TUBA1A MICROTUBULES ESTABLISH THE FOUNDATIONS FOR NEURONAL FUNCTION by GEORGIA CHRISTINE BUSCAGLIA B.S., Syracuse University, 2014 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Neuroscience Program 2020 This thesis for the Doctor of Philosophy degree by Georgia Christine Buscaglia has been approved for the Neuroscience Program by Abigail Person, Chair Jeffrey Moore Santos Franco Lee Niswander Matthew Kennedy Wendy Macklin Emily Bates, Advisor Date: May 22, 2020 ii Buscaglia, Georgia Christine (PhD., Neuroscience Program) Tuba1a Microtubules Establish the Foundations for Neuronal Function Thesis directed by Associate Professor Emily Bates ABSTRACT Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of a/b- tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment and in adulthood. The mechanisms by which neurons regulate microtubule network assembly and organization to respond appropriately to external cues are largely unknown. Cells express different a- and b-tubulin genes, or isotypes, providing one potential level of microtubule regulation. TUBA1A is the most abundant a-tubulin isotype in the developing brain and mutations to TUBA1A in humans cause severe brain malformations. The role of TUBA1A in the adult brain is largely unknown. We use a loss-of-function mutation to Tuba1a, Tuba1aN102D, to investigate requirements for Tuba1a in developing and adult neurons. Tuba1aND substitution reduced the amount of Tuba1a protein and prevented incorporation of Tuba1a into cellular microtubule polymers. Tuba1aND/+ neurons with reduced Tuba1a had altered microtubule dynamics and slower neuron outgrowth compared to controls. Reduced Tuba1a a-tubulin impacted axon extension and impaired formation of forebrain commissures in vivo, supporting a role for Tuba1a in axon guidance. Neurons deficient in Tuba1a failed to localize microtubule associated protein- iii 1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Further, we show that neurons with reduced Tuba1a had diminished axonal microtubule density and impaired intracellular transport. We demonstrate that Tuba1a is a major component of adult brain a-tubulin, implicating Tuba1a in adult neuronal microtubule function. Tuba1aND/+ heterozygous mice had approximately half the amount of brain a-tubulin as wild- type at birth, but normal a-tubulin abundance in adult Tuba1aND/+ brains. Despite this, Tuba1aND/+ neuromuscular junction synapses deteriorated with age, leading to adult-onset behavioral deficits. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance. We further show that the Tuba1a-rich microtubule tracks assembled during development are essential for mature neuron function and maintenance of synapses over time. These data provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment, and investigate novel functions of TUBA1A in the adult nervous system. Approved: Emily A. Bates iv For my father, John (Buzzy) Buscaglia. Thank you for always encouraging me, whether you knew it or not, you inspired me to do this. You are deeply missed. v ACKNOWLEDGEMENTS I would like to acknowledge the many people who have helped me through the years, without all of you I wouldn’t be where I am today. I would like to thank my thesis advisor, Dr. Emily Bates, for sharing her enthusiasm for discovery, constantly being my personal cheerleader, and supporting me through all endeavors. I would like to thank my thesis advisory committee, Dr. Abigail Person, Dr. Jeffrey Moore, Dr. Santos Franco, Dr. Matthew Kennedy, Dr. Wendy Macklin, and Dr. Lee Niswander for their support, feedback, and enthusiastic discussion throughout this process. I would also like to thank Dr. Jayne Aiken for creating the Tuba1a-His6 constructs, for sharing the GFP-MACF43 plasmids, and for her expertise and insightful conversations. Thank you to Mark Gutierrez and Dr. Santos Franco (University of Colorado Anschutz) for sharing the Myr-TdTomato plasmid. Thank you to Dr. John Caldwell (University of Colorado Anschutz) for his assistance with rodent hindlimb muscle dissections and his kind gift of the rhodamine-labeled a-Bungarotoxin, used for NMJ-labeling. Thank you to Kyle Northington (University of Colorado Anschutz) for his aid with the qRT-PCR experiments in Chapters II and III. Thank you to Katie Hoff (University of Colorado Anschutz) for her assistance with the Tuba1a- His6 rat neuron experiments in Chapter II. Thank you to Danae Mitchell (University of Colorado Anschutz) for sectioning and staining spinal cord sections used in Chapter III. Thank you to Emily Sullivan Gibson, Alexandra Lucas, Dr. Matthew Kennedy and Dr. Mark Dell’Acqua (University of Colorado Anschutz) for providing rat P0-P2 cortex and use of the AMAXA nucleofector. Thank you to Dr. Jennifer Bourne and the Electron Microscopy Core (University of Colorado Anschutz). Finally, I would like to thank my boyfriend, family and friends for their unending encouragement of my scientific pursuits. Thank you for supporting me through a crazy five years, and for making sure I had some fun along the way. vi TABLE OF CONTENTS CHAPTER I. MICROTUBULES IN DEVELOPMENT AND DISEASE OF THE NERVOUS SYSTEM .......................................................................................................................... 1 Introduction ............................................................................................................. 1 Microtubule Properties and Regulation .................................................................. 5 Microtubule Structure and Dynamic Instability ......................................... 5 Microtubule Polymerization ....................................................................... 6 Mechanisms of Microtubule Regulation: MAPs and the Tubulin Code ................. 8 Microtubule-Associated Proteins ................................................................ 8 Tubulin Post-Translational Modifications .................................................. 9 Tubulin Isotypes and the “Tubulin Code” ................................................ 13 TUBA1A: The Neuronal a-tubulin ....................................................................... 16 TUBA1A Identity ....................................................................................... 16 Tuba1a Expression Pattern ....................................................................... 19 Mechanisms Regulating TUBA1A Expression ........................................ 22 Tuba1a in Regeneration ............................................................................ 24 Microtubules in Developing and Adult Neurons .................................................. 26 Microtubules in Neural Development ....................................................... 26 Adult Neuronal Microtubule Function ...................................................... 28 Microtubules in Nervous System Disease ............................................................ 30 vii Tubulinopathies Reveal Essential Role of TUBA1A in Brain Formation and Function .............................................................................................. 30 Mouse Models Provide Insight into neuronal microtubule function ........ 33 Microtubules in Neurodegeneration ......................................................... 36 Conclusions ........................................................................................................... 40 II. TUBA1A PLAYS AN ESSENTIAL ROLE IN AXON PATHFINDING ..................... 41 Introduction ........................................................................................................... 41 Materials and Methods .......................................................................................... 43 Results ................................................................................................................... 50 TUBA1A is required for formation of midline commissural structures ... 50 Tuba1aND a-tubulin does not incorporate into neuronal microtubules .... 53 Reduced Tuba1a alters microtubule dynamic properties in neurons ........ 58 Tuba1a dictates neurite extension and cytoskeletal organization in growth cone ........................................................................................................... 60 Tuba1aND neurons fail to localize critical developmental proteins to growth cone ............................................................................................... 65 Multi-faceted functions of TUBA1A during neurodevelopment .............. 67 Discussion ............................................................................................................. 67 Studying a-tubulin isotypes in vivo .......................................................... 67 TUBA1A influences microtubule density, dynamics, and function in cells ................................................................................................................... 70 Models of Tuba1a-dysfunction reveal potential roles in developmental signaling ...................................................................................................