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La neurodégénérescence fait partie intégrante de la maladie de Huntington (MH) dont les premiers symptômes moteurs et cognitifs apparaissent vers l’âge de 30 à 40 ans. Cette maladie incurable est causée par une mutation dans le gène codant pour la protéine huntingtin (htt). L’activation de la caspase-6 (casp6) est observée au stade présymptomatique chez l’humain et les modèles murins MH faisant de la casp6 un joueur majeur dans la neurodégénérescence précoce associé à la MH. De plus, le clivage de htt mutant par la casp6 produit un fragment N-terminal neurotoxique essentielle au développement de la MH. Des résultats préliminaires ont permis de révéler l’interaction et le clivage des protéines proapoptotiques Serine/Threonine Kinase 3 (STK3), Death-Domain Associated Protein (DAXX) par la casp6. Des effets proapoptotiques sont associés à leurs fragments et leur production par les caspases pourrait influencer la neurodégénérescence observée dans diverses maladies neurodégénératives et dans le vieillissement normal. Nos résultats dans les souris C57Bl/6 démontrent que l’expression de DAXX varie fortement avec l’âge selon l’organe analysé. Ses divers fragments ne suivent pas la même tendance d’un organe à l’autre suggérant des fonctions différentielles à travers l’organisme et une importante régulation de ses fonctions par des modifications post-traductionnelles. Chez les modèles murins de la MH, les souris YAC128, nous avons constaté une augmentation des fragments à 65 et 70 kDa dans le cortex et une diminution de DAXX entier et du fragment à 70 kDa dans le cervelet soulignant la possibilité de fonctions spécifiques selon les régions cérébrales. Nous avons aussi démontré pour la première fois le clivage de STK3 par la caspase-7 et la production différentielle de fragments par les caspase-3, 6 et 7. L’expression protéique de STK3 augmente globalement à travers l’organisme avec l’âge et dans le cervelet des souris YAC128. Par contre, une diminution de l’expression de STK3 est observée dans le cortex des individus atteints de la MH et des souris YAC128. Finalement, par l’induction de différents stress cellulaires, nous avons constaté la présence d’un mécanisme adaptatif des neurones modèles de la MH impliquant STK3. En conclusion, l’expression de DAXX et STK3 varie avec l’âge à travers l’organisme et est altérée dans la maladie de Huntington. Plus particulièrement, STK3semble être impliqué dans un mécanisme protégeant les neurones de la mort cellulaire dans la MH.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.