O peptídeo idêntico ao C-terminal da proteína S100A9 murina (pS100A9mH92-G110) inibe a hiperalgesia inflamatória induzida pela carragenina. Em adição, este peptídeo inibe a hiperalgesia inflamatória induzida por tripsina, uma serino protease capaz de ativar receptores ativados por protease do tipo 2 (PAR2). O objetivo inicial deste trabalho foi caracterizar a relação estrutura/ efeito do pS100A9m, a fim de determinar a menor seqüência peptídica dotada de atividade antinociceptiva. Ainda, como parte dos objetivos, neste trabalho foram investigados os mecanismos envolvidos no efeito antinociceptivo do pS100A9m e da menor seqüência ativa sobre a hiperalgesia induzida pela ativação de PAR2. Diferentes seqüências peptídicas homólogas ao pS100A9m foram sintetizadas e avaliadas em ratos submetidos ao modelo de hiperalgesia mecânica induzida por carragenina. Dentre todas as seqüências peptídicas investigadas, o peptídeo denominado AcE97-G102 foi determinado como a menor seqüência ativa com efeito semelhante ao pS100A9m. Com relação aos estudos sobre a ativação de PAR2, os resultados obtidos demonstraram que o pS100A9m bem como o AcE97-G102 inibem a hiperalgesia térmica e mecânica decorrentes da ativação de PAR2 (induzida por um peptídeo agonista deste receptor ? PAR2AP). A análise por imuno-histoquímica demonstrou que a ativação de PAR2 aumenta a expressão da proteína Egr-1 em neurônios nociceptivos, sendo o pS100A9m capaz de inibir este efeito. Em adição, ambos pS100A9m e AcE97-G102 inibiram o influxo de cálcio induzido por PAR2AP ou tripsina, em neurônios sensoriais do gânglio da raiz dorsal da medula espinhal (DRG). Por outro lado, nenhum dos peptídeos apresentou efeito sobre a mobilização de cálcio em células HEK-293, que naturalmente expressam PAR2, ou em células KNRK transfectadas com este tipo de receptor, sugerindo que o efeito tanto do pS100A9m quanto do AcE97-G102, sobre a ativação de PAR2, seja específico para neurônios sensoriais. O pS100A9m e o AcE97-G102 inibiram o influxo de cálcio nos neurônios DRG estimulados com bradicinina, capsaicina ou KCl. Ainda, o pS100A9m inibiu a liberação de substância P induzida por PAR2. Os resultados obtidos com o tratamento de neurônios DRG com tapsigaragina ou com ionóforo de cálcio sugerem um efeito direto do pS100A9m sobre os canais de cálcio. Desta forma, foi avaliada atividade do pS100A9m e do AcE97-G102 sobre culturas de células HEK-tsA transfectadas com canais de cálcio dependente de voltagem do tipo N ou do tipo L. Os resultados obtidos demonstraram que ambos peptídeos inibirem o influxo de cálcio em células transfectadas com receptores do tipo N. Em conjunto, os dados aqui obtidos demonstram que o efeito do C-terminal da proteína S100A9 murina sobre a nocicepção experimental é devido a uma inibição de canais de cálcio do tipo N, por uma ação direta em neurônios sensoriais. Ainda, a seqüência responsável por este efeito está localizada na porção E97-G102 do domínio C-terminal da proteína S100A9 murina. / Peptide identical to the C-terminus of S100A9 protein (mS100A9pH92-G110) inhibits inflammatory hyperalgesia induced by carrageenan and trypsin, a serine protease that activates protease-activated receptors 2 (PAR2). The aim of this work was to characterize the relationship between structure and function of mS100A9p in order to identify the shortest peptide sequence endowed with antinociceptive effect. Furthermore, the mechanisms involved on the antinociceptive effect of both mS100A9p and the shortest homologous sequence on PAR2-induced hyperalgesia were also evaluated. Different peptide sequences homologous to mS100A9p were synthesized and evaluated in rats submitted to the carrageenan-induced mechanical hyperalgesia model. Among all evaluated sequences, the peptide AcE97-G102 was found to be the shortest sequence that showed an antinociceptive effect similar to that induced by mS100A9p. In regard to PAR2 activation, data obtained herein demonstrated that both mS100A9p and AcE97-G102 inhibit PAR2-induced mechanical and thermal hyperalgesia, induced by the selective agonist peptide ? PAR2AP. Imunohistochemical evaluation demonstrated that PAR2 activation increased Egr-1 protein expression on sensory neurons and mS100A9p inhibited this effect. In addition, both mS100A9p and AcE97-G102 inhibited PAR2- and trypsin-induced calcium influx in dorsal root ganglia neurons (DRG). On the other hand, no effect on the calcium influx of the peptides were observed on HEK-293 cells or KNRK-PAR2 transfected cells, suggesting that the effects of mS100A9p and AcE97-G102 on PAR2 activation are specific for sensory neurons. Both mS100A9p and AcE97-G102 inhibited DRG calcium flux when cells were stimulated with bradykinin, capsaicin or KCl. Also, mS100A9p inhibited PAR2-induced substance P release in DRG. Treatment of DRG with either thapsigargin or calcium ionophore suggest a direct effect of mS100A9p on calcium channels. To evaluate this hypothesis the effects of mS100A9p and AcE97-G102 were evaluated on N-type or L-type voltage-dependent calcium channel transfected HEK-tsA cells. Both peptides inhibited calcium influx of N-type transfected cells. In conclusion, data presented herein demonstrate that the C-terminus of murine S100A9 protein inhibits experimental nociception through a block of N-type voltage-dependent calcium channels, directly on sensory neurons. Also, the domain involved in this effect is localized on the sequence E97-G102 of the C-terminus of murine S100A9 protein.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Genetic associations have been discovered for many human complex traits, and yet for most associated loci the causal variants and molecular mechanisms remain unknown. Studies mapping quantitative trait loci (QTLs) for molecular phenotypes, such as gene expression, RNA splicing, and chromatin accessibility, provide rich data that can link variant effects in specific cell types with complex traits. These genetic effects can also now be modeled in vitro by differentiating human induced pluripotent stem cells (iPSCs) into specific cell types, including inaccessible cell types such as those of the brain. In this thesis, I explore a range of approaches for using QTLs to identify causal variants and to link these with molecular functions and complex traits. In Chapter 2, I describe QTL mapping in 123 sensory neuronal cell lines differentiated from human iPSCs. I observed that gene expression was highly variable across iPSC-derived neuronal cultures in specific gene categories, and that a portion of this variability was explained by commonly used iPSC culture conditions, which influenced differentiation efficiency. A number of QTLs overlapped with common disease associations; however, using simulations I showed that identifying causal regulatory variants with a recall-by- genotype approach in iPSC-derived neurons is likely to require large sample sizes, even for variants with moderately large effect sizes. In Chapter 3, I developed a computational model that uses publicly available gene expression QTL data, along with molecular annotations, to generate cell type-specific probability of regulatory function (PRF) scores for each variant. I found that predictive power was improved when the model was modified to use the quantitative value of annotations. PRF scores outperformed other genome-wide scores, including CADD and GWAVA, in identifying likely causal eQTL variants. In Chapter 4, I used PRF scores to identify relevant cell types and to fine map potential causal variants using summary association statistics in six complex traits. By examining individual loci in detail, I showed how the enrichments contributing to a high PRF score are transparent, which can help to distinguish plausible causal variant predictions from model misspecification.

La ciguatéra est une forme d’intoxication faisant suite à l’ingestion de poissons contaminés par des toxines appelées « ciguatoxines ». Cette intoxication endémique des régions tropicales est un problème économique et de santé non négligeable qui tend à prendre de plus en plus d’ampleur. L’essor du tourisme, le réchauffement climatique et la hausse des exportations internationales de poissons tropicaux favorisent l’expansion de la ciguatéra aux parties du globe au climat tempéré, jusqu’alors peu concernées par cette maladie. Les enjeux thérapeutiques et économiques de la ciguatéra sont de taille puisqu’il n’existe aucun moyen rapide et fiable de détecter un poisson contaminé et qu’aucun traitement efficace permettant sa prise en charge n’a actuellement été établi.Le prurit, terme médical désignant les démangeaisons est un symptôme notamment associé aux maladies de peau qui impacte grandement la qualité de vie des patients qui en souffrent.Ces dernières décennies, de nombreuses études scientifiques ont permis de mieux comprendre sa physiopathologie. Le prurit est un symptôme fréquemment observé chez les personnes atteintes de ciguatéra, d’où l’appellation « la Gratte », souvent employée pour faire référence à la pathologie.Dans le but d’étudier les mécanismes cellulaires à l’origine du prurit et des autres troubles sensoriels cutanés survenant lors de la ciguatéra, nous avons étudié l’effet des ciguatoxines sur un modèle in vitro de neurones sensoriels cocultivés avec des kératinocytes. Nous avons mis en évidence la libération dans le surnageant des neuropeptides de l'inflammation neurogène, SP et CGRP. L'effet d'antagonistes sélectionnés a été testé afin mettre en évidence les médiateurs impliqués dans la libération de neuropeptides. Par ailleurs, la signalisation cellulaire sous-jacente de cette sécrétion de neuropeptides a été étudiée par des expériences d’imagerie calcique réalisées sur neurones et kératinocytes.Les résultats obtenus valident notre coculture en tant que modèle de choix pour l’étude in vitro des mécanismes cellulaires, et plus généralement, dans les troubles neurocutanés impliqués dans le prurit ciguatérique. Parmi les antagonistes testés, une molécule s’est avérée particulièrement intéressante pour inhiber les effets constatés de la toxine. Ces résultats originaux, obtenus avec l’antagoniste d’un médiateur du prurit, présentent une perspective thérapeutique nouvelle et prometteuse, pour répondre à un enjeu de santé publique futur. / Ciguatera fish poisoning (CFP) is a seafood poisoning occurring after contaminated fish fleshes ingestion containing toxins called « ciguatoxines » (CTXs). This illness, originating from tropical and subtropical areas, is an economic and health problems which becomes substantial in relation to international tropical fishes export and tourism development, as well as global warming rise. Those factors contribute to CFP sprouting in non-endemic temperate climate regions which were not until then concerned by CFP. Economic and health stakes of CFP are important since no reliable and ready-to-use detection system for CTXs in fishes have been developed, along with no relevant cure has been established to treat CFP.Pruritus, medical term refer to itch, is a clinical sign usually associated to skin diseases which strongly alter patients’ quality of life. Last decades, several studies allowed to better understand pruritus pathophysiology. Interestingly, people suffering from CFP frequently present pruritus, hence designation “La Gratte” or “La Gratel (le)” employed in endemic areas.The aim of these works was to study cellular and molecular mechanisms of CTXs at the root of neurological cutaneous troubles occurring in CFP. Here, we evaluated CTXs effects on in vitro model composed of sensory neurons cocultived with primary keratinocytes, quantifying neuropeptides known to be involved in pruritus. Compiling knowledges about CFP and pruritus pathophysiology, some antagonists were tested to neutralize CTXs-mediated neuropeptide release. To deal with signaling pathways in depth of neuropeptide exocytosis induced by CTXs, mechanism known to be accurately regulated by calcium homeostasis, calcium imaging experiments were performed.Results obtained in this project confirm the use of such a model to elucidate cellular mechanism of CFP pruritus, but also constitute an alternative in vitro tool to study chemicals inducing abnormal cutaneous senses. Among antagonists tested, one stands out from the crowd and was proved to be effective to inhibit CTXs-evoked effects studied. Those originals results, collected with antagonist of pruritus mediator, show new and promiscuous therapeutic prospects for future health concern.

Ce travail se situe dans le cadre d'un projet pluridisciplinaire, visant à utiliser un nouveau mode de stimulation des neurones sensoriels par l'infrarouge (IR) à 1875 nm. Actuellement les prothèses cochléaires et visuelles utilisent la stimulation électrique qui permet certes de visualiser des objets et de suivre une conversation mais avec une résolution qui pourrait certainement être améliorée par un autre mode de stimulation, notamment l'infrarouge. Nous avons d'abord démontré qu'une telle technique était possible dans les cellules ganglionnaires de la rétine ainsi que celles du ganglion de Scarpa (vestibule). Les réponses biologiques obtenues sous forme de variations transitoires de calcium intracellulaire et de potentiel d'action, (enregistrées par les techniques d'imagerie calcique et de patch-clamp) nous ont permis d'approfondir cette étude. En effet, de précédents travaux ont montré la faisabilité de la stimulation optique par IR des nerfs périphériques. Mais le mécanisme à l‟origine de la réponse évoquée par IR dans le tissu biologique n'a jamais été décrit jusqu'ici. Nous décrivons pour la première fois le mécanisme moléculaire qui conduit à la genèse de VVEL (variation de potentiel de membrane évoquée par laser IR). L'élément déclencheur de ce mécanisme au niveau membranaire a été révélé à l'aide d'une approche pharmacologique. Le blocage des canaux-récepteurs thermosensibles de la famille de 'Transient Receptor Potential' (Vanilloides) par le rouge de ruthénium et le RN1734, inhibe les VVELs. Nous démontrons que le mécanisme fait intervenir des canaux sodiques et calciques dépendants du voltage, dont l'activation lors d'une stimulation par l'IR est dépendante de l'ouverture des canaux thermosensibles TRPV4. / Infrared (IR) laser irradiation has been established as an appropriate stimulus for primary sensory neurons under conditions where sensory receptor cells are impaired or lost. Yet, development of clinical applications has been impeded by lack of information about the molecular mechanisms underlying the laser induced neural response. Here, we first demonstrate that retinal and vestibular ganglion cells generate biological responses evoked by mid laser irradiation. Then, we directly address this question through pharmacological characterization of the biological response evoked by mid infrared irradiation of isolated retinal and vestibular ganglion cells from rodents. Whole-cell patch-clamp recordings reveal that both voltage-gated calcium and sodium channels contribute to the laser evoked neuronal voltage variations (LEVV). In addition, selective blockade of the LEVV by micromolar concentrations of ruthenium red and RN1734 identifies thermo-sensitive TRPV4 channels as the primary effectors of the chain reaction triggered by mid infrared laser irradiation.

Indiana University-Purdue University Indianapolis (IUPUI) / ABSTRACT Yue Wang Neurofibromin, nerve growth factor and Ras: their roles in controlling the excitability of mouse sensory neurons Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. It is likely that sensory neurons with reduced levels of neurofibromin have augmented Ras-GTP activity. In a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-), the patch-clamp recording technique is used to investigate the role of neurofibromin in controlling the state of neuronal excitability. Sensory neurons isolated from adult Nf1+/- mice generate more APs in response to a ramp of depolarizing current compared to Nf1+/+ mice. In order to elucidate whether the activation of Ras underlies this augmented excitability, sensory neurons are exposed to nerve growth factor (NGF) that activates Ras. In Nf1+/+ neurons, exposure to NGF increases the production of APs. To examine whether activation of Ras contributes to the NGF-induced sensitization in Nf1+/+ neurons, an antibody that neutralizes Ras activity is internally perfused into neurons. The NGF-mediated augmentation of excitability is suppressed by the Ras-blocking antibody in Nf1+/+ neurons, suggesting the NGF-induced sensitization in Nf1+/+ neurons depends on the activation of Ras. Surprisingly, the excitability of Nf1+/- neurons is not altered by the blocking antibody, suggesting that this enhanced excitability may depend on previous activation of downstream effectors of Ras. To determine the mechanism giving rise to augmented excitability of Nf1+/- neurons, isolated membrane currents are examined. Consistent with the enhanced excitability of Nf1+/- neurons, the peak current density of tetrodotoxin-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents (INa) are significantly larger than in Nf1+/+ neurons. Although the voltage for half-maximal activation (V0.5) is not different, there is a significant depolarizing shift in the V0.5 for steady-state inactivation of INa in Nf1+/- neurons. In summary, these results demonstrate that the enhanced production of APs in Nf1+/- neurons results from a larger current amplitude and a depolarized voltage dependence of steady-state inactivation of INa that leads to more sodium channels being available for the subsequent firing of APs. My investigation supports the idea that regulation of channels by the Ras cascade is an important determinant of neuronal excitability. Grant D. Nicol, Ph.D, Chair

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are peptides implicated in the inflammatory response. They are released in increased amounts during inflammation and induce thermal hyperalgesia. Whether these molecules directly affect the sensitivity of primary nociceptive sensory neurons is unknown. This information could provide a link between increased inflammation-induced release of GFLs and their ability to promote inflammatory hyperalgesia. These molecules bind to one of four GFRα receptor subtypes, and this GFL-GFRα complex often translocates to the receptor tyrosine kinase, Ret. The focus of this dissertation was to determine whether GFLs modulate the stimulated-release of calcitonin gene-related peptide (CGRP). Isolated sensory neurons and freshly dissociated spinal cord tissue were used to examine the enhancement in stimulated-release of CGRP, a measure of sensitization. Exposure of isolated sensory neurons to GDNF, neurturin, and artemin, enhanced the capsaicin stimulated-release of immunoreactive CGRP (iCGRP). Sensitization by GFLs occurred in freshly dissociated spinal cord tissue. Persephin, another member of the GFL family, did not enhance stimulated-release of iCGRP. These results demonstrate that specific GFLs are mediators of neuronal sensitivity. The intracellular signaling pathways responsible for this sensitization were also evaluated. Inhibition of the mitogen activated protein kinase (MAPK)/extracellular signal-related kinase 1/2 (Erk 1/2) pathway selectively abolished the enhancement of CGRP release by GDNF. NTN-induced sensitization was abolished by inhibition of the phosphatidylinositol-3-kinase (PI-3K) pathway. Reduction in Ret abolished the GDNF-induced sensitization, but did not fully inhibit NTN or ART-induced sensitization. Inhibition of other cell surface receptors (neural cell adhesion molecule (NCAM), and Integrin β-1) had distinct effects on the sensitization capability of each of the GFLs. Ret and NCAM inhibition in combination abolished ART-induced sensitization. It was necessary to inhibit Ret, NCAM, and Integrin β-1 to prevent the NTN-induced sensitization. These data demonstrate that the GFLs use distinct signaling mechanisms to induce the sensitization of nociceptive sensory neurons. The work presented in this thesis provides the first evidence for these novel and distinct Ret-independent pathways for GFL-induced actions and provides insight into the mechanism of sensory neuronal sensitization in general.

No description available.

La douleur est perçue par des neurones spécialisés, les nocicepteurs, dont le corps cellulaire est localisé, au niveau du tronc, dans les ganglions de la racine dorsale (DRG). Ces neurones détectent les informations sensorielles en périphérie (peau, muscles ou viscères) et les transmettent aux neurones spinaux qu'ils connectent au niveau de la corne dorsale de la moelle épinière. D'un point de vue morphologique, anatomique, physiologique, mais également moléculaire, une hétérogénéité importante de ces neurones est observée. Le but principal du laboratoire est de trouver de nouvelles molécules impliquées dans les mécanismes moléculaires qui spécifient les différentes sous-populations neuronales des DRG. Dans ce cadre, il a été possible d'identifier et valider plusieurs gènes présentant un profil d'expression très particulier et spécifiant des populations neuronales bien distinctes au sein des DRG. Parmi ces gènes, tafa4 est principalement exprimé dans des neurones non-peptidergiques de type C. Tafa4 est une petite protéine sécrétée proche des chemokines de type CC dont la fonction est jusqu'à présent inconnue, et dont l'expression dans les DRG n'a encore jamais été décrite. Au cours de ce travail, j'ai pu identifier Tafa4 comme un nouveau marqueur d'une sous-population de neurones sensoriels des DRG : les C-LTMRs (C-Low Threshold MechanoReceptor). La génération d'une lignée de souris Tafa4 KO dans laquelle le gène tafa4 a été remplacé par la protéine fluorescente Vénus, nous a permis de mettre en évidence que la population de neurones tafa4+ projette en central dans la lamina II interne de la moelle épinière et en périphérique exclusivement au niveau de la peau poilue. / The perception of pain is initiated by the detection of noxious stimuli by the peripheral endings of primary nociceptive neurons. They are a specialized group of small-diameter pseudounipolar neurons with cell bodies in the dorsal roots ganglia (DRG). They give rise to thinly myelinated (Ad-fibers) or unmyelinated (C-fibers) afferent fibers, which convey the signal from the periphery to the dorsal horn of the spinal cord. Our laboratory is interested in molecular mechanisms which underlie the specification of somatic sensory neurons and their properties. In order to find novel molecular factors involved in this process, we identified several new nociceptor subtype specific genes by microarray experiments. Among these genes, tafa4 which encodes a small secreted protein distantly related to CC chemokine with unknown function, appears to have a DRG-specific expression from early developmental stages and becomes restricted to a subset of C-fibers non-peptidergic nociceptors in adult DRG. By using transgenic mice, we show that Tafa4 neurons specifically project to the dorsal horn lamina IIi and innervate the hairy skin. They have electrophysiological signature of C-Low-threshold mechanoreceptors (C-LTMRs), a population of sensory neurons implicated in the injury-induced mechanical hyper-sensitivity as well as in the affective component of touch. Mutant mice lacking Tafa4 do not present developmental defects and specify Tafa4 population correctly. However, despite no obvious molecular changes in Tafa4 mutants, these mice display significant increase in tissue injury induced hyper-sensitivity which could be reduced by intrathecally applied Tafa4 protein.