National concern over traumatic brain injury (TBI) is growing rapidly. Recent focus is on mild TBI (mTBI), which is the most prevalent injury level in both civilian and military demographics. A preeminent sequelae of mTBI is cognitive network disruption. Advanced neuroimaging of mTBI victims supports this premise, revealing alterations in activation and structure-function of excitatory and inhibitory neuronal systems, which are essential for network processing. However, clinical neuroimaging cannot resolve the cellular and molecular substrates underlying such changes. Therefore, to understand the full scope of mTBI-induced alterations it is necessary to study cortical networks on the microscopic level, where neurons form local networks that are the fundamental computational modules supporting cognition. Recently, in a well-controlled animal model of mTBI, we demonstrated in the excitatory pyramidal neuron system, isolated diffuse axonal injury (DAI), in concert with electrophysiological abnormalities in nearby intact (non-DAI) neurons. These findings were consistent with altered axon initial segment (AIS) intrinsic activity functionally associated with structural plasticity, and/or disturbances in extrinsic systems related to parvalbumin (PV)-expressing interneurons that form GABAergic synapses along the pyramidal neuron perisomatic/AIS domains. The AIS and perisomatic GABAergic synapses are domains critical for regulating neuronal activity and E-I balance. In this dissertation, we focus on the neocortical excitatory pyramidal neuron/inhibitory PV+ interneuron local network following mTBI. Our central hypothesis is that mTBI disrupts neuronal network structure and function causing imbalance of excitatory and inhibitory systems. To address this hypothesis we exploited transgenic and cre/lox mouse models of mTBI, employing approaches that couple state-of-the-art bioimaging with electrophysiology to determine the structural- functional alterations of excitatory and inhibitory systems in the neocortex.

Myelination of axons in the central and peripheral nervous system (CNS and PNS) is required for saltatory propagation of nerve impulses. Myelinated axons are organized in functionally distinct membrane domains and the correct formation and maintenance of these domains is fundamental for the correct propagation of the electrical impulse; however, the underlying mechanisms by which these domains are specified are just starting to be unravelled. The paranodal junctions (PNJs) have been shown to contribute to node formation in the CNS and to domain maintenance both in the CNS and PNS. In this thesis I have studied the function of the linkage of the PNJs to the axonal cytoskeleton in regulating these processes by using a combination of knock out and transgenic rescue strategies. Further, I have initiated studies on the live imaging of node assembly. I have shown that the link between the PNJ and the axonal cytoskeleton is required for both the correct timing of oligodendrocyte process migration and for clustering nodal proteins at heminodes, before nodes of Ranvier are formed. Failure to correctly regulate these events during development results in shorter internodes in adults. Further, I have shown the importance of the axonal paranodal cytoskeleton in the maintenance of the node of Ranvier, both in CNS and PNS. In the absence of a link between the PNJ and the axonal cytoskeleton, paranodes disassemble, which causes disruption of both nodal and juxtaparanodal domains. Electron microscopy shows that, despite paranodal disruption, transverse bands are preserved when the anchorage of the PNJ to the axonal cytoskeleton is removed. Surprisingly, the preservation of these structures is associated with the amelioration of the neurological defects seen in mice lacking PNJs. In order to study nodal assembly, I have initiated studies on the transport of the nodal proteins Neurofascin186 and β1Nav tagged with fluorescent tags in transgenic mice, in order to analyse axonal transport during development. I have exploited the triangularis muscle explant preparation and have analysed transport of nodal proteins in young and adult mice. I have shown that transport speeds decrease with age and that the two proteins are transported at different speeds in young animals, but these differences do not persist in adults. This suggests that during myelination these proteins are transported in different vesicles and that this may change during development.

Les mutations du gène SPG4 codant la spastin sont responsables de la forme la plus fréquente de Paraplégies Spastiques Héréditaires (PSH), des maladies neurologiques caractérisées par une dégénérescence des faisceaux cortico-spinaux. La spastin, ainsi que son homologue p60-katanin sont des enzymes de cassure des microtubules (MSE) essentielles à la croissance des neurones moteurs spinaux (NMS) chez l'embryon de poisson-zèbre mais dont le rôle dans les processus de guidage axonal également dépendant des microtubules (MTs) demeurent énigmatiques. Les principaux objectifs de ma thèse ont consisté à préciser le rôle et le degré de redondance fonctionnelle existant entre ces deux MSE lors de l'établissement des circuits moteurs chez ce téléoste et de clarifier les mécanismes pathogéniques à l¿origine des PSH liées au gène SPG4.J'ai tout d'abord contribué à montrer que la p60-Katanin contrôle la trajectoire des axones des NMS et la mobilité des larves de façon dose-dépendante et non redondante avec la spastin. De plus, notre étude identifie la polyglutamylation des MTs par TTLL6 comme un élément clé de l'activité de la p60-Katanin lors de ce processus. Sur le même modèle, j'ai révélé un rôle différentiel des isoformes majoritaires de la spastin (résultant d¿une traduction alternative, M1 et M61) au cours du développement des NMS en démontrant un rôle coopératif de M1 et d'autres protéines de PSH dans l'inhibition de la voie des BMPs et révélant un rôle pour M61 en aval de la signalisation Neuropilin-1. Ces données suggèrent que l'altération de ces deux grandes voies de signalisation essentielles au développement des NMS pourrait contribuer à la pathogénèse des formes SPG4. / Mutations in SPG4, encoding spastin cause the major form of Hereditary Spastic Paraplegias (HSP), a paralytic disorder characterised by the degeneration of the corticospinal tracts. Spastin and its close homologue p60-katanin are microtubule-severing enzymes (MSE) required for spinal motor neuron (SMN) axon extension during zebrafish development. However, their roles in SMN axon navigation which also rely on microtubules (MTs) remain elusive. My PhD work aimed at refining the functional specificity and redundancy of these MSE during motor circuit wiring and clarifying the physiopathology of SPG4-linked HSP. I have first contributed to show that p60-Katanin controls SMN axon targeting and larval locomotion in a dose-dependent manner. We also demonstrated that Spastin and p60-Katanin play differential roles in SMN navigation and identified TTLL6-mediated MT polyglutamylation as a key event in regulating p60-Katanin activity in this process. Concomitantly, I have conducted a functional analysis of spastin main isoforms (resulting from alternative translation, M1 and M61) during zebrafish development, which reveals their critical and specific involvement in two distinct signalling pathways that are both essential for motor circuit wiring and locomotor behaviours. This study has provided compelling evidences for a concerted role for M1 and other HSP proteins in the down-regulation of the BMP pathway and reveals a specific role for M87 as a downstream effector of Neuropilin-1 signalling. Altogether, our study emphasizes defective BMP signalling as a key pathogenic mechanism in HSP, and shows that dysregulation of the Neuropilin-1 pathway may equally contribute to SPG4-linked HSP.

Dans le système nerveux des bilatériens, certains neurones, appelés commissuraux, étendent leurs axones au-delà de la ligne médiane pour se connecter à des neurones controlatéraux. La Nétrine-1, une protéine sécrétée, guide ces axones jusqu'à la ligne médiane ventrale. Son récepteur DCC, exprimé dans les axones commissuraux, est considéré comme le relais de cette signalisation. Cependant, chez la souris, le récepteur Robo3 participe également à cette signalisation et est nécessaire pour que les axones commissuraux franchissent la ligne médiane ventrale. Dans ce travail, nous avons étudié la conservation des récepteurs Robo3 et DCC chez les vertébrés. De façon surprenante, nous avons mis en évidence que le gène Dcc est absent chez deux groupes d'oiseaux. Toutefois, cette perte ne semble pas s'accompagner de défaut de guidage des axones commissuraux chez ces oiseaux. Le gène Robo3 est, quant à lui, présent chez tous les vertébrés, mais présente des particularités structurales et fonctionnelles chez les mammifères, notamment la perte de liaison aux Slits (les ligands canoniques des récepteurs Robo). Chez les amniotes, Robo3 est spécifiquement exprimé dans les axones commissuraux avant le croisement de la ligne médiane. En revanche, chez les autres vertébrés, son expression est plus vaste et sa fonction dans le guidage commissural reste ambigüe. Enfin, la restauration de la signalisation Robo3-Slits chez la souris et sans effet sur la mise en place des commissures, et son rôle chez les autres vertébrés reste inconnu. Dans l'ensemble, ces données apportent un nouvel éclairage sur la diversité des commissures et de leurs systèmes de guidage chez les vertébrés. / In most bilateria, some neurons, called commissural neurons, extend their axons across midline to connect with contralateral targets. Netrin-1, a protein secreted at the ventral midline by the floor plate, atracts commissural axons and guide them towards midline. It is thought that the DCC receptor mediates Netrin-1 attraction towards ventral midline. However, in the mouse, Robo3 receptor also participate to Netrin-1 attractive signaling, and is necessary for commissural axons to reach ventral mdline. In this work, we have been studiing Robo3 and DCC receptors conservation in vertebrates. Strickingly, we found that birds withinh two major groups, the Galliforms and Passeriforms, do not have a Dcc gene. However, our comparative analysis of commissure development in birds revealed no difference between birds having or not DCC. On the other hand, Robo3 gene is present in all vertebrates studied, but is divergent in mammals. Especially Robo3 does not bind its cananical lignads Slits in these group. In amniotes, Robo3 is specifically expressed in commissural axons before they cross the midline. In contrast, Robo3 expression in other vertebrates is broder, and its exact function in commissural axon guidance remains unclear. Finally, restoring Slit binding to Robo3 in mammals does not affect commissural guidance, and the rôle of Robo3-Slit signaling in other vertebrates remains unclear. These data shed a new light on commissures diversity and their guidance mechanism among vertebrates.

SARM1 is an injury-induced nicotinamide adenine dinucleotide nucleosidase (NADase) that was previously shown to promote axonal degeneration in response to traumatic, toxic, and excitotoxic stressors. This raises the question of whether a SARM1-dependent program of axonal degeneration is central to a common pathway contributing to disease burden in neurological disorders. The degree to and mechanism by which SARM1 inactivation decreases the pathophysiology of such disorders is of interest to establish the rationale to pursue SARM1 as a therapeutic target. In this study, we compare the course and pathology of experimental autoimmune encephalomyelitis (EAE) in Sarm1-knockout (KO) mice and wild-type littermates to test the contribution of SARM1-dependent axonal degeneration specifically in the context of chronic, immune-mediated central nervous system (CNS) inflammation. The question of whether SARM1 loss in Sarm1-KO mice would inhibit, promote, or have a negligible impact on EAE-induced axonal degeneration and more broadly CNS inflammation was explored using a variety of analyses: quantification of clinical score in a chronic EAE model, CNS immune infiltrate profile, axon initial segment morphology in layer V cortical neurons, axonal transport disruption and transection in the lumbar spinal cord. Additionally, we have proposed a method for detecting SARM1 activation in situusing a novel SARM1-mCitrine bimolecular fluorescence complementation (BiFC) technique. Successful implementation of such a molecular tool would allow for a detailed, mechanistic approach to enhance our understanding of upstream intracellular signals that trigger SARM1 activation.

acase@tulane.edu

The current focus of research efforts in the area of the biomechanics of traumatic brain injury is the development of numerical (finite element) models of the human head. A validated numerical model of the human head may lead to better head injury criteria than those used currently in crashworthiness studies. A critical step in constructing a validated finite element model of the head is determining the mechanical threshold, should it exist, for various types of injury to brain tissue. This thesis describes a biomechanical study of axonal injury in the anaesthetised sheep. The study used the measurements of the mechanics of an impact to the living sheep, and a finite element model of the sheep skull and brain, to investigate the mechanics of the resulting axonal injury. Sheep were subjected to an impact to the left lateral region of the skull and were allowed to survive for four hours after the impact. The experiments were designed specifically with the numerical model in mind; sufficient data were collected to allow the mechanics of the impact to be faithfully reproduced in the numerical model. The axonal injury was identified using immunohistological methods and the injury was mapped and quantified. Axonal injury was produced consistently in all animals. Commonly injured regions included the sub-cortical and deep white matter, the hippocampi and the margins of the lateral ventricles. The degree of injury was closely related to the peak impact force and to kinematic measurements, particularly the peak change in linear and angular velocity. There was significantly more injury in animals receiving fractures. A three-dimensional finite element model of the sheep skull and brain was constructed to simulate the dynamics of the brain during the impact. The model was used to investigate different regimes of material properties and boundary conditions, in an effort to produce a realistic model of the skull and brain. Model validation was attempted by comparing pressure measurements in the experiment with those calculated by the model. The distribution of axonal injury was then compared with the output of the finite element model. The finite element model was able to account for approximately thirty per cent of the variation in the distribution and extent of axonal injury, using von Mises stress as the predictive variable. Logistic regression techniques were used to construct sets of curves which related the extent of injury, to the predictions of the finite element model, on a regional basis. The amount of observable axonal injury in the brains of the sheep was clearly related to the severity of the impact, and was related to the predictions of a finite element model of the impact. Future improvements to the fidelity of the finite element model may improve the degree to which the model can explain the variation in injury throughout the brain of the animal and variations between animals. This thesis presents results, and a methodological framework, that may be used to further our understanding of the limits of human endurance, in the tolerance of the brain to head impact. All experiments reported herein conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. / Thesis (Ph.D.)--Mechanical Engineering, 2000.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.