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Anatomy of the Axon: Dissecting the role of microtubule plus-end tracking proteins in axons of hippocampal neurons ISBN: 978-90-393-7095-7 On the cover: Interpretation of Rembrandt van Rijn’s iconic oil painting The Anatomy Lesson of Dr. Nicolaes Tulp (1632). Instead of a human subject, a neuron is being dissected under the watchful eye of members of the Hoogenraad & Akhmanova labs circa 2018. The doctoral candidate takes on the role of Dr. Nicolaes Tulp. The year in which this thesis is published, 2019, marks the 350th anniversary of Rembrandt’s passing. It is therefore declared Year of Rembrandt. Cover photography: Jasper Landman (Bob & Fzbl Photography) Design and layout: Dieudonnée van de Willige © 2019; CC-BY-NC-ND. Printed in The Netherlands. Anatomy of the Axon: Dissecting the role of microtubule plus-end tracking proteins in axons of hippocampal neurons Anatomie van de Axon: Ontleding van de rol van microtubulus-plus-eind-bindende eiwitten in axonen van hippocampale neuronen (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 22 mei 2019 des middags te 2.30 uur door Dieudonnée van de Willige geboren op 24 oktober 1990 te Antwerpen, België Promotoren: Prof. dr. C.C. Hoogenraad Prof. dr. A. Akhmanova Voor Hans & Miranda Index Preface 7 List of non-standard abbreviations 9 Microtubule plus-end tracking proteins 10 in neuronal development 1 Cellular & Molecular Life Sciences, 2016 Dynein regulator NDEL1 controls polarized 56 cargo transport at the axon initial segment 2 Neuron, 2016 Cytolinker Gas2L1 regulates axon branching 104 and outgrowth via microtubule-dependent actin stabilization 3 Submitted manuscript Exploring the roles of newly identified 150 plus-end tracking proteins in neuronal development 4 Collection of preliminary data 5 General discussion 206 Appendix: 218 CV & List of publications 220 Non-expert summary in English and Dutch 224 & Acknowledgements 236 Preface The complexity of the human brain has fascinated scientists since the late 19th century. According to estimations made in recent years, the male adult brain contains approximately 86 billion neurons [1, 2]. Perhaps more fascinating than the sheer number of nerve cells – there are over ten times as many neurons in a single brain as there are people on earth – is the assumption that each of those neurons is shaped uniquely. For neurons, form and function are tightly linked. A neuron’s morphology directly underlies its ability to participate in neural networks by forming connections with other neurons. Fortunately for the field of molecular neuroscience, the majority of these morphologies are established according to a common framework. Multipolar neurons, the most prevalent type of neurons in the vertebrate central nervous system and the cells on which the work in this thesis is based, are divided into two distinct subcellular compartments. The somatodendritic compartment encompasses the soma or cell body, which contains the nucleus, and multiple dendrites, which are thick, branched and relatively short processes extending from the soma. Dendrites act as receivers of chemically encoded information. The second neuronal subcompartment, the axon, is the focus of this thesis. Axons transmit information by propagating action potentials, resulting in neurotransmitter release at terminals along the axon. Multipolar neurons typically extend one axon, which is much longer and thinner than dendrites. The axon is the first neurite to mature in dissociated neuron cultures. It forms a complex branched arbour that reaches impressive combined lengths of millimetres. The cytoskeleton plays a crucial role in establishing neuronal morphology and polarity. It facilitates vesicle and organelle transport, allowing for the delivery of nutrients and macromolecules to build and maintain the neuronal processes, and offers structural support. The work in this thesis provides insight into the role and regulation of cytoskeletal processes during axon maturation, with a special focus on microtubules (MTs) and the proteins that bind to and regulate MTs from growing MT plus-ends (called +TIPs). Specifically, at the start of the work that resulted in this thesis, our goal was to identify new roles for +TIPs in neurodevelopment. 7 Chapter 1 features a review that familiarizes the reader with the various morphological changes associated with establishing neuronal polarity. It details the role that MTs play in this process and presents a discussion of what +TIPs had been implicated in neurodevelopment at the time of publishing. In Chapter 2, we zoom in on a region located in the proximal axon. The so-called axon initial segment (AIS) is a gatekeeper of neuronal polarity. One of its functions is to sort cargo that enters from the soma: vesicles that are destined for dendrites reverse out of the axon, while cargo destined for the axon proceeds past the AIS. This chapter clears up some of the molecular mechanisms behind cargo filtering at the AIS. It describes how the MT-based motor protein dynein drives the reversal of mis-sorted vesicles, and how its regulator NDEL1 localizes to the AIS cortex to locally stimulate dynein activity. LIS1, a second dynein regulator that can also associate with the MT plus-end, is also shown to participate in vesicle filtering. Chapter 3 features Gas2L1, a less well characterized +TIP that mediates crosstalk between the actin and MT cytoskeletons. We find that Gas2L1 promotes axon branching and balances axon outgrowth during neurodevelopment. In doing so, Gas2L1 functions as an actin stabilizer, but it utilizes a regulatory mechanism that places this function under direct control of MTs. We describe how Gas2L1 is inhibited via an intramolecular interaction, and how its autoinhibition is relieved by simultaneous binding of both MTs and actin filaments. A number of other promising findings are presented in Chapter 4. This chapter hosts a collection of preliminary data. In addition to more insights into Gas2L1, we identify the +TIP NCKAP5L as a putative novel regulator of axon formation. Furthermore, we describe a link between MT plus-end dynamics and phosphorylation of the axonal MT-associated protein tau, which is associated with Alzheimer’s disease. We reflect on the suggested role of a family of plus-end tracking tau kinases in this process. Surprisingly, these kinases appear to be native to the axon initial segment, raising questions about their function in healthy neurons. Finally, we place these chapters in the context of recent literature in a summarizing discussion in Chapter 5. 8 Preface References 1. Azevedo, F.A., et al., Equal numbers 2. von Bartheld, C.S., J. Bahney, and S. of neuronal and nonneuronal cells make the Herculano-Houzel, The search for true numbers human brain an isometrically scaled-up primate of neurons and glial cells in the human brain: brain. J Comp Neurol, 2009. 513(5): p. 532-41. A review of 150 years of cell counting. J Comp Neurol, 2016. 524(18): p. 3865-3895. List of non-standard abbreviations throughout this thesis Full name Classification Full name Classification ABD Actin-binding domain Dom MCAK Mitotic centromere-associated P Abi Abelson interacting protein P kinesin Abl Abelson kinase Kin MT Microtubule Struc ACF7 Actin crosslinking factor 7 + MTA Microtubule-targeting agent R AD Alzheimer’s Disease D NAV Neuron Navigator + AIS Axon initial segment Struc NCKAP5L NCK Associated Protein 5 Like + AnkG AnkyrinG AIS + NDE1 Nuclear distribution element 1 Moto AP Adaptor protein P NDEL1 Nuclear distribution-like element AIS Moto APC Adenomatosis Polyposis Coli + 1 AT8 Ser202/Thr205 phosphorylation NF-186 Neurofascin-186 AIS sites on tau protein NMDA N-Methyl-D-aspartate BDNF Brain-derived neurotrophic factor PAEZ Pre-axonal exclusion zone Struc P PCM1 Pericentriolar material 1 P CAMSAP Calmodulin-regulated spectrin- PHF Paired helical filament Struc P associated protein PI3K Phosphoinositide 3-kinase Kin CAP-Gly Cytoskeletal-associated protein PKC Protein kinase C Kin Dom glycine-rich SCA11 Spinocerebellar ataxia type 11 D cdk Cyclin-dependent kinase Kin SOCE Store operated calcium entry Struc CDK5RAP CDK5 Regulatory Subunit + STIM1 Stromal interaction molecule 1 + 2 Associated Protein 2 SVs Synaptic vesicles Struc CEP/CP Centrosomal protein P +TIP Microtubule plus-end tracking + CFEOM1 Congenital fibrosis of the protein D extraocular muscles type 1 TANC2 Tetratricopeptide Repeat, CH Calponin homology Dom Ankyrin Repeat And Coiled-Coil Moto CLASP Cytoplasmic linker protein- Containing 2 + associated protein TDP-43 Transactive response DNA P CLIP Cytoplasmic linker protein + binding protein of 43 kDa CNS Central nervous system TfR Transferrin receptor P DIV Days-in-vitro TOG Tumor overexpressed gene Dom DRG Dorsal root ganglia TRIM46 Tripartite Motif Containing 46 AIS EB End-binding protein + TRIO Trio Rho Guanine Nucleotide + EFA-6 Exchange factor for Arf6 P Exchange Factor EM Electron microscopy T TTBK1/2 Tau-tubulin kinase 1/2 + Kin ER Endoplasmic reticulum Struc VAPA/B VAMP Associated Protein A/B P FRAP Fluorescence recovery after T photobleaching Legend* GAR Growth arrest-specific 2 protein- Dom Microtubule plus-end tracking protein + related region Protein native to the axon initial segment AIS Gas2L1/2 Growth arrest-specific 2-like 1/2 + Kinase Kin GSK3ß Glycogen synthase kinase 3 beta Kin Motor protein (or regulator thereof) Moto HMN7B Hereditary motor neuropathy 7B D Other type of protein P IFT Intraflagellar transport Struc Protein domain Dom IP3R Inositol 1,4,5-triphosphate P receptor Intracellular structure/mechanism Struc Jasp Jasplakinolide R Technique T KIF1A Kinesin-like protein KIF1A Moto Disease D LatB Latrunculin B R Reagent R LIS1 Lissencephaly-1 Protein Moto + * in context of this thesis; classifications are non-exhaustive MACF1/2 Microtubule-actin crosslinking + factor 1/2 MAP Microtubule-associated protein P 9 1 Microtubule plus-end tracking proteins in neuronal development Dieudonnée van de Willige1, Casper C.
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