It is known that guidance cues play important roles in neuronal outgrowth and axonal path-finding. These guidance cues, when bound to the receptors on nerve growth cones, trigger a series of signal transduction pathways that result in dynamic rearrangement of cytoskeleton in neuronal cells. Although enormous studies have been suggested that intracellular calcium concentration is crucial in regulation of neurite outgrowth and cytoskeleton rearrangement, the precise molecular mechanism underlying growth cone guidance is not well understood. The aim of our studies is to set up an evaluation system, growth cone turning assay, which is suitable and reproducible for exploration of the molecular mechanism of guidance cue-induced growth cone turning. The 1-day-old cultured spinal neurons of Xenopus laevis and the approved attractive guidance cue, cAMP, were used in our growth cone turning assay. We evaluate the effects of neuronal developmental stage, size of pressure, frequency and duration in cAMP-induced growth cone turning. We found application of cAMP with a pressure of 2 Hz in frequency and 20 ms in duration can elicit a reliable growth cone attractive response in cultured spinal motoneurons with 14~18 hours in age. Depletion of intracellular calcium store with thapsigargin in calcium-free Ringer could significantly abolish cAMP-induced growth cone turning. The cAMP-induced growth cone turning was not blunted when calcium was omitted from extracellular fluid or bath application of calcium channel inhibitor suggesting calcium influx is not responsible for the growth cone attractive effect of cAMP. Application of membrane-permeable inhibitors of ryanodine receptors but not inositol 1,4,5-trisphosphate (IP3) effectively occluded the attractive effect elicited by cAMP. The cAMP-induced growth cone turning was unapparent under the presence of Li+, a blocker of IP3 formation. It has been suggest besides block IP3 formation, Li+ is also involved in modulation of several signaling pathways including GSK3-£] dependent signaling. However, bath application of GSK3-£] inhibitor has no significant effect on cAMP-induced growth cone turning. Taken together, our results suggest ryanodine-mobilized intracellular Ca2+ store play a major role in cAMP-induced growth cone turning. Moreover, our result on surveying various embryonic stage, drug application protocol also establish a good condition for neuronal growth cone turning assay, which can be used in exploring the molecular mechanism of growth cone path-finding.

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Thesis advisor: Laura Anne Lowery / Thesis advisor: David Burgess / While XMAP215 (CKAP5 / ch-TOG) has been best characterized for its microtubule polymerase function, recent studies have highlighted a novel role for XMAP215 in facilitating an interaction between microtubules and F-actin in the embryonic neuronal growth cone, a critical structure involved in neuronal outgrowth and guidance mechanisms. Microtubule and F-actin cytoskeletal cross talk and reorganization are important aspects of axonal guidance mechanisms, but how associated proteins facilitate this function largely remains a mystery. In addition, it has long been established that neuronal growth cone navigation depends on changes in microtubule (MT) and F-actin architecture downstream of guidance cues. However, the mechanisms by which MTs and F-actin are dually coordinated remain a fundamentally unresolved question. Here, I report that the well-characterized MT polymerase, XMAP215 (also known as ch-TOG / CKAP5), plays an important role in mediating MT–F-actin interactions within the growth cone. I demonstrate that XMAP215 regulates MT–F-actin alignment through its N-terminal TOG 1–5 domains. Additionally, I show that XMAP215 directly binds to F-actin in vitro and co-localizes with F-actin in the growth cone periphery. By working with lab colleagues, we also find that XMAP215 is required for regulation of growth cone morphology and response to the guidance cue, Ephrin A5. Our findings provide the first strong evidence that XMAP215 coordinates MT and F-actin interaction in vivo. It is here that I suggest a model in which XMAP215 regulates MT extension along F-actin bundles into the growth cone periphery and that these interactions may be important to control cytoskeletal dynamics downstream of guidance cues. Furthermore, I then go on to study this dual microtubule and F-actin role, diving deeper into the mechanism behind this novel ability of XMAP215. Here, I report that XMAP215 is capable of spatially localizing populations of microtubules into distinct domains in the growth cone through its less well-characterized microtubule-lattice binding activity. In addition, through the use of purified proteins and biochemical assays, I show that XMAP215 is capable of binding directly to F-actin, facilitated by its unique TOG5 domain. Finally, through biochemical means and super resolution imaging, I show that this novel function of XMAP215 is mediated by polymerase-incompetent mutants of XMAP215. Taken together, my findings show strong evidence of a non-microtubule-polymerase function of XMAP215, providing mechanistic insights into how microtubule populations can be guided by interaction with the F-actin cytoskeleton. In conclusion, I explore a novel and functionally important role for XMAP215 in facilitating interactions between microtubule and actin cytoskeletons, bridging the two structural components of the cell together. In this way, XMAP215 is now known as a distinct microtubule/F-actin regulator that governs microtubule exploration through the help of actin in neurons, in addition to its previously characterized function as a microtubule polymerase. While this thesis explores the very groundwork of XMAP215’s new novel function, there is still a great deal more to learn about the overall mechanism occurring, as well as an understanding of its role in various cell types. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The nervous system is required for human cognition, motor function, and sensory interaction. A complex network of neuronal connections, or synapses, carries out these behaviors, and defects in neural connectivity can result in developmental and degenerative diseases. In vertebrate nervous systems, synapses most commonly occur at axon terminals. Upon reaching their synaptic targets, growth cones lose their motility and become boutons specialized for neurotransmitter release. I am studying this process in R7 photoreceptors in the

The development of a stereotyped pattern of neural connectivity depends upon the behavior of growth cones, motile structures at the tips of axons that propel axon growth and steer the axon to its targets. When growth cones reach their appropriate target cells, they halt and ultimately remodel into stationary presynaptic boutons. The influence of extracellular cues in directing growth cones to their targets is well studied, but cell-intrinsic factors are also increasingly appreciated for their role in driving much of growth cone behavior. Dual leucine zipper kinases (DLKs) promote growth cone motility and must be kept in check to ensure normal development. PHR (Pam/Highwire/RPM-1) ubiquitin ligases therefore target DLK for proteosomal degradation unless axon injury occurs. Overall DLK levels decrease during development, but how DLK levels are regulated within a developing growth cone has not been examined. We analyzed the expression of the fly DLK Wallenda (Wnd) in R7 photoreceptor growth cones as they halt at their targets and as they remodel into presynaptic boutons. We found that Wnd protein levels are repressed by the PHR protein Highwire (Hiw) during R7 growth cone halting, as has been observed in other systems. However, during remodeling, Wnd levels are further repressed by a temporally-expressed transcription factor, Tramtrack69 (Ttk69). Previously unobserved negative feedback from JNK also contributes to Wnd repression. We conclude that maturing neurons progressively deploy additional mechanisms to keep DLK off and thereby protect their connectivity. We use live imaging to directly probe the effects of Wnd and Ttk69 on remodeling R7 growth cones and conclude that Ttk69 coordinates multiple regulators of this process. Preliminary results indicate that excess Wnd signaling requires the transcription factor Fos to disrupt growth cone remodeling in R7s. This opens up new strategies to identify how Wnd exerts its motility-promoting effects on growth cone cytoskeletons. Additional findings point to a later requirement for Wnd in normal R7 synapse development, suggesting that Wnd expression is not fully silenced in R7s. Further investigation into these findings would greatly advance our understanding of how the neuronal cytoskeleton is regulated as neurons undergo profound morphological and functional changes while developing. This dissertation includes both unpublished and published co-authored material. This dissertation also includes supplemental movie files, which can be found online and are described in Appendix B.

The activity of the growth cone is necessary for neuron axon elongation during neuron development and regeneration. This is a highly dynamic process of cytoskeletal recorganization that requires a significant amount of energy provided by the mitochondria. The localization of a sufficient number of mitochondria at the growth cone is essential to support its activity during neuron development and regeneration. Both promotion and inhibition of the motility of growth cones can be induced by intrinsic factors of neuron itself such as cytoskeleton dynamics and motor protein activity, as well as extracellular molecules in the vicinity of the neuron such as nerve growth factor (NGF) and components of the extracellular matrix. The proposed hypothesis is that some of these factors have an impact on the localization and morphology of mitochondria. My work in this project is aimed to determine which of these factors have the greatest impact on mitochondria in neurons. Using sensory neurons isolate / Biomedical Sciences

Throughout neuronal development, bouts of spontaneous electrical activity are critical for the proper wiring of neuronal connections. Alterations in firing activity can affect growth cones, which tip developing and regenerating neurites and are responsible for the integration of extracellular guidance cues into pathfinding behaviors. While growing evidence implicates gaseous signaling molecules, nitric oxide (NO) and carbon monoxide (CO), as modulators of neuronal firing activity, less is understood about how they affect growth cone motility. Therefore, in this dissertation, I focus on how NO and CO affect electrical activity of developing and regenerating neurons and how these effects translate into changes at the growth cone level. The specific goals of this dissertation were to investigate 1) the neuron-type-specific effects of NO on growth cone motility; 2) the role of CO in the regulation of neuronal firing activity and excitability; and 3) the role CO plays in the regulation of growth cone motility. Using the well-established developmental model, Helisoma trivolvis, neurons were isolated in single-cell culture allowing for the maximal control over environmental conditions for the direct characterization of NO and CO. In the study of NO, differences in B5 and B19 growth cone responses to NO were due to neuron-type-specific differences in action potential duration. Moreover, the non-responsive B19 growth cones could be made responsive to NO treatment upon the pharmacological broadening of its action potentials. While NO has been found to increase firing activity, the study of CO revealed that CO had the opposite effect on electrical activity, silencing spontaneous firing activity and decreasing neuronal excitability. The study of CO on growth cone motility showed that CO increased growth cone filopodial length through a soluble guanylyl cyclase/protein kinase G/ryanodine receptor mediated pathway without inducing robust increases in growth cone calcium concentration. Taken together, this dissertation reveals new insight into how NO and CO regulate electrical activity and growth cone motility, providing evidence for these gases as important signaling messengers during for the development and regeneration of nervous system.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

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.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.