by Ling Lin. / "November 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 149-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

During development of the nervous system, formation of specific connections between nerve cells depends on the stability of growing axons to reach appropriate target cells and form synapses. In culture, hippocampal neurons form numerous synapses by developing axonal and dendritic extensions. To elucidate principles of neuronal signaling and network establishment, creation of neuronal networks in which connectivity and pathways can be experimentally controlled is of great interest. In the present study we used a microcontact printing technique to control and study neurite outgrowth of hippocampal neurons in vitro. My preliminary results show that hippocampal neurons follow the microcontact printed pattern of poly-D-lysine (PDL). In doing so, neurons retain their morphology with normal subcellular distribution of various cell adhesion and synaptic molecules. However, the distribution of various axonal or dendrite components is altered. Hence we have developed a system in which isolated axons and dendrites align with inputs from very few neurons. With this technique we intend to study axon-dendrite communications on a spatially restricted and defined substrate.

Glial cells represent the most abundant cell population in the central nervous system (CNS), and yet, have historically been thought of as merely support cells for neurons. Over the past few decades, however, the number of identified roles that glial cells play in the CNS has expanded at an exponential rate, revealing new and exciting functions in neuron-glial communication. At synapses, astrocytes are now recognized as part of a "tripartite" complex with pre- and postsynaptic structures and can modulate synaptic transmission and plasticity. Accumulating evidence has also revealed new roles for oligodendrocytes in regulating axon diameter and integrity, and ion channel clustering. Despite our knowledge of the physiological connections between neurons and glia, relatively little is known about the morphological interplay of these cells during development and in the mature brain. The results presented in this thesis reveal the extent and time-course of rapid remodelling of astrocytes and oligodendrocytes in close proximity to dendritic spines and axons respectively. These findings provide further evidence that glia play an important role in regulating the structural plasticity of the brain. The methodology developed also provides a powerful system for the study of neuron-glial structural dynamics and may contribute to the development of novel therapeutic strategies for diseases affecting the central nervous system.

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.

Le claustrum est une structure télencéphalique sous-corticale présente chez tous les mammifères. Il est réciproquement interconnecté avec tout le cortex cérébral. Le claustrum serait impliqué dans la synchronisation d’oscillations corticales et le maintien de l’attention. Les projections corticales sensorielles au claustrum de la souris forment des bandes longitudinales dédiées à chaque modalité sensorielle, qui peuvent également se chevaucher. Actuellement, il n'y a aucune étude de la morphologie des axones des projections corticales au claustrum. Les paramètres quantitatifs morphométriques des axones uniques permettent de comprendre comment l’information est distribuée à l’intérieur d’une structure. Pour visualiser les axones de la projection visuelle au claustrum, des injections iontophorétiques du traceur neuronal antérograde, la leucoagglutinine Phaseolus vulgaris (PHA-L) ont été réalisées dans le cortex visuel de souris adultes C57BL/6J. La révélation immunohistochimique du PHA-L a été réalisée pour reconstruire les axones individuels des projections des cortex visuels au claustrum en utilisant Neurolucida 360 (MBF Biosciences). Ces projections ont été montrées dans la région centrale le long de l’axe dorso-ventral du claustrum ipsi- et controlatéral. Il y a trois types de morphologie des axones : certains parcourent toute la longueur du claustrum sans ramification, d’autres sont longs et branchés, et d’autres sont courts. Les arborisations locales pourraient suggérer des modules plus petits au sein de l'organisation longitudinale du claustrum. Certaines collatérales de l'axone sortent du domaine visuel central pour se ramifier dans des domaines des autres modalités sensorielles. La projection du cortex visuel au claustrum est ainsi constituée d’axones dont la morphologie est peu diversifiée. Des axones individuels acheminent l’information sur toute l’étendue rostro-caudale du claustrum. La bifurcation des axones suggère que l’information visuelle peut être acheminée vers des territoires de d’autres modalités sensorielles dans le claustrum. La distribution des varicosités suggère que les contacts synaptiques s’établissent aléatoirement tout au long du trajet des axones. / The claustrum is a subcortical telencephalic structure present in all mammals. It is widely and reciprocally interconnected with the entire cerebral cortex. It has been suggested to be involved in the synchronization of cortical oscillations and in attention. The claustrum in mice exhibits a clear dorsoventral organization of longitudinal bands dedicated to different sensory cortical projections, that can also overlap. Presently, there are no studies of the cortical projections to the claustrum at the single axon level. Single axon quantitative morphometric parameters help understanding how information is distributed within a structure and how it interacts with the entire brain. To reveal axons of the visual cortex projection to the claustrum, iontophoretic injections of the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were performed in the visual cortex of adult C57BL/6J mice. Individual axons of the projections from the visual cortices to the claustrum were reconstructed using Neurolucida 360 (MBF Biosciences). These projections were shown in central region of the dorsoventral axis in the ipsi- and contralateral claustra. There were three morphological types of axon: some traveled all the length of the claustrum without significant branching, some were long and branched, and others were short. Local arborizations might suggest smaller modules within the longitudinal organization of the claustrum. Some collaterals of the main axon exit the central visual domain to branch into other sensory modalities. The visual projection to the claustrum is formed by poorly diversified axon regarding to their morphology. Individual axons route the information through all the rostro-caudal length of the claustrum. Axonal bifurcations suggest that visual information can be directed to other territories associated to other sensory modalities. The varicosity distribution suggest that synapses are established randomly.

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