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.

The sciatic nerves of rats were crushed with fine forceps and allowed to survive for 3 or 7 days, at which time the 5th lumbar dorsal root ganglion was injected with [3H]glucosamine. Animals were killed 18 h later and the nerves proximal and distal to the crush site were cut into 3 mm segments. Gangliosides were purified from these segments, and radioactivity was separately measured in gangliosides, neutral glycolipids and glycoprotein. For all 3 fractions, radioactivity was distributed similarly between the crush site the point of maximum axonal elongation. A second smaller peak of ganglioside radioactivity was seen to span a few segments immeidately distal to the point of maximum axonal elongation. We propose two possible explanation for this: (1) it represents ganglioside synthesis by Schwann cells (from blood-borne [3H]glucosamine) as part of the mitogenic response of these cells to the reappearance of axons; or (2) recently synthesized, transported gangliosides are released from the growth cone and taken up by adjacent mitogenic Schwann cells.

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The sense of smell relies upon the detection of odorants by neurons located in the nasal cavity. These neurons, referred to as olfactory sensory neurons (OSNs), line the olfactory epithelium and extend axons that make synaptic connections with mitral/tufted cells in the olfactory bulb. The mechanisms by which these synaptic connections form remain largely unknown. The development of these synaptic connections relies on the axons of immature OSNs innervating the olfactory bulb. The primary goal of this dissertation was to identify components of the mechanisms used by immature OSN axons to innervate the olfactory bulb. To accomplish this goal, a knockout mouse model was used. OSN axons, of Emx2 knockout mice fail to innervate the olfactory bulb. As EMX2 is a transcription factor, this model was used investigate the possible causes of the defective OSN axon growth. To gain a better understanding of OSN axon growth, differences in expression of axon growth and guidance genes in immature and mature OSNs was investigated. This analysis revealed that many axon growth and guidance genes are differential expressed, and helped to identify immature OSN specific genes. The data also revealed a previously unrecognized developmental stage, termed nascent OSNs, identified by the expression of Cxcr4. Analysis of Emx2-/- mice revealed that EMX2 is necessary for OSN survival, odorant receptor expression and expression of the axonogenesis related gene Ablim1. EMX2 is necessary for the expression of many odorant receptor genes; however the loss of odorant receptor expression does not explain the axon growth defects. Apoptosis is increased in Emx2-/- mice, an outcome that may be due to the failed axon growth. Analysis of axon guidance gene expression identified a large reduction in Ablim1 expression in Emx2-/- mice. Ablim1 is expressed by immature OSNs, placing it in the proper cell type to regulate OSN axon growth. The loss of Ablim1 expression in Emx2-/- mice indicates defective signaling in the axon growth cone and a possible mechanism regulating OSN axon growth into the olfactory bulb. The data presented in this dissertation provide new insight into the regulation of odorant receptor gene expression and OSN axon growth.

Adaptation is one of the basic phenomena of biology, while adaptability is an important feature for neural network. Young barn owl can well adapt its visual and auditory integration to the environmental change, such as prism wearing. At first, a mathematical model is introduced by the related study in biological experiment. The model well explained the mechanism of the sensory map realignment through axongenesis and synaptogenesis. Simulation results of this model are consistent with the biological data. Thereafter, to test the model’s application in hardware, the model is implemented into a robot. Visual and auditory signals are acquired by the sensors of the robot and transferred back to PC through bluetooth. Results of the robot experiment are presented, which shows the SC model allowing the robot to adjust visual and auditory integration to counteract the effects of a prism. Finally, based on the model, a silicon Superior Colliculus is designed in VLSI circuit and fabricated. Performance of the fabricated chip has shown the synaptogenesis and axogenesis can be emulated in VLSI circuit. The circuit of neural model provides a new method to update signals and reconfigure the switch network (the chip has an automatic reconfigurable network which is used to correct the disparity between signals). The chip is also the first Superior Colliculus VLSI circuit to emulate the sensory map realignment.

During the development of the nervous system, neurons migrate to their final location and extend neurites that navigate long distances in the extracellular environment to reach their synaptic targets. The proper functioning of the nervous system depends on correct connectivity, and mistakes in the wiring of the nervous system lead to brain abnormalities and mental illness. Growth cones are motile structures located at the tip of extending neurites that sense and respond to guidance cues encountered along the path toward their targets. Binding of these cues to receptors located on growth cone filopodia and lamellipodia triggers intracellular signaling pathways that regulate growth cone cytoskeletal dynamics. Although studies on extracellular cues and their effects on neuronal guidance are well documented, less is known about the intracellular signaling mechanisms that regulate growth cone motility. This dissertation focuses on two signaling pathways and describes how they might be involved in determining growth cone morphology during neuronal development. The specific aims of this work address: (1) the role of phosphatidylinositol-3-kinase (PI-3K) and its downstream signaling pathway in regulating growth cone motility, and (2) the effect of nitric oxide (NO) release from a single cell on growth cone morphology of neighboring neurons. This study employs defined neurons from the pond snail, Helisoma trivolvis, to demonstrate that inhibition of PI-3K induces a concomitant increase in filopodial length and a decrease in the rate at which neurites advance. These effects are mediated through the lipid and protein kinase activities of PI-3K, and filopodial elongation is due to an increase in the rate at which filopodia elongate and the time that individual filopodia spend extending. Additionally, this study demonstrates that NO release from a single cell can affect growth cone dynamics on neighboring neurons via soluble guanylyl cyclase (sGC), and that NO has a physiological effect up to a distance of 100 ìm. Overall this study provides new information on cellular mechanisms regulating growth cone motility, and suggests a potential role of PI-3K and NO in neuronal pathfinding in vivo.

Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

This research focuses on the nanoscopic investigation of the three-dimensional surface morphology of the neural growth cones from the snail Helisoma trivolvis, and InN and InGaN semiconductor material systems using Atomic Force Microscopy (AFM). In the analysis of the growth cones, the results obtained from AFM experiments have been used to construct a 3D architecture model for filopodia. The filopodia from B5 and B19 neurons have exhibited different tapering mechanisms. The volumetric analysis has been used to estimate free Ca2+ concentration in the filopodium. The Phase Contrast Microscopy (PCM) images of the growth cones have been corrected to thickness provided by AFM in order to analyze the spatial refractive index variations in the growth cone. AFM experiments have been carried out on InN and InGaN epilayers. Ternary InGaN alloys are promising for device applications tunable from ultraviolet (Eg[GaN]=3.4 eV) to near-infrared (Eg [InN]=0.7 eV). The real-time optical characteristics and ex-situ material properties of InGaN epilayers have been analyzed and compared to the surface morphological properties in order to investigate the relation between the growth conditions and overall physical properties. The effects of composition, group V/III molar ratio and temperature on the InGaN material characteristics have been studied and the growth of high quality indium-rich InGaN epilayers are demonstrated.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18169号 / 医博第3889号 / 新制||医||1003(附属図書館) / 31027 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 大森 治紀, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM