Thrombin signaling and HIV dementia

描述(申请人提供)：病理证据表明，凝血酶及其主要受体--蛋白酶激活受体-1(PAR-L)的水平与艾滋病毒脑炎有关。凝血酶可以通过受损的血脑屏障进入中枢神经系统，也可以由脑实质的免疫激活细胞产生。凝血酶的潜在作用可能在HIVD的发病机制中起重要作用，包括其降解支持细胞黏附和生存的基质蛋白的能力，以及其通过PARs传递信号的能力。内皮细胞PARs的激活与细胞形状的改变、趋化因子释放的增加和黏附分子的表达增加有关。神经元PAR的激活也已被证实，并被认为是凝血酶对这些细胞的一些先前报道的影响的基础，包括神经毒性和轴突回缩。有趣的是，HIV痴呆(HIVD)与轴突回缩的证据有关，包括树突简化和突触密度降低。然而，负责任的机制并没有得到很好的理解。 在目前的R21应用中，我们建议测试凝血酶作用于特定神经元部分，通过激活糖原合成酶激酶3b(GSK3b)来刺激轴突回缩的假设，GSK3b是细胞形状和存活的关键中介。GSK3b已被证明可以磷酸化包括tau在内的微管相关蛋白，并且在一些实验范例中，tau的磷酸化增加与轴突回缩有关。我们还将确定凝血酶对GSK3b影响的潜在机制。虽然以前的研究表明，通常与Rho GTP酶活性变化相关的G蛋白亚型可以影响GSK3b的活性，但没有研究表明与腺苷环化酶活性降低相关的G蛋白亚型可以起到同样的作用。因此，我们将测试这一假设，即凝血酶刺激神经元既影响依赖于PKA的丝氨酸磷酸化的减少，也影响依赖于RhoA的GSK3b酪氨酸磷酸化的增加。这些实验的结果应该会促使未来的研究，以确定其他与HIVD相关的刺激是否针对类似的路径。最终，我们希望更好地了解在HIVD背景下导致神经元形态改变的机制，并确定靶向GSK3b和/或特定G蛋白偶联受体是否有用。

DESCRIPTION (provided by applicant): Pathological evidence suggests that levels of both thrombin and its principal receptor, proteinase activated receptor-1 (PAR-l), are increased in associated with HIV encephalitis. Thrombin can enter the CNS through a damaged blood brain barrier, and it is also produced by immune activated cells of the brain parenchyma. Potential effects of thrombin which may be important to the pathogenesis of HIVD include its ability to degrade matrix proteins that support cell adhesion and survival, as well as its ability to signal through PARs. Activation of endothelial cell PARs has been linked to changes in cell shape, increased chemokine release, and increased adhesion molecule expression. Activation of neuronal PARs has also been demonstrated and is thought to underlie some of the previously reported effects of thrombin on these cells, including neurotoxicity and neurite retraction. Of interest, HIV dementia (HIVD) has been associated with evidence of neurite retraction including dendritic simplification and reduced synaptic density. The responsible mechanisms, however, are not well understood. In the present R21 application, we propose to test the hypothesis that thrombin acts on specific neuronal PARs to stimulate neurite retraction through the activation of glycogen synthase kinase 3b (GSK3b), a critical mediator of cell shape and survival. GSK3b has been shown to phosphorylate microtubule-associated proteins including tau, and increased phosphorylation of tau has been linked to neurite retraction in a number of experimental paradigms. We will also identify the mechanisms that underlie thrombin's effects on GSK3b. While previous studies have suggested that G protein subtypes typically associated with alterations in Rho GTPase activity can affect the activity of GSK3b, none have shown that those linked to decreased adenylate cyclase activity can do the same. We will therefore test the hypothesis that thrombin stimulation of neurons affects both a decrease in PKA-dependent serine phosphorylation and an increase in RhoA dependent tyrosine phosphorylation of GSK3b. The results from these experiments should prompt future studies to determine whether other HIVD related stimuli that cause neurite retraction target similar pathways. Ultimately, we hope to gain a better understanding of mechanisms that contribute to altered neuronal morphology in the setting of HIVD, and to determine whether it may be useful to target GSK3b and/or specific G protein coupled receptors.