EphrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors can be expressed in both pre-and post-synaptic membranes. Previous studies show that interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), suggesting that reverse signaling through postsynaptic ephrinBs may be required for learning and memory. Our collaborative studies demonstrate that the cytoplasmic domain of ephrinB3, and hence reverse signaling, is not required for ephrinB-dependent learning and memory tasks or for LTP of these synapses. We demonstrate that ephrinB3 null mutants show changes in several synaptic proteins including reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3lacZ reverse signaling mutants, supporting an Eph receptor forward signaling role for ephrinB3 in these processes. NMDA receptors are important in regulating synaptic functions and plasticity in the adult hippocampus, and Eph receptors have been shown to cluster NMDA receptors to the cell membrane. These studies show that ephrinB3 interacts with EphA4 to regulate plasma membrane levels of NR1 in Cos-1 cells and primary hippocampal neurons. In the absence of ephrinB3, NR1 levels are decreased in synaptosomal membranes, increased in microsomal tissues, but not changed in total extracts. This suggests that ephrinB3 regulates NR1 levels through protein trafficking and not gene transcription. Analysis of protein trafficking confirmed that ephrinB3 specifically interacts with EphA4 receptor to regulate NR1 exocytosis but not endocytosis in both transfected Cos-1 cells and primary hippocampal neurons. We postulate that ephrin-Eph receptor interactions are important mediators of synaptic formation and function, in part, through their regulation of NMDA receptors in the hippocampal synapse. In addition, we find that both ephrinB3KO and ephrinB3lacZ mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling through Eph receptors to modulate NMDA receptor trafficking, LTP and learning.

Ephrins and Eph receptors have been implicated in a number of developmental processes including axon growth and guidance. One important guidepost is the central nervous system midline, where ephrins and Eph receptors have been implicated. At the embryonic midline, axons either cross into the contralateral central nervous system (CNS) targeting appropriate partners on the opposite side or remain ipsilateral extending either rostrally or caudally. In these studies, we examine a major forebrain commissure called the corpus callosum (CC). Agenesis of the CC is a rare birth defect that occurs in isolated conditions and in combination with other developmental cerebral abnormalities. Recent identification of families of growth and guidance molecules has generated interest in the mechanisms that regulate callosal growth. One family, ephrins and Eph receptors, has been implicated in mediating midline pathfinding decisions; however, the complexity of these interactions has yet to be unraveled. This dissertation sheds light on which B-class ephrins and Eph receptors function to regulate CC midline growth, and how these molecules interact with important guideposts during development. We also show that multiple Eph receptors (B1, B2, B3, and A4) and B-class ephrins (B1, B2, and B3) are present and function in developing forebrain callosal fibers based on both spatial and temporal expression patterns and analysis of gene-targeted knockout mice. Defects are most pronounced in the combination double knockout mice, suggesting that compensatory mechanisms exist for several of these family members. Furthermore, these CC defects range from mild hypoplasia to complete agenesis and Probst's bundle formation. Further analysis of the ephrinB3 gene revealed that Probst's bundle formation may reflect aberrant glial formations which alter the normal architecture of midline glia resulting in one potential mechanism of this abnormal phenotype. Another potential mechanism we discovered is a role for EphB1 receptor in the altered sensitivity of CC axons to midline guidance cues. Removal of this receptor resulted in cortical axons responding to GW guidepost cells with increased sensitivity. Our results support a significant role for ephrins and Eph receptors in CC development and may provide insight to possible mechanisms involved in axon midline crossing as well how failed molecular and genetic mechanisms may contribute to human CC disorders. Lastly, we show that one fiber tract that remains ipsilateral in the forebrain may use distinct midline guideposts to regulate proper growth and guidance. These findings implicate additional ephrins and Eph receptors in CC midline guidance than previously known and reveal novel mechanisms in mice, which may be pertinent to human disease states that result in agenesis of the CC.

La conversion de la protéine prion cellulaire PrPC en une isoforme pathologique, la protéine prion scrapie PrPSc, est à l'origine d'un groupe de maladies neurodégénératives, les Encéphalopathies Spongiformes Transmissibles (EST). De nombreux travaux indiquent que la toxicité de la PrPSc implique une déviation de la fonction normale de la PrPC, cependant le rôle physiologique de la protéine prion n’est que partiellement compris. Dans ce travail, nous nous sommes attachés à identifier des voies de signalisation mobilisées par la PrPC qui pourraient à la fois rendre compte du rôle de cette protéine dans le développement du système nerveux et être impliquées dans la pathogénèse des EST. Nous montrons que la protéine prion contrôle l’activité de la voie Notch, une voie de signalisation qui joue un rôle majeur dans le développement mais également dans l’homéostasie du système nerveux central et la plasticité synaptique. Dans des modèles ex vivo et in vivo d’EST, nous mettons en évidence une diminution de l’activité de la voie Notch, ainsi que de l’expression des récepteurs de la famille Eph - connus pour leur implication dans l’activité synaptique. Cette diminution des Eph est retrouvée dans des cellules dépourvues de PrPC. Ainsi, l’observation d’un profil similaire entre la perte d’expression de la PrPC et l’infection par les prions renforce l’idée d’une déviation de la fonction normale de la PrPC par la PrPSc. Des inhibiteurs de l’activité histone désacétylase (HDAC) permettent de rétablir l’expression des acteurs de la voie Notch et des récepteurs Eph aussi bien dans les cellules déplétées en PrPC que dans celles infectées par les prions, suggérant que des mécanismes épigénétiques sont impliqués dans le contrôle transcriptionnel de ces gènes par la protéine prion. Ce travail fournit les bases pour évaluer un effet bénéfique des inhibiteurs de HDAC dans un modèle de souris infectées par les prions et ainsi déterminer si les HDAC pourraient constituer de nouvelles cibles thérapeutiques pour combattre les EST. / The conversion of the cellular prion protein PrPC into a pathogenic isoform, the scrapie prion protein PrPSc, lies at the root of a group of neurodegenerative disorders known as Transmissible Spongiform Encephalopathies (TSEs). Several lines of evidence indicate that PrPSc-mediated toxicity involves a subversion of PrPC normal function, however, our knowledge of PrPC physiological role is still far from complete. In this work, we sought to identify signalling pathways mobilized by PrPC that could accommodate both its role in central nervous system development and its implication in TSE pathogenesis. We show that the prion protein controls the activity of the Notch pathway, which plays an overriding role during embryonic development as well as central nervous system homeostasis and synaptic plasticity. In both ex vivo and in vivo models of TSE, we monitored a decrease in Notch activity, together with reduced expression of Eph receptors, which are key players in synaptic activity. The reduction in Eph is also found in PrPC-depleted cells. Hence, our observation of a similar signature of PrPC depletion and prion infection strengthens the view that PrPSc diverts PrPC function. We found a restoration of Notch and Eph effectors expression in response to histone deacetylase (HDAC) inhibitors, both in PrPC-depleted and prion-infected cells, suggesting that epigenetic mechanisms are involved in the PrP-dependent transcriptional control of these genes. This work provides a foundation for assessing a beneficial effect of HDAC inhibition in prion-infected mice and thereby defining whether HDAC could represent novel therapeutic targets to combat TSEs.

An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future. / Ph.D. / Traumatic brain injuries (TBIs) impact millions of individuals each year in the United States, making it a significant cause of death and disability. Furthermore, TBI has been linked to other comorbidities such as Alzheimers Disease, mood disorders, and epilepsy. Since the primary impact of a TBI cannot be predicted or prevented, research focuses on the secondary injury response as a therapeutic target to improve the outcomes following brain insult. Blood brain barrier (BBB) disruption is a well described consequence of TBI and has been correlated to a worse prognosis. The BBB normally provides a barrier between the circulating blood and the brain as protection and to maintain homeostasis. It is understood that decreased BBB integrity leads to subsequent edema, inflammatory response, and glial excitotoxicity, however, the mechanisms regulating this response remain to be investigated. Recent focus has been on a family of receptor tyrosine kinases, Eph receptors, that are unregulated following brain injury. Utilizing a mouse model, we can manipulate the temporal and spatial expression of Eph receptors to understand their role in the secondary injury cascade. Findings indicated that ablation of Eph receptors specifically on endothelial cells (ECs) resulted in preservation of BBB integrity at 1-, 4-, and 7- days following injury. Based on these results, we hypothesize that Eph receptor signaling on ECs negatively mediates BBB function and recovery following TBI. To test this hypothesis, we performed a comparative analysis between wild type (WT) and knockout (KO) mice on the expression of genes integral to BBB integrity, functional motor deficits, and loss of tissue in the lesion site following injury. We discovered significant decreases in lesion volume correlating with improvements in motor function in the KO mice compared to the WT. Moreover, KO mice showed increased expression of genes important for BBB maintenance such as Occludin and Tie2. To further discern the mechanism for these effects, we blocked Tie2 in the KO mice and observed similar negative prognostic indicators as in the WT. Future studies are warranted to understand the downstream signaling of Eph receptors on the Tie2 pathway. This data provides evidence that Eph signaling influences the BBB negatively following TBI through the Tie2 pathway and may be exploited for therapeutic means in the future.

In the intestinal epithelium, Wnt signaling drives the expression of the genes encoding tyrosine kinase receptors EphB2 and EphB3 and represses the expression of their membrane-tethered ligands, ephrin-Bs. Eph-ephrin interactions result in cellular repulsion and are involved in boundary formation. The project of this thesis is to understand the mechanism by which EphB−ephrin-B signals restrict cell positioning of cell types (cell sorting) in the normal intestinal epithelium and suppress colorectal cancer (CRC) progression beyond the earliest stages. We have demonstrated that at the onset of CRC EphB receptors impair the expansion of tumor cells through a mechanism dependent on E-cadherin–mediated adhesion. We show that EphB-mediated compartmentalization restricts the spreading of EphB+ tumor cells into ephrin-B1+ territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1+ intestinal cells at the onset of tumorigenesis. We have discovered that cell sorting is the outcome of two integrated mechanisms: cell contraction/repulsion and differential cell adhesion. The latter is the driving force to induce EphB/ephrin-B−mediated cell compartmentalization. We have developed in vitro models to analyze the mechanisms that induce E-cadherin remodeling upon EphB activation. We found RhoA, p120-catenin and the metalloproteinase ADAM10 as downstream effectors of EphB signaling involved in the control of cell sorting in CRC cells. / A l'epiteli intestinal, la ruta de senyalització Wnt indueix l'expressió dels gens que codifiquen per als receptors tirosina kinasa EphB2 i EphB3 i reprimeixen la dels seus lligands transmembrana, efrines de tipus B. Les interaccions Eph-efrina causen repulsió cel·lular i estan implicades en la formació de fronteres entre compartiments. La finalitat d'aquesta tesi és entendre el mecanisme pel qual la senyalització per EphB−efrina-B restringeix el posicionament dels diferents tipus cel·lulars a l'epiteli intestinal normal i suprimeix la progressió del càncer colorectal (CRC) en els primer estadis. Hem demostrat que, a l’inici del CRC, els receptors EphB restringeixen l'expansió de les cèl·lules tumorals a través d'un mecanisme depenent d'adhesió intercel·lular a través d’E-cadherina. En aquest treball es mostra in vitro i in vivo que la compartimentalització mitjançada per la senyalització dels receptors EphB restringeix l’invasió de les cèl·lules tumorals EphB+ als territoris efrina-B+. Aquests resultats indiquen que les cèl·lules de CRC han de silenciar l’expressió d'EphB per evitar les interaccions repulsives imposades per les cèl·lules intestinals normals efrina-B+ circumdants al començament del procés de tumorigènesi. Hem pogut discernir que el reordenament cel·lular per senyals EphB−efrina-B és el resultat de dos mecanismes integrats: la contracció/repulsió intercel·lular i l’adhesió diferencial entre diferents poblacions cel·lulars. Aquesta última és la força principal que condueix a la compartimentalització cel·lular mitjançada per EphB−efrina-B. Hem desenvolupat models in vitro per analitzar els mecanismes que provoquen el remodelament de la E-cadherina sota la senyalització per EphB. Presentem RhoA, p120-catenina i ADAM10 com a efectors de la senyalització de la ruta EphB implicats en el control de la compartimentalització cel·lular en el CRC.

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