related VPS10 domain containing receptor 2 (SorCS2). Electrophysiological recordings and behavioural testing in SorCS2 -/- mice in this study revealed decreased memory function and traits resembling human neurodevelopmental disorders such as schizophrenia (Glerup et al., 2016). Although part of this phenotype can be attributed to the interaction of SorCS2 with TrkB, which has been shown to mediate long term potentiation (LTP), it is possible that p75NTR plays a role in the formation and maintenance of the reported synaptic plasticity. Furthermore, in a recent study that used RNAscope and a proximity ligation assay to detect low p75NTR expression levels with high spatial and temporal resolution, it has been reported that cortical PV interneurons retain p75NTR expression into adulthood (Baho et al., 2019). Interestingly, by specifically deleting expression in PV interneurons, this study also found that p75NTR signaling is required for the timing and maturation of the connectivity of these cells, potentially by regulating the formation of cell perisomatic innervation and perineural nets. Therefore, p75NTR regulates the development of these late-maturing inhibitory neurons, thereby potentially controlling the formation and plasticity of cortical circuits. Finally, cerebellar Purkinje cells maintain p75NTR expression throughout life and conditional knockout of this receptor in mice has been associated with an autism-like behavioural phenotype (Lotta et al., 2014). Knockout animals in this study showed decreased dendritic complexity and defects in arborization, indicating that developmental expression of p75NTR in these cells is required for proper integration into cerebellar networks. Taken together, p75NTR expression in neurons of the mature nervous system is limited but plays an important role in the functional maturation of the affected cell types as well as their integration into neuronal networks. Loss of p75NTR function in such contexts can, at least in mice, induce behavioural phenotypes that are reminiscent of prominent human neurodevelopmental disorders, such as schizophrenia and autism.

3.1.3 p75NTR expression in progenitors

In contrast to the adult brain, expression of p75NTR during development is more widespread and highly dynamic, with several cell types expressing the receptor at different times during neurodevelopment. While in early embryogenesis p75NTR is ubiquitously expressed in neural crest cells and is considered a marker for this cell type, substantial expression is only retained in certain populations of later stage progenitors. The spatial and temporal- dependent expression of p75NTR is not surprising, given the large range of developmental functions that can be regulated by its signaling pathways (see Chapter 1, section 1.2). However, why certain cell types depend on p75NTR for crucial cellular functions such as

36 survival or cell cycle exit, while others do not, remains an enigma. Furthermore, to date p75NTR function has only been studied in isolated populations of progenitors, and, to our knowledge, comprehensive spatiotemporal mapping of p75NTR expression in the developing mouse brain had not previously been reported (Meier et al., 2019; this thesis).

3.1.4 p75NTR expression in other cell types

In addition to neurons and their precursors, glial cells constitute another major cell population in the CNS. Glia are highly specialized cells that support neuronal function and can be distinguished into 2 types of macroglia, oligodendrocytes and astrocytes, and microglia. While expression of p75NTR in oligodendrocytes has been reported before (Cohen et al., 1996), the evidence in current literature of p75NTR expression in oligodendrocytes and astrocytes is less clear. To further investigate p75NTR expression in these cell types, publicly available expression databases were mined. In recent years, technological advancement in the fields of large-scale proteomics and transcriptomics made it possible to cover tissue expression patterns with high accuracy and increasing resolution. With the emergence of single cell sequencing method, the resolution of transcriptomics methods has now reached the cellular level, allowing detection of differential gene transcription programs in distinct cell types at different stages of development (Kanter and Kalisky, 2015). However, transcript abundance often poorly correlate with their respective protein levels, which are the main functional components of the cell (Liu et al., 2016b). Therefore, to study protein function, proteomics data are more useful. While mass spectrometry based methods have not yet reached the same resolution as single cell sequencing methods, efforts have been made to comprehensively analyse protein expression in distinct brain regions and in pure cultures of individual cell types. A recent study using liquid chromatography mass spectrometry (LC MS/MS) based proteomics aimed at in-depth characterization of the proteome of the major regions in the mouse brain and in cultures of individual neuronal and neuron-associated cell types (Sharma et al., 2015). Mining this data for p75NTR confirmed the lack of expression in most adult brain regions analysed, with the exception of the cerebellum (data not shown). p75NTR peptides were also absent from isolated astrocytes (Fig. 3.1). Expression in microglia isolated from young and adult mice was detected in only one of 3 independent replicates and is therefore of low confidence. Robust expression was detected in cultured oligodendrocytes and cortical neurons measured at 3 different timepoints over the course of development (Fig. 3.1).

Figure 3.1: Relative p75NTR protein abundance in different cell types of the mouse CNS measured with label-free fractionation mass spectrometry (Sharma et al., 2015). 3 independent samples were measured of either cells isolated from mice (microglia, astrocytes) or pure in vitro cultures (oligodendrocytes, neurons). Data plotted as log2 of the label-free quantification (LFQ) intensity. div = day in vitro

Development of the nervous system is also dependent on non-neuronal and non-glial cell types that express p75NTR, such as vascular endothelial cells and mesenchymal cells (Huber and Chao, 1995; Nikam et al., 1995; Slowik et al., 1993). Indeed, a complete p75NTR knockout mouse has been reported to show defects in the vascular system (von Schack et al., 2001a); however, it was not reported whether this phenotype impacted brain development of these mice. A more comprehensive and in vivo assessment of p75NTR expression during development was therefore undertaken.

38 3.2 Results

3.2.1 The p75 neurotrophin receptor is expressed transiently in newly born neurons during brain development

To assess spatiotemporal changes in p75NTR expression, we first analysed p75NTR protein levels at different stages of embryonic development by immunohistochemical staining of mouse brain sections. To capture p75NTR expression during neurogenesis, we collected embryonic tissue of wild type mice (p75fl/fl) from E11.5 – E16.5. p75NTR protein could be detected as early as E11.5, with positive immunostaining visible in the ventral part of the telencephalon (Fig. 3.2), and predominant staining colocalising with the early differentiation marker beta III tubulin (Jiang and Oblinger, 1992). This indicates that p75NTR expression is highest in post-mitotic neurons, although some expression was observed in the proliferative zones. The number of p75NTR-positive cells in the ventral telencephalon increased throughout the early stages of development as more neurons were born. In the cortex, p75NTR expression was detected in postmitotic neurons only from E14.5, where it was present in radially projecting neurons of the CP and in migrating neurons within the intermediate zone (IZ). At E16.5, p75NTR expression in the ventral telencephalon was decreased, coincident with the disappearance of the transient ganglionic eminences. Notable exceptions were a ventrolaterally localized neuronal population and their neurites projecting laterally to the cortex (Fig. 3.2), and the cells of the presumptive basal forebrain (data not shown).

Figure 3.2: Spatiotemporal mapping of p75NTR expression in the developing telencephalon. Schematic representations of coronal embryonic mouse brain sections from E11.5-E16.5 and corresponding immunostaining for p75NTR and beta III tubulin (E11.5). Arrows indicate the same cell co-stained for both markers. Scale bars: 200 µm, V= ventricle

40 3.2.2 p75NTR is expressed in a subpopulation of intermediate progenitors

The p75NTR expression pattern in the telencephalon clearly showed high levels of p75NTR protein in post-mitotic neurons, indicating that p75NTR could play a role in the lineage progression of these cells, with p75NTR upregulation occurring at the transition between progenitor and neuron. To further investigate whether p75NTR was expressed in a specific type of progenitor, we co-stained for p75NTR and Ascl1, a marker of IPCs, in the ventral telencephalon of E12.5 wild type mice (p75fl/fl). While expression was low in Ascl1-positive cells in the VZ and SVZ, a proportion of p75NTR -positive IPCs were observed at the border of the SVZ and the marginal zone (MZ), from where immature post-mitotic neurons start their migration to their respective target region (Fig. 3.3A, B). In rare cases, cells in the VZ and other progenitor types, such as RPGs, were also found to express p75NTR (Fig. 3.3B).

Figure 3.3: p75NTR expression in progenitors. Immunostaining for p75NTR and Ascl1 in a wild type (p75fl/fl) control mouse at E12.5. Nuclei are counterstained with DAPI (blue). A) Image of the medial ganglionic eminences (MGE) subventricular and marginal zone as indicated by the red box on the top left. Arrowheads indicate Ascl1-positive progenitors that co-stain with p75NTR. Scale bar: 50 µm. Box indicates position of zoom image on the right. Scale bar of zoom image: 10 µm. B) Image of the MGE ventricular zone as indicated by the red box on the top left. Arrows indicate an example of a cell at the ventricular zone in the MGE exhibiting the typical radial glial progenitor morphology and staining positive for p75NTR. Scale bars: 30 µm, V = ventricle

42 3.2.3 p75NTR is expressed in a subpopulation of neurogenic progenitors

To test the possibility that p75NTR is expressed in cells that are about to exit the cell cycle and differentiate into neurons, we used a Tis21-GFP reporter line that was crossed to our p75fl/fl (wild type) strain. Tis21 is an anti-proliferative gene expressed in neurogenic progenitor types (Iacopetti et al., 1999). We assessed co-expression of p75NTR with GFP, which is expressed under control of the Tis21 promoter, in E12.5 embryonic mouse brain tissue to determine whether neurogenic progenitors are p75NTR-positive. We chose the medial ganglionic eminence as point of analysis since p75NTR expression is highest in this area (Fig. 3.4). As expected, most GFP-positive cells were detected in the VZ and SVZ, with few neurogenic progenitors being observed in the mantle zone (MZ) (Fig. 3.4). Therefore, Tis21-positive cells exhibit an expression pattern roughly opposite of that of p75NTR. Intriguingly however, a subpopulation of neurogenic progenitors located at the SVZ/MZ border stained positive for p75NTR (Fig. 3.4). Therefore, at least some neurogenic progenitors start expressing p75NTR before differentiating into neurons. According to their location away from the VZ they likely identify as IPCs and therefore may mark the transition of this progenitor type into neurons. This is further supported by the fact that a population of Ascl1- positive IPCs express p75NTR (Fig. 3.3). An alternative hypothesis is that the double positive cells had already but recently exited the cell cycle and the GFP had not yet been degraded. These results indicate that p75NTR expression is upregulated in a subpopulation of IPCs shortly before or immediately after completion of their final cell cycle and differentiation into neurons.

Figure 3.4: A subset of neurogenic progenitors at the border of the subventricular zone express p75NTR. Immunostaining for p75NTR (purple) and GFP (green) expressed under control of the Tis21 promoter in an E12.5 p75fl/fl Tis21-GFP mouse. Arrows indicate the same cell co-stained for both markers. p75NTR is also expressed by endothelial cells, and p75NTR-positive blood vessels are marked with an asterisk in the zoom image (bottom right). Dotted line indicates the border between the subventricular zone and the mantle zone determined by morphological features. Scale bars: 100 µm, V= ventricle, MGE = medial ganglionic eminences.

44 3.2.4 p75NTR is expressed in tangentially migrating neurons

Intriguingly, as development progresses, p75NTR expression is downregulated in the majority of cells and marks increasingly distinct neuronal populations. Around E14.5 tangentially projecting fibers are observed that project laterally into the cortex (Fig. 3.2). This route is used by cells that migrate tangentially from the ventral to the dorsal telencephalon to incorporate into target regions such as the neocortex or hippocampus. It is therefore conceivable that p75NTR plays a role in the migration of these cells. To test whether p75NTR is expressed in migrating neurons as well as the fiber substrate, we performed lineage tracing of ventral telencephalic neurons using a mouse strain harbouring a cre recombinase fused to a tamoxifen-inducible estrogen receptor (creERT2) expressed under the Ascl1 promoter, and a flox-stop-flox tdTomato gene cassette inserted in the ROSA26 locus (Ascl1- creERT2 tdTomfl/fl). Upon tamoxifen administration, creERT2 translates into the nucleus of Ascl1-positive cells and induces constitutive expression of the tdTomato marker gene (Feil et al., 1997). To investigate whether migrating neurons derived from Ascl1-positive progenitors express p75NTR, pregnant females were injected with tamoxifen at E11.5 and the embryos collected for analysis at E16.5, when the p75NTR-positive fiber tracks were obvious. While the majority of tdTomato-positive cells remained in the ventral structures of the telencephalon, presumably forming the striatum and other nuclei of the basal ganglia, a small but significant proportion of neurons was detected that were migrating, or had already migrated, into the neocortex (Fig. 3.5A, B). Clearly visible were the two migratory streams of interneurons along the SVZ and along the lower IZ of the neocortex; GABAergic interneurons are known to migrate via distinct and independent streams before diving and incorporating into the neocortex, and gene expression is thought to differ between migratory streams (Antypa et al., 2011; Tamamaki et al., 1997). Interestingly, while neurons migrating along the basal stream in the SVZ neither expressed p75NTR themselves, nor were following p75NTR-expressing fibers, neurons in the apical stream were observed forming tight contact with these fiber tracks (Fig. 3.5B’). Furthermore, neurons found in the upper intermediate and lower MZ of the neocortex were usually p75NTR-positive themselves, indicating that they were not only following p75NTR-expressing substrates but are also capable of intrinsic p75NTR signalling (Fig. 3.5B’’).

Figure 3.5: p75NTR is expressed in dorsolaterally projecting fiber tracks and in tangentially migrating neurons in the cortical intermediate and marginal zone. Representative immunohistochemical stainings for p75NTR and tdTomato in E16.5 Ascl1-creERT2 tdTomato flox- stop-flox (Ascl1-creERT2 tdTomfl/fl) mice, 5 days after the administration of tamoxifen. A, B) Low resolution images of tdTomato (A) and tdTomato co-stained with p75NTR (B) in the dorsal area of the lateral ganglionic eminence and the ventral region of the neocortex. Asterisks mark an accumulation of tdTomato-positive cells in the ventral telencephalon from where neurons migrate. White and red arrowheads in A) mark the basal and apical migratory streams of GABAergic interneurons, respectively. Red dotted outline in B) indicates region of high-resolution image in B’. Scale bars: 500 µm. B’) High-resolution image of migrating interneurons in the cortical intermediate and marginal zone. White arrowheads indicate cells that are interacting with their leading edge with p75NTR-positive fibers tracks, red arrowheads mark cells that are both positive for tdTomato and for p75NTR. Red dotted outline indicates zoom region displayed in B’’). Scale bar: 50 µm. B’’) Representative image of tdTomato-positive neurons in the cortical marginal zone that express p75NTR. Dotted outlines indicate the typical morphology of tangentially migrating interneurons. Scale bar: 10 µm.

46 3.3 Discussion

This chapter reviewed the literature on spatiotemporal p75NTR expression pattern in the developing and adult mouse brain and defined its expression in different cell types in the early developing mouse brain using immunohistochemistry. We found that p75NTR expression is scarce in RPGs and increases during the progression from IPCs to neurons. Specifically, p75NTR is upregulated in a subpopulation of Ascl1-positive IPCs at the border of the SVZ/MZ of the ganglionic eminences, a location that correlates with that of beta III tubulin-positive p75NTR-positive neurogenic progenitors. Strongest expression of p75NTR is found in post-mitotic neurons, some of which migrate tangentially in the IZ/MZ of the neocortex along p75NTR-positive fibers. The precise identity of the cell population forming the p75NTR-positive fiber tracks remains to be determined, but the pattern of innervation strongly indicates that they are part of early thalamocortical connections. Studying the formation of these connections in a Tau-GFP-expressing mouse line revealed that cortical subplate neurons innervate the thalamus as early as E14.5, and their pattern resembles that of p75NTR-positive fiber tracks (reviewed in Grant et al., 2012). Furthermore, transient cortical subplate neurons are known to express p75NTR (Allendoerfer et al., 1990), and it is therefore likely that the p75NTR-positive fiber tracks belong to cortical subplate axons ingrowing the thalamus. Change in expression levels during transition from progenitor to neuron can have different causes. Principally, the upregulation of p75NTR levels in post-mitotic cells compared with progenitors can mean that either a) certain pathways regulated by p75NTR need stronger activation in neurons, b) p75NTR fulfils a different function after neurogenic divisions are complete, or c) p75NTR upregulation in neurogenic progenitors is needed for terminal differentiation. Indeed it has been proposed that specific protein levels are more important for function than their mere presence or absence (Geiger et al., 2012). The expression pattern described in this chapter as well as the large amount of evidence on p75NTR function in cell cycle regulation and differentiation make option c) the most likely. The following chapters explore the function that p75NTR plays in progenitors of the developing mouse brain as well as the mechanism by which p75NTR may regulate neuronal differentiation.

Contribution to this chapter by others:

Sample preparation, Nissl staining, and measurements of the whole brain section images included in Fig. 4.4A-C were performed by Dr. Fabienne Alfonsi according to the methods described in Chapter 2, section 2.5. Dr. Nyoman Kurniawan performed the small animal magnetic resonance imaging and assisted with the analysis of MRI data included in Fig. 4.4D-I, according to the methods described in Chapter 2, section 2.9. The Golgi Cox staining of the samples used for analysis in Fig. 4.5 were performed by Michael Milne, according to the methods described in Chapter 2, section 2.7. Sample preparation, immunohistochemical staining, and analysis of the data presented in Fig. 4.7 were performed by Dr. Fabienne Alfonsi. Conception and design of the experiments (except for data included in Fig. 4.7), and interpretation of the results were performed by the thesis author. Prof. Elizabeth Coulson contributed intellectually to data interpretation and conclusions.

Chapter 4: Cell-autonomous and non cell-autonomous effects of p75NTR signaling regulate normal formation of the neocortex and basal forebrain

4.1 Introduction

4.1.1 Chapter overview

In the previous chapter, we reported that p75NTR is expressed in the developing mouse telencephalon, with expression in the ventral brain starting ~2 days prior to the neocortex. The dorsal progenitor zones as well as the ganglionic eminences give rise to cortical neurons; however, it is unclear what role(s) p75NTR plays during cortical development. As the functional outcome of p75NTR signaling is highly dependent on cellular context, we examined the in vivo effect of conditional p75NTR gene deletion from neural precursors from E10.5 using our p75NTR floxed mouse strain (p75fl/fl; Boskovic et al., 2014) crossed to three different cre deleter lines. In these novel conditional knockout strains, p75NTR expression is either deleted in all neuronal progenitors, in dorsal progenitor zones only, or exclusively in the MGE and POA. To characterize the phenotypical outcome of p75NTR deletion in the adult brain, we measured the volume of different brain regions, counted the number of neuronal subpopulations, and assessed neuronal morphology in conditional p75NTR knockout and control mice. Particularly, the focus of this study was p75NTR function in the development of the neocortex.

4.1.2 Knockout strategy and conditional p75NTR knockout mouse strains

Nestin-Cre p75in/in mice To achieve p75NTR knockout in all neuronal progenitors and to assess the effect of nervous system-specific conditional deletion, we crossed our p75fl/fl mice to a Nestin-Cre deleter line (Nestin-Cre; Dubois et al., 2006). Nestin is a type VI intermediate filament that is expressed in progenitor cells of the neuronal lineage in the developing and adult brain (Lendahl et al., 1990). Nestin-cre-driven recombination is observed from around E10.5 onward (Dubois et al., 2006), resulting in knockout of p75NTR in progenitor cells of the neuronal lineage from early embryogenesis onwards.

Emx1-iCre p75in/in mice To assess the effect of intrinsic p75NTR loss in progenitors of excitatory cortical projection neurons, we crossed p75fl/fl mice to an Emx1-iCre deleter line (Kessaris et al., 2006).

51 Expression of the Emx1 homeobox gene transcription factor is mostly restricted to the dorsal progenitor zones of the telencephalon (Simeone et al., 1992). Emx1 transcripts are first detectable around E9.5 (Gulisano et al., 1996), therefore allowing cre-driven recombination to occur roughly at the same developmental time point as in Nestin-Cre p75in/in mice, but limited to the cortical progenitor population.

Nkx2.1-iCre p75wt/in mice The second major neuron subtype in the cortex besides projection neurons are GABAergic inhibitory interneurons that are generated in the ganglionic eminences of the ventral telencephalon. To assess the effect of p75NTR knockout on interneuron progenitors, we crossed p75fl/fl mice to an Nkx2.1-iCre deleter line (Kessaris et al., 2006). Nkx2.1 is a homeobox transcription factor expressed in the MGE and POA, the birthplace of cortical PV and SST interneurons that together account for ~70% of all cortical interneurons (Rudy et al., 2011). By crossing Nkx2.1-iCre mice to our p75fl/fl strain, a heterozygous conditional deletion of p75NTR in progenitors of the MGE and POA from E9.5 onwards was achieved. Heterozygous p75NTR knockout animals (Nkx2.1-iCre+/-; p75wt/in) were infertile and therefore no homozygous animals were generated. All analyses were performed using the heterozygous knockout mice.

52 4.1.3 Chapter aims and hypothesis

This chapter explores the phenotypic outcome of early embryonic p75NTR deletion in adult conditional knockout mice. Specifically, we aimed to answer the following questions:

 Is p75NTR expression in neuronal progenitors required for normal formation of the brain and are all brain regions equally dependent on p75NTR signaling during development?

 What effect does p75NTR gene deletion have on different neuronal populations?

 Does p75NTR signaling play a generic role in neuronal progenitors or are there cell type specific differences?

Based on the expression data from chapter 3, we hypothesised that p75NTR plays a crucial role in the development of the brain. This chapter explores the consequences of p75NTR knockout during early neurogenesis in all neuronal progenitors as well as distinct progenitor subpopulations.

53 4.2 Results

4.2.1 p75NTR deletion in conditional knockout mouse strains is efficient and specific

The efficiency of the knockout in Nestin-Cre p75in/in, Emx1-iCre p75in/in, and Nkx2.1-iCre p75wt/in mice was assessed by staining for the marker mCherry in embryonic brain sections at E12.5 and E14.5 (Fig. 4.1). In Nestin-Cre p75in/in mice, mCherry expression was detected as early as E11.5 (data not shown). By E12.5, robust recombination was observed in all progenitor zones of the ventral telencephalon, including the ganglionic eminences, POA, and septum (Fig. 4.1A). Similar to the pattern of wild type p75NTR expression, mCherry signal was also detectable in the neocortex from E14.5. Concurrent with the appearance of mCherry, loss of p75NTR signal was observed in neuronal progenitors, but not in non- neuronal tissues such as the mesenchyme (Fig. 4.1A), indicating that p75NTR knockout strategy is efficient as well as specific. In Emx1-iCre p75in/in mice, p75NTR knockout was assessed at E14.5. Robust expression of mCherry was detected in the neocortex while p75NTR expression was lost reliably (Fig. 4.1B). Interestingly, mCherry was also expressed in fibers projecting dorso-laterally from the ventral telencephalon to the neocortex, indicating that they originate from an Emx1-positive cell population distinct from progenitors found in the neocortex (Fig. 4.1B). Previous lineage tracing analyses of Emx1-positive progenitors have identified a striatal subpopulation that, in contrast to the ventro-dorsal migratory stream of interneurons, migrate from the developing neocortex into the LGE (Cocas et al., 2009). They are primarily localised to the dorsal striatum and connect to neurons in layer V of the neocortex. The Emx1-lineage- derived population of neurons extending p75NTR-positive fibers towards the neocortex may therefore be part of the cortico-striatal axis. Other ventrally localised mCherry-positive cell populations were not observed. In contrast to Nestin-Cre p75in/in and Emx1-iCre p75in/in mice, partial p75NTR expression was retained in Nkx2.1-iCre p75wt/in mice due to infertility of heterozygous knockout animals. The majority of mCherry-positive cells were detected in the MGE and POA, indicating that the knockout was restricted to Nkx2.1-positive progenitors, as expected (Fig. 4.1C). Although mCherry expression was also observed in isolated cells in the LGE, the morphology of these cells with clearly distinguishable leading and trailing edge suggest that they represent a population of migrating interneurons that have left the MGE after recombination took place rather than a non-specific event in Nkx2.1-negative progenitors (Fig. 4.1C). Fewer mCherry- expressing cells were also detected in the septum, representing a population of cholinergic neurons and interneurons of the presumptive basal forebrain nuclei (Fig. 4.1C).

55 Figure 4.1: Qualitative assessment of p75NTR expression loss in three conditional knockout strains. Representative images of immunohistochemical stainings for p75NTR and mCherry in different brain regions of Nestin-Cre p75in/in, Emx1-iCre p75in/in, and Nkx2.1-iCre p75wt/in mice. A) mCherry expression and loss of p75NTR signal in the ventral telencephalon at E12.5 and in the neocortex at E14.5 in Nestin-Cre p75in/in mice. Arrows indicate mesenchymal cells in which p75NTR expression was retained. Scale bars: 200 µm. B) mCherry expression and loss of p75NTR signal in the neocortex of Emx1-iCre p75in/in mice at E14.5. Arrows indicate mCherry-positive dorsolateral fibers. Scale bars: 100 µm. C) mCherry expression in the MGE and POA (top panel) and mCherry and p75NTR expression the basal forebrain (lower panel) at E12.5 in Nkx2.1-iCre p75wt/in mice. Arrows indicate mCherry-positive cells that migrated out of the MGE. Scale bars: 100 µm. V= ventricle

56 4.2.2 Nestin-Cre p75in/in mice have decreased brain volume with a disproportionate reduction of the neocortex and basal ganglia

To assess the phenotypic outcome of p75NTR knockout in neuronal progenitors in adult mice, we performed basic characterization of Nestin-cre p75in/in mice at 2-6 months of age. Although born in the expected Mendelian ratio and equal sex ratio (Fig. 4.2A, B), Nestin-cre p75in/in mice were noticeably smaller than their p75fl/fl littermates and animals of the Nestin- Cre parental strain (Fig. 4.2C, D). However, no behavioural differences were observed in basic testing of anxious behaviour (elevated plus maze test), and cognition (Y-maze novel arm recognition test) (Fig. 4.3A, B). Furthermore, with the exception of weaker hind paw grip strength in Nestin-cre p75in/in mice, no overt motor function deficits were observed compared to controls (Fig. 4.3C), and adult mice were fertile. Histological analysis of adult Nestin-Cre p75in/in mice revealed a significantly decreased brain volume, with a decrease in the thickness of the cortex (-29%) and the volume of the caudate putamen (-42%) compared to control p75fl/fl animals (Fig. 4.4A-C) and those from the Nestin-Cre single line (data not shown). As expected, p75NTR protein expression was undetectable in the basal forebrain of the adult Nestin-Cre p75in/in animals, further confirming the success of the knockout strategy in neuronal cells (data not shown; see Boskovic et al., 2014). In order to characterise the altered brain architecture and accurately measure the brain volume of the Nestin-Cre p75in/in mice, 3D images of knockout and control brains were acquired by ex vivo MRI. The volume of the whole brain and individual areas, including the cerebral cortex, hippocampus, basal forebrain, diencephalon (including the thalamus, epithalamus, and hypothalamus), and cerebellum, were measured using registered C57/BL6 MRI atlases. Analysis of MRI imaging revealed structural abnormalities in the brain of adult p75NTR knockout animals, including dilated lateral and 4th ventricles and a thalamic fissure along the midline, that were not present in control mice (Fig. 4.4D-F). Consistent with the results of histological analyses, the volumetric measurements revealed that the whole brain volume was significantly reduced in Nestin-Cre p75in/in animals compared to controls (-31.9 ±2.2%; Fig. 4.4G). The subregions were also reduced in volume, although to varying extents (Fig. 4.4H). The neocortex and basal forebrain were the most severely affected, with a reduction of 40.0 ±2.2% and 41.2 ±3.6%, respectively, whereas the cerebellum was found to be only 27.4 ±1.2% smaller than in control animals. Given that Nestin-Cre p75in/in mice were stunted in growth, the volumes of the measured brain areas were normalized to the whole brain volume (Fig. 4.4I). This revealed that the volumes of the neocortex and the basal forebrain of knockout mice were disproportionately smaller relative to the entire brain, whereas the cerebellum, despite being smaller than the control, was proportionally larger

57 when measured against the entire brain. The difference in severity of the affected brain areas likely reflects different roles of p75NTR in the development of each structure.

Figure 4.2: Nestin-Cre (NesCre) p75in/in mice show reduced growth and are born in Mendelian ratios. A, B) Distribution of genotypes (A) and gender (B) of p75fl/fl x NesCre p75fl/wt offspring. Data collected from 20 litters, N=125. Deviation from expected percentage is not statistically significant (Chi2 value=4.7097 for p=0.05 and 3 degrees of freedom). C) Weight measurement of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice at 6 months of age. Student’s t-test, * p<0.05. D) Images of control (p75fl/fl), cre-control (NesCre), and knockout (NesCre p75in/in) mice at P60, depicting the difference in size.

60 Figure 4.3: Basic behavioural analysis of adult Nestin-Cre (NesCre) p75in/in mice. A) Elevated plus maze test. Mice were allowed to explore the maze for 5 min and the total time spent in each zone (cumulative duration), the frequency of entering each zone, the time spent moving / not moving, the frequency of alternating between moving / not moving, total distance moved, and the latency to first entry into open and closed arms was measured. B) Y-maze novel arm recognition test. On the first day, mice were placed in one arm of the maze (home arm) with one arm closed off and allowed to explore for 10 min. The next day, mice were placed in the home arm of the maze with both arms open for 5 min. The duration spent in the home and novel arm and the centre, the frequency of entering each zone, and the latency of first entry into each zone was measured. C) Basic motor function assessment using rotarod and grip strength of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice. The mice were placed on a rotating rod. The rotation speed was gradually increased and the time until the mice fell off the rod was measured. Each mouse was tested 3 times and the average time to the first fall was calculated. Grip strength was measured by evenly pulling the mice across a bar attached to a grip strength meter, either with hind- or forepaws. Each mouse was tested 3 times and the average grip strength for each limb pair was calculated. Grip strength units in N. Mice were tested at 6 months of age, N=6 for all experiments, mean ± SEM, Student’s t-test.

Figure 4.4: Reduced brain volume in Nestin-Cre (NesCre) p75in/in mice. A) Nissl staining of control (p75fl/fl) and knockout (NesCre p75in/in) mouse coronal brain sections illustrating reduced brain size and altered structures of the knockout mice. Scale bars: 1 mm. B, C) Quantification of cortical thickness and the volume of the caudate putamen (CPu) from histological sections (N=4 mice per genotype). Student’s t-test. D-F) MRI contrast images illustrating the atrophic brain structure and enlarged ventricles in a NesCre p75in/in mouse compared to a control mouse at 3 months of age. Scale bars: 2 mm. D) Axial view comparing the 4th ventricle (arrowhead) and lateral ventricles (arrow) in NesCre p75in/in and control mice. E) Coronal view highlighting the thalamic fissure (arrow) present in a NesCre p75in/in mouse. F) Sagittal view showing tissue reduction and large internal cavity (arrows) in a NesCre p75in/in mouse. G) Quantification of the whole brain volume of control and NesCre p75in/in mice. H) Reduction in volumes of individual brain regions in NesCre p75in/in mice as a percentage of controls. I) Volume of individual brain regions normalized to whole brain volume showing relative change compared to control. N=3 mice, Student’s t-test * p<0.05 ** p<0.01 *** p<0.001

62 4.2.3 p75NTR is required for the development of cortical interneurons and upper-layer pyramidal neurons

To determine the reason for the reduced cortical volume, coronal brain sections of Nestin- Cre p75in/in and littermate control mice were stained using the Golgi Cox silver method. Low- resolution images suggested a reduced number and disorganization of pyramidal neurons in the upper layers of the cortex (Fig. 4.5A). The morphology of the pyramidal neurons was also altered (Fig. 4.5B). Quantification and Sholl analyses of total dendrites, soma size, and number of dendritic trees revealed no significant differences between genotypes (Fig. 4.5C, D). However, when analysed separately, the apical dendrites of layer V neurons in Nestin- Cre p75in/in mice showed increased overall dendritic complexity compared to those of p75fl/fl mice, displaying longer total dendritic length (+50.3 ±7.3%) and a higher total number of dendritic nodes (+48.5 ±10.7%) and endings (+43.3 ±10.3%; Fig. 4.5E-G). Nonetheless, these results suggested that a reduction in cell number rather than cell complexity may be the primary reason for the reduction in cortical volume in Nestin-Cre p75in/in mice. Next, we investigated whether the six layered neocortical laminar architecture in the primary somatosensory cortex was affected in p75NTR knockout mice. p75fl/fl and Nestin-Cre p75in/in brains were stained with the nuclear marker DAPI (Fig 4.6A). Although the thickness of each layer in the knockout mice was significantly reduced compared to that in the control animals, the change was most pronounced in layers II/III and IV, which were reduced by 37.7 ±1.2% and 42.6 ±6.7% respectively, whereas the deeper layers (V, VI) were less affected (Fig. 4.6B). When normalized to absolute cortical thickness, layers II/III and IV of Nestin-Cre p75in/in cortices remained reduced by 25.4 ±1.3% and 31.2 ±6.6%, respectively, compared to those of controls (Fig. 4.6C). No significant difference in the normalized thickness of layers I or VI was observed between genotypes, whereas the relative thickness of layer V of Nestin- Cre p75in/in mice was increased by 9.9 ±0.9% compared to that in control animals (Fig. 4.6C), potentially reflecting the increased apical dendritic complexity of pyramidal neurons residing in this layer. To determine the pyramidal cell number in the upper and lower layers, histological sections of the somatosensory cortex were stained with the layer markers Tbr1 and Ctip2, respectively. Tbr1 identifies neurons in layers II/III and V/VI, while Ctip2 labels neurons residing in layers V/VI (Arlotta et al., 2005; Bulfone et al., 1995; Rubenstein et al., 1999). Apart from the obviously reduced upper layer thickness, the laminar architecture of pyramidal neuronal subtypes was grossly normal in the knockout mice (Fig. 4.6D). Furthermore, the density of DAPI-stained nuclei in layers II/III, as well as the number of nuclei and Ctip2-positive neurons in layer V were not significantly different (Fig. 4.6E, F).

63 This indicates that the reduction in layer thickness is due to a decrease in absolute cell number. In particular, the number of later-born pyramidal cells within the upper cortical layers was more severely reduced by the developmental loss of p75NTR than the number of early-born deep layer pyramidal neurons.

Figure 4.5: Golgi silver stain showing changed neuronal morphology in Nestin-Cre (NesCre) p75in/in mice. A) Low resolution light microscopy image of a Golgi silver-stained coronal section of the neocortex of adult control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice at 6 months of age. Scale bars: 500 µm. B) Representative tracing images of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) layer V neurons. C) Sholl analysis of the number of intersections of total dendritic trees of layer V pyramidal neurons. D) Quantification of total number of dendritic trees and soma size of layer V pyramidal neurons. E) Sholl analysis of the number of intersections and F) total length of layer V pyramidal neuron apical dendrites. G) Quantification of number of nodes and endings, complexity (arbitrary values), and total length of layer V pyramidal neuron apical dendrites. N=20 (4 animals were analyzed, 5 neurons traced per animal, mean ± SEM). Sholl analyses were examined using a two-way ANOVA test. An alpha level of 0.05 was considered significant. Significant effects and interactions were further analyzed using the Holm-Sidak's post hoc multiple comparisons test. Student’s t-tests were used for comparisons of single parameters between the two groups. * p<0.05

Figure 4.6: Immunohistochemical analysis of cortical layering in Nestin-Cre (NesCre) p75in/in mice. A) Image of DAPI-stained coronal sections of the somatosensory cortex of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice at 3 months of age. Cellular density changes between layers were used to delineate layer boundaries. Scale bars: 400 µm. B) Quantification of layer thickness (absolute) of the somatosensory cortex of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice. 5 matched sections per brain were measured. C) Quantification of layer thickness normalized to whole cortical thickness. D) Coronal sections of the brains of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice immunostained for the cortical layer markers Ctip2 and Tbr1. Scale bars: 400 µm. The cell density in layer II/III (E) and layer IV (F) was measured by counting DAPI-positive nuclei (layer II/III) or Ctip2-positive cells (layer IV) and normalizing to area size. Cell counts were taken in 5 square areas per section in 3 sections per mouse. N=3 mice per genotype, mean ± SEM, Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001, n.s: not significant

66 4.2.4 p75NTR is required for the development of neurons of the ventral telencephalic lineage

We next analyzed the number of GABAergic interneurons, the second major neuronal type, in the cortex. Brain sections of adult control and Nestin-Cre p75in/in mice were stained for the interneuron markers PV, SST, NPY and calretinin (Fig. 4.7A). The number of interneurons of all subtypes analyzed were significantly reduced in the cortex of Nestin-Cre p75in/in mice compared to control mice (Fig. 4.7B). In particular, interneuron subtypes that are derived from the MGE and POA were found to be most severely affected, with a reduction of 50.1 ±4.8% for PV and 44.1 ±3.0% for SST interneurons, respectively. To assess whether other neurons derived from the MGE and POA (Anderson et al., 1997; Marin et al., 2000; Olsson et al., 1998; Zhao et al., 2003) were affected by the loss of p75NTR, the number of cholinergic basal forebrain neurons, striatal cholinergic neurons, and striatal PV-positive interneurons were counted (Fig. 4.7C, D). The absolute number of cholinergic basal forebrain neurons was significantly reduced (-32.7 ±9.5% compared to control), as was the number of striatal neurons (-64.0 ±3.6% and -26.7 ±3.8% for PV-positive neurons and cholinergic neurons, respectively) (Fig. 4.7E, F). However, when adjusted for the size of the respective brain areas, the number of cholinergic neurons was not changed in the basal forebrain compared to control, and the number of ChAT-positive neurons in the striatum was proportionally increased in the knockout animals (+18.5 ±3.8%) (Fig. 4.7E, F). In contrast, the number of PV-positive interneurons in the striatum was proportionally decreased compared to the control. These findings demonstrate that the loss of p75NTR in nestin-expressing neural stem cells culminates in deficits to both dorsal and ventral telencephalic lineages, highlighting the critical role of p75NTR in cortical development.

Figure 4.7: Nestin-Cre (NesCre) p75in/in mice have reduced numbers of interneurons and cholinergic neurons. A) Representative images of coronal brain sections of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice immunostained for the interneuron markers parvalbumin (PV), somatostatin (SST), neuropeptide Y (NPY) and calretinin (CR) at P60. B) Quantification of interneuron subtype numbers in the somatosensory cortex in a 400 µm wide region of interest spanning all layers. C) Representative images of basal forebrain sections immunostained for choline acetyltransferase (ChAT) and p75NTR (in control) or mCherry (in Nestin-Cre p75in/in mice). D) Representative images of coronal sections at the level of the striatum of control (p75fl/fl) and knockout (Nestin-Cre p75in/in) mice immunostained for ChAT and PV. E) Quantification of the total number of cholinergic basal forebrain neurons (total cell count of every 3rd section) and absolute cell number normalized to the average basal forebrain volume of each strain measured by MRI. F) Quantification of cholinergic neurons and PV-positive interneurons in the striatum (total cell count of every 3rd section) and cell number normalized to the average striatal volume of each strain measured by MRI. N=5 mice, mean ± SEM, Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001, scale bars: 100 µm

68 4.2.5 Heterozygous knockout of p75NTR in MGE derived progenitors reduces cortical layer thickness and the number of PV interneurons

Since p75NTR deletion in Nestin-Cre p75in/in mice affects all neuronal progenitors, it is likely that some of the observed phenotypes are the results of non cell-autonomous effects. We therefore investigated how deletion in specific neuronal populations affected their respective development while p75NTR expression in other neuronal subtypes remained unchanged. To investigate the effect of p75NTR deletion on cortical projection neurons and cortical interneurons, we crossed our p75fl/fl mice to an Emx1-iCre or a Nkx2.1-iCre deleter line. In both strains, recombination is initiated around E10 and gene deletion is largely restricted to distinct progenitor zones of either the neocortex or MGE and POA, respectively. The resulting homozygous Emx1-iCre p75in/in mice were indistinguishable in appearance from their wild type littermates and were fertile. Heterozygous Nkx2.1-iCre p75wt/in mice however were not born in mendelian ratios, yielding only ~8% iCre-positive animals when a ratio of 50% was expected. Furthermore, iCre-positive animals were noticeably overweight compared to p75wt/fl littermate controls (Fig. 4.8) and did not breed. Since neither the Nkx2.1- iCre nor the p75fl/fl parental strain expressed any of the observed phenotypes (increased embryonic lethality, obesity, and infertility), haploinsufficiency of p75NTR in tissues such as the thyroid or lung may have caused these effects in Nkx2.1-iCre p75wt/in mice. We therefore used the heterozygous strain for all further analyses.

Figure 4.8: Image of control (p75wt/fl), and heterozygous knockout (Nkx2.1-iCre p75wt/in) mice at P60, illustrating the increased body mass of Nkx2.1-iCre p75wt/in mice.

69 To assess whether the Emx1-iCre p75in/in and the Nkx2.1-iCre p75wt/in strains partially recapitulated the cortical phenotype observed in Nestin-Cre p75in/in mice, brains of adult p75fl/fl, Emx1-iCre p75in/in, and Nkx2.1-iCre p75wt/in mice were stained for the nuclear marker DAPI, the cortical layer marker Tbr1, and the interneuron marker PV. Cortical thickness and neuron number were measured in the dorsal and lateral area of the somatosensory cortex (Fig. 4.9A-D). Total cortical thickness was not significantly changed in either strain compared to p75fl/fl controls (Fig. 4.9E, F). However, measuring individual layers revealed that deep layer V of the dorsal somatosensory cortex was reduced significantly in Nkx2.1-iCre p75wt/in mice compared to controls (-20.6 ±6.9%, Fig. 4.9G). In the lateral somatosensory cortex, the upper layers II-IV were also reduced in thickness, albeit to a smaller degree (-11.0 ±2.7%, Fig. 4.9H). Interestingly, loss of p75NTR in cortical projection neuron progenitors in Emx1-iCre p75in/in mice did not affect cortical thickness, indicating that this phenotype in Nestin-Cre p75in/in mice is not solely caused by intrinsic loss of expression in the dorsal telencephalon. To assess whether cortical interneurons derived from the MGE and POA were affected by loss of p75NTR expression in our knockout strains, the number of PV interneurons in the whole width of the cortex as well as in individual cortical layers was counted (Fig. 4.9I-L). In Nkx2.1-iCre p75wt/in mice, the total number of PV neurons was reduced by 28.6 ±3.0% dorsally and 35.2 ±4.5% laterally, compared to controls. When analysed according to cortical layers, PV interneuron number in the dorsal region of interest was most significantly reduced in deep layer V (-39.9 ±5.8%), potentially reflecting the reduction in thickness observed in this layer. In the lateral region of interest, PV interneurons were reduced in the upper layers II-IV (-40.3 ±7.2%) as well as the deep layers V and VI (-28.4 ±8.1% and -39.0 ±5.1%, respectively). These results indicate that p75NTR expression in the MGE and POA is required for development of the appropriate number of cortical interneurons and that the number of interneurons may correlate with cortical layer thickness. Interestingly, while PV interneuron number was not significantly changed in layers of the dorsal somatosensory cortex in Emx1- iCre p75in/in mice, a slight albeit significant reduction was observed in the upper layers of the lateral somatosensory cortex (-22.4 ±6.7%). This suggests that, although PV interneuron number is largely normal in these mice, loss of p75NTR in dorsal progenitors affects a subpopulation of interneurons in upper layers. Overall, this study suggests that the effect of heterozygous p75NTR knockout in ventral progenitors has a profound impact on cortical development and that intrinsic expression in dorsal progenitors is not required for regulation of cortical neuronal number.

71 Figure 4.9: Deep layer thickness and number of PV interneurons is decreased in Nkx2.1-iCre p75wt/in mice. A, B) Schematic representations of coronal plane used for analysis. Region of dorsal (A) and lateral (B) measurements indicated in red. C, D) Coronal sections of the brains of control (p75fl/fl) and knockout (Emx1-iCre p75in/in and Nkx2.1-iCre p75wt/in) mice immunostained for the cortical layer marker Tbr1 and interneuron marker parvalbumin (PV) in both regions of interest. Scale bars: 200 µm. E, F) The total cortical thickness was measured from the pial to the apical surface in both regions of interest. 1-way ANOVA followed by Dunnett’s post-hoc test. G, H) Tbr1 staining was used to identify the borders of layers I, II-IV, V, and VI, and the thickness of each layer was measured in both regions of interest. 2-way ANOVA followed by Dunnett’s post-hoc test. I, J) The total number of PV interneurons was measured from the pial to the apical surface in a 500 µm wide area of the cortex in both regions of interest. 1-way ANOVA followed by Dunnett’s post-hoc test. K, L) Tbr1 staining was used to identify the borders of layers I, II-IV, V, and VI, and the number of PV interneurons in each layer was counted in a 500 µm wide region of the cortex. 2-way ANOVA followed by Dunnett’s post-hoc test. Cell counts were taken in 3 sections per mouse. N=4 mice per genotype, mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

72 4.2.6 Heterozygous loss of p75NTR in Nkx2.1-positive progenitors leads to a reduced number of cholinergic basal forebrain neurons but not cortical SST interneurons

In addition to PV interneurons, the progenitor zones of the MGE and POA also give rise to SST interneurons and a subpopulation of cholinergic basal forebrain neurons (Magno et al., 2017). To investigate whether other neuronal types derived from Nkx2.1-positive progenitors are affected by heterozygous p75NTR knockout, we counted the number of SST and calretinin interneurons in the cortex and cholinergic neurons in basal forebrain nuclei (medial septum (MS), vertical diagonal band of Broca (VBD), horizontal diagonal band of Broca (HDB), Fig. 4.10A) of adult Nkx2.1-iCre p75wt/in mice. The area of brain sections of Nkx2.1-iCre p75wt/in mice was not reduced compared to controls indicating that volume of the forebrain was largely normal (Fig. 4.10B). However, the number of cholinergic basal forebrain neurons in the VDB and HDB was significantly reduced in Nkx2.1-iCre p75wt/in mice (-53.5 ±7.7% in VDB and -59.6 ±10.3% in HDB, respectively, Fig. 4.10C, D), indicating that development of these neurons are also dependent on p75NTR expression. Interestingly, no change was found in the number of SST interneurons (Fig. 4.10E, F). The number of calretinin interneurons, which are not derived from Nkx2.1-positive progenitors, was also unchanged (Fig. 4.10E, F). These results suggest that reduction of p75NTR expression in Nkx2.1- positive progenitors during early neurogenesis selectively affects the number of cortical PV interneurons and cholinergic basal forebrain neurons, but not that of SST interneurons.

74 Figure 4.10: Nkx2.1-iCre p75wt/in mice have reduced numbers of cholinergic neurons and PV interneurons. A) Schematic representation of a coronal mouse brain section at the level of the basal forebrain. The basal forebrain is outlined in green and arrows indicate the nuclei of the medial septum (MS), and the vertical and horizontal diagonal band of Broca (VDB/HDB). B) Quantification of section area measured in control (p75fl/fl, N=6) and knockout (Nkx2.1-iCre p75wt/in, N=5) mice. 3 coronal sections per mouse were measured in the rostral region shown in (A). Student’s t-test. C) Representative images of basal forebrain sections immunostained for choline acetyltransferase (ChAT) in control (p75fl/fl) and knockout (Nkx2.1-iCre p75wt/in) mice. Scale bars: 500 µm. D) Quantification of the total number of cholinergic basal forebrain neurons (total cell count of every 3rd section), and per subregion in control (p75fl/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) mice. 2-way ANOVA followed by Sidak's post-hoc test, N=3 mice per genotype. E) Representative images of a region of interest in the somatosensory cortex of control (p75fl/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) mice immunostained for the interneuron markers parvalbumin (PV), somatostatin (SST), and calretinin (CR). Scale bars: 200 µm. F) Quantification of interneuron subtype numbers in the somatosensory cortex in an equally sized region of interest across all sections. Cell counts were taken in 3 sections per mouse. Student’s t-test, N=6 mice per genotype. Mean ± SEM, *** p<0.001, *** p<0.0001

75 4.3 Discussion

Nestin-Cre p75in/in mice Our investigation of the neurodevelopmental function of p75NTR, using a mouse strain in which deletion of the p75NTR gene is specific to neuronal progenitors shortly before the onset of neurogenesis (Dubois et al., 2006), revealed a range of phenotypes. The most obvious phenotype observed in adult Nestin-Cre p75in/in mice was their reduced size. A similar decrease has been reported upon deletion of p75NTR exon IV (von Schack et al., 2001b), but not in p75NTR exon III-deficient mice (Lee et al., 1994b). However the latter mice can retain expression of a functional truncated version of p75NTR, which may account for the discrepancy in these observations (Boskovic et al., 2014; von Schack et al., 2001b). Although nestin is predominantly expressed in the progenitors of the developing brain, there is evidence for its expression in other tissues, including developing cardiomyocytes, presomitic mesoderm, myotome and dermatone and endothelial cells of developing blood vessels (Wiese et al., 2004). It is therefore conceivable that a secondary effect of p75NTR knockout in other non-neuronal tissues, such as defects in the vascular system, may have contributed to the reduced body size of the Nestin-Cre p75in/in mice. Alternatively, the parental Nestin-Cre strain has been reported to have reduced body size, thought to be the result of mild hypopituitarism (Declercq et al., 2015). Although this phenotype was not apparent in our parental strain or Nestin-Cre p75wt/in heterozygous mice, it is possible that the reduced body size is unrelated to the loss of p75NTR, and the overall smaller brain in the mutant mice was an indirect effect. To correct for this possibility, the relative change in brain region sizes was assessed by normalizing the data to the whole brain, revealing disproportionate reduction in the volume of the cortex and basal ganglia, correlating with reduced numbers of cortical pyramidal neurons, interneurons, and striatal and basal forebrain neurons. The multitude of phenotypes observed in the Nestin-Cre p75in/in mice likely reflects different roles that p75NTR plays in the development of these structures and indicates that its expression is required for their adequate formation. As the neocortex was one of the structures that was most severely affected by the embryonic loss of p75NTR, and a specific role for p75NTR in the development of this area has not been described, we focussed our study on the changes that occurred during early cortical development. The neocortex of adult Nestin-Cre p75in/in mice contained signficantly fewer later-born, upper layer neurons than control mice, but equivalent numbers of early-born pyramidal neurons when normalized to total cortical thickness. Cortical layers are formed in an inside-out fashion during development, creating the deep layer neurons first, followed by the upper layers (Gilmore and Herrup, 1997). A reduction in upper layer size therefore reflects a defect

76 in later-born neurons rather than early-born populations. However, deeper layer pyramidal neurons displayed increased apical dendritic complexity. Increased dendritic complexity has previously been reported for hippocampal neurons lacking p75NTR (Zagrebelsky et al., 2005), and this phenotype could therefore be due to an intrinsic lack of p75NTR in these cells, although other, non cell-autonomous effects, such as reduced inhibitory control or compensation for the reduced number of upper layer pyramidal neurons, are also possible explanations for the dendritic complexity phenotype. Furthermore, the number of PV and SST expressing cortical interneurons, as well that of cholinergic neurons derived from the ventral lineage, were significantly reduced in Nestin- Cre p75in/in mice. However, when normalized to the volume of the respective brain areas, the number of cholinergic basal forebrain neurons was unchanged in the basal forebrain, whereas in the striatum it was proportionally increased in the knockout animals. It has previously been shown that a conditional knockout of p75NTR in a ChAT-Cre p75in/in mouse strain leads to an increase in cholinergic neurons in the basal forebrain due to inhibition of naturally occurring cell death (Boskovic et al., 2014). Therefore, the loss of progenitors of cholinergic neurons might be partially compensated during postnatal development. In contrast, the number of PV-positive interneurons in the striatum was proportionally decreased compared to the control, suggesting that the loss of interneuron progenitors cannot be compensated by a similar mechanism.

Emx1-iCre p75in/in mice Given that cortical projection neurons, cortical interneurons, and cholinergic neurons of the basal forebrain and striatum originate from spatially and transcriptionally distinct progenitor zones, these results suggest that p75NTR could fulfil a generic role in the production, differentiation, and/or survival of these neuronal progenitors. To investigate whether intrinsic loss of p75NTR signaling affected the development of specific neuronal populations while expression in other cell types remains normal, we generated two conditional mouse strains in which deletion of the p75NTR gene is specific to either cortical projection neurons (Emx1- iCre p75in/in) or cortical interneuron progenitors (Nkx2.1-iCre p75wt/in), respectively. To our surprise, the Emx1-iCre p75in/in mice did not recapitulate the cortical phenotype observed in Nestin-Cre p75in/in mice. Cortical layer thickness was unchanged, and number of Tbr1- positive neurons was normal. Therefore, the reduced cortical thickness and number of upper layer cortical neurons in Nestin-Cre p75in/in mice was likely caused by non cell-autonomous effects of p75NTR knockout in other cells that are not derived from Emx1-expressing progenitors. A mild reduction of PV interneurons was found in the upper layers of the lateral

77 somatosensory cortex Emx1-iCre p75in/in mice, which is unlikely a direct result of p75NTR knockout. p75NTR expressed by neuronal fiber tracks has been shown to regulate migratory properties of gonadotropin-releasing hormone-1 (GnRH) and olfactory ensheathing cells that use these tracks as a substrate (Raucci et al., 2013). A small number of Emx1-positive progenitors located in the ventrolateral telencephalon connects the striatal area with the neocortex via p75NTR-expressing fibers, which serve as a substrate for a subpopulation of migrating interneurons (see Chapter 3, section 3.2.4). It is therefore possible that loss of p75NTR in these fiber tracks in Emx1-iCre p75in/in mice affected the migration of a proportion of cortical interneurons and led to apoptosis or suboptimal positioning of the affected cells. Another possibility is that, while generation of neurons from Emx1-positive progenitors is not directly dependent on intrinsic p75NTR signaling, loss of p75NTR in these cells affects the differentiation of interneurons non cell-autonomously. Indeed, it has been shown that p75NTR expression in cultured neurons stimulates the differentiation towards a GABAergic phenotype of surrounding cells via a secreted factor (Lin et al., 2007). However, the nature of this factor is as of yet unknown, and it is unclear if differentiation of interneurons is also regulated by a similar mechanism in vivo.

Nkx2.1-iCre p75wt/in In contrast to Emx1-iCre p75in/in mice, the phenotype of the Nkx2.1-iCre p75wt/in mouse line was much more severe, despite harbouring a deletion of only one p75NTR allele. Generation of homozygous knockout animals was not possible, and heterozygous animals were clearly distinguishable from their wild type littermates by their increased body weight. While primarily known as a master regulator of interneuron progenitors, Nkx2.1 is also expressed in other organs, such as the thyroid and lung (source: proteinatlas.org). The thyroid is a crucial hormone gland involved in the regulation of body growth and metabolic function via release of triiodothyronine (T3) and tetraiodothyronine (T4) hormones. An underactive thyroid gland, which could potentially be caused by loss of p75NTR expression during development of this organ, leads to a decrease in the basic metabolic rate and weight gain, and has been proposed to negatively impact male fertility (Aiceles and da Fonte Ramos, 2016; Aiceles et al., 2017). While it is an intriguing thought that p75NTR might play a role in thyroid development and / or function, investigating this possibility was beyond the scope of this thesis. However, Nkx2.1-iCre p75wt/in mice also showed changes in the brain, which partially recapitulated the phenotype seen in Nestin-Cre p75in/in mice. The most striking phenotype was the dramatic reduction of cortical PV interneurons in the adult animal, which correlated with a decreased thickness of the corresponding cortical layer.

78 Interestingly, while in Nestin-Cre p75in/in mice the upper cortical layers were disproportionally reduced, loss of p75NTR expression in the MGE and POA affected the development of deep layers more severely. This indicates that heterozygous p75NTR knockout in the MGE and POA leads to a reduced number of interneurons, and that this reduction may indirectly affect cortical development. Surprisingly, in contrast to PV interneurons, the number of the SST- expressing subtype was not changed in Nkx2.1-iCre p75wt/in mice. SST interneurons are the second major type of cortical interneurons produced by Nkx2.1-positive progenitors in the MGE and POA. There are several possible explanations for the differential effect that heterozygous p75NTR knockout has on the development of interneuron subpopulations. Firstly, it is possible that limitations of the mouse model are responsible for the observed discrepancy. Although Nkx2.1 is expressed evenly throughout the MGE, iCre expression under the control of the Nkx2.1 promotor is more consistently observed in the ventral than in the dorsal part (Kessaris et al., 2006; supplementary figure). Importantly, SST interneurons are derived from a slightly different location in the MGE than PV interneurons. Transplantation experiments with GFP-expressing mice have shown that, while PV interneurons are mainly derived from the ventral MGE, the SST subtype shows a bias towards the dorsal MGE (Wonders et al., 2008). Therefore, it is possible that heterozygous p75NTR knockout was not efficient enough in dorsal MGE progenitors to significantly affect SST interneuron number. Alternatively, region and cell type specific effects influence p75NTR signaling within the MGE itself. In addition to differences in SST and PV progenitor location, the timing of production of these two interneuron subgroups also differ, with SST interneurons being born ~1 day earlier than PV interneurons (Inan et al., 2012). Consistent with this observation, it has been proposed that apically located RGPs in the VZ give rise predominantly to SST interneurons, while basal IPC progenitors residing in the SVZ are biased towards producing the PV expressing subtype (Petros et al., 2015). Therefore, p75NTR expression might play a role specifically in the survival or differentiation that is specific to IPCs in the ventral telencephalon.

79 4.4 Conclusion

In this chapter we revealed that conditional knockout of the p75NTR gene in neuronal progenitors in Nestin-cre p75in/in mice results in structural abnormalities of the brain, including a marked decrease in brain volume. However, p75NTR deficiency did not affect all brain areas to the same extent; when normalized to the whole brain volume, the basal forebrain and neocortex were most significantly reduced. The numbers of adult cortical projection neurons and interneurons were decreased, which likely accounts for the total reduction in cortical volume. Given that these two major classes of cortical neurons originate from spatially and transcriptionally distinct progenitor zones, we investigated whether p75NTR plays a generic role in neuronal development by deleting the p75NTR gene in dorsal or ventral progenitors of projection neurons or cortical interneurons, respectively. While homozygous loss of p75NTR expression in dorsal progenitors did not severely affect cortical development, heterozygous knockout of the gene in interneuron progenitors was sufficient to significantly reduce the number of PV interneurons generated. Together, these results indicate that intrinsic expression of p75NTR is required for the development of cortical PV interneurons and that loss of p75NTR expression in neuronal progenitors affects cortical development by both cell-autonomous and cell-non autonomous effects. The next chapter investigates the underlying cause of the reduced neuronal number in p75NTR knockout strains and elucidates a mechanism by which p75NTR signaling could control PV interneuron development.

Contribution to this chapter by others:

Sample preparation, immunohistochemical staining, and data analysis included in Fig. 5.6D- F were performed by Lidia Madrid as part of her Master’s research project, under supervision of the thesis author.

Chapter 5: p75NTR function in progenitor survival and neuronal differentiation

5.1 Introduction

5.1.1 Chapter overview

In the previous chapter, we demonstrated that conditional knockout of p75NTR in neuronal progenitors results in variable effects on the brain, including a marked decrease in brain volume and reduced number of both late-born upper layer cortical projection neurons and cortical interneurons. A reduction in cell number can be caused by apoptosis of progenitors and/or neurons, or a change in cell cycle regulation that lowers the rate of neurogenesis. We therefore next investigated the underlying cause of the resulting phenotype in our conditional p75NTR knockout mice. To achieve this, we assessed survival, proliferation, and differentiation of neuronal progenitors in the first half of embryonic development, when the majority of cortical neurons are produced, and prior to the gliogenic switch. Furthermore, we investigated the impact of p75NTR deletion on progenitors and newly born neurons in the developing mouse brain.

5.1.2 Cell survival and cell death

Programmed cell death is a process that occurs naturally at low rates during embryonic development and ensures that the adequate number of cells is produced. This is important to control the size of all organs and tissues, including the nervous system. In contrast, if a cell loses the ability to undergo apoptosis, over-proliferation can occur, leading to growth defects and tumorigenesis. Therefore, the fate of a cell needs to be tightly regulated and is controlled by both intrinsic and extrinsic factors. An example for extrinsic control is the secretion of survival factors such as FGF2 and epidermal growth factor (EGF), the absence of which can lead to apoptosis of neuronal progenitors (Reynolds and Weiss, 1996). Intrinsic factors include improper DNA replication or distribution of chromosomes during mitosis, defects that can be detected by the cell and lead to initiation of cell death pathways (‘mitotic catastrophe’) (Ianzini and Mackey, 1998; Prokhorova et al., 2019). Cell death in tissues can be identified based on cell morphology or biochemically through the expression of apoptotic markers. The structural changes include condensation of chromatin (‘pyknosis’), shrinkage of the cytoplasm and nucleus, and, ultimately, cell fragmentation followed by phagocytosis by neighbouring cells (Kerr et al., 1972). Of the molecular machinery that executes apoptosis, the major form of programmed cell death, activation of the caspases plays a

83 critical role. Pro-apoptotic signals trigger a cascade of signaling pathways that lead to cleavage and subsequent activation of executioner caspases. Caspases are cysteine- dependent aspartate-directed proteases that coordinate the proteolysis of cellular components, DNA fragmentation and, ultimately, cell disassembly (Thornberry and Lazebnik, 1998). Cleaved caspases are therefore a hallmark of apoptosis and can be used as a chemical marker to identify dying cells in tissues and to assess if the rate of apoptosis is elevated above naturally occurring levels.

5.1.3 Cortical neuron lineage markers in dorsal and ventral progenitor zones

The progression from RGPs into IPCs and, subsequently, differentiation into neurons, is thought to be a generic principle common to most neuronal lineages. However, in dorsal and ventral lineages, the transition from one cell type to another is controlled by the activation of partially different subsets of genes. Expression analysis of different molecular cues in specific cell types can be used to systematically study their developmental fate. The following paragraphs provide an overview of a range of markers that characterize the major neuronal lineages and can be used to identify cellular subtypes.

Radial glial progenitors RGPs are one of at least two major neuron-producing progenitor types in the developing brain. They are located in the VZ and can be identified by their distinct morphology characterized by long radial processes that extend from the apical to the pial surface of the brain (Hartfuss et al., 2001), as well as expression of the glutamate transporter GLAST (Shibata et al., 1997), the intermediate filament protein vimentin (Dahl et al., 1981), and the transcription factors hairy and enhancer of split-1 (Hes1) and Hes5 (Kageyama et al., 2008). Common cellular characteristics of mitotic progenitors, such as the proliferation marker Ki67, can be used to distinguish RGPs and IPCs from differentiated neurons (Gerdes et al., 1984). In addition, RGPs located in the dorsolateral VZ of the neocortex, but not RGPs of the ventral germinal zones, specifically express the transcription factor Pax6, which plays an important role in their differentiation (Gotz et al., 1998).

Intermediate progenitors The second major neuronal progenitor type are IPCs, which are produced from RGPs and form the SVZ. They have a limited capacity for proliferation and eventually pause during their final cell cycle before dividing symmetrically into two neurons (Miyata et al., 2004). The anti-proliferative gene Tis21 is expressed in all cells undergoing neurogenic divisions and

84 marks IPCs during their final cell cycle. However, although the majority of Tis21-expressing cells are thought to be IPCs during the peak of neurogenesis, Tis21 also marks a subset of RGPs that undergo neurogenic asymmetric divisions (Attardo et al., 2008). In the neocortex, the generation of IPCs from RGPs coincides with the downregulation of Pax6 and the upregulation of the transcription factor Tbr2 (Fig. 5.1; Englund et al., 2005). Conversely, IPCs in the ventral telencephalon are characterized by high expression of the transcription factor Ascl1, which is essential for neuronal differentiation (Kelly et al., 2018).

Neurons Neurons are post-mitotic cells and therefore negative for the proliferation marker Ki67. Instead, neuronal differentiation induces the expression of the neuron-specific cytoskeletal element beta III tubulin, which remains highly expressed throughout a neuron’s lifespan. Immature neurons also express doublecortin (DCX), a microtubule-associated protein that promotes neurite outgrowth and cell migration (Francis et al., 1999; Lee et al., 1990). In the neocortex, downregulation of Tbr2 and upregulation of the transcription factor Tbr1 marks the transition from IPCs to post-mitotic neurons (Fig. 5.1; Englund et al., 2005).

Figure 5.1: Schematic representation of sequential marker expression in the dorsal neuronal lineage. Radial glial progenitors (RGPs) located in the ventricular zone (VZ) express Pax6, while Tbr2 marks intermediate progenitor cells (IPCs) located in the subventricular zone (SVZ), and Tbr1 mature neurons located in the cortical plate (CP).

85 5.1.4 Chapter aims and hypothesis

Using different lineage markers and methods to assess the rate of proliferation, apoptosis, and neurogenesis, this chapter aims to answer the following specific research questions:

 Does loss of p75NTR affect survival, differentiation, and proliferation of neuronal progenitors?

 Is this effect specific to a particular cell type and does it affect distinct neuronal lineages differently?

 Are p75NTR-mediated effects cell-autonomous or non cell-autonomous?

Based on the phenotype in Nestin-Cre p75in/in mice, we expect that loss of p75NTR impairs progenitor survival and/or embryonic neurogenesis. This is hypothesised to be a combination of cell-autonomous (in MGE progenitors) as well as cell-non autonomous (in cortical progenitors) effects.

86 5.2 Results

5.2.1 p75NTR is required for the survival of cortical neuron progenitors and production of later born neurons

In order to investigate the cause of the reduced number of cortical neurons in the Nestin- Cre p75in/in mice, embryonic brains were collected at timepoints from E11.5 to E16.5, and sections were stained for the proliferation marker Ki67 and the apoptotic marker cleaved caspase 3. Whereas cleaved caspase 3-positive staining was rarely seen in the p75fl/fl mice, the brains of embryonic Nestin-Cre p75in/in mice displayed high numbers of apoptotic cells in all areas of the rostral telencephalon, particularly in the cortex, MGE and POA, consistent with the regions where p75NTR expression is first detected (Fig. 5.2A; Fig. 5.2A-C). Increased numbers of apoptotic cells were first observed at E11.5, and were seen throughout the peak of neurogenesis, until approximately E16.5 (data not shown). Interestingly, although p75NTR expression was primarily in post-mitotic neurons in wildtype mice, the apoptotic cells in Nestin-Cre p75in/in mice were situated in the proliferative zones that co-stained with the proliferation marker Ki67 (Fig. 5.2D-F), as well as in the IZ of the cortex. Fewer dying neurons were observed in the CP where p75NTR expression is highest, suggesting that p75NTR expression and function may begin prior to cell cycle exit, neuronal differentiation and/or migration into the CP. To explore this possibility, the progenitor-to-neuron ratio was assessed during early development in p75fl/fl and Nestin-Cre p75in/in mice. Dams were injected with BrdU 1 hour prior to embryo collection to mark cells in the S-phase of mitosis. Neuronal lineage progression was then assessed in brain sections with immunostaining for BrdU and Ki67, or the cortical IPC marker Tbr2 and neuronal marker Tbr1 (Englund et al., 2005; Fig. 5.3A, B). At E14.5, a significant reduction in proliferating S-phase progenitors was observed in Nestin- Cre p75in/in mice compared to controls (-16.9 ±2.8%; Fig. 5.3C). Similarly, a lower number of Tbr2-positive progenitors was observed in the knockout mice (-18.5 ±6.5%; Fig. 5.3D). In contrast, the number of Tbr1-positive neurons was not significantly reduced (Fig. 5.3E). Consistent with this result, at E12.5, the number of BrdU-positive cells that were Ki67- negative, i.e. had exited the cell cycle within the past 24 hours (Kee et al., 2002), was unchanged in Nestin-Cre p75in/in mice compared to controls (Fig. 5.3F, G). However, in E14.5 embryos collected 24 hours after BrdU administration, the number of newly produced neurons in Nestin-Cre p75in/in cortices was reduced by 41.4 ±4.9% compared to that in control brains, indicating that fewer later born neuronal populations were being produced (Fig. 5.3H, I). This indicates that loss of p75NTR affects the rate of neurogenesis in the second half of cortical neuron develoment. A similar reduction in the

87 number of newborn neurons was also seen in the ganglionic eminences, as indicated by smaller size of the mantle zone (Fig. 5.4). This phenotype is consistent with the reduced number of cortical interneurons and later-born upper layer neurons of adult Nestin-Cre p75in/in mice.

Figure 5.2: Increased cleaved caspase 3 activation in the telencephalon of Nestin-Cre (NesCre) p75in/in mice. A) Representative images of E12.5 control (p75fl/fl) and knockout (Nestin- Cre p75in/in) brain sections stained for cleaved caspase 3 and DAPI. Scale bars: 200 µm. The boxed regions illustrate cleaved caspase 3-positive cells in the medial ganglionic eminences (MGE) and neocortex. B) Schematic representation indicating areas in which apoptotic cells were quantified. C) Quantification of apoptotic (cleaved caspase 3-positive) cells in the cortex, MGE and lateral ganglionic eminences (LGE) in E12.5 control (p75fl/fl) and knockout (Nestin-Cre p75in/in) brain sections normalized to the total area. Student’s t-test. D) Image of E14.5 NesCre p75in/in cortical section stained for cleaved caspase 3 and Ki67, showing the distribution of apoptotic cells in the ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP). Scale bar: 150 µm. E) Quantification of apoptotic (cleaved caspase 3-positive) cells per mm2 in each zone. 2-way ANOVA followed by Tukey’s post-hoc test. F) Distribution of apoptotic cells across cortical areas as a percentage of total cleaved caspase 3-positive cells. N=3 mice, mean ± SEM, * p<0.05 ** p<0.01 *** p<0.001

90 Figure 5.3: Reduced number of proliferative cells and newborn neurons in Nestin-Cre (NesCre) p75in/in cortices. A) Representative images of E14.5 control (p75fl/fl) and knockout (Nestin- Cre p75in/in) cortical sections stained for BrdU and Ki67. BrdU was administered 1 h before embryo collection to label S-phase cells. B) Representative images of E14.5 control (p75fl/fl) and knockout (Nestin-Cre p75in/in) cortical sections stained for the intermediate progenitor marker Tbr2 and neuronal marker Tbr1. C) Quantification of S-phase (BrdU-positive) cells in the cortical sections of control and knockout mice at E14.5 in a 200 µm wide region of interest. N=3, Student’s t-test. D, E) Quantification of Tbr2-positive intermediate progenitor cells (D) and Tbr1-positive neurons (E) in cortical sections of control and knockout mice at E14.5 in a 200 µm wide region of interest. N=4, Student’s t-test. F) Representative images of E12.5 control (p75fl/fl) and knockout (Nestin-Cre p75in/in) cortical sections stained for BrdU and Ki67. BrdU was administered 24 h before embryo collection to label newborn neurons. G) Quantification of post-mitotic (BrdU-positive, Ki67-negative) cells at E12.5 in a 200 µm wide region of interest. N=3, Student’s t-test. H) Representative images of E14.5 control (p75fl/fl) and knockout (Nestin-Cre p75in/in) cortical sections stained for BrdU and Ki67. BrdU was administered 24 h before embryo collection to label newborn neurons. I) Quantification of post- mitotic (BrdU-positive, Ki67-negative) cells at E14.5 in a 200 µm wide region of interest. N=3 mice per genotype, mean ± SEM, Student’s t-test, * p<0.05, ** p<0.01. Scale bars: 100µm

Figure 5.4: Reduced size of the developing telencephalon in Nestin-Cre (NesCre) p75in/in mice. Representative images of E12.5 control (p75fl/fl) and knockout (Nestin-Cre p75in/in) coronal mouse brain sections stained for the proliferation marker Ki67 and BrdU (24 hours post-injection). Comparison of rostral and midbrain sections. Scale bars: 400µm in whole section images and 150µm in septal region of interest. Arrows indicate the reduced size of the mantle zone in the Nestin-Cre p75in/in ventral telencephalon, and apparent reduction in the number of newborn BrdU-positive neurons in this area of the septum.

92 5.2.2 Effect of embryonic p75NTR expression loss on survival and production of different subpopulations of cortical neurons

To investigate whether conditional knockout of p75NTR in precursors of only the cortex or the MGE/POA phenocopies the Nestin-Cre p75in/in mice, we first compared the rate of apoptosis in embryos of Emx1-iCre p75in/in and Nkx2.1-iCre p75wt/in mice to cre-negative littermate controls. An increased number of cleaved caspase 3-positive cells was observed in the embryonic cortex of Emx1-iCre p75in/in mice at E13.5 and E14.5, and in the MGE/POA as well as the septum of Nkx2.1-iCre p75wt/in mice, where the number of dying cells peaked between E11.5 and E12.5 (Fig. 5.5A, B). In all strains, cleaved caspase 3-positive cells were found almost exclusively in Ki67-positive progenitor zones, as well as in the IZ in the cortex of Emx1-iCre p75in/in mice (Fig. 5.5A, B). Similar to Nestin-Cre p75in/in mice, fewer dying neurons were observed in the mantle zone and the CP where post-mitotic neurons reside and p75NTR expression is highest. In these mice, dying cells continued to be observed, but at lower numbers until E15.5, and the cleaved caspase 3 signal subsided coincidentally with the disappearance of the transient ganglionic eminences (data not shown). The fact that more apoptotic cells were found in both Emx1-positive and Nkx2.1-positive progenitors of conditional p75NTR knockout mice than in their respective controls suggests that p75NTR plays, at least in part, a survival function that is generic to both progenitor populations. However, while the morphology of the embryonic and adult cortex of Emx1- iCre p75in/in mice was grossly normal, the volume of the MGE in Nkx2.1-iCre p75wt/in mice at E12.5 was reduced significantly compared to littermate controls (-41.7 ±9.6%), potentially reflecting the higher rate of apoptosis found in these mice (Fig. 5.5C, D). To investigate whether the increased rate of apoptosis correlated with a reduction in the number of IPCs or post-mitotic cells in the cortex, we assessed the number of Tbr2-positive progenitors and Tbr1-positive neurons in the dorsal cortex of Nkx2.1-iCre p75wt/in and Emx1- iCre p75in/in mice at E14.5. The MGE-specific heterozygous p75NTR knockout did not significantly change the number of either cell type in the cortex at this time point (Fig. 5.6A- C). However, the numbers of Tbr2-positive progenitors and Tbr1-positive neurons and the progenitor-to-neuron ratio was also unchanged in Emx1-iCre p75in/in mice (Fig. 5.6D-F). This was contrary to the phenotype observed in the Nestin-Cre p75in/in mice. Together these results suggests that production of cortical projection neurons from IPCs in Nestin-Cre p75in/in mice through additional non cell-autonomous effects.

Figure 5.5: Apoptosis in embryonic Emx1-iCre p75in/in and Nkx2.1-iCre p75wt/in mice correlating with reduced MGE size in Nkx2.1-iCre p75wt/in mice. A) Representative images of immunohistochemical staining for cleaved caspase 3 (purple, A’) and Ki67 (green, A’’) in the cortex of Emx1-iCre p75in/in mice at E14.5. The dashed line indicates the border of the proliferative zone as identified by Ki67 staining. Scale bar: 150 µm. B) Representative images of immunohistochemical staining for cleaved caspase 3 (purple, B’) and Ki67 (green, B’’) in the cortex of Nkx2.1-iCre p75wt/in mice at E12.5. The dashed line indicates the border of the proliferative zone as identified by Ki67 staining. Scale bar: 150 µm. C) Representative low-resolution images of E12.5 coronal brain sections of control mice (p75wt/fl) and MGE-specific heterozygous p75NTR knockout strain (Nkx2.1-iCre p75wt/in) counterstained with DAPI to illustrate the embryonic brain morphology. Scale bar: 500 µm. D) Quantification of MGE area in control (p75wt/fl) and MGE-specific heterozygous p75NTR knockout mice (Nkx2.1-iCre p75wt/in) at E12.5. The area was measured in 3 consecutive sections per brain. N=6 mice per genotype, mean ± SEM, Student’s t-test, ** p<0.01.

Figure 5.6: Loss of p75NTR expression in ventral and dorsal progenitors does not affect the number of neocortical intermediate progenitor cells and newly born neurons. A) Representative images of E14.5 control (p75wt/fl) and MGE-specific heterozygous knockout (Nkx2.1- iCre p75wt/in) cortical sections stained for the intermediate progenitor marker Tbr2 and neuronal marker Tbr1. Scale bar: 100µm. B, C) Quantification of Tbr2-positive intermediate progenitor cells (B) and Tbr1-positive neurons (C) in the cortical sections of control (p75wt/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) mice at E14.5 in a 200 µm wide region of interest. N=4, Student’s t- test. D) Representative images of E14.5 control (p75fl/fl) and neocortex-specific knockout (Emx1-iCre p75in/in) cortical sections stained for the intermediate progenitor marker Tbr2 and neuronal marker Tbr1. Scale bar: 100µm. E, F) Quantification of Tbr2-positive intermediate progenitor cells (E) and Tbr1-positive neurons (F) in cortical sections of control (p75fl/fl) and knockout (Emx1-iCre p75in/in) mice at E14.5 in a 200 µm wide region of interest. N=5 mice per genotype, Student’s t-test. Mean ±SEM.

95 5.3 Discussion

The effect of p75NTR loss on surival, proliferation, and differentiation of cortical neuron progenitors In this chapter we explored the embryonic development of conditional p75NTR knockout strains to identify the underlying cause of the reduced number of cortical neurons found in adult animals. We found that the reduction of neuronal number in Nestin-Cre p75in/in mice can be explained, at least partially, by the observed apoptosis of telencephalic progenitors during early stages of neurogenesis. Increased cell death was detected throughout the developing Nestin-Cre p75in/in brain from E11.5 to about E16.5, with the most obvious increase in apoptotic cells found in the ventral part of the telencephalon, notably in the MGE and POA. The ganglionic eminences are transient structures that have been identified as the birthplace of interneurons (Anderson et al., 1997), and the MGE also gives rise to basal forebrain neurons (Zhao et al., 2003). Similarly, an increased number of apoptotic cells was also evident in the proliferative zones of the developing cortex, coincidental with a decreased number of S-phase cells and Tbr2-positive IPCs at E14.5. In contrast, apoptosis was rarely observed in the CP where mature neurons reside, and the number of Tbr1-positive neurons did not change significantly. Nonetheless, a marked decrease in the production of mature neurons was apparent during the peak of neurogenesis at E14.5, when neurons destined for the late-born upper cortical layers are being produced. However, in addition to apoptosis, other non cell-autonomous effects of p75NTR may have also contributed to the reduction in cortical neuronal number in Nestin-Cre p75in/in mice. The reduced number of S-phase cells in the cortex could indicate that the cell cycle length of progenitor cells is affected, as has been reported for other neural populations (Vilar et al., 2006; Zanin et al., 2016; Zhang et al., 2009). The hypothesis that the phenotype in Nestin-Cre p75in/in mice is indeed a combination of cell-autonomous and non cell-autonomous effects is supported by data from our conditional mouse strains that harbour a cell type-specific deletion of the p75NTR gene in projection neuron progenitors or interneuron progenitors, respectively. Although both strains phenocopy the increased rate of apoptosis during early development in Nestin-Cre p75in/in mice, we did not find a significant reduction in the number of Tbr2-positive intermediate progenitors or Tbr1-positive neurons at E14.5 in the cortex of the progenitor subpopulation- specific knockout mice. In particular, the increased rate of apoptosis observed in cortices of Emx1-iCre p75in/in mice at E14.5 did not translate into a similar reduction of cortical neurons in the adult animal (Chapter 4, section 4.2.5). This suggests (1) that while p75NTR may

96 promote survival of neuronal progenitors cell-autonomously, the effect on proliferation and differentiation is non cell-autonomous, and (2) that the reduced production of projection neurons in Nestin-Cre p75in/in mice can be largely attributed to the death or loss of function of secondary supporting cell types rather than the projection neuron progenitors themselves.

Non cell-autonomous regulation of cortical development Several possibilities exist that could account for non cell-autonomous effects impacting cortical progenitor expansion. Thalamocortical innervation during early development has been shown to be cruical for cortical development and to influence the proliferation of cortical precursors via secretion of the basic fibroblast growth factor (bFGF). In the absence of thalamic afferents, the duration of the cell cycle and G1 phase was found to be increased in cortical precursors in vitro as well as in organotypic culture of cortical explants, which significantly reduced their proliferative ability (Dehay et al., 2001). In Nestin-Cre p75in/in mice, postnatal thalamic midline fusion is impaired resulting in abnormal thalamic mophology in the adult (Chapter 4, section 4.2.2; Meier et al., 2019). Therefore, loss of thalamic neurons in Nestin-Cre p75in/in mice might affect cortical progenitor expansion as a secondary effect. Other cell types known to regulate cortical development are subplate neurons and Cajal- Retzius cells, two transient heterogeneous populations of cells that belong to the earliest generated neurons in the developing brain (between E11 and E13 in mice, Griveau et al., 2010; Luskin and Shatz, 1985; Super et al., 1998). Subplate neurons form the subplate, which is located between the IZ and the CP, and guide axons connecting layer IV of the neocortex with the thalamus to their respective targets (Allendoerfer and Shatz, 1994). Cajal-Retzius cells on the other hand are located in the MZ of the cortex and secrete signals that affect cortical neurons, such as Reelin, a glycoprotein that governs cortical architecture (Frotscher et al., 2009). Both subplate neurons and Cajal-Retzius cells also express p75NTR (Allendoerfer et al., 1990; Blanquie et al., 2017; Ghosh and Shatz, 1993) and play an important role in the laminar organization and subsequent maturation of the neocortex (Super et al., 1998). Furthermore, Cajal-Retzius cells have been shown to regulate proliferation of neuronal progenitors (Griveau et al., 2010). Although, Cajal-Retzius cells are thought to arise from different regions in the pallium, including the cortical hem and the ventral cortex, all of which are Emx1-positive regions (Bielle et al., 2005). A defect in Cajal- Retzius cells caused by loss of p75NTR would therefore be expected to manifest in a phenotype in Emx1-iCre p75in/in mice, similar to that of Nestin-cre p75in/in mice. In contrast, while some subplate neurons may be generated from Emx1-positive progenitors, they at least partially originate from subpallial, Emx1-negative regions during early development

97 (Shinozaki et al., 2002). Loss of p75NTR in subplate neurons could therefore affect cortical development in the Nestin-Cre p75in/in, but not the Emx1-iCre p75in/in mice, by stalling neocortical progenitor expansion. However, previous studies in embryonic mice in which the subplate was deleted also found a pronounced defect in the laminar architecture in the cortex, a phenotype we did not observe in the Nestin-Cre p75in/in mice (Xie et al., 2002). This suggests that although the subplate in Nestin-Cre p75in/in mice might be affected, its function is at least partially retained.

Indeed, the extrinsic cues that control cortical progenitor proliferation are manifold and complex. Studies in the ferret have shown that RGPs and IPCs can sense growing axons (such as thalamic afferents) as well as migrating neurons via their cell bodies and extended processes, and that this cell-to-cell contact impacts their development (Reillo et al., 2017). One study identified a population of glutaminergic neurons that originates from the PSPB and migrates into the cortex at E12.5, thereby regulating cortical progenitor expansion in a non cell-autonomous manner (Teissier et al., 2010). More recently, it has been reported that the proliferation of pyramidal neuron progenitors is controlled by the rate of cortical invasion of migrating interneurons, with higher levels of proliferation observed if more interneurons invade at the same time (Silva et al., 2018). Similarly, a mouse strain harbouring a deletion of the transcription factor Nkx2.1, a master regulator of interneuron development, showed attenuated proliferation of pyramidal neuron progenitors, consistent with the idea that the number of pyramidal neurons is fine-tuned and adjusted during development according to the number of invading interneurons, thereby ensuring the proper formation of local networks (Butt et al., 2008; Silva et al., 2018). Given the significant reduction in interneurons in the Nestin-Cre p75in/in mice, this could be a parsimonious explanation. However, although our mice harbouring a mono-allelic deletion of p75NTR gene specific to the MGE/POA (Nkx2.1-iCre p75wt/in) show a reduction of ~20% in the number of PV interneurons and a relative reduction in the thickness of deeper cortical layers, we did not observe any obvious differences in the number of cortical IPCs or newly born neurons at E14.5. This could be due to the relatively small effect size in heterozygous mice, the fact that other interneuron populations are not affected, or due to different timing in developmental events. Nevertheless, contribution of reduced neuronal invasion from the subpallium in Nestin-Cre p75in/in mice is a viable hypothesis to explain at least in part the different rate of proliferation compared to cortex specific p75NTR knockout in Emx1-Cre p75in/in mice.

98 Another possible site of extrinsic regulation of neuronal progenitor development is the choroid plexus, a structure that controls the composition of cerebrospinal fluid (CSF) and secretes a variety of signalling factors. These factors include mitogens such as Shh and Fgf2, which could influence proliferation and differentiation of neuronal progenitors in the VZ that are in direct contact with the CSF. Indeed, secretory molecules derived from the choroid plexus have been shown to directly influence the development of neuroepithelial cells (Huang et al., 2010; Johansson et al., 2013). We observed that p75NTR is highly expressed in the choroid plexus at early stages of development (data not shown). Loss of p75NTR expression in choroid plexus cells in Nestin-Cre p75in/in mice could potentially alter CSF composition, indirectly influencing the proliferative ability of neuronal progenitor cells.

Lastly, other non-neuronal tissues such as the vascular system can impact neuronal progenitor behaviour by shaping the environment of the stem cell niche. Vascularization of the nervous system starts around the same time as neuronal production (Hogan et al., 2004). In addition to supplying nutrients and oxygen to neuronal progenitors, vascular endothelial cells secrete soluble factors that directly regulate proliferation and differentiation of neuronal stem cells (Gama Sosa et al., 2007). Furthermore, physical interaction of neuronal stem cells with vascular endothelial cells via cell-adhesion molecules can lead to activation of the Notch and mTOR signalling pathways, enhancing their self-renewing capacity (Rosa et al., 2016). Interestingly, in addition to neuronal stem cells, it has been shown that vascularization of the SVZ also affects the proliferation of Tbr2-positive cortical IPCs, although the direct mechanism of this effect remains to be determined (Javaherian and Kriegstein, 2009). Nestin and p75NTR have been shown to be expressed in endothelial cells, and, although we did not observe any obvious vascular defects in Nestin-Cre p75in/in mice, loss of p75NTR in the vascular system may impact the development of neuronal progenitors dependent on its support (Wiese et al., 2004).

Taken together, the reduction in neuronal number observed in Nestin-Cre p75in/in mice may reflect a combination of cell-autonomous and non cell-autonomous effects, resulting in the impaired survival and proliferative capacity of cortical progenitors. It remains to be determined to what extent each of these factors contribute to the phenotype.

99 5.4 Conclusion

In summary, we have found that knockout of p75NTR causes increased apoptosis of neurogenic progenitors and reduced rates of proliferation and neurogenesis in Nestin-Cre p75in/in mice, which together likely account for the total reduction in adult cortical volume observed for these animals. While apoptosis is a generic phenotype occurring in different populations of neuronal progenitors in which p75NTR expression was lost, the effect on cortical pyramidal neuron progenitor proliferation appears to be caused primarily by non cell- autonomous effects. The next chapter investigates the identity of apoptotic cells in the MGE and provides a molecular mechanism by which p75NTR normally facilitates survival of these cells.

100

Chapter 6: p75NTR induces NF-κB phosphorylation and regulates the survival and differentiation of intermediate progenitor cells

6.1 Introduction

6.1.1 Chapter overview

In the previous chapter, we demonstrated that loss of p75NTR expression in neuronal progenitors leads to apoptosis via caspase activation. While p75NTR deletion in all neuronal progenitors affected cortical development via a variety of cell-autonomous and non cell- autonomous effects, it is unclear at what stage of development MGE progenitors die, and how p75NTR normally regulates the survival of these cells. Therefore, in this chapter, we investigated whether p75NTR plays a MGE-cell-autonomous role, which MGE cell type(s) predominantly undergo apoptosis, and which signaling pathways are affected by loss of p75NTR. Specifically, we investigated whether p75NTR knockout in the MGE leads to cell cycle dysregulation and changes in the NF-κB pathway. Lastly, we asked whether neurotrophic signaling via Trk receptors is impaired.

6.1.2 p75NTR in cell survival

Historically, p75NTR function in survival was attributed solely to its ability to enhance Trk receptor function and, in absence of Trk, p75NTR activation was thought to induce apoptosis. However, it has emerged that p75NTR can function in survival and differentiation of neuronal progenitors in the absence of Trk receptors, such as by activation of the NF-κB pathway. The NF-κB family of transcription factors consists of the five subunits: p65 (RelA), p50, c- Rel, RelB, and p52 that can dimerize and translocate into the nucleus to activate genes involved in cell cycle regulation, proliferation, and survival (Hayden and Ghosh, 2004). They can be activated by a range of stimuli, and neurotrophin binding to p75NTR has been shown to act via the p65 subunit in neurons (Hamanoue et al., 1999; Hayden and Ghosh, 2012; Khursigara et al., 2001). NF-κB signaling directly induces transcription of anti-apoptotic genes such as B-cell lymphoma 2 (Bcl-2) homologs Bcl-xL and Bfl1/A1, inhibitors of apoptosis that prevent depolarization of mitochondrial membranes and cytochrome c release (Lee et al., 1999; Luo et al., 2005; Zong et al., 1999). Phosphorylation events of various components of the NF-κB pathway play a crucial role in its activation and regulation. When inactive, the NF-κB subunits p65 and p50 are sequestered in the cytoplasm bound to the inhibitory protein IκBα. Upon activation, a complex of two kinases, IκB kinase (IKK) α and IKKβ, phosphorylates IκBα, leading to its recognition by an E3 ligase and subsequent

103 ubiquitin-dependent proteasomal degradation (Christian et al., 2016). NF-κB pathway activation can therefore be assessed based on the phosphorylation levels of its components.

6.1.3 p75NTR in cell cycle regulation

Studies in the PC12 neuron-like cell line has shown that p75NTR cell surface expression oscillates and that signaling in response to neurotrophin binding is cell cycle phase-specific (Urdiales et al., 1998). It has since been shown that p75NTR is able to interact with several cell cycle regulatory molecules such as NRIF, NRAGE, and SC-1, all of which can lead to cell cycle arrest (reviewed in Lopez-Sanchez and Frade, 2002). The small GTPase RhoA can also interact with p75NTR and may directly be able to influence the cell cycle (Villalonga and Ridley, 2006; Yamashita et al., 1999). Furthermore, in addition to genes involved in neuronal survival, the NF-κB pathway has also been shown to induce cell cycle regulatory genes. DNA binding of p65-p50 homodimers increases during G0/G1 transition phase in mouse fibroblasts and promotes expression of cyclin D1, thus initiating proliferation (Guttridge et al., 1999). However, it has also been demonstrated that activation of the NF-κB pathway can also lead to cell cycle arrest in epithelial cells via induction of the CDK inhibitor p21 (Seitz et al., 2000). More recently, canonical NF-κB signaling via p65 phosphorylation has been implicated in cell cycle arrest and differentiation in human embryonic stem cells (Yang et al., 2010), murine neuronal stem cells (Zhang et al., 2012), and Schwann cells (Limpert et al., 2013), pointing towards a role of NF-κB activation in neuronal differentiation.

104 6.1.4 Chapter aims and hypothesis

To investigate the cellular and molecular mechanisms underlying apoptosis and cortical malformation in p75NTR knockout mice, we asked the following specific research questions:

 Does p75NTR play a cell-autonomous role in MGE progenitor survival?

 Which cell type predominantly undergoes apoptosis upon p75NTR gene deletion?

 Which molecular mechanisms are most likely involved in the observed phenotype?

The previous chapters showed that p75NTR expression is predominantly found in post-mitotic neurons and is detectable in the ventral telencephalon at E11.5, ~2 days prior before becoming evident in the dorsal telencephalon. Furthermore, conditional heterozygous p75NTR knockout in MGE progenitors reduced the number of PV interneurons, which are thought to be derived from basal progenitors. We therefore hypothesise that intrinsic p75NTR expression in IPCs is required for their survival and/or appropriate cell cycle exit, and that this is independent of its ability to enhance Trk signaling. Furthermore, we hypothesise that p75NTR function is mediated by the NF-κB pathway and that its activation will be changed in the MGE of heterozygous p75NTR knockout mice.

105 6.2 Results

6.2.1 Intrinsic p75NTR expression is required for survival of interneuron progenitors

The absence of a cortical phenotype in Emx1-iCre p75in/in mice strongly suggests that the proliferation and differentiation of cortical progenitors is regulated by p75NTR via non cell- autonomous effects. We therefore first asked whether p75NTR regulates interneuron progenitor survival directly or indirectly via non cell-autonomous ways. To remove the influence of other cell types on interneuron development, we dissected the MGE at E13.5 and allowed MGE progenitors to differentiate for 15 days in culture. The anti-mitotic drug AraC was added to the medium at DIV 4 to prevent the growth of non-neuronal cells such as glia and endothelial cells that could potentially impact the survival of the cultured interneurons. Nkx2.1-iCre embryos heterozygous for the ROSA::tdTomato construct were used to assess the specificity and purity of the culture system. At DIV 15, the neurons in culture were found both positive for tdTomato and GAD67 (GAD67 is essential for the production of GABA and is therefore used a marker of inhibitory interneuron fate), confirming their MGE origin and their GABAergic identity, respectively (Fig. 6.1). We then used this system to compare the survival and cell fate specification of control (p75wt/fl) and heterozygous p75NTR knockout (Nkx2.1-iCre p75wt/fl) progenitors in vitro. After 15 days in culture, both control and heterozygous knockout neurons co-stained for both beta III tubulin and GAD67, identifying them as predominantly GABAergic neurons (Fig. 6.2A). However, as the MGE also gives rise to a subpopulation of cholinergic neurons of the basal forebrain we measured the ratio of beta III tubulin and GAD67 expression in both control and heterozygous knockout cultures at DIV 15 by Western blot (Fig. 6.2B). After loading an equal amount of protein from cell lysates from MGE of both genotypes, GAD67 was normalized to the beta III tubulin signal. If the proportion of GABAergic neurons produced by heterozygous knockout cultures was changed without affecting cholinergic neurons, the ratio of beta III tubulin to GAD67 would be different from that of control cultures. This would indicate either a change in fate, or a selectiveness of p75NTR for cell type-specific survival. However, no change was found in the ratio for heterozygous knockout cultures compared to controls, indicating that an equal proportion of GABAergic neurons was produced from heterozygous p75NTR knockout progenitors (Fig. 6.2C). However, cultures from heterozygous p75NTR knockout MGE progenitors produced obviously and significantly fewer neurons compared to control cultures (-41.2 ±4.7% Fig 6.2D). These results suggest that heterozygous loss of p75NTR affects the survival of MGE-derived precursors and/or neurons cell-autonomously without inducing a change in fate.

106

Figure 6.1: MGE-derived GABAergic neurons can be cultured in vitro. Representative immunocytochemical stainings for tdTomato and GAD67 in control interneuron cultures (Nkx2.1- iCre-postive; p75NTR wildtype; tdTomato-positive). Interneuron cultures were fixed after 15 days in vitro (DIV). Arrowhead indicate neuronal clusters co-staining for both tdTomato and GAD67. Scale bar: 500 µm.

107

Figure 6.2: Heterozygous deletion of p75NTR expression reduces the ability of interneurons to survive in vitro. A) Representative images of immunocytochemical staining for the GABAergic marker GAD67 and the generic neuronal marker beta III tubulin in control (p75wt/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) MGE cultures at day in vitro (DIV) 15. Scale bar: 500 µm. B) Western blot analysis of beta III tubulin and GAD67 expression in interneuron cultures at DIV 15. 20 µg of protein were loaded per sample, 3 mice per genotype were analysed. Each lane was loaded with the lysates from a different control (p75wt/fl) or heterozygous knockout (Nkx2.1-iCre p75wt/in) interneuron culture. C) Quantification of Western blot analysis. The GAD67 signal was normalized to beta III tubulin to compare the ratio of GABAergic cells and neurons in control (p75wt/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) cultures. D) DAPI-counterstained cells were counted in the field of vision of control (p75wt/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) cultures at DIV 15. 5 images of equal size were counted per sample, N=3 for each genotype, mean ± SEM, Student’s t-test, ** p<0.01

108 6.2.2 p75NTR is required for the survival of neurogenic intermediate progenitor cells in the medial ganglionic eminence

Next the identity of the dying MGE cells was investigated. The location of the majority of apoptotic cells in the Nestin-Cre p75in/in knockout strain were predominanty observed in the SVZ and IZ, rather than the MZ. Furthermore, the pattern of p75NTR expression indicated upregulation in post-mitotic cells. Similarly, In the MGE of Nkx2.1-iCre p75wt/in mice, cleaved caspase 3-positive cells were almost exclusively found in locations where proliferative progenitors reside, as shown when co-stained with Ki67 and BrdU (given 1 hour prior to sacrifice) further suggesting they are progenitors (Fig. 6.3A). Together this suggested that loss of p75NTR leads to death of IPCs. However, it was also possible that loss of p75NTR causes death of neurogeneic RGPs or newly born neurons that have not yet migrated out of the proliferative zones. To distinguish between these possibilities, we first considered whether the morphology of dying cells resembled proliferating progenitors or post-mitotic neurons. The cleaved caspase 3-positive cells in Nkx2.1-iCre p75wt/in mice at E11.5 frequently displayed a binucleate morphology, resembling cells in mitosis or during the process of cytokinesis (Fig. 6.3A-C). In addition, we frequently observed multinucleated cellular structures in the subpallium of both Nkx2.1-iCre p75wt/in and Nestin-Cre p75in/in mice (Fig. 6.4). These enlarged cells were mCherry-positive and did not occur in control animals or Nkx2.1- negative zones in Nkx2.1-iCre p75wt/in mice, suggesting that their generation results as a direct consequence of p75NTR loss (Fig. 6.4A, B). Even though most of these enlarged cells did not stain positive for cleaved caspase 3 (Fig. 6.4A), their nuclei frequently showed the condensed phenotype of pyknosis, indicating that cell death was taking place albeit perhaps through caspase-independent mechanisms (Fig. 6.4B).

109

Figure 6.3: Apoptotic cells in Nkx2.1-iCre p75wt/in mice display the morphological features of proliferating progenitors. A) Representative images of E11.5 heterozygous knockout (Nkx2.1-iCre p75wt/in) coronal mouse brain section stained for Ki67 (green), BrdU (1 hour post-injection, red), and cleaved caspase 3 (yellow). The outline indicates the region of interest magnified in A’ and A’’. Apoptotic cells were located in the within the proliferative zone (Ki67-positive, BrdU-positive) at the outer area of the SVZ (arrowheads in A’) and frequently displayed a binucleate morphology (arrowheads in A’’). Scale bars: 400 µm for A, 50 µm for A’ and 20 µm for A’’. VZ: ventricular zone, SVZ: subventricular zone, MZ: mantle zone. B, C) High-resolution microscopy images of cleaved caspase 3-positive cell duplets. Arrowheads indicate individual cell bodies. Scale bar: 10 µm.

110

Figure 6.4: Enlarged multinucleic mCherry-positive cells in Nkx2.1-iCre p75wt/in mice undergo cell death. A) Representative images of immunohistochemical stainings for cleaved caspase 3 (purple) and mCherry (green) at E11.5 in the MGE of Nkx2.1-iCre p75wt/in mice. Arrowheads indicate enlarged multinucleated mCherry-positive cells, the dashed rectangle indicates the location of the area magnified in A’. Scale bars: 50 µm for A, 10 µm for A’. B) High resolution microscopy image of mCherry-positive (green) multinucleated structure negative for cleaved caspase 3 (purple). Scale bar: 10 µm.

111 To further test the hypothesis that the majority of apoptotic cells in heterozygous p75NTR knockout mice are indeed progenitors rather than immature neurons, we crossed our Nkx2.1-iCre mice to a p75fl/fl Tis21-GFP reporter line. Tis21 is an anti-proliferative gene expressed in all cells about to undergo neurogenic divisions. To assess whether Tis21- positive cells undergo cell death, we collected embryos at E11.5, when the rate of apoptosis was highest in Nkx2.1-iCre p75wt/in mice, and analysed the percentage of cleaved caspase 3-positive cells that co-stained either with GFP (indicative of a neurogenic progenitor identity) or with beta III tubulin (indicative of a neuronal identity). We found that 68.7 ±4.9% of the total cleaved caspase 3 signal co-localized with GFP and only 23.9 ±5.9% with beta III tubulin. These results indicate that loss of p75NTR in MGE precursors, predominantly leads to the death of progenitors undergoing neurogenic cell cycle exit (Fig. 6.5).

112

Figure 6.5: The majority of apoptotic cells in Nkx2.1-iCre p75wt/in mice are neurogenic progenitors. A) Representative images of immunohistochemical staining for GFP (green), cleaved caspase 3 (red), and beta III tubulin (purple) in the MGE of Nkx2.1-iCre p75wt/in Tis21-GFP+/- mice at E11.5. SVZ: subventricular zone, MZ: medial zone. Scale bar: 100 µm. B) Result of the cleaved caspase 3 co-localization analyses, illustrating the majority of the signal localized with GFP. C) Quantification of the percentage of total cleaved caspase 3 signal co-localized with GFP and beta III tubulin, respecitively. N=6 mice, mean ± SEM, Student’s t-test, *** p<0.001.

113 Both RGPs and IPCs can also undergo neurogenic divisions and this subpopulation of precursors will therefore be Tis21-positive. To futher investigate whether loss of p75NTR causes death of IPCs, we crossed Ascl1-creERT2; tdTomfl/fl mice to our p75fl/fl strain (p75fl/fl Ascl1-creERT2 tdTomfl/fl). Ascl1 is expressed by more fate restricted IPCs that reside in the SVZ and therefore allows distinction between neurogenic RGPs and IPCs (Berninger et al., 2007). The recombination driving p75NTR knockout and expression of the tdTomato reporter was initiated by tamoxifen administration at E11.5, and embryos were collected 48 hours post-injection. Apoptotic, cleaved caspase 3-positive cells were counted and compared to cre-negative littermate controls. Although some cell death was found in control animals, potentially induced by tamoxifen injection of the mother, preliminary data suggest that the number of apoptotic cells was significantly increased in heterozygous p75NTR knockout mice. Analysis of cleaved caspase 3 signal of littermate control and heterozygous knockout embryos indicate a 150% increase in apoptosis (Fig. 6.6; data obtained from one litter only), consistent with the results from Nestin-Cre p75in/in and Nkx2.1-iCre p75wt/in mice.This indicates that loss of p75NTR triggered cell death in MGE-dervied IPCs cell-automomously.

114

Figure 6.6: Preliminary data suggesting increased apoptosis upon p75NTR expression loss in ventral IPCs. A) Representative images of immunohistochemical staining for leaved caspase 3 in the GE of control (p75fl/fl) and knockout (p75in/in Ascl1-creERT2) mice at E13.5, 48 hours after tamoxifen induced recombination. CreERT2-positive and negative mice were analysed from the same litter to correct for low leves of tamoxifen-induced apoptosis in controls. Dotted square indicates region of interest in the MGE shown in enlarged images. Scale bars: 200 µm. B) Quantification of cleaved caspase 3 signal plotted as a percentage of control. N=2 mice per genotype (1 litter), mean ± SEM

115 6.2.3 Heterozygous loss of p75NTR decreases phosphorylation of p65 S536 and expression of the cell cycle regulatory protein p27 in MGE progenitors

To investigate the molecular mechanism by which p75NTR controls survival and development of MGE progenitors, we collected MGE tissue of p75wt/fl control and heterozygous Nkx2.1- iCre p75wt/in knockout embryos at E13.5 and analysed the tissue with Western blotting. p75NTR can principally promote survival via activation of two distinct pathways: the NF-κB pathway via intracellular interaction with RIP2, or the Erk1/2 pathway via enhancement of Trk receptor signaling (Hamanoue et al., 1999; Khursigara et al., 2001; Mahadeo et al., 1994). Furthermore, p75NTR signaling can regulate proliferation and differentiation, and cell cycle perturbations when p75NTR expression is lost could potentially also lead to apoptosis. To distinguish between these two possibilities, and to test whether the cell cycle of cells within the MGE is affected by the knockout, we analysed the level of phosphorylation of p65 and Erk1/2, the expression of IκBα, and expression of the cell cycle regulatory protein p27 (Fig. 6.7A). p27 is a CDK inhibitor and belongs to the Cip/Kip family. Its expression is low in proliferating cells and high when they are in a quiescent state (Coats et al., 1996; Nourse et al., 1994). Normalising the signal of phosphorylated forms of p65 and Erk1/2 to total expression of p65 and Erk1/2, respectively, showed that phosphorylation on serine 536 (S536) of p65 was decreased by -26.2 ±7.4% while levels of phosphorylated Erk1/2 were not significantly different between heterozygous p75NTR knockout and control MGE tissues (Fig. 6.7B). The levels of total p65 protein were not changed significantly between groups. This indicates that loss of p75NTR expression results in alteration of the p65 activation cascade rather than affecting expression levels either directly or due to the resulting cell death. However, we found that total Erk (1/2) protein was increased by 37.5 ±10.9% in heterozygous knockout MGEs compared to controls, potentially reflecting a compensatory mechanism to maintain constant levels of Trk signaling if levels of p75NTR are insufficient to promote activation (Fig. 6.7B). Interestingly, the protein levels of IκBα, which sequesters NF- κB subunits in the cytoplasm and gets degraded upon phosphorylation by the IKK complex (Mercurio et al., 1997), were unchanged in the heterozygous knockout mice compared to controls (Fig. 6.7B). Strikingly, the levels of p27 were reduced by 40.8 ±7.9% compared to controls, indicating that the cell cycle is indeed dysregulated in precursors of heterozygous p75NTR knockout mice MGE tissue (Fig. 6.7B).

116

Figure 6.7: Heterozygous knockout of p75NTR in MGE progenitors decreases phosphorylation of p65 and lowers p27 expression but does not affect phosphorylation of Erk1/2. A) Representative Western blot images for total p65, phospho p65 (p-p65; Serine 536), NF-κB inhibitor α (IκBα), mitogen-activated protein kinase 3 (MAPK3/Erk1) and MAPK1/Erk2, phospho Erk1 and Erk2 (p-Erk1/2), p27, and GAPDH, of control (p75wt/fl) and heterozygous knockout (Nkx2.1-iCre p75wt/in) E13.5 mouse MGE tissue. For each sample, the MGEs of 3 embryos (knockout or control) were pooled and loaded on two gels (MGE of 1.5 embryos per lane). Each lane for control or knockout represents samples from independent litters. B) Quantification of Western blot analysis. The signal of each antibody was normalised to GAPDH signal to correct for smaller MGE size of knockout embryos. The normalised signal of p-p65 and p-Erk1/2 was also normalised to total p65 or total Erk1/2, respectively. N=6 samples for each genotype, MGE of 3 embryos per sample, 1.5 MGEs per lane. Each lane represents embryos from independent litters. Mean ± SEM, Student’s t-test, ** p<0.01, *** p<0.001

117 6.3 Discussion

The results in this chapter provide evidence that homozygous p75NTR expression is required cell-autonomously for MGE IPCs to successfully progress through the last neurogenic division, a process that activates NF-κB signaling and p27 activity. p75NTR function in the survival of intermediate progenitors Principally, the reduced number of interneurons observed in Nkx2.1-iCre p75wt/in mice could have resulted from a change in the fate of dividing RGPs that either lead to depletion of the stem cell pool through a reduction of self-renewing divisions, or to a decrease in the production of IPCs, both of which would result in fewer later born neurons being generated. An alternative explanation is that while the production of neurons and IPCs from RGPs is normal, the IPCs have reduced capacity for division or differentiation. Our observations, that the majority of apoptotic cells were found in the SVZ and IZ of the MGE rather than the VZ, is more indicative of an IPC phenotype and therefore consistent with the latter explanation. Moreover, using an MGE-specific p75NTR heterozygous knockout mouse that was crossed to a Tis21-GFP reporter line (Nkx2.1-iCre p75wt/in; Tis21-GFP), we observed a significantly higher rate of apoptosis in Tis21-GFP-positive p75NTR-deficient progenitors than in beta III tubulin-positive neurons. This finding is consistent with other reports that suggest p75NTR can play a pivotal role in neurogenic divisions (Bernabeu and Longo, 2010; Catts et al., 2008; Colditz et al., 2010; Young et al., 2007; Zanin et al., 2016). Furthermore, preliminary results of a conditional mouse line that harbours an inducible cre recombinase expressed under the Ascl1 promoter indicates that loss of p75NTR causes death of IPCs. The morphology of the dying cells in p75NTR-deficient mice also indicates that they are IPCs undergoing failed division or differentiation and that p75NTR upregulation in these cells prior to, or immediately after, their final neuerogeneic division is therefore important for normal MGE neurogenesis. These data strongly support the hypothesis that p75NTR expression is crucial for neurogenic fate restricted progenitors to undergo neurogenic divisions.

Survival signaling and cell cycle regulation by p75NTR Apoptosis of neuronal progenitors by reduced p75NTR expression can either be caused by decreased survival signaling, or indirectly by failed cell cycle regulation (Pucci et al., 2000). It is well established that p75NTR can enhance neurotrophin binding to Trk receptors and lead to increased activation of Trk downstream signaling pathways. However, knockout of Trk receptors in neuronal progenitors does not affect their survival (Polleux et al., 2002) and phosphorylation of Erk1/2, a readout for Trk receptor activation, was not changed in the

118 MGE of our conditional heterozygous p75NTR knockout mice. This suggests that apoptosis of cells with reduced levels of p75NTR is caused independently from its function as a Trk co- receptor. However, Akt phosphorylation in heterozygous knockout MGEs was not assessed, and although Erk1/2 and Akt pathways are co-activated by Trk receptors, it remains to be determined if reduced p75NTR levels affected Akt-mediated survival signaling. p75NTR can also induce survival signaling upon binding of RIP2 and activation of the NF-κB pathway. For example, it has been shown that p75NTR-mediated NF-κB activation enhances survival of trigeminal neurons and cerebellar granule neurons in response to NGF (Hamanoue et al., 1999; Khursigara et al., 2001; Kisiswa et al., 2018). A marked decrease in S536 p65 phosphorylation was detected in heterozygous p75NTR knockout MGEs, suggesting a decrease in the activity of the NF-κB pathway. The phosphorylation of NF-κB directly affects its function via regulating subunit stability and transcriptional activity. It has been proposed that specific phosphorylation signature could direct gene specific transcriptional activity of NF-κB subunits (Christian et al., 2016), and 31 different phosphorylation sites were identified on p65 alone (source: phosphosite.org). In contrast, the levels of the p65 binding partner and activity regulator IκBα was unchanged between p75NTR-deficient and wildtype MGE tissue. However, serine phosphorylation S536 has been shown to regulate nuclear translocation and transcription initiation independently from IκBα in T-cells (Sasaki et al., 2005). Furthermore, p65 S536 phosphorylation mediated by other members of the TNF receptor family has been shown to increase transcriptional activity (Sakurai et al., 1999; Sakurai et al., 2003). Therefore, p75NTR signaling might activate NF- κB via p65 S536 phosphorylation independent of IκBα degradation.

In addition to survival, NF-κB signaling has been shown to regulate the cell cycle. While NF- κB-induced cell cycle progression and proliferation is associated with a range of cancer types, it has been shown to initiate differentiation in other cell types. Indeed, several studies suggest that NF-κB signaling in neuronal progenitors plays a role in differentiation rather than survival. A study using neuronal stem cells derived from E14 mouse ganglionic eminences, in which NF-κB nuclear translocation was blocked short term, suggested that activation of this pathway drives neurogenesis (Shingo et al., 2001). Furthermore, an investigation using MGE neurosphere cultures from p65-deficient mice did not find an effect on survival but observed a decreased number of neurons being generated from these cultures (Young et al., 2006). Cell cycle arrest is an important prerequisite for neuronal differentiation (Caviness et al., 2003). While the link between NF-κB and cell cycle progression is well established and has

119 been shown to depend on upregulation of cyclin D1 (Takebayashi et al., 2003), the downstream components of NF-κB activation linked to differentiation are less well understood. The downregulation of the cell cycle inhibitor p27 in the heterozygous p75NTR knockout MGEs, concurrently observed with a decrease in p65 S536 phosphorylation, suggests that NF-κB does not promote proliferation in this case. One study that investigated the downstream genes regulated by NF-κB activation in the context of neuronal differentiation reported that expression of C/EBPβ, a transcription factor promoting proliferation (Legraverend et al., 1993), was decreased upon p65 binding to its promoter (Zhang et al., 2012). Furthermore, gene expression profiling of primary neuronal progenitors treated with an IKKβ inhibitor revealed that most genes with significant changes were involved in neurogenesis (Zhang et al., 2012). Therefore, reduced NF-κB activity may lead to failure of neurogenic programs and uncontrolled cell cycle progression, followed by induction of apoptosis and mitotic catastrophe. Eventually, this leads to depletion of the progenitor pool and subsequent decrease in the production of neurons.

The current hypothesis is that p75NTR-mediated cell cycle regulation and survival in ventral progenitors are linked; however, both processes could also be independent of each other. Previously, p75NTR has been shown to promote cell cycle exit of granule cell progenitors upon binding to NT-3, and that removal of p75NTR expression during cerebellar development resulted in increased proliferation, leading to an increase in the volume of the adult cerebellum with functional consequences (Zanin et al., 2016). In this case, p75NTR deletion did not lead to apoptosis of granule cell progenitors. More recently, a follow up study using primary cerebellar cultures from p75NTR knockout rats showed that upon binding of pro-NT- 3 to p75NTR, RhoA activity decreases together with the rate of proliferation (Zanin et al., 2019). Rho GTPases are important regulators of the cytoskeleton that can directly affect the cell cycle by inducing gene expression of cyclins and CDKs, and play an important function during the process of cytokinesis (reviewed in David et al., 2012). Importantly, there have also been reports of abnormally occurring multinucleated neurons when RhoA signaling is disturbed (Harding et al., 2016). It is therefore possible that, in MGE progenitors, p75NTR mediates cell cycle regulation via RhoA activation, while NF-κB signaling is required for neuronal progenitor survival.

120 6.4 Conclusion

In this chapter, we showed that p75NTR plays a critical role in the survival and cell cycle regulation of ventral IPCs. Partial loss of p75NTR expression in the MGE during early development leads to death of progenitors via apoptosis and mitotic catastrophe, a process that likely contributes to the reduction in PV interneuron number found in adult mice. Both, ineffective survival signaling as well as dysregulation of the cell cycle could potentially result in cell death. The underlying molecular mechanism in both processes may involve decreased activity of the NF-κB pathway, which may normally be induced by p75NTR independent of its function as a Trk co-receptor. However, alternative mechanisms of cell cycle regulation via p75NTR are also possible.

121

Chapter 7: Thesis summary and conclusions

7.1 Significance

The cortex is the part of the mammalian brain that mediates higher cognitive functions and complex behaviour. Neurodevelopmental disorders that result in reduced cortical thickness in humans can manifest in severe mental disabilities and neuropsychiatric disorders such as schizophrenia (Kuperberg et al., 2003). Reduced interneuron number, in particular the PV-expression subtype, has also been reported in schizophrenia patients (Kaar et al., 2019), in a rat model of schizophrenia (Lodge et al., 2009), and in other severe cognitive disorders such as autism (Hashemi et al., 2017). Furthermore, de novo missense variants in neurotrophin signaling pathway genes may increase vulnerability for schizophrenia (Kranz et al., 2015). Targeted exome capture in 48 schizophrenia patients of genes encoding for Trk receptors, p75NTR, the neurotrophins, and interactors of both Trk and p75NTR, revealed missense coding variants of at least one of these genes in 37 of 48 cases. Of these cases, two demonstrated novel variants in the p75NTR-interacting Rho-GEF TRIO, and, in a different case, a novel missense variant occurred in the intracellular domain of p75NTR gene itself (Kranz et al., 2015). These findings suggest that rare missense variants in neurotrophic signaling pathways provide additional genetic risk factors for schizophrenia. It remains to be determined to which extent these variants, in particular p75NTR and its interactors, may contribute to the disease phenotype.

Therefore, in order to better understand complex neurodevelopmental disorders, it is important to learn how the brain is normally formed, and, in particular, to uncover mechanisms underpinning cortical neurogenesis. In this study, we show for the first time that neurotrophic signaling mediated by p75NTR is directly involved in the normal formation of the cortex and is required for production of the appropriate number of cortical projection neurons and interneurons.

125 7.2 Summary of findings and conclusions

We demonstrate that p75NTR expression in the developing brain is strongest in the MZ, IZ, and CP, where newly born neurons reside. Using Tis21GFP and Ascl1-creERT2 tdTomfl/fl marker mice, we also identified a subpopulation of ventral neurogenic progenitors and migrating immature interneurons that are p75NTR-positive, suggesting that p75NTR expression is upregulated during neuronal differentiation. The data presented in this thesis provides evidence for an important function of p75NTR in the development of the brain, in particular the neocortex and basal ganglia. Firstly, we demonstrate that loss of p75NTR expression in neuronal progenitors during early neurogenesis leads to a number of phenotypes in the adult, including a marked reduction in cortical volume, reduced number of cortical interneurons and cholinergic basal forebrain neurons, as well as decreased neuronal complexity of cortical pyramidal neurons. Secondly, we show that p75NTR regulates normal formation of the neocortex via a range of cell-autonomous and non cell-autonomous effects (Fig. 7.1). Regulation of proliferation and survival of cortical projection neuron progenitors is independent of intrinsic p75NTR signaling, and is controlled by non cell- autonomous factors including invasion of interneurons into the cortex and innervation by thalamic afferents. In contrast, the establishment of adequate cortical PV interneuron number depends on intrinsic p75NTR expression in MGE progenitors. In the absence of p75NTR, interneuron progenitor survival is decreased and neurogenic divisions are disrupted, leading to a depletion of the progenitor pool and subsequently fewer neurons being produced. In addition to phenotypical assessments, we carried out a number of functional studies that revealed a novel role for p75NTR in the survival of ventral telencephalic progenitors. We provide evidence that neurogenic IPCs are particularly vulnerable to loss of p75NTR expression. Firstly, the majority of apoptotic cells were Tis21-positive and located at the SVZ/MZ border, rather than the VZ where neurogenic RGPs reside. Secondly, dying cells were often observed in duplicates or displayed a multinucleated morphology, indicating that cell death occurred during or shortly after cell division. Lastly, loss of p75NTR in more fate restricted Ascl1-positive IPCs is sufficient to induce apoptosis in these cells.

126 The molecular mechanisms that could lead to this phenotype are manifold and complex. Dysregulation of the cell cycle in MGE progenitors of p75NTR knockout mice was evident by the reduced expression of the cell cycle inhibitory protein p27. However, reduced expression of genes involved in cell survival may also have contributed to the increased rate of apoptosis. One major signaling cascade activated by p75NTR that is involved in both cell survival and cell cycle regulation is the NF-kB pathway. We found that S536 phosphorylation of the p65 subunit is reduced in the MGE of p75NTR knockout mice compared to controls, suggesting a downregulation of NF-kB signaling in the absence of p75NTR. This effect seems to be independent of Trk receptor signaling and therefore mediated solely by p75NTR. It remains to be determined whether p75NTR action is dependent on other co-receptors, such as sortilin, and whether the presence of specific ligands is required to mediate survival in this context. Based on this data, we propose a mechanism in ventral IPCs in which p75NTR regulates timely cell cycle exit and differentiation. In the absence of p75NTR expression, the cell cycle is shortened and proliferation increased, which may ultimately lead to induction of apoptotic programs and mitotic catastrophe. This results in fewer cortical interneurons, specifically the PV-expressing type, and in turn affects cortical layer thickness. It is therefore possible that changes in p75NTR expression, or somatic mutations in p75NTR in precursors (Kranz et al., 2016), could underlie neurodevelopmental disorders such as schizophrenia, and is a promising avenue for future research.

127

Figure 7.1: Diagram illustrating the combination hypothesis of intrinsic and extrinsic factors influencing cortical development of p75NTR knockout mice. During early stages of development, neurons are predominantly generated by radial glial progenitors (RGPs). At later stages, neurogenesis switches to a two-stage process, with intermediate progenitor cells (IPCs) being produced first (Molyneaux et al., 2007). p75NTR expression is upregulated in IPCs prior to neurogenic divisions, and loss of p75NTR in MGE progenitors reduces their survival cell-autonomously. In contrast, the proliferation and survival of cortical projection neuron progenitors is controlled cell non- autonomously by invasion of ventral telencephalic lineage neurons, and by other cell types such as thalamic neurons that innervate the cortex (green arrows).

128 7.3 Additional thoughts

Although this thesis aimed to determine the role of p75NTR in brain development, the investigation was restricted to neurons and neuronal progenitors. Nonetheless, p75NTR is known to play important roles in glial cells. Related effects in other cell types than neurons are likely to play a role in the Nestin-Cre p75in/in phenotype. The early onset of apoptosis observed in conditional p75NTR knockout strains, prior to the gliogenic switch, as well as the resulting decrease in neuronal number, strongly indicates a direct effect of p75NTR function on neurogenesis. However, an additional effect on the development of micro and macroglia of the CNS cannot be excluded, and is indeed an intriguing possibility, albeit beyond the scope of this thesis. While astrocytes do not seem to express p75NTR, expression can be detected in cultured microglia and oligodendrocytes at various stages of development (Chapter 3, section 3.1.4). Furthermore, a defect in glial production might have contributed to the neuronal phenotype in Nestin-Cre p75in/in mice due to secondary effects. Lastly, while the majority of glial cells are produced during late embryogenesis and early postnatal development, there is evidence for waves of early born oligodendrocytes that emerge from the MGE and migrate through the forebrain as early as E14.5 (Kessaris et al., 2006). These early oligodendrocytes compete for space with later born populations and are subsequently eliminated, raising questions about their purpose. Therefore, although we have shown that intrinsic loss of p75NTR in Ascl1-positive IPCs and GAD67-positive interneuron progenitors leads to cell death, an effect on early oligodendrocytes cannot be excluded. It would be interesting to investigate whether oligodendrocyte survival is impaired by p75NTR deletion and whether this contributes to the phenotype observed in p75NTR knockout mice.

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139 Vaccarino, F. M., Schwartz, M. L., Raballo, R., Rhee, J. and Lyn-Cook, R. (1999). Fibroblast growth factor signaling regulates growth and morphogenesis at multiple steps during brain development. Curr Top Dev Biol 46, 179-200. Vicario, A., Kisiswa, L., Tann, J. Y., Kelly, C. E. and Ibanez, C. F. (2015). Neuron-type-specific signaling by the p75NTR death receptor is regulated by differential proteolytic cleavage. J Cell Sci 128, 1507-1517. Vilar, M., Murillo-Carretero, M., Mira, H., Magnusson, K., Besset, V. and Ibanez, C. F. (2006). Bex1, a novel interactor of the p75 neurotrophin receptor, links neurotrophin signaling to the cell cycle. EMBO J 25, 1219-1230. Villalonga, P. and Ridley, A. J. (2006). Rho GTPases and cell cycle control. Growth Factors 24, 159-164. von Schack, D., Casademunt, E., Schweigreiter, R., Meyer, M., Bibel, M. and Dechant, G. (2001a). Complete ablation of the neurotrophin receptor p75NTR causes defects both in the nervous and the vascular system. Nat Neurosci 4, 977-978. von Schack, D., Casademunt, E., Schweigreiter, R., Meyer, M., Bibel, M. and Dechant, G. (2001b). Complete ablation of the neurotrophin receptor p75NTR causes defects both in the nervous and the vascular system. Nature Neuroscience 4, 977. Wiese, C., Rolletschek, A., Kania, G., Blyszczuk, P., Tarasov, K. V., Tarasova, Y., Wersto, R. P., Boheler, K. R. and Wobus, A. M. (2004). Nestin expression--a property of multi-lineage progenitor cells? Cell Mol Life Sci 61, 2510-2522. Wonders, C. P., Taylor, L., Welagen, J., Mbata, I. C., Xiang, J. Z. and Anderson, S. A. (2008). A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence. Dev Biol 314, 127-136. Xie, Y., Skinner, E., Landry, C., Handley, V., Schonmann, V., Jacobs, E., Fisher, R. and Campagnoni, A. (2002). Influence of the embryonic preplate on the organization of the cerebral cortex: a targeted ablation model. J Neurosci 22, 8981-8991. Yamashita, T., Tucker, K. L. and Barde, Y. A. (1999). Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron 24, 585-593. Yang, C., Atkinson, S. P., Vilella, F., Lloret, M., Armstrong, L., Mann, D. A. and Lako, M. (2010). Opposing putative roles for canonical and noncanonical NFkappaB signaling on the survival, proliferation, and differentiation potential of human embryonic stem cells. Stem Cells 28, 1970-1980. Young, K. M., Bartlett, P. F. and Coulson, E. J. (2006). Neural progenitor number is regulated by nuclear factor-kappaB p65 and p50 subunit-dependent proliferation rather than cell survival. J Neurosci Res 83, 39-49. Young, K. M., Merson, T. D., Sotthibundhu, A., Coulson, E. J. and Bartlett, P. F. (2007). p75 neurotrophin receptor expression defines a population of BDNF-responsive neurogenic precursor cells. J Neurosci 27, 5146-5155. Yun, K., Potter, S. and Rubenstein, J. L. (2001). Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128, 193-205. Yushkevich, P. A., Piven, J., Hazlett, H. C., Smith, R. G., Ho, S., Gee, J. C. and Gerig, G. (2006). User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage 31, 1116-1128. Zagrebelsky, M., Holz, A., Dechant, G., Barde, Y. A., Bonhoeffer, T. and Korte, M. (2005). The p75 neurotrophin receptor negatively modulates dendrite complexity and spine density in hippocampal neurons. J Neurosci 25, 9989-9999. Zanin, J. P., Abercrombie, E. and Friedman, W. J. (2016). Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor. Elife 5. Zanin, J. P., Verpeut, J. L., Li, Y., Shiflett, M. W., Wang, S. S., Santhakumar, V. and Friedman, W. J. (2019). The p75NTR Influences Cerebellar Circuit Development and Adult Behavior via Regulation of Cell Cycle Duration of Granule Cell Progenitors. J Neurosci 39, 9119-9129. Zhang, W., Zeng, Y. S., Wang, J. M., Ding, Y., Li, Y. and Wu, W. (2009). Neurotrophin-3 improves retinoic acid-induced neural differentiation of skin-derived precursors through a p75NTR- dependent signaling pathway. Neurosci Res 64, 170-176.

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141 Appendix

Documents included in the appendix:

 Link to the publication included in this thesis: https://dev.biologists.org/content/146/18/dev181933.long

 The animal ethics certificates QBI/566/18, QBI/135/18/BREED, QBI/534/15, and QBI/084/15/NHMRC/ARC/BREED

142 Office of Research Ethics Director Nicole Shively Animal Ethics Approval Certificate 20-Mar-2019 Please check all details below and inform the Animal Ethics Unit within 10 working days if anything is incorrect.

Activity Details Chief Investigator: Professor Elizabeth Coulson, Queensland Brain Institute Title: Molecular mechanisms of neuronal differentiation survival and death (renewal 2018). AEC Approval Number: QBI/566/18 Previous AEC Number: QBI/534/15/NHMRC Approval Duration: 20-Feb-2019 to 20-Feb-2022 Funding Body: NHMR, QBI Group: Anatomical Biosciences Other Staff/Students: Bree Rumballe, Melanie Flint, Gabriela Acosta, Xiangyu Zhou, Mark Bellingham, Karine Mardon, Daniel Blackmore, Trent Woodruff, Nyoman Kurniawan, Dhanisha Jhaveri, Stephen Williams, Lee Fletcher, Amanda Chiam Xu Wen, Lei Qian, Oliver Rawashdeh, Kai-Hsiang Chuang, Angelo Tedoldi, Michael Milne, Jacinta Conroy, Sonja Meier, Hsu-Lei Lee, Zengmin Li, Leda Kasas, Kym French, Trish Hitchcock Location(s): St Lucia Bldg 79 - Queensland Brain Institute

Summary Subspecies Strain Class Gender Source Approved Remaining Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 71 71 modified Breeding Colony Mice - genetically ChAT-Cre Adults Mix Institutional 105 105 modified Breeding Colony Mice - genetically ChAT-Cre x Adults Mix Institutional 250 250 modified ChR2-YFP Breeding Colony Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 170 170 modified x ChR2-YFP Breeding Colony Mice - genetically Hugo Adults Mix Institutional 146 146 modified Breeding Colony Mice - genetically Hugo x Emx-iCre Adults Mix Institutional 6 6 modified Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 6 6 modified iCre Breeding Colony Mice - genetically Free Chopper x Adults Mix Institutional 6 6 modified ChAT-Cre Breeding Colony Mice - genetically Free Chopper X Adults Mix Institutional 6 6 modified Hugo X ChAT- Breeding Colony Cre Mice - genetically Hif1alphaflox/flox Adults Mix Institutional 40 40 modified Breeding Colony Mice - genetically P75-GVP Adults Mix Institutional 100 100 modified Breeding Colony Mice - genetically p75Nglyco Adults Mix Institutional 20 20 modified Breeding Colony

Permits

Provisos

Approval Details

Description Amount Balance

Mice - genetically modified (APP/PS1, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 136 136 Mice - genetically modified (Ascl1-CreERT2 x tdTomato, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 14 14 Mice - genetically modified (Ascl1-CreERT2 x tdTomato, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 19 Feb 2019 Initial approval 112 112 Mice - genetically modified (ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 105 105 Mice - genetically modified (ChAT-Cre x ChR2-YFP, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 250 250 Mice - genetically modified (Free Chopper , Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 20 20

Page 2 of 4 Mice - genetically modified (Free Chopper x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 6 6 Mice - genetically modified (Free Chopper X Hugo X ChAT-Cre , Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 6 6 Mice - genetically modified (Hif1a x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 20 20 Mice - genetically modified (Hif1alphaflox/flox, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 40 40 Mice - genetically modified (Hugo, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 131 131 20 Mar 2019 Mod #2 15 146 Mice - genetically modified (Hugo x Ascl1-CreERT2 x tdTomato , Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 20 20 Mice - genetically modified (Hugo x Ascl1-CreERT2 x tdTomato, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 19 Feb 2019 Initial approval 160 160 20 Mar 2019 Mod #2 160 320 Mice - genetically modified (Hugo x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 71 71 Mice - genetically modified (Hugo x ChAT-Cre x ChR2-YFP, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 170 170 Mice - genetically modified (Hugo x Emx-iCre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 6 6 Mice - genetically modified (Hugo x Nestin–cre, Mix, Adults, Institutional Breeding Colony) 20 Mar 2019 Mod #2 15 15 Mice - genetically modified (Hugo x Nestin–cre, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 20 Mar 2019 Mod #2 40 40 Mice - genetically modified (Hugo x Nkx2.1-iCre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 6 6 Mice - genetically modified (Hugo x Nkx2.1-iCre x Tis21-GFP, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 19 Feb 2019 Initial approval 120 120 20 Mar 2019 Mod #2 160 280 Mice - genetically modified (Hugo x Tis21-GFP, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 75 75 20 Mar 2019 Mod #2 60 135 Mice - genetically modified (Nkx2.1-iCre, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 75 75 20 Mar 2019 Mod #2 60 135 Mice - genetically modified (P75-GVP, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 100 100 Mice - genetically modified (p75Nglyco, Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 20 20

Page 3 of 4 Mice - non genetically modified (C57BL/6J , Mix, Adults, Institutional Breeding Colony) 19 Feb 2019 Initial approval 367 367 20 Mar 2019 Mod #2 24 391 Mice - non genetically modified (C57BL6J, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 19 Feb 2019 Initial approval 800 800 20 Mar 2019 Mod #2 160 960

Please note the animal numbers supplied on this certificate are the total allocated for the approval duration

Please use this Approval Number: 1. When ordering animals from Animal Breeding Houses 2. For labelling of all animal cages or holding areas. In addition please include on the label, Chief Investigator's name and contact phone number. 3. When you need to communicate with this office about the project.

It is a condition of this approval that all project animal details be made available to Animal House OIC. (UAEC Ruling 14/12/2001)

The Chief Investigator takes responsibility for ensuring all legislative, regulatory and compliance objectives are satisfied for this project. This certificate supersedes all preceding certificates for this project (i.e. those certificates dated before 20-Mar-2019)

Activity Details Chief Investigator: Professor Elizabeth Coulson, Queensland Brain Institute Title: Molecular mechanisms of neuronal differentiation, survival and death (3) AEC Approval Number: QBI/534/15/NHMRC Previous AEC Number: QBI/407/12/NHMRC Approval Duration: 16-Mar-2016 to 16-Mar-2019 Funding Body: NHMR, QBI Group: Anatomical Biosciences Other Staff/Students: Bree Rumballe, Daniel Blackmore, Trent Woodruff, Nyoman Kurniawan, Brett Collins, Yunpeng Wang, Karine Mardon, Tess Onraet, Dhanisha Jhaveri, Thomas Burne, Suzy Alexander, Zoran Boskovic, Stephen Williams, Iris Wang, Lee Fletcher, Aung Aung Kywe Moe, Lei Qian, Ramon Martinez-Marmol, Kai-Hsiang Chuang, Kyna-Anne Conn, Michael Milne, Sonja Meier, Peter Li, Sajini Kiru, Mia Langguth, Xiangyu Zhou, Chai Chee Ng, Trish Hitchcock Location(s): St Lucia Bldg 79 - Queensland Brain Institute St Lucia Bldg 64 - Sir William MacGregor St Lucia Bldg 57 - Centre of Advanced Imaging St Lucia Bldg 76 - Chemistry (SCMB) St Lucia Bldg 65 - Skerman St Lucia Bldg 81 - Otto Hirschfeld

Summary Subspecies Strain Class Gender Source Approved Remaining Mice - genetically ChAT-Cre Adults Mix Institutional 297 294 modified Breeding Colony Mice - genetically p75Nglyco X Adults Mix Institutional 249 249 modified ChAT-Cre Breeding Colony Mice - genetically Free Chopper X Adults Mix Institutional 727 724 modified ChAT-Cre Breeding Colony Mice - genetically ChAT-Cre Adults Male Institutional 245 200 modified Breeding Colony Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 520 481 modified Breeding Colony Mice - genetically TrkA-Cre Adults Mix Institutional 69 69 modified Breeding Colony Mice - genetically Free Chopper X Adults Mix Institutional 117 102 modified TrkA-Cre Breeding Colony Mice - genetically p75Nglyco x Adults Male Institutional 96 96 modified Hugo x ChAT-Cre Breeding Colony

Animal Ethics Unit Cumbrae-Stewart Building +61 7 336 52925 (Enquiries) [email protected] Office of Research Ethics Research Road +61 7 334 68710 (Enquiries) uq.edu.au/research The University of Queensland St Lucia Qld 4072 Australia +61 7 336 52713 (Coordinator) Page 1 of 11 Mice - genetically p75Nglyco x Free Adults Male Institutional 96 96 modified Chopper Breeding Colony Mice - genetically Hugo Adults Male Institutional 212 212 modified Breeding Colony Mice - genetically floxed TrkB Adults Male Institutional 144 144 modified Breeding Colony Mice - genetically Hugo x ChAT-Cre Adults Male Institutional 192 184 modified Breeding Colony Mice - genetically Free Chopper X Adults Male Institutional 96 96 modified Hugo Breeding Colony Mice - genetically Free Chopper Adults Male Institutional 192 166 modified Breeding Colony Mice - genetically p75Nglyco x Adults Male Institutional 96 96 modified Hugo Breeding Colony Mice - genetically p75Nglyco X Adults Male Institutional 96 96 modified ChAT-Cre Breeding Colony Mice - genetically p75Nglyco x Adults Mix Institutional 117 117 modified Hugo x ChAT-Cre Breeding Colony Mice - genetically Hugo X Emx-iCre Adults Mix Institutional 48 20 modified Breeding Colony Mice - genetically p75Nglyco x Free Adults Mix Institutional 48 48 modified Chopper x ChAT- Breeding Colony Cre Mice - genetically p75Nglyco Adults Mix Institutional 63 41 modified Breeding Colony Mice - genetically Free Chopper X Adults Mix Institutional 48 48 modified Hugo X ChAT- Breeding Colony Cre Mice - genetically Hugo Adults Mix Institutional 52 52 modified Breeding Colony Mice - genetically BDNF Adults Mix Institutional 132 117 modified (Val66Met) Breeding Colony Mice - genetically APP/PS1 Adults Male Institutional 166 150 modified Breeding Colony Mice - genetically Hugo x TrkA-Cre Adults Mix Institutional 116 116 modified Breeding Colony Mice - genetically APP/PS1 x BDNF Adults Mix Institutional 56 42 modified fl/fl Breeding Colony Mice - genetically APP/PS1 x Free Adults Mix Institutional 240 240 modified Chopper x ChAT- Breeding Colony Cre Mice - genetically APP/PS1 x Adults Mix Institutional 68 66 modified SNX27 Breeding Colony Mice - genetically APP/PS1 X PI3K Adults Mix Institutional 68 65 modified delta Breeding Colony Mice - genetically APP/PS1 Adults Mix Institutional 434 372 modified Breeding Colony Mice - genetically APP/PS1 x Hugo Adults Mix Institutional 240 210 modified x ChAT-Cre Breeding Colony Mice - genetically Free Chopper X Adults Male Institutional 192 177 modified TrkA-Cre Breeding Colony Mice - genetically p75Nglyco Adults Male Institutional 198 183 modified Breeding Colony Mice - genetically pR5 Adults Mix Institutional 72 72 modified Breeding Colony

Page 2 of 11 Mice - genetically p75Nglyco x Juvenile / Weaners Unknown Institutional 424 424 modified Hugo X TrkA-Cre / Pouch animal Breeding Colony Mice - genetically Hugo Juvenile / Weaners Unknown Institutional 424 424 modified / Pouch animal Breeding Colony Mice - genetically Free Chopper X Juvenile / Weaners Unknown Institutional 424 424 modified TrkA-Cre / Pouch animal Breeding Colony Mice - genetically Hugo x TrkA-Cre Juvenile / Weaners Unknown Institutional 424 424 modified / Pouch animal Breeding Colony Mice - genetically SNX27 Juvenile / Weaners Unknown Institutional 424 424 modified / Pouch animal Breeding Colony Mice - genetically Free Chopper Juvenile / Weaners Unknown Institutional 424 424 modified / Pouch animal Breeding Colony Mice - genetically p75Nglyco Juvenile / Weaners Unknown Institutional 424 417 modified / Pouch animal Breeding Colony Mice - genetically Free Chopper X Adults Mix Institutional 16 16 modified HB9-Cre Breeding Colony Mice - genetically Hugo x ChAT-Cre Adults Female Institutional 90 84 modified Breeding Colony Mice - genetically p75Nglyco Adults Female Institutional 90 66 modified Breeding Colony Mice - genetically BDNF Adults Female Institutional 90 89 modified (Val66Met) Breeding Colony Mice - genetically floxed TrkB Adults Female Institutional 90 90 modified Breeding Colony Mice - genetically SNX27 Prenatal / Embryo Unknown Institutional 448 448 modified Breeding Colony Mice - genetically APP/PS1 Prenatal / Embryo Unknown Institutional 448 448 modified Breeding Colony Mice - genetically floxed TrkB Prenatal / Embryo Unknown Institutional 448 448 modified Breeding Colony Mice - genetically APP/PS1 X PI3K Prenatal / Embryo Unknown Institutional 448 448 modified delta Breeding Colony Mice - genetically SNX27 Adults Female Institutional 90 90 modified Breeding Colony Mice - genetically APP/PS1 X PI3K Adults Female Institutional 90 87 modified delta Breeding Colony Mice - genetically APP/PS1 Adults Female Institutional 90 51 modified Breeding Colony Mice - genetically Hugo X ChAT- Prenatal / Embryo Unknown Institutional 448 448 modified Cre Breeding Colony Mice - genetically p75Nglyco Prenatal / Embryo Unknown Institutional 448 442 modified Breeding Colony Mice - genetically BDNF Prenatal / Embryo Unknown Institutional 448 448 modified (Val66Met) Breeding Colony Mice - genetically Ex-breeders Adults Mix Institutional 30 26 modified Breeding Colony Mice - genetically Hugo x Nestin-cre Adults Mix Institutional 254 208 modified Breeding Colony Mice - genetically Hugo x Nestin-cre Prenatal / Embryo Mix Institutional 320 240 modified Breeding Colony Mice - genetically Hugo x Emx1- Adults Mix Institutional 200 193 modified iCre Breeding Colony Mice - genetically Hugo x Emx1- Prenatal / Embryo Mix Institutional 320 240 modified iCre Breeding Colony

Page 3 of 11 Mice - genetically Hugo x Nkx2.1- Prenatal / Embryo Mix Institutional 320 320 modified iCre Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 200 158 modified iCre Breeding Colony Mice - genetically Hugo Inverted Adults Mix Institutional 200 141 modified Breeding Colony Mice - genetically Hugo Inverted Prenatal / Embryo Mix Institutional 320 240 modified Breeding Colony Mice - genetically ChAT-Cre X Adults Mix Institutional 96 96 modified APP/PS1 Breeding Colony Mice - genetically TrkA-Cre x floxed Adults Mix Institutional 48 38 modified TrkB Breeding Colony Mice - genetically Hugo x Nestin-cre Neonates Mix Institutional 0 0 modified Breeding Colony Mice - genetically ChAT-cre; ChR2- Adults Mix Institutional 160 160 modified YFP Breeding Colony Mice - genetically SOD1 x ChAT- Adults Mix Institutional 144 141 modified cre x Free Breeding Colony Chopper Mice - genetically Hugo Inverted Neonates Mix Institutional 320 320 modified Breeding Colony Mice - genetically p75Nglyco KI Adults Mix Institutional 40 40 modified Breeding Colony Mice - non C57BL/6 Adults Mix Commercial 595 534 genetically breeding colony modified Mice - non C57BL/6 Adults Male Commercial 144 67 genetically breeding colony modified Mice - non C57BL/6 Juvenile / Weaners Unknown Commercial 424 424 genetically / Pouch animal breeding colony modified Mice - non C57BL/6 Adults Female Commercial 56 46 genetically breeding colony modified Mice - non C57BL/6 Prenatal / Embryo Unknown Commercial 448 448 genetically breeding colony modified

Permits

Provisos

Approval Details

Description Amount Balance

Mice - genetically modified (APP/PS1 , Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -39 51 Mice - genetically modified (APP/PS1 , Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 166 166 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -16 150 Mice - genetically modified (APP/PS1 , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 430 430

Page 4 of 11 13 Jul 2016 Mod #4 4 434 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -62 372 Mice - genetically modified (APP/PS1 , Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (APP/PS1 x BDNF fl/fl, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 56 56 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -14 42 Mice - genetically modified (APP/PS1 x Free Chopper x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 240 240 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 240 Mice - genetically modified (APP/PS1 x Hugo x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 240 240 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -30 210 Mice - genetically modified (APP/PS1 X PI3K delta, Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -3 87 Mice - genetically modified (APP/PS1 X PI3K delta, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 68 68 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -3 65 Mice - genetically modified (APP/PS1 X PI3K delta, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (APP/PS1 x SNX27, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 68 68 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -2 66 Mice - genetically modified (BDNF (Val66Met), Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -1 89 Mice - genetically modified (BDNF (Val66Met), Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 132 132 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -15 117 Mice - genetically modified (BDNF (Val66Met), Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (ChAT-Cre, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 245 245 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -45 200 Mice - genetically modified (ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 297 297 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -3 294 Mice - genetically modified (ChAT-Cre X APP/PS1, Mix, Adults, Institutional Breeding Colony)

Page 5 of 11 13 Jul 2016 Mod #6 96 96 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (ChAT-cre; ChR2-YFP, Mix, Adults, Institutional Breeding Colony) 11 Aug 2016 Mod #10 160 160 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 160 Mice - genetically modified (Ex-breeders, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Modification #2 30 30 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -4 26 Mice - genetically modified (floxed TrkB, Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 90 Mice - genetically modified (floxed TrkB, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 144 144 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 144 Mice - genetically modified (floxed TrkB, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (Free Chopper , Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 192 192 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -26 166 Mice - genetically modified (Free Chopper , Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (Free Chopper X ChAT-Cre , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 667 667 11 Aug 2016 Mod #12 60 727 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -3 724 Mice - genetically modified (Free Chopper X HB9-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 16 16 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 16 Mice - genetically modified (Free Chopper X Hugo, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 96 96 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (Free Chopper X Hugo X ChAT-Cre , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 48 48 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 48 Mice - genetically modified (Free Chopper X TrkA-Cre , Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 192 192 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -15 177 Mice - genetically modified (Free Chopper X TrkA-Cre , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 117 117

Page 6 of 11 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -15 102 Mice - genetically modified (Free Chopper X TrkA-Cre , Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (Hugo , Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 212 212 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 212 Mice - genetically modified (Hugo , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 48 48 13 Jul 2016 Mod #4 4 52 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 52 Mice - genetically modified (Hugo, Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (Hugo Inverted, Mix, Adults, Institutional Breeding Colony) 11 May 2016 Mod #3 40 40 13 Jul 2016 Mod #7 20 60 14 Sep 2016 Mod #14 10 70 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -59 11 20 Jun 2017 Mod #19 130 141 Mice - genetically modified (Hugo Inverted, Mix, Neonates, Institutional Breeding Colony) 14 Sep 2016 Mod #15 320 320 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 320 Mice - genetically modified (Hugo Inverted, Mix, Prenatal / Embryo, Institutional Breeding Colony) 11 May 2016 mod #3 60 60 14 Sep 2016 Mod #14 20 80 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -80 0 20 Jun 2017 Mod #19 240 240 Mice - genetically modified (Hugo x ChAT-Cre, Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -6 84 Mice - genetically modified (Hugo x ChAT-Cre, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 192 192 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -8 184 Mice - genetically modified (Hugo x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 413 413 13 Jul 2016 Mod #4 7 420 11 Aug 2016 Mod #12 60 480 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -39 441 20 Jun 2017 Mod #25 40 481 Mice - genetically modified (Hugo X ChAT-Cre, Unknown, Prenatal / Embryo, Institutional Breeding Colony)

Page 7 of 11 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (Hugo x Emx1-iCre, Mix, Adults, Institutional Breeding Colony) 11 May 2016 Mod #3 40 40 13 Jul 2016 Mod #7 20 60 14 Sep 2016 Mod #14 10 70 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -7 63 20 Jun 2017 Mod #19 130 193 Mice - genetically modified (Hugo x Emx1-iCre, Mix, Prenatal / Embryo, Institutional Breeding Colony) 11 May 2016 mod #3 60 60 14 Sep 2016 Mod #14 20 80 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -80 0 20 Jun 2017 Mod #19 240 240 Mice - genetically modified (Hugo X Emx-iCre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 48 48 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -28 20 Mice - genetically modified (Hugo x Nestin-cre, Mix, Adults, Institutional Breeding Colony) 11 May 2016 Mod #3 40 40 13 Jul 2016 Mod #7 20 60 14 Sep 2016 Mod #14 10 70 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -46 24 20 Jun 2017 Mod #24 54 78 20 Jun 2017 Mod #19 130 208 Mice - genetically modified (Hugo x Nestin-cre, Mix, Neonates, Institutional Breeding Colony) 13 Jul 2016 Mod #9 320 320 14 Sep 2016 Mod #15 -320 0 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 0 Mice - genetically modified (Hugo x Nestin-cre, Mix, Prenatal / Embryo, Institutional Breeding Colony) 11 May 2016 Mod #3 60 60 14 Sep 2016 Mod #14 20 80 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -80 0 20 Jun 2017 Mod #19 240 240 Mice - genetically modified (Hugo x Nkx2.1-iCre, Mix, Adults, Institutional Breeding Colony) 11 May 2016 Mod #3 40 40 13 Jul 2016 Mod #7 20 60 14 Sep 2016 Mod #14 10 70 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -42 28 20 Jun 2017 Mod #19 130 158 Mice - genetically modified (Hugo x Nkx2.1-iCre, Mix, Prenatal / Embryo, Institutional Breeding Colony) 11 May 2016 Mod #3 60 60 14 Sep 2016 Mod #14 20 80 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 80

Page 8 of 11 20 Jun 2017 Mod #19 240 320 Mice - genetically modified (Hugo x TrkA-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 116 116 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 116 Mice - genetically modified (Hugo x TrkA-Cre, Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (p75Nglyco, Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -24 66 Mice - genetically modified (p75Nglyco, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 198 198 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -15 183 Mice - genetically modified (p75Nglyco, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 63 63 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -22 41 Mice - genetically modified (p75Nglyco, Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -7 417 Mice - genetically modified (p75Nglyco, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -6 442 Mice - genetically modified (p75Nglyco KI, Mix, Adults, Institutional Breeding Colony) 20 Jun 2017 Mod #25 40 40 Mice - genetically modified (p75Nglyco X ChAT-Cre, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 96 96 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (p75Nglyco X ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 249 249 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 249 Mice - genetically modified (p75Nglyco x Free Chopper, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 96 96 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (p75Nglyco x Free Chopper x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 48 48 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 48 Mice - genetically modified (p75Nglyco x Hugo, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 96 96 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (p75Nglyco x Hugo x ChAT-Cre, Male, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 96 96

Page 9 of 11 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 96 Mice - genetically modified (p75Nglyco x Hugo x ChAT-Cre, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 117 117 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 117 Mice - genetically modified (p75Nglyco x Hugo X TrkA-Cre, Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (pR5, Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 72 72 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 72 Mice - genetically modified (SNX27, Female, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 90 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 90 Mice - genetically modified (SNX27, Unknown, Juvenile / Weaners / Pouch animal, Institutional Breeding Colony) 9 Mar 2016 Initial approval 424 424 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - genetically modified (SNX27, Unknown, Prenatal / Embryo, Institutional Breeding Colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448 Mice - genetically modified (SOD1 x ChAT-cre x Free Chopper, Mix, Adults, Institutional Breeding Colony) 11 Aug 2016 Mod #11 144 144 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -3 141 Mice - genetically modified (TrkA-Cre , Mix, Adults, Institutional Breeding Colony) 9 Mar 2016 Initial approval 69 69 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 69 Mice - genetically modified (TrkA-Cre x floxed TrkB, Mix, Adults, Institutional Breeding Colony) 13 Jul 2016 Mod #8 48 48 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -10 38 Mice - non genetically modified (C57BL/6, Female, Adults, Commercial breeding colony) 9 Mar 2016 Initial approval 56 56 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -10 46 Mice - non genetically modified (C57BL/6, Male, Adults, Commercial breeding colony) 9 Mar 2016 Initial approval 144 144 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -77 67 Mice - non genetically modified (C57BL/6, Mix, Adults, Commercial breeding colony) 9 Mar 2016 Initial approval 547 547 13 Jul 2016 Mod #4 8 555 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) -61 494 20 Jun 2017 Mod #25 40 534 Mice - non genetically modified (C57BL/6, Unknown, Juvenile / Weaners / Pouch animal, Commercial breeding colony) 9 Mar 2016 Initial approval 424 424

Page 10 of 11 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 424 Mice - non genetically modified (C57BL/6, Unknown, Prenatal / Embryo, Commercial breeding colony) 9 Mar 2016 Initial approval 448 448 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25729) 0 448

Please note the animal numbers supplied on this certificate are the total allocated for the approval duration

It is a condition of this approval that all project animal details be made available to Animal House OIC. (UAEC Ruling 14/12/2001)

The Chief Investigator takes responsibility for ensuring all legislative, regulatory and compliance objectives are satisfied for this project. This certificate supercedes all preceeding certificates for this project (i.e. those certificates dated before 27-Jun-2017)

Activity Details Chief Investigator: Professor Elizabeth Coulson, Queensland Brain Institute Title: Neurotrophic related transgenic mouse breeding colony AEC Approval Number: QBI/135/18/BREED Previous AEC Number: QBI/084/15/NHMRC/ARC/BREED Approval Duration: 18-Jun-2018 to 18-Jun-2021 Funding Body: ARC, NHMRC, Uniquest Group: Anatomical Biosciences Other Staff/Students: Michael Milne, Bree Rumballe, Sonja Meier, Ramon Martinez-Marmol, Kym French, Trish Hitchcock Location(s): St Lucia Bldg 79 - Queensland Brain Institute St Lucia Bldg 76 - Chemistry (SCMB)

Summary Subspecies Strain Class Gender Source Approved Remaining Mice - genetically APP/PS1 (breed) Adults Mix Institutional 180 172 modified Breeding Colony Mice - genetically APP/PS1 (cull) Adults Mix Institutional 360 356 modified Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 90 82 modified P13Kdelta (breed) Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 120 80 modified P13Kdelta (cull) Breeding Colony Mice - genetically Asc11-Cre ERT2 Adults Mix Institutional 72 71 modified x tdTomato Breeding Colony (breed) Mice - genetically Asc11-Cre ERT2 Adults Mix Institutional 1152 1151 modified x tdTomato (cull) Breeding Colony Mice - genetically ChAT-Cre (breed) Adults Mix Institutional 465 444 modified Breeding Colony Mice - genetically ChAT-Cre (cull) Adults Mix Institutional 266 223 modified Breeding Colony Mice - genetically ChAT-Cre x ChR2 Adults Mix Institutional 56 42 modified -YFP (breed) Breeding Colony

Mice - genetically ChAT-Cre x ChR2 Adults Mix Institutional 74 71 modified -YFP (cull) Breeding Colony Mice - genetically ChR2-YFP Adults Mix Institutional 270 249 modified (breed) Breeding Colony Mice - genetically ChR2-YFP (cull) Adults Mix Institutional 135 92 modified Breeding Colony

Animal Ethics Unit Cumbrae-Stewart Building +61 7 336 52925 (Enquiries) [email protected] Office of Research Ethics Research Road +61 7 334 68710 (Enquiries) uq.edu.au/research The University of Queensland St Lucia Qld 4072 Australia +61 7 336 52713 (Coordinator) Page 1 of 9 Mice - genetically Free Chopper Adults Mix Institutional 270 263 modified (breed) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 135 49 modified (cull) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 132 129 modified :ChaT-cre (breed) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 189 174 modified :ChaT-cre (cull) Breeding Colony Mice - genetically Free Chopper x Adults Mix Institutional 94 82 modified Hugo x Chat Cre Breeding Colony (breed) Mice - genetically Free Chopper x Adults Mix Institutional 73 5 modified Hugo x Chat Cre Breeding Colony (cull) Mice - genetically Hugo (breed) Adults Mix Institutional 544 526 modified Breeding Colony Mice - genetically Hugo (cull) Adults Mix Institutional 290 247 modified Breeding Colony Mice - genetically Hugo : ChAT Cre Adults Mix Institutional 138 122 modified (breed) Breeding Colony Mice - genetically Hugo : ChAT Cre Adults Mix Institutional 136 93 modified (cull) Breeding Colony Mice - genetically Hugo Inverted Adults Mix Institutional 15 14 modified (cull) Breeding Colony Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 274 266 modified x ChR2-YFP Breeding Colony (breed) Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 476 425 modified x ChR2-YFP Breeding Colony (cull) Mice - genetically Hugo x Emx1- Adults Mix Institutional 175 150 modified iCre (cull) Breeding Colony Mice - genetically Hugo x Nestin-cre Adults Mix Institutional 23 22 modified (breed) Breeding Colony Mice - genetically Hugo x Nestin-cre Adults Mix Institutional 189 184 modified (cull) Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 132 132 modified icre (breed) Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 135 135 modified icre (cull) Breeding Colony Mice - genetically Nestin Cre (cull) Adults Mix Institutional 20 20 modified Breeding Colony Mice - genetically Nkx2.1-iCre Adults Mix Institutional 150 146 modified (breed) Breeding Colony Mice - genetically Nkx2.1-iCre (cull) Adults Mix Institutional 113 75 modified Breeding Colony Mice - genetically TrkB fl/fl (breed) Adults Mix Institutional 90 82 modified Breeding Colony Mice - genetically TrkB fl/fl (cull) Adults Mix Institutional 60 24 modified Breeding Colony Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 28 16 modified Tis21-GFP (breed Breeding Colony Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 404 327 modified Tis21-GFP (cull) Breeding Colony

Page 2 of 9 Mice - genetically p75-GVP (breed) Adults Mix Institutional 180 174 modified Breeding Colony Mice - genetically p75-GVP (cull) Adults Mix Institutional 240 240 modified Breeding Colony Mice - genetically p75Nglyco x Adults Mix Institutional 270 270 modified Hugo x Chat Cre Breeding Colony (breed) Mice - genetically p75Nglyco x Adults Mix Institutional 456 413 modified Hugo x Chat Cre Breeding Colony (cull) Mice - genetically p75Nglycos Adults Mix Institutional 270 253 modified (breed) Breeding Colony Mice - genetically p75Nglycos (cull) Adults Mix Institutional 135 106 modified Breeding Colony Mice - genetically Hif1a (breed) Adults Mix Institutional 42 42 modified Breeding Colony Mice - genetically Hif1a : ChAT-cre Adults Mix Institutional 132 132 modified (breed) Breeding Colony Mice - genetically Hif1a : ChAT-cre Adults Mix Institutional 96 96 modified (cull) Breeding Colony Mice - genetically Hif1a (cull) Adults Mix Institutional 42 42 modified Breeding Colony Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 78 69 modified Asc11-creERT2; Breeding Colony tdTomato (breed) Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 1152 1118 modified Asc11-creERT2; Breeding Colony tdTomato (cull) Mice - genetically Tis21-GFP (breed) Adults Mix Institutional 24 24 modified Breeding Colony Mice - genetically Tis21-GFP (cull) Adults Mix Institutional 384 384 modified Breeding Colony Mice - genetically ArchT-EGFP Adults Mix Institutional 276 276 modified (breed) Breeding Colony Mice - genetically ArchT-EGFP Adults Mix Institutional 135 135 modified (cull) Breeding Colony Mice - genetically ChAT-Cre X Adults Mix Institutional 50 50 modified ArchT-EGFP Breeding Colony (breed) Mice - genetically ChAT-Cre X Adults Mix Institutional 114 114 modified ArchT-EGFP Breeding Colony (cull) Mice - genetically Hugo x Emx-iCre Adults Mix Institutional 18 18 modified (breed) Breeding Colony Mice - genetically ALZ17 (breed) Adults Mix Institutional 24 24 modified Breeding Colony Mice - genetically ALZ17 (cull) Adults Mix Institutional 120 120 modified Breeding Colony Mice - genetically ALZ17 x ChAT- Adults Mix Institutional 24 24 modified Cre (breed) Breeding Colony Mice - genetically ALZ17 x ChAT- Adults Mix Institutional 360 360 modified Cre (cull) Breeding Colony Mice - Inbred C57BL/6 (breed) Adults Mix Commercial 160 152 breeding colony

Permits

Page 3 of 9 Provisos 1) The amount of animals shown on this certificate only includes your Founder(s) and projected culled animal limit. 2) Progeny used for Colony maintenance should be reported on this project. 3) Estimated progeny are required on the application however the final numbers of progeny transferred to Research Project(s) must be reported only on the Research Project. ABS AEC Executive approval on 29/05/2019 for Amendment #6, which is scheduled to be ratified at the ABS June AEC meeting. Approval Details

Description Amount Balance

Mice - genetically modified (ALZ17 (breed), Mix, Adults, Institutional Breeding Colony) 20 Dec 2019 Amend. #13 24 24 Mice - genetically modified (ALZ17 (cull), Mix, Adults, Institutional Breeding Colony) 20 Dec 2019 Amend #13 120 120 Mice - genetically modified (ALZ17 x ChAT-Cre (breed), Mix, Adults, Institutional Breeding Colony) 20 Dec 2019 Amend. #13 24 24 Mice - genetically modified (ALZ17 x ChAT-Cre (cull), Mix, Adults, Institutional Breeding Colony) 20 Dec 2019 Amend. #13 360 360 Mice - genetically modified (APP/PS1 (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 180 180 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -8 172 Mice - genetically modified (APP/PS1 (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 360 360 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -4 356 Mice - genetically modified (APP/PS1 x P13Kdelta (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 90 90 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -8 82 Mice - genetically modified (APP/PS1 x P13Kdelta (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 120 120 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -40 80 Mice - genetically modified (ArchT-EGFP (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2018 Mod #5 6 6 21 Dec 2018 Mod #5 270 276 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 276 Mice - genetically modified (ArchT-EGFP (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2018 Mod #5 135 135 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 135 Mice - genetically modified (Asc11-Cre ERT2 x tdTomato (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 72 72 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -1 71 Mice - genetically modified (Asc11-Cre ERT2 x tdTomato (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 1152 1152 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -1 1151 Mice - genetically modified (ChAT-Cre (breed), Mix, Adults, Institutional Breeding Colony)

Page 4 of 9 18 Jun 2018 initial approval 450 450 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -21 429 27 Nov 2019 Amend. #11 12 441 20 Dec 2019 Amend. #13 3 444 Mice - genetically modified (ChAT-Cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 inititial approval 225 225 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -43 182 27 Nov 2019 Amend. #11 26 208 20 Dec 2019 Amend. #13 15 223 Mice - genetically modified (ChAT-Cre X ArchT-EGFP (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2018 Mod #5 8 8 21 Dec 2018 Mod #5 42 50 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 50 Mice - genetically modified (ChAT-Cre X ArchT-EGFP (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2018 Mod #5 24 24 21 Dec 2018 Mod #5 90 114 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 114 Mice - genetically modified (ChAT-Cre x ChR2-YFP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 42 42 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -14 28 20 Sep 2019 Amend. #9 14 42 Mice - genetically modified (ChAT-Cre x ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 34 34 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -3 31 20 Sep 2019 Amend. #9 40 71 Mice - genetically modified (ChR2-YFP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -21 249 Mice - genetically modified (ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 135 135 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -43 92 Mice - genetically modified (Free Chopper (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -7 263 Mice - genetically modified (Free Chopper (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 135 135 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -86 49 Mice - genetically modified (Free Chopper :ChaT-cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 132 132 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -3 129 Mice - genetically modified (Free Chopper :ChaT-cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 189 189

Page 5 of 9 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -15 174 Mice - genetically modified (Free Chopper x Hugo x Chat Cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 90 90 24 Aug 2018 Mod #2 4 94 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -12 82 Mice - genetically modified (Free Chopper x Hugo x Chat Cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 53 53 24 Aug 2018 Mod #2 20 73 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -68 5 Mice - genetically modified (Hif1a (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 42 42 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 42 Mice - genetically modified (Hif1a (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 42 42 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 42 Mice - genetically modified (Hif1a : ChAT-cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 132 132 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 132 Mice - genetically modified (Hif1a : ChAT-cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 96 96 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 96 Mice - genetically modified (Hugo (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 540 540 24 Aug 2018 Mod #2 4 544 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -18 526 Mice - genetically modified (Hugo (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270 24 Aug 2018 Mod #2 20 290 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -43 247 Mice - genetically modified (Hugo : ChAT Cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 132 132 24 Aug 2018 Mod #2 6 138 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -16 122 Mice - genetically modified (Hugo : ChAT Cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 96 96 24 Aug 2018 Mod #2 40 136 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -43 93 Mice - genetically modified (Hugo Inverted (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 15 15 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -1 14 Mice - genetically modified (Hugo x ChAT-Cre x ChR2-YFP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270

Page 6 of 9 24 Aug 2018 Mod #2 4 274 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -8 266 Mice - genetically modified (Hugo x ChAT-Cre x ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 456 456 24 Aug 2018 Mod #2 20 476 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -51 425 Mice - genetically modified (Hugo x Emx1-iCre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 40 40 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -25 15 20 Dec 2019 Amend. #12 135 150 Mice - genetically modified (Hugo x Emx-iCre (breed), Mix, Adults, Institutional Breeding Colony) 20 Dec 2019 Amend. #12 18 18 Mice - genetically modified (Hugo x Nestin-cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 14 14 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -1 13 3 Oct 2019 Amend. #8 9 22 Mice - genetically modified (Hugo x Nestin-cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 45 45 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -5 40 3 Oct 2019 Amend. #8 75 115 27 Nov 2019 Amend. #10 69 184 Mice - genetically modified (Hugo x Nkx2.1-icre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 132 132 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 132 Mice - genetically modified (Hugo x Nkx2.1-icre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 135 135 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 135 Mice - genetically modified (Nestin Cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 20 20 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 20 Mice - genetically modified (Nkx2.1-iCre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 150 150 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -4 146 Mice - genetically modified (Nkx2.1-iCre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 113 113 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -38 75 Mice - genetically modified (p75 fl/fl (Hugo) x Asc11-creERT2; tdTomato (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 72 72 24 Aug 2018 Mod #2 6 78 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -9 69 Mice - genetically modified (p75 fl/fl (Hugo) x Asc11-creERT2; tdTomato (cull), Mix, Adults, Institutional Breeding Colony)

Page 7 of 9 18 Jun 2018 initial approval 1152 1152 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -34 1118 Mice - genetically modified (p75 fl/fl (Hugo) x Tis21-GFP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 24 24 24 Aug 2018 Mod #2 4 28 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -12 16 Mice - genetically modified (p75 fl/fl (Hugo) x Tis21-GFP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 384 384 24 Aug 2018 Mod #2 20 404 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -77 327 Mice - genetically modified (p75-GVP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 180 180 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -6 174 Mice - genetically modified (p75-GVP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 240 240 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 240 Mice - genetically modified (p75Nglyco x Hugo x Chat Cre (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 270 Mice - genetically modified (p75Nglyco x Hugo x Chat Cre (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 456 456 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -43 413 Mice - genetically modified (p75Nglycos (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 270 270 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -17 253 Mice - genetically modified (p75Nglycos (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 135 135 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -29 106 Mice - genetically modified (Tis21-GFP (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 24 24 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 24 Mice - genetically modified (Tis21-GFP (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 384 384 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) 0 384 Mice - genetically modified (TrkB fl/fl (breed), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 90 90 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -8 82 Mice - genetically modified (TrkB fl/fl (cull), Mix, Adults, Institutional Breeding Colony) 18 Jun 2018 initial approval 60 60 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -36 24 Mice - Inbred (C57BL/6 (breed), Mix, Adults, Commercial breeding colony) 18 Jun 2018 initial approval 150 150

Page 8 of 9 31 Dec 2018 Use in 2018 (from 2019 MAR; AEMAR49721) -8 142 20 Dec 2019 Amend #13 10 152

Please note the animal numbers supplied on this certificate are the total allocated for the approval duration

It is a condition of this approval that all project animal details be made available to Animal House OIC. (UAEC Ruling 14/12/2001)

Activity Details Chief Investigator: Professor Elizabeth Coulson, Queensland Brain Institute Title: Neurotrophic related transgenic mouse breeding colony AEC Approval Number: QBI/084/15/NHMRC/ARC/BREED Previous AEC Number: QBI/029/12/NHMRC/ARC/BREED Approval Duration: 18-Jun-2015 to 18-Jun-2018 Funding Body: ARC, NHMRC, Uniquest Group: Anatomical Biosciences Other Staff/Students: Marie lou Camara, Michael Milne, Trish Hitchcock

Location(s): St Lucia Bldg 79 - Queensland Brain Institute

Summary Subspecies Strain Class Gender Source Approved Remaining Mice - genetically P75NGFR B6 Adults Mix Institutional 10 10 modified (cull) Breeding Colony Mice - genetically Hugo (breed) Adults Mix Institutional 120 87 modified Breeding Colony Mice - genetically Hugo (cull) Adults Mix Institutional 280 6 modified Breeding Colony Mice - genetically Hugo Inverted Adults Mix Institutional 90 49 modified (breed) Breeding Colony Mice - genetically Hugo Inverted Adults Mix Institutional 270 17 modified (cull) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 90 62 modified (breed) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 270 2 modified (cull) Breeding Colony Mice - genetically p75Nglycos Adults Mix Institutional 90 24 modified (breed) Breeding Colony Mice - genetically p75Nglycos (cull) Adults Mix Institutional 450 82 modified Breeding Colony Mice - genetically BDNF Adults Mix Institutional 90 23 modified (Val66Met) Breeding Colony (breed) Mice - genetically BDNF Adults Mix Institutional 270 115 modified (Val66Met) (cull) Breeding Colony Mice - genetically BDNF fl/fl Adults Mix Institutional 90 90 modified (breed) Breeding Colony Mice - genetically BDNF fl/fl (cull) Adults Mix Institutional 270 190 modified Breeding Colony

Animal Ethics Unit Cumbrae-Stewart Building +61 7 336 52925 (Enquiries) [email protected] Office of Research Ethics Research Road +61 7 334 68710 (Enquiries) uq.edu.au/research The University of Queensland St Lucia Qld 4072 Australia +61 7 336 52713 (Coordinator) Page 1 of 16 Mice - genetically P13K Delta Adults Mix Institutional 90 90 modified (breed) Breeding Colony Mice - genetically P13K Delta (cull) Adults Mix Institutional 270 270 modified Breeding Colony Mice - genetically ChAT-Cre (breed) Adults Mix Institutional 45 0 modified Breeding Colony Mice - genetically ChAT-Cre (cull) Adults Mix Institutional 270 14 modified Breeding Colony Mice - genetically TrkA-Cre (breed) Adults Mix Institutional 45 25 modified Breeding Colony Mice - genetically TrkA-Cre (cull) Adults Mix Institutional 270 144 modified Breeding Colony Mice - genetically Nkx2.1-iCre Adults Mix Institutional 45 14 modified (breed) Breeding Colony Mice - genetically Nkx2.1-iCre (cull) Adults Mix Institutional 270 48 modified Breeding Colony Mice - genetically Emx1-iCre Adults Mix Institutional 45 45 modified (breed) Breeding Colony Mice - genetically Emx1-iCre (cull) Adults Mix Institutional 270 270 modified Breeding Colony Mice - genetically NestinERT2-Cre Adults Mix Institutional 45 45 modified (breed) Breeding Colony Mice - genetically NestinERT2-Cre Adults Mix Institutional 270 270 modified (cull) Breeding Colony Mice - genetically Nestin Cre (breed) Adults Mix Institutional 45 29 modified Breeding Colony Mice - genetically Nestin Cre (cull) Adults Mix Institutional 270 204 modified Breeding Colony Mice - genetically Hugo : ChAT Cre Adults Mix Institutional 120 19 modified (breed) Breeding Colony Mice - genetically Hugo : ChAT Cre Adults Mix Institutional 660 154 modified (cull) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 120 73 modified :ChaT-cre (breed) Breeding Colony Mice - genetically Free Chopper Adults Mix Institutional 660 139 modified :ChaT-cre (cull) Breeding Colony Mice - genetically Hugo Adults Mix Institutional 120 120 modified :NestinERT2-cre Breeding Colony (breed) Mice - genetically Hugo Adults Mix Institutional 660 660 modified :NestinERT2-cre Breeding Colony (cull) Mice - genetically Hugo x TrkA-cre Adults Mix Institutional 210 210 modified (breed) Breeding Colony Mice - genetically Hugo x TrkA-cre Adults Mix Institutional 1050 1045 modified (cull) Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 210 200 modified icre (breed) Breeding Colony Mice - genetically Hugo x Nkx2.1- Adults Mix Institutional 1050 1006 modified icre (cull) Breeding Colony Mice - genetically Hugo x Emx1- Adults Mix Institutional 210 138 modified iCre (breed) Breeding Colony Mice - genetically Hugo x Emx1- Adults Mix Institutional 1050 800 modified iCre (cull) Breeding Colony

Page 2 of 16 Mice - genetically Hugo x Nestin-cre Adults Mix Institutional 210 173 modified (breed) Breeding Colony Mice - genetically Hugo x Nestin-cre Adults Mix Institutional 1050 846 modified (cull) Breeding Colony Mice - genetically Free Chopper : Adults Mix Institutional 210 180 modified TrkA-cre (breed) Breeding Colony Mice - genetically Free Chopper : Adults Mix Institutional 1050 894 modified TrkA-cre (cull) Breeding Colony Mice - genetically p75Nglyco x Adults Mix Institutional 120 120 modified Hugo x Chat Cre Breeding Colony (breed) Mice - genetically p75Nglyco x Adults Mix Institutional 780 780 modified Hugo x Chat Cre Breeding Colony (cull) Mice - genetically Free Chopper x Adults Mix Institutional 120 80 modified Hugo x Chat Cre Breeding Colony (breed) Mice - genetically Free Chopper x Adults Mix Institutional 780 497 modified Hugo x Chat Cre Breeding Colony (cull) Mice - genetically APP/PS1 Adults Mix Institutional 110 72 modified (breeder) Breeding Colony Mice - genetically APP/PS1 (cull) Adults Mix Institutional 360 42 modified Breeding Colony Mice - genetically pR5 (P301L) Adults Mix Institutional 90 90 modified (breed) Breeding Colony Mice - genetically pR5 (P301L) Adults Mix Institutional 180 141 modified (cull) Breeding Colony Mice - genetically SNX27-/+ (breed) Adults Mix Institutional 90 79 modified Breeding Colony Mice - genetically SNX27-/+ (cull) Adults Mix Institutional 180 85 modified Breeding Colony Mice - genetically APP/PS1 x BDNF Adults Mix Institutional 90 75 modified fl/fl (breed) Breeding Colony Mice - genetically APP/PS1 x BDNF Adults Mix Institutional 360 334 modified fl/fl (cull) Breeding Colony Mice - genetically pR5 (P301L) x Adults Mix Institutional 90 79 modified BDNF fl/fl Breeding Colony (breed) Mice - genetically pR5 (P301L) x Adults Mix Institutional 180 72 modified BDNF fl/fl (cull) Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 90 81 modified SNX27-/+ (breed) Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 90 8 modified SNX27-/+ (cull) Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 120 49 modified P13Kdelta (breed) Breeding Colony Mice - genetically APP/PS1 x Adults Mix Institutional 780 498 modified P13Kdelta (cull) Breeding Colony Mice - genetically APP/PS1 : Hugo Adults Mix Institutional 90 82 modified (breeder) Breeding Colony Mice - genetically APP/PS1 : Hugo Adults Mix Institutional 180 112 modified (cull) Breeding Colony Mice - genetically APP/PS1 x Hugo Adults Mix Institutional 120 112 modified x ChAT-Cre Breeding Colony (breed)

Page 3 of 16 Mice - genetically APP/PS1 x Hugo Adults Mix Institutional 390 230 modified x ChAT-Cre (cull) Breeding Colony Mice - genetically APP/PS1 x Free Adults Mix Institutional 90 90 modified Chopper (breed) Breeding Colony Mice - genetically APP/PS1 x Free Adults Mix Institutional 180 180 modified Chopper (cull) Breeding Colony Mice - genetically APP/PS1 x Free Adults Mix Institutional 120 120 modified Chopper x ChAT- Breeding Colony cre (breed) Mice - genetically APP/PS1 x Free Adults Mix Institutional 390 390 modified Chopper x ChAT- Breeding Colony cre (cull) Mice - genetically TrkB fl/fl (breed) Adults Mix Institutional 180 141 modified Breeding Colony Mice - genetically TrkB fl/fl (cull) Adults Mix Institutional 150 49 modified Breeding Colony Mice - genetically ChAT-cre KI/KI Adults Mix Institutional 75 75 modified (breed) Breeding Colony Mice - genetically ChAT-Cre X Adults Mix Institutional 120 120 modified APP/PS1 (cull) Breeding Colony Mice - genetically TrkA-cre KI/WT Adults Mix Institutional 10 9 modified (Breed) Breeding Colony Mice - genetically TrkA-cre KI/WT- Adults Mix Institutional 25 0 modified TrkBfl/WT Breeding Colony (breed) Mice - genetically TrkA-cre x TrkBfl Adults Mix Institutional 165 35 modified (cull) Breeding Colony Mice - genetically SOD1 G93A X Adults Mix Institutional 55 11 modified Free Chopper X Breeding Colony ChAT-Cre (breed) Mice - genetically SOD1 G93A X Adults Mix Institutional 42 0 modified Free Chopper X Breeding Colony ChAT-Cre (cull) Mice - genetically ChR2-YFP Adults Mix Institutional 164 149 modified (breed) Breeding Colony Mice - genetically ChAT-cre; ChR2- Adults Mix Institutional 30 28 modified YFP (cull) Breeding Colony Mice - genetically ChR2-YFP (cull) Adults Mix Institutional 108 74 modified Breeding Colony Mice - genetically p75-GVP (breed) Adults Mix Institutional 36 36 modified Breeding Colony Mice - genetically p75-GVP (cull) Adults Mix Institutional 50 50 modified Breeding Colony Mice - genetically Asc11-Cre ERT2 Adults Mix Institutional 36 36 modified x tdTomato Breeding Colony (breed) Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 24 24 modified Asc11-creERT2; Breeding Colony tdTomato (breed) Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 672 672 modified Asc11-creERT2; Breeding Colony tdTomato (cull) Mice - genetically Tis21-GFP Adults Mix Institutional 36 36 modified (breed) Breeding Colony Mice - genetically Tis21-GFP (cull) Adults Mix Institutional 384 384 modified Breeding Colony

Page 4 of 16 Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 30 30 modified Tis21-GFP Breeding Colony (breed) Mice - genetically p75 fl/fl (Hugo) x Adults Mix Institutional 384 384 modified Tis21-GFP (cull) Breeding Colony Mice - genetically ChAT-Cre x Adults Mix Institutional 30 18 modified ChR2-YFP Breeding Colony (breed) Mice - genetically Hugo x ChAT-Cre Adults Mix Institutional 10 10 modified x ChR2-YFP Breeding Colony (cull) Mice - genetically Asc11-Cre ERT2 Adults Mix Institutional 384 384 modified x tdTomato (cull) Breeding Colony Mice - Inbred C57BL/6 (breed) Adults Mix Institutional 700 650 Breeding Colony

Permits

Provisos 1) The amount of animals shown on this certificate only includes your Founder(s) and projected culled animal limit. 2) Progeny used for Colony maintenance should be reported on this project. 3) Estimated progeny are required on the application however the final numbers of progeny transferred to Research Project(s) must be reported only on the Research Project. Approval Details

Description Amount Balance

Mice - genetically modified (APP/PS1 (breeder), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -11 79 13 Apr 2016 Mod #5 20 99 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -10 89 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -17 72 Mice - genetically modified (APP/PS1 (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) -34 146 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -146 0 8 Mar 2017 Mod #10 180 180 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -138 42 Mice - genetically modified (APP/PS1 : Hugo (breeder), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 86 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -4 82 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 82 Mice - genetically modified (APP/PS1 : Hugo (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) -45 135 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -20 115 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -3 112 Mice - genetically modified (APP/PS1 x BDNF fl/fl (breed), Mix, Adults, Institutional Breeding Colony)

Page 5 of 16 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -15 75 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 75 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 75 Mice - genetically modified (APP/PS1 x BDNF fl/fl (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 360 360 31 Dec 2015 Use in 2015 (from 2016 MAR) -11 349 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -15 334 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 334 Mice - genetically modified (APP/PS1 x Free Chopper (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 90 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 90 Mice - genetically modified (APP/PS1 x Free Chopper (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 180 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 180 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 180 Mice - genetically modified (APP/PS1 x Free Chopper x ChAT-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 120 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 120 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 120 Mice - genetically modified (APP/PS1 x Free Chopper x ChAT-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 390 390 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 390 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 390 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 390 Mice - genetically modified (APP/PS1 x Hugo x ChAT-Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) -2 118 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 118 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -6 112 Mice - genetically modified (APP/PS1 x Hugo x ChAT-Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 390 390 31 Dec 2015 Use in 2015 (from 2016 MAR) -9 381 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -50 331 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -101 230 Mice - genetically modified (APP/PS1 x P13Kdelta (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120

Page 6 of 16 31 Dec 2015 Use in 2015 (from 2016 MAR) -12 108 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -33 75 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -26 49 Mice - genetically modified (APP/PS1 x P13Kdelta (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 780 780 31 Dec 2015 Use in 2015 (from 2016 MAR) -32 748 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -103 645 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -147 498 Mice - genetically modified (APP/PS1 x SNX27-/+ (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -1 89 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -8 81 8 Mar 2017 Mod #10 - colony closed 0 81 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 81 Mice - genetically modified (APP/PS1 x SNX27-/+ (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -12 78 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -70 8 8 Mar 2017 Mod #10 - colony closed 0 8 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 8 Mice - genetically modified (Asc11-Cre ERT2 x tdTomato (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #15 12 12 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 12 18 Apr 2018 Mod #18 24 36 Mice - genetically modified (Asc11-Cre ERT2 x tdTomato (cull), Mix, Adults, Institutional Breeding Colony) 18 Apr 2018 Mod #18 384 384 Mice - genetically modified (BDNF (Val66Met) (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -20 70 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -47 23 8 Mar 2017 Mod #10 - colony closed 0 23 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 23 Mice - genetically modified (BDNF (Val66Met) (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -45 225 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -110 115 8 Mar 2017 Mod #10 - colony closed 0 115 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 115 Mice - genetically modified (BDNF fl/fl (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 90

Page 7 of 16 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 90 Mice - genetically modified (BDNF fl/fl (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -80 190 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 190 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 190 Mice - genetically modified (ChAT-Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) -16 29 11 Aug 2016 Mod #8 30 59 7 Nov 2016 administrative adjustment -30 29 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -29 0 Mice - genetically modified (ChAT-Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -46 224 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -150 74 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -60 14 Mice - genetically modified (ChAT-cre KI/KI (breed), Mix, Adults, Institutional Breeding Colony) 13 Apr 2016 Mod #5 45 45 11 Aug 2016 Mod #8 30 75 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 75 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 75 Mice - genetically modified (ChAT-Cre X APP/PS1 (cull), Mix, Adults, Institutional Breeding Colony) 13 Apr 2016 Mod #5 120 120 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 120 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 120 Mice - genetically modified (ChAT-Cre x ChR2-YFP (breed), Mix, Adults, Institutional Breeding Colony) 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -12 -12 3 Jan 2018 Mod #13 30 18 Mice - genetically modified (ChAT-cre; ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 11 Aug 2016 Mod #8 20 20 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 20 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -2 18 3 Jan 2018 Mod #13 10 28 Mice - genetically modified (ChR2-YFP (breed), Mix, Adults, Institutional Breeding Colony) 11 Aug 2016 Mod #8 30 30 12 Oct 2016 Mod #9 134 164 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 164 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -15 149 Mice - genetically modified (ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 12 Oct 2016 Mod #9 108 108 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 108

Page 8 of 16 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -34 74 Mice - genetically modified (Emx1-iCre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 45 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 45 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 45 Mice - genetically modified (Emx1-iCre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 270 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 270 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 270 Mice - genetically modified (Free Chopper (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 86 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -12 74 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -12 62 Mice - genetically modified (Free Chopper (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -32 238 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -130 108 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -106 2 Mice - genetically modified (Free Chopper : TrkA-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 210 210 31 Dec 2015 Use in 2015 (from 2016 MAR) -12 198 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -18 180 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 180 Mice - genetically modified (Free Chopper : TrkA-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 1050 1050 31 Dec 2015 Use in 2015 (from 2016 MAR) -39 1011 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -107 904 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -10 894 Mice - genetically modified (Free Chopper :ChaT-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) -5 115 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -24 91 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -18 73 Mice - genetically modified (Free Chopper :ChaT-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 660 660 31 Dec 2015 Use in 2015 (from 2016 MAR) -83 577 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -91 486 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -347 139 Mice - genetically modified (Free Chopper x Hugo x Chat Cre (breed), Mix, Adults, Institutional Breeding Colony)

Page 9 of 16 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 120 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -11 109 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -29 80 Mice - genetically modified (Free Chopper x Hugo x Chat Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 780 780 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 780 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -40 740 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -243 497 Mice - genetically modified (Hugo (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -6 84 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -15 69 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -12 57 3 Jan 2018 Mod #13 30 87 Mice - genetically modified (Hugo (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -60 210 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -134 76 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -80 -4 3 Jan 2018 Mod #13 10 6 Mice - genetically modified (Hugo : ChAT Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) -21 99 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -39 60 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -41 19 Mice - genetically modified (Hugo : ChAT Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 660 660 31 Dec 2015 Use in 2015 (from 2016 MAR) -199 461 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -227 234 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -80 154 Mice - genetically modified (Hugo :NestinERT2-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 120 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 120 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 120 Mice - genetically modified (Hugo :NestinERT2-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 660 660 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 660 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 660 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 660 Mice - genetically modified (Hugo Inverted (breed), Mix, Adults, Institutional Breeding Colony)

Page 10 of 16 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -8 82 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -20 62 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -13 49 Mice - genetically modified (Hugo Inverted (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -65 205 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -53 152 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -135 17 Mice - genetically modified (Hugo x ChAT-Cre x ChR2-YFP (cull), Mix, Adults, Institutional Breeding Colony) 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 0 3 Jan 2018 Mod #13 10 10 Mice - genetically modified (Hugo x Emx1-iCre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 210 210 31 Dec 2015 Use in 2015 (from 2016 MAR) -23 187 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -36 151 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -13 138 Mice - genetically modified (Hugo x Emx1-iCre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 1050 1050 31 Dec 2015 Use in 2015 (from 2016 MAR) -20 1030 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -114 916 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -116 800 Mice - genetically modified (Hugo x Nestin-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 210 210 31 Dec 2015 Use in 2015 (from 2016 MAR) -5 205 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -12 193 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -20 173 Mice - genetically modified (Hugo x Nestin-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 1050 1050 31 Dec 2015 Use in 2015 (from 2016 MAR) -85 965 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -75 890 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -44 846 Mice - genetically modified (Hugo x Nkx2.1-icre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 210 210 31 Dec 2015 Use in 2015 (from 2016 MAR) -5 205 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -5 200 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 200 Mice - genetically modified (Hugo x Nkx2.1-icre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 1050 1050 31 Dec 2015 Use in 2015 (from 2016 MAR) -2 1048 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -42 1006 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 1006

Page 11 of 16 Mice - genetically modified (Hugo x TrkA-cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 210 210 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 210 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 210 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 210 Mice - genetically modified (Hugo x TrkA-cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 1050 1050 31 Dec 2015 Use in 2015 (from 2016 MAR) -5 1045 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 1045 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 1045 Mice - genetically modified (Nestin Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 45 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -8 37 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -8 29 Mice - genetically modified (Nestin Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 270 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -43 227 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -23 204 Mice - genetically modified (NestinERT2-Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 45 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 45 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 45 Mice - genetically modified (NestinERT2-Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 270 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 270 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 270 Mice - genetically modified (Nkx2.1-iCre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 41 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -13 28 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -14 14 Mice - genetically modified (Nkx2.1-iCre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -37 233 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -73 160 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -112 48 Mice - genetically modified (P13K Delta (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90

Page 12 of 16 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 90 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 90 Mice - genetically modified (P13K Delta (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 270 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 270 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 270 Mice - genetically modified (p75 fl/fl (Hugo) x Asc11-creERT2; tdTomato (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #15 24 24 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 24 Mice - genetically modified (p75 fl/fl (Hugo) x Asc11-creERT2; tdTomato (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #15 672 672 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 672 Mice - genetically modified (p75 fl/fl (Hugo) x Tis21-GFP (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #16 30 30 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 30 Mice - genetically modified (p75 fl/fl (Hugo) x Tis21-GFP (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #16 384 384 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 384 Mice - genetically modified (p75-GVP (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #14 36 36 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 36 Mice - genetically modified (p75-GVP (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #14 50 50 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 50 Mice - genetically modified (P75NGFR B6 (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 10 10 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 10 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 10 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 10 Mice - genetically modified (p75Nglyco x Hugo x Chat Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 120 120 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 120 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 120 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 120 Mice - genetically modified (p75Nglyco x Hugo x Chat Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 780 780 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 780 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 780 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 780

Page 13 of 16 Mice - genetically modified (p75Nglycos (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -12 78 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -34 44 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -20 24 Mice - genetically modified (p75Nglycos (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -155 115 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -154 -39 8 Mar 2017 Mod #10 180 141 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -59 82 Mice - genetically modified (pR5 (P301L) (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 90 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 90 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 90 Mice - genetically modified (pR5 (P301L) (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) -39 141 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 141 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 141 Mice - genetically modified (pR5 (P301L) x BDNF fl/fl (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -6 84 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -5 79 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 79 Mice - genetically modified (pR5 (P301L) x BDNF fl/fl (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) -52 128 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -56 72 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 72 Mice - genetically modified (SNX27-/+ (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 90 90 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 86 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -7 79 8 Mar 2017 Mod #10 - colony closed 0 79 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 79 Mice - genetically modified (SNX27-/+ (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 180 180 31 Dec 2015 Use in 2015 (from 2016 MAR) -51 129 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -44 85 8 Mar 2017 Mod #10 - colony closed 0 85

Page 14 of 16 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 85 Mice - genetically modified (SOD1 G93A X Free Chopper X ChAT-Cre (breed), Mix, Adults, Institutional Breeding Colony) 3 Aug 2016 Modification #7 55 55 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -20 35 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -24 11 Mice - genetically modified (SOD1 G93A X Free Chopper X ChAT-Cre (cull), Mix, Adults, Institutional Breeding Colony) 3 Aug 2016 Modification #7 42 42 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -25 17 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -17 0 Mice - genetically modified (Tis21-GFP (breed), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #16 36 36 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 36 Mice - genetically modified (Tis21-GFP (cull), Mix, Adults, Institutional Breeding Colony) 21 Dec 2017 Mod #16 384 384 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 384 Mice - genetically modified (TrkA-Cre (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 45 45 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 41 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -8 33 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -8 25 Mice - genetically modified (TrkA-Cre (cull), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 270 270 31 Dec 2015 Use in 2015 (from 2016 MAR) -49 221 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -43 178 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -34 144 Mice - genetically modified (TrkA-cre KI/WT (Breed), Mix, Adults, Institutional Breeding Colony) 13 Apr 2016 Mod #4 10 10 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 10 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -1 9 Mice - genetically modified (TrkA-cre KI/WT-TrkBfl/WT (breed), Mix, Adults, Institutional Breeding Colony) 13 Apr 2016 Mod #4 25 25 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -25 0 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) 0 0 Mice - genetically modified (TrkA-cre x TrkBfl (cull), Mix, Adults, Institutional Breeding Colony) 13 Apr 2016 Mod #4 165 165 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -113 52 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -17 35 Mice - genetically modified (TrkB fl/fl (breed), Mix, Adults, Institutional Breeding Colony) 14 Oct 2015 Modification #1 150 150 31 Dec 2015 Use in 2015 (from 2016 MAR) -4 146 13 Apr 2016 Mod #4 30 176

Page 15 of 16 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -20 156 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -15 141 Mice - genetically modified (TrkB fl/fl (cull), Mix, Adults, Institutional Breeding Colony) 14 Oct 2015 Modification #1 150 150 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 150 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) -59 91 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -42 49 Mice - Inbred (C57BL/6 (breed), Mix, Adults, Institutional Breeding Colony) 10 Jun 2015 initial approval 660 660 31 Dec 2015 Use in 2015 (from 2016 MAR) 0 660 31 Dec 2016 Use in 2016 (from 2017 MAR; AEMAR25725) 0 660 21 Dec 2017 Mod #14 40 700 31 Dec 2017 Use in 2017 (from 2018 MAR; AEMAR37769) -50 650

Please note the animal numbers supplied on this certificate are the total allocated for the approval duration

It is a condition of this approval that all project animal details be made available to Animal House OIC. (UAEC Ruling 14/12/2001)