Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. Differences in the magnitude and characteristics of the peripheral-derived immune cell response to TBI are key contributors to the secondary cascades of damage following brain trauma, and means of modifying this response to improve clinical outcome are a current area of active research. Our work elucidated the peripheral immune response to TBI by characterizing the transcriptomic profile of juvenile vs adult peripheral immune cells following TBI as well as discovering a novel role for the tyrosine kinase receptor EphA4 in the peripheral-derived immune response to brain trauma. Previous work has demonstrated significant differences in recovery from TBI in young vs adult animals, and some studies have indicated that the immune response contributes to these differences. We utilized next-generation sequencing to compare gene expression profiles of blood cell fraction samples in juvenile and adult mice. Our work demonstrated that juvenile peripheral immune cells show a more dynamic response to TBI than adult and that pattern recognition receptor signaling is a cornerstone of these differences. To assess the specific mechanisms involved in the peripheral response to TBI, we utilized a bone marrow chimeric mouse model lacking EphA4 in the hematopoietic compartment. These studies found decreased lesion infiltration of peripheral immune cells, specifically activated macrophages, in the absence of EphA4. We also showed that EphA4 interacts with the Tie2/Angiopoietin signaling axis to regulate macrophage phenotype on the M1/2 continuum. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the peripheral-derived immune cell response to TBI. / Traumatic brain injury (TBI) is a major health concern in the United States and worldwide and effective treatment options are limited. While the blood-brain barrier (BBB) excludes immune cells in the blood from entering the healthy brain, brain trauma compromises BBB integrity and allows massive infiltration of peripheral neutrophils, macrophages, and other immune cells. This circulating immune cell response to TBI contributes to damage following brain trauma, and means of modifying this response to improve recovery are a current area of active research. Our work explored the circulating immune cell response to TBI by comparing the gene expression profile of young vs adult circulating immune cells following TBI as well as discovering a novel role for the EphA4 protein in the circulating immune cell response to brain trauma. Previous work has found significant differences in recovery from TBI in young vs adult animals and that the immune response contributes to these differences. To explore this, we compared gene expression profiles of blood immune cells in young and adult mice and found that young immune cells show a more dynamic response to TBI than adult. To assess the specific pathways involved in the circulating immune cell response, we used a mouse model lacking EphA4 in these cells. Our studies found decreased numbers of immune cells, specifically macrophages, entering the injury area in the absence of EphA4. We also showed that EphA4 interacts with the Tie2 protein and its Angiopoietin protein binding partners. Originally studied as an important contributor to blood vessel function, Tie2 has recently been found to play a role in the function of macrophages. Our work demonstrated that EphA4 interacts with Tie2 to regulate pro-recovery vs proinflammatory characteristics in macrophages. Overall, our work demonstrated a novel role for EphA4, mediated by Tie2, as a pro-inflammatory regulator of the circulating immune cell response to TBI.

Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke. / Doctor of Philosophy / Stroke is the fifth leading cause of death in the United States. Ischemic stroke is the most common type of stroke and occurs when blood flow to part of the brain is impeded. Lack of blood results in cell death and tissue damage in the brain. In an effort to restore blood flow, specialized blood vessels in the brain called collaterals remodel and become larger to allow re-routed blood to the blood-deprived region of the brain. The duration it takes to remodel these remarkable blood vessels and re-route blood varies in humans, and sometimes is not able to prevent adequate tissue damage. The current work explores novel therapeutic targets to accelerate collateral remodeling in an effort to reduce tissue loss after stroke. We present studies which show that a protein called EphA4, found on endothelial cells restricts remodeling, and when inhibited in the brain can increase collateral remodeling and reduced adverse effects after ischemic stroke.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Traumatic Brain Injury (TBI) mainly affects young persons in traffic accidents and the elderly in fall accidents. Improvements in the clinical management have significantly improved the outcome following TBI but survivors still suffer from depression, memory problems, personality changes, epilepsy and fatigue. The initial injury starts a series of events that give rise to a secondary injury process and despite several clinical trials there is no drug available for clinical use that targets secondary brain injury mechanisms. Some recovery of function is seen during the first months following injury but is usually limited and there are no drugs that stimulate the recovery of lost function. Some of the recovery is attributed to plasticity, the brains ability to adapt to new circumstances, and enhancing plasticity via increased axonal growth has the potential to partly restore lost function. In this thesis mice were subjected to the controlled cortical impact model of TBI and functional outcome was evaluated using Morris water maze, the cylinder test and the rotarod. Brain tissue loss was measured in all Papers but the additional histological analyses differ among the Papers. Attempts to increase axonal growth were made by interfering with Nogo receptor function in Paper I and by conditional knockout of ephA4 in Paper II. Contrary to the hypothesis cognition was impaired in Paper I but otherwise no effects of treatment were detected in Paper I and II. Much is still unknown about plasticity and despite the discouraging results of Papers I and II this treatment approach is still worth further exploration. It is firmly established that TBI results in an inflammatory response and some aspects of it may damage brain tissue. In Papers III and IV the inflammatory response was attenuated using an IL-1β directed antibody which resulted in reduced tissue loss and edema while improving cognitive function. The results from Papers III and IV are encouraging and the possibility to find a treatment based on IL-1β inhibition appears promising.

No description available.

No description available.

Neuronal networks are the central functional units of the nervous system. Knowledge about the identity of participating neurons and the assembly of these during development is crucial for the understanding of CNS function. A promising system to dissect the development and functionalities of a neuronal network is the central pattern generator (CPG) for locomotion. We used screening approaches to identify spinal neuronal subpopulations by their specific gene expression, potentially involved in CPG function. Amongst others we found paired-like homeodomain transcription factor 2 (Pitx2) as a cholinergic interneuron marker for partition cells, with a possible role in the spinal network for locomotion. In addition, we present two genes, Chondrolectin (Chodl) and Estrogen-related receptor beta (ERRβ) as novel markers for fast and slow motor neurons, respectively. The neuronal components of the CPG integrate three key functions; rhythm generation, ipsilateral flexors/extensors coordination and bilateral coordination over the midline. Commissural interneurons (CINs) are considered to participate in the latter. During development axons are guided to their targets by the help of axon guidance molecules. Netrin-1 and its receptor DCC (Deleted in Colorectal Cancer) have been shown to play an important role for spinal cord neurons in axon-pathfinding and migration towards the midline. We show that loss of netrin-1 functionally results in a switch from alternating to synchronous left-right locomotor activity and deletion of DCC surprisingly leads to a different phenotype, best described as uncoordination. Thus, during development, netrin-1 and DCC play a critical role for the establishment of a functional balanced CPG. Further we show a selective loss of CINs, predominantly from dorsally originating subtypes, not affecting the ventral-most V3 subtype in netrin-1 mutant mice, but a loss of CINs from all progenitor domains in Dcc mutant mice. Together, our data suggest a netrin-1-independent mechanism for DCC in axon guidance and a role of the most ventral originating CINs as part of the neuronal network controlling synchronous activities over the midline. Another pair of axon guidance molecules, EphA4 and ephrinB3, has been shown to cooperate in preventing ipsilateral interneurons from crossing the spinal midline and if either molecule is deleted in mice, this will result in a defect in left-right coordination of locomotion. We provide in vivo and in vitro evidence that the GTPase-activating protein α2-chimerin, as a downstream molecule of EphA4 signaling, is essential in axon guidance decisions involved in the correct formation of the spinal circuitry for locomotion.

Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.

Les noyaux supraoptiques (NSO) et paraventriculaires (NPV) de l’hypothalamus montrent un phénomène réversible de plasticité structurale neurono-gliale dans diverses conditions physiologiques telles que la parturition, l’allaitement ou lors d’une surcharge en sel. En effet, les feuillets astrocytaires qui enveloppent normalement les somas et dendrites des neurones à ocytocine (OT) ou à vasopressine (AVP) se rétractent alors, autour des neurones à OT, laissant place à la formation de nouvelles synapses, surtout GABAergiques. Nous avons émis l’hypothèse voulant que ces mouvements cellulaires soient régulés par des molécules connues pour leurs rôles dans l’adhérence et la motilité cellulaires, notamment les récepteurs Eph et les éphrines (Efn). Nous avons étudié le rôle de l’un de ces récepteurs, EphA4, un récepteur à tyrosine kinase reconnaissant l’ensemble des Efn, A ou B, puis tenté d’identifier les Efn partenaires dans le NSO, à la suite d’une surcharge en sel. Pour démontrer la présence d’EphA4 dans le NSO et déterminer l’effet d’une surcharge en sel sur son expression et sa localisation, nous avons utilisé l’hybridation in situ et l’immunohistochimie en microscopie électronique, sur des coupes de cerveaux de souris ou rats traités ou non à l’eau salée pendant 1-7 j, avec des ribosondes ou des anticorps spécifiques pour EphA4. Ces travaux ont démontré une augmentation de l’expression d’EphA4 dans le NSO, notamment dans des dendrites, après le régime salé. La distribution de cette expression correspondait à celle des neurones OT et était absente de la glia limitans. Nous avons ensuite déterminé l’effet d’une absence d’EphA4 sur les mouvements astrocytaires et la synaptogènese autour des dendrites à OT et AVP, en utilisant des souris EphA4 knockouts et des souris de type sauvage des mêmes portées. Nous avons ainsi mesuré la couverture astrocytaire des dendrites OT ou AVP, identifiées par immunocytochimie anti-OT ou anti-AVP, en microscopie électronique. Ces mesures ont confirmé la rétraction des feuillets astrocytaires et la synaptogenèse autour des dendrites OT, mais pas autour des dendrites AVP, chez les souris de type sauvage, et démontré que la rétraction des feuillets astrocytaires et la synaptogenèse sur les dendrites OT ne se produisait pas chez les souris knockouts soumises à la surcharge en sel. L’ensemble de ces résultats démontre un rôle d’EphA4 dans cette plasticité structurale neurono-gliale. Afin d’identifier l’Efn partenaire d’EphA4 dans cette fonction, nous avons utilisé l’hybridation in situ et l’immunohistochimie pour les EfnB3 et -A3. L’hybridation in situ n’a pas démontré d’expression de l’EfnB3 dans le NSO, tandis que les résultats pour l’EfnA3 restent à quantifier. Cependant, l’immunohistochimie anti-EfnA3 montre un marquage d’astrocytes dans le NSO et la glia limitans, marquage qui semble augmenter après surcharge en sel, mais il reste à démontrer que l’anticorps anti-EfnA3 est bien spécifique et à quantifier les éventuels changements sur un plus grand nombre d’animaux. L’ensemble de ces observations démontre un rôle du récepteur EphA4 dans les mécanismes à la base des changements structuraux neurono-gliaux du NSO et pointe vers l’EfnA3 comme partenaire d’EphA4 dans ce modèle. / The supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus display reversible neurono-glial structural plasticity in various physiological conditions, such as parturition, lactation, or following salt loading. In such conditions, astrocytic leaflets that normally envelop the somas and dendrites of ocytocin (OT) or arginin-vasopressin (AVP) neurons retract from OT processes where they are replaced by new synapses, mainly GABAergic. Our hypothesis proposes that these cellular movements are regulated by molecules known for their roles in cell adhesion and motility, notably Eph receptors and ephrins (Efn). We have examined the role of one of these receptors, EphA4, a tyrosine-kinase receptor recognizing all ephrins, A or B, and then tried to identify the Efn interacting with EphA4 in these functions, following salt-loading. To demonstrate the presence of EphA4 in the SON and determine the effect of salt loading on its expression, we used in situ hybridization and immunohistochemistry in light and electron microscopy, on brain sections from rats or mice treated with salted water during 1-7 d, using riboprobes and antibodies specific for EphA4. These experiments demonstrated that EphA4 is expressed in the SON, with a distribution of its mRNA similar to that of OT neurons, and that it was absent from the glia limitans. Its expression increased following salt loading, particularly in dendrites. We then tested the effect of an absence of EphA4 on astrocytic process retraction and on synaptogenesis, using EphA4 kockout mice and wild-type littermates. We measured the ratio of astrocytic contact, and counted the number of synapses on the circumference of OT and AVP dendrites, identified in electron microscopy by immunocytochemistry, after 7 d of salt loading. The results confirmed the retraction of astrocytic processes from OT dendrites in wild-type animals after salt loading, and no change around AVP dendrites. However, there was no retraction from OT dendrites in EphA4 knockout mice, following salt loading. Altogether, these results constitute strong evidence for a role of EphA4 in the astrocyte leaflet retraction and accompanying synaptogenesis, specifically around OT dendrites. In order to identify the Efn interacting with EphA4 in this function, we used in situ hybridization and immunohistochemistry for EfnB3 and –A3. The in situ hybridization did not show the presence of EfnB3 in the SON, while the results for EfnA3 are currently being quantified. Nevertheless, anti-EfnA3 immunohistochemistry showed labelling in astrocytes and in the glia limitans of the SON, a labelling that seemed to increase following salt loading, although the specificity of the anti-EfnA3 antibody remains to be demonstrated on EfnA3 knockout mice, and its expression requires to be measured on a larger number of mice. The latter observations indicate EfnA3 as the potential partner (receptor/ligand) for EphA4 in the neurono-glial structural plasticity occurring in the SON following salt loading.