Les cellules de Schwann (CS) sont les cellules gliales myélinisantes du Système Nerveux Périphérique (SNP). Il existe une communication étroite entre ces cellules et les axones auxquels elles s’associent et ce dès les stades les plus précoces de leur développement. Elles migrent tout en se divisant le long des axones; cette division migratoire est suivie d’une deuxième division post-migratoire dans le but d’établir un ratio 1:1 avec les axones pour ensuite les myéliniser. Ce travail vise à analyser, in vivo, le comportement des CS chez le poisson zèbre au cours de leurs divisions.Nous avons remarqué que les CS se divisent parallèlement aux axones le long du nerf de la Ligne Latérale Postérieure (PLL). En analysant les deux mutants has et nok, nous avons montré que les gènes de polarité apicale aPKC et pals1 ne sont pas requis pour la migration et la division des CS, ni pour leur capacité à myéliniser. Nous avons mis en évidence, en analysant le mutant cassiopeia qui présente des défauts d’organisation du fuseau mitotique et en utilisant l’agent pharmacologique le nocodazole, que l’assemblage du fuseau mitotique au cours de la division des CS est essentiel pour la myélinisation.En parallèle, nous avons analysé le rôle du gène rgs4 (regulator of G-protein Signaling 4) dans le développement du SNP chez le poisson zèbre. Nous avons généré un mutant stable rgs4 par la technique CRISPR/Cas9 et montré un rôle de ce gène dans le développement du ganglion de la PLL et des motoneurones, et ce en agissant en amont de la voie PI3K/Akt/mTOR.Contrairement à l’inhibition pharmacologique qui suggère un rôle de rgs4 dans la myélinisation périphérique, le mutant ne présente pas de défauts de myéline. / Schwann cells (SCs) are the myelinating glial cells of the Peripheral Nervous System (PNS). They derive from neural crest cells during development, then migrate and divide along the axons of the peripheral nerves. This migratory division is followed by a post-migratory division in order to radially sort the axons in a 1:1 ratio and wrap them with a myelin sheath. This work provides an analysis of the polarity of SC divisions, in vivo, in intact zebrafish embryos.We showed that SCs divide parallel to the axons along the Posterior Lateral Line nerve (PLL). By analyzing the two mutants has and nok, we revealed that the apical polarity genes aPKC and pals1, are neither required for the migration and division of SCs, nor for their capacity to myelinate. By studying the cassiopeia mutant that shows defects in mitotic spindle, we revealed that the assembly of the mitotic spindle is essential for SC myelination.We have also analysed the role of rgs4 (regulator of G-protein Signaling 4) in PNS development. We generated a stable rgs4 mutant using the CRISPR/Cas9 technology. We showed that rgs4 plays an essentiel role in PLLg and motoneurons development by acting upstream of PI3K/Akt/mTOR pathway. Pharmacological analysis suggested a role for rgs4 in peripheral myelination, however, the rgs4 mutant do not show any myelin defects.

Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling. / text

No description available.

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs that form various dimers with 12 Gγs and a 5th Gβ that dimerizes with the four regulator of G protein signaling (RGS) proteins of the R7 family. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1-4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results of Chapter 3 show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations, and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way. Chapter 4 discusses PhLP2, a recently discovered essential protein, and member of the Pdc family that does not play a role in G protein signaling. Several studies have indicated that PhLP2 acts as a co-chaperone with CCT in the folding of actin, tubulin, and several cell cycle and pro-apoptotic proteins. In a proteomics screen for PhLP2A interacting partners, α-tubulin, 14-3-3, elongation factor 1α, and ribosomal protein L3 were found. Further proteomics studies indicated that PhLP2A is a phosphoprotein that is phosphorylated by CK2 at threonines 47 and 52.

Schizophrenia is a severe neuropsychiatric disorder of largely unknown etiology that is often accompanied by high rates of cigarette smoking, reduced quit success, and high relapse rates. Dysregulated dopamine signaling and aberrant synaptic plasticity in the mesocorticolimbic pathway are implicated in the pathophysiology of schizophrenia and conferred substance abuse disorder and relapse vulnerability. Genetic factors are presumed to play a significant role in the development of schizophrenia, with a 40-50% concordance rate for monozygotic twins, although genetic markers are inconsistent. As such, epigenetic factors have instead been implicated. Specifically, there is strong evidence to suggest DNA methylation at several candidate genes contributes significantly to the pathophysiology of schizophrenia. To investigate this heritable component, our laboratory has developed a novel heritable model of drug abuse vulnerability in psychosis. This model is the first to show heritable increases in dopamine D2 receptor sensitivity via several behavioral and neurobiological markers, including enhanced behavioral responding to nicotine and changes in D2 signaling cascades in brain regions associated with psychosis and comorbid drug abuse. Increased D2 receptor sensitivity is the most consistent biomarker of psychosis found in preclinical animal models and postmortem brain tissue of individuals diagnosed with schizophrenia, lending considerable strength to the validity of the model. This study aimed to further validate the model as a useful and valuable tool for better understanding the pathophysiology of comorbid nicotine use and relapse in psychosis, and to explore more effective therapeutic targets than current antipsychotic medications. Results reveal DNA methylation as an epigenetic mechanism conferring heritability of the psychosis-like phenotype in the model. We additionally demonstrate altered relapse-like behavior, clinically consistent with reduced quit success and elevated relapse vulnerability. Interestingly, changes in relapse-like behavior were correlated to elevated protein levels of brain derived neurotrophic factor (BDNF), a marker of activity-dependent plasticity, in brain areas associated with drug reward. Further, modulation of the metabotropic glutamate type 5 (mGlu5) receptor alleviates the enhanced nicotine conditioned place preference observed in the model. Mechanistically, mGlu5 modulation restores normal dopamine D2 signaling and mitigates aberrant plasticity responses that are thought to drive the behavior in a region-specific manner.

Major histocompatibility complex molecules (MHCII) are cell surface glycoproteins that present extracellular antigens to CD4+ T lymphocytes and initiate adaptive immune responses. Apart from their protective role, overexpression of MHCII contributes to autoimmune disorders where the immune system attacks our own tissues. Autoimmune diseases are characterized by self-reactive responses to autoantigens, promoting tissue damage, inflammation mediated by proinflammatory cytokines, autoreactive lymphocytes, and autoantibodies. MHCII molecules are tightly regulated at the level of transcription by Class II transactivator (CIITA). CIITA associates with an enhanceosome complex at MHCII promoters and regulates the expression of MHCII. It is thus crucial to understand the regulation of CIITA expression in order to regulate MHCII in autoimmune diseases. Our lab has shown that the 19S ATPases of the 26S proteasome associate with MHCII and CIITA promoters and play important roles in gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles of the 19S ATPases Sug1, S7, and S6a in expression of CIITApIV genes. These ATPases are recruited to CIITApIV promoters and coding regions, interact with the elongation factor PTEFb, and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITApIV transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by knockdown of 19S ATPases. Alternatively, inflammation is also suppressed via the Regulator of G-protein signaling 10 (RGS10) in microglial cells which express high levels of RGS10 and promote homeostasis in the central nervous system. However, chronic activation of microglial cells leads to release of cytokines which cause neuroinflammation. Our investigation of roles played by RGS10 in chronically activated microglial cells indicates that RGS10 binds to promoters of IL-1β, and TNF-α and regulates these genes, while the molecular mechanism remains to be investigated. Together, our observations indicate roles for the UPS in modulating gene expression and for RGS10 in regulating proinflammatory cytokines in microglial cells, each of which provides novel therapeutic targets to combat inflammation in autoimmune and neurodegenerative diseases.

To survive, animals must adapt to a complex and challenging world in a way that is flexible and responsive, while maintaining internal homeostasis. Neuromodulators provide a means to systemically alter behavioral or physiological state based on intrinsic or extrinsic cues, however dysregulated neuroendocrine signaling has negative consequences for fitness and survival. Here I examine neuroendocrine function and dysfunction using the escape response in Caenorhabditis elegans. The RFamide neuropeptide FLP-18 is a co-transmitter with the monoamine tyramine and functions both synergistically and antagonistically to tyramine in coordinating escape behavior. Using behavioral analysis and calcium imaging, I show that FLP-18 functions primarily through the G-protein coupled receptor (GPCR) NPR-5 to increase calcium levels in muscle, enhancing locomotion rate, bending and reversal behavior during the escape response. Furthermore, I examine the relationship between persistent acute stress and resilience using repeated activation of the escape response as a model of neuroendocrine dysregulation. Repeated activation of the escape response shortens lifespan and renders animals more susceptible to thermal, oxidative, and nutritional stress. Tyramine release is necessary and sufficient for this effect and activity of the tyraminergic RIM neurons is differentially regulated by acute versus long-term stressors. Impaired stress resistance requires both the GPCR TYRA-3 in the intestine and intestinal neuropeptide release. Activation of the insulin receptor DAF-2 is downstream of TYRA-3 and inhibits the transcription factors DAF-16/FOXO, SKN-1/Nrf2 and HSF-1, linking monoamine signaling in acute stress to the insulin signaling pathway and impaired resilience to long-term stressors.