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Damage to the adult mammalian central nervous system (CNS), either by traumatic injury or disease, usually results in permanent sensory and/or motor deficits. Regeneration of neural circuits is limited both by the lack of growthpromoting molecules and by the presence of growth-inhibitory molecules in the mature brain and spinal cord. The research described here examines the therapeutic potential of viral vectors and neuronal transplants to reconstruct damaged neural pathways in the CNS. Experimental neural transplantation techniques often fall short of expectations because of limited transplant survival and insufficient neurite outgrowth to repair connections and induce behavioral recovery. These shortcomings are addressed in the current studies by virus-mediated expression of cell-specific neurotrophic and guidance molecules in the host brain prior to cell transplantation. The initial proof-of-principle studies show that viral vectors can be used to create axon-guidance pathways in the adult mammalian brain. With such pathways in place, subsequent transplantation of neurons leads to longdistance, targeted outgrowth of neurites. Application of this technique to a rat model of Parkinsons disease demonstrates that circuit reconstruction leads to functional recovery. For this study, rats were lesioned on one side of their brain with 6-hydroxydopamine to produce a hemiparkinsonian state. The motor deficit was confirmed by amphetamine-induced rotation testing and spontaneous motor asymmetry testing. The rats were then divided into experimental groups to receive lentivirus injections along a path between the substantia nigra (SN) and the striatum to express glial cell-line derived neurotrophic factor (GDNF), GDNF family receptor alpha-1 (GFR1), netrin-1 or green fluorescent protein (GFP, control). One group received combination injections of lenti-GDNF and lenti-GFR1. One week after virus injections, animals received transplants of embryonic midbrain dopaminergic neurons into their SNs. They were tested for motor asymmetry every two weeks for a total of eight weeks and then brain tissue was harvested for immunohistochemical analysis. Results demonstrate that virus-induced expression of GDNF and GFR1 supports growth of dopaminergic fibers from cells transplanted into the SN all the way to the striatum, and these animals have a significant reduction in both drug-induced and spontaneous motor asymmetry.

An important mechanism during nervous system development is the guidance of axons by chemical gradients. The structure responsible for responding to chemical cues in the embryonic environment is the axonal growth cone -- a structure combining sensory and motor functions to direct axon growth. In this thesis, we develop a series of mathematical models for the gradient-based guidance of axonal growth cones, based on the idea that growth cones might be optimised for such a task. In particular, we study axon guidance from the framework of Bayesian decision theory, an approach that has recently proved to be very successful in understanding higher level sensory processing problems. We build our models in complexity, beginning with a one-dimensional array of chemoreceptors simply trying to decide whether an external gradient points to the right or the left. Even with this highly simplified model, we can obtain a good fit of theory to experiment. Furthermore, we find that the information a growth cone can obtain about the locations of its receptors has a strong influence on the functional dependence of gradient sensing performance on average concentration. We find that the shape of the sensitivity curve is robust to changes in the precise inference strategy used to determine gradient detection, and depends only on the information the growth cone can obtain about the locations of its receptors. We then consider the optimal distribution of guidance cues for guidance over long range, and find that the same upper limit on guidance distance is reached regardless of whether only bound, or only unbound receptors signal. We also discuss how information from multiple cues ought to be combined for optimal guidance. In chapters 5 and 6, we extend our model to two-dimensions, and to explicitly include temporal dynamics. The two-dimensional case yields results which are essentially equivalent to the one dimensional model. In contrast, explicitly including temporal dynamics in our leads to some significant departures from the one-dimensional and two-dimensional models, depending on the timescales over which various processes operate. Overall, we suggest that decision theory, in addition to providing a useful normative approach to studying growth cone chemotaxis, might provide a framework for understanding some of the biochemical pathways involved in growth cone chemotaxis, and in the chemotaxis of other eukaryotic cells.

xv, 83 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / This dissertation describes research to identify genes involved in specification, patterning and development of zebrafish primary motoneurons. We first examined the spatiotemporal expression patterns of retinoic acid and retinoid X receptor mRNAs to determine whether particular ones might be involved in motoneuron specification or patterning. Retinoic acid and retinoid X receptor mRNAs are expressed at the right time to pattern motoneurons, but the expression patterns did not suggest roles for particular receptors. In contrast, netrin mRNAs are expressed in specific motoneuron intermediate targets and knockdown experiments revealed an important role in development of VaP motoneurons. Two identified motoneurons, CaP and VaP, initially form an equivalence pair. CaPs extend long axons that innervate ventral muscle. VaPs extend short axons that stop at muscle fibers called muscle pioneers; VaPs later typically die. Previous work showed that during extension, CaP axons pause at several intermediate targets, including muscle pioneers, and that both CaP and muscle pioneers are required for VaP formation. We found that mRNAs for different Netrins are expressed in intermediate targets before CaP axon contact: netrin 1a in muscle pioneers, netrin 1b in hypochord, and netrin 2 in ventral somite. We show that Netrins are unnecessary to guide CaP axons but are necessary to prevent VaP axons from extending into ventral muscle. Netrin 1a is necessary to stop VaP axons at muscle pioneers, Netrin 1a and Netrin 2 together are necessary to stop VaP axons near the hypochord, and Netrin 1b appears dispensable for CaP and VaP development. We also identify Deleted in colorectal carcinoma as a Netrin receptor that mediates the ability of Netrin 1a to cause VaP axons to stop at muscle pioneers. Our results suggest Netrins refine axon morphology to ensure final cell-appropriate axon arborization. To learn whether Netrin proteins diffuse away from their sources of synthesis to function at a distance, we are developing Netrin antibodies. If successful, the antibodies will provide the research community at large with a new tool for understanding in vivo Netrin function. This dissertation includes both my previously published and unpublished coauthored material. / Committee in charge: Monte Westerfield, Chairperson, Biology Judith Eisen, Advisor, Biology; Victoria Herman, Member, Biology; John Postlethwait, Member, Biology; Clifford Kentros, Outside Member, Psychology

Thesis advisor: Laura Anne Lowery / Axon guidance is a critical process in forming the connections between a neuron and its target. Development of a properly functioning nervous system relies heavily on how accurately an axon is guided to the right target. Defects in the guidance machinery may result in neurological disorders. The growth cone that is formed at the tip of a growing axon is responsible for navigating axons to their final targets. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues received by membrane-associated receptors at the growth cone periphery. Upon receiving guidance cues, a number of intracellular signal transduction pathways are initiated downstream of the guidance receptors, that can promote or halt growth cone advance. The growth cone generates these responses by remodeling its cytoskeletal components, which are actin network in the periphery and microtubules in the growth cone center. In this thesis, we focus on understanding the role of microtubule dynamics regulation within the growth cone as it makes guidance decisions. Specifically, we examine the role of TACC3 as a microtubule plus-end binding protein during axon outgrowth and guidance. We show that TACC3 localizes at microtubule plus-ends in embryonic Xenopus laevis growth cones and regulates microtubule growth parameters. We also show that TACC3 is important for promoting axon outgrowth in cultured neural tube explants. Furthermore, our data suggests that TACC3 affects axon guidance in vivo and ex vivo. Examination of embryos depleted of TACC3 revealed guidance defects in the spinal cord neurons, while TACC3-overexpressing cultured spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Finally, in an attempt to delineate the mechanism behind TACC3-mediated axon guidance under Slit2, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that retaining phosphorylatable tyrosines within the TACC domain is important for its microtubule plus-end tracking behavior and its impact on microtubule dynamics regulation, axon outgrowth and guidance. Together, this thesis contributes new insights to the understanding of the role of TACC3 as a microtubule plus-end binding protein and identifies TACC3 as a potential regulator of axon outgrowth and guidance during Xenopus laevis embryonic development. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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