EXACERBATION OF AD NEUROPATHOLOGY BY ASTROGLIAL FACTORS

大量的发展研究已经证明了 星形胶质细胞对神经元迁移和突起的支持 外延生长。这些研究已经呈现出意想不到的新维度 近年来通过对伴随着的明显相关现象的观察 阿尔茨海默病(AD)的神经退行性改变。一个标志 阿尔茨海默病的细胞外沉积是一种潜在的神经毒性 多肽，(β-淀粉样蛋白，由交替裂解产生 更大的膜相关应用程序。这种蛋白质与蛋白质的相关性 神经胶质-神经元发育相互作用是正常分裂的 而分泌型APP与蛋白酶Nexin II(PNII)同源，1 连接蛋白家族的成员--神经胶质来源的分泌分子，通过结合 抑制神经元表面蛋白水解酶，介导轴突生长 在开发过程中。 反应性胶质细胞增多症和存活神经元的流产发芽都是 通常在阿尔茨海默病大脑的受累区域观察到。这些发现促使人们 考虑到星形胶质细胞重现的可能性 分泌促进轴突生长的因子，包括PMII/APP和层粘连蛋白， 可能会导致流产的萌芽，导致神经元的额外周期 退化。经历流产的萌发尝试的神经元可能 产生更多的APP的淀粉样变性片段。 我们已经开发了一个神经元细胞培养模型来测试这些和相关的 假设。初步研究表明，治疗 星形胶质条件培养液(CM)培养的未分化神经细胞 或纯化的APP可诱导神经发生，且无毒性。相比之下，CM- 或APP-治疗长期分化的(老化的)神经细胞， 如果减少或消除了差异剂(DBcAMP)， 导致败育萌发和退化。CM和APP是良性的 这些老化的培养物在萌发时被阻止，方法是在 浓度很高。我们之前的研究表明， 钙蛋白酶(钙激活的蛋白酶)和蛋白质的过度激活 这些细胞中的激酶C(PKC)可诱导ALZ-50免疫反应。我们会 确定CM和APP诱导的神经毒性是否伴随 ALZ-50诱导，以及神经元产生β-淀粉样蛋白是否涉及 细胞内递送特定的钙激活蛋白和蛋白激酶C 钙蛋白酶和蛋白激酶C激活剂和抑制物。的潜在作用 神经退行性变中的异常磷酸化tau(产生ALZ-50) 将通过反义寡核苷酸介导的tau抑制来检测 在CM和APP治疗前的合成，并确定是否 神经退化减轻；这些分析将提供信息 关于tau是导致神经变性还是它是神经退行性变的副产品。 认为神经退行性变是重述的结果的假设 将通过反义寡核苷酸进一步探索发芽- 介导生长相关蛋白GAP-43的下调 是在这些细胞中发芽所必需的。潜在的关键 与细胞外基质成分的相互作用将通过 交替培养中层粘连蛋白的包涵体。 这项提案中包含的研究并不是为了 全面纳入所有神经胶质来源的生长因子 在宪法上或在发展和再生期间表现出来的。 他们也没有试图阐明公元时期的启蒙事件(S)。 然而，通过探索神经胶质细胞加剧AD的可能性 神经病理学，希望对治疗有新的见解 方法可能会发生变化。

Numerous developmental studies have documented the roles played by astroglial cells in the support of neuronal migration and neurite outgrowth. These studies have taken on an unexpected new dimension in recent years by observations of apparently related phenomena accompanying the neurodegenerative events of AIzheimer's disease (AD). One hallmark of AD is the extracellular deposition of a potentially neurotoxic peptide, (beta-amyloid, which is generated by an alternate cleavage of the larger membrane-associated APP. The relevance of this protein to glial-neuronal developmental interactions is that the normally-cleaved and secreted form of APP is homologous with protease nexin II (PNII), one of a family of nexins - glial-derived secreted molecules that, by binding to and suppressing neuronal surface proteases, mediate neurite outgrowth during development. Both reactive gliosis and abortive sprouting of surviving neurons are routinely observed in affected areas of AD brains. These findings prompt consideration of the possibility that a recapitulation of astroglial secretion of neurite-promoting factors, including PMII/APP and laminin, may prompt abortive sprouting, leading to additional cycles of neuronal degeneration. Neurons undergoing abortive sprouting attempts may generate increased amounts of amyloidogenic fragments of APP. We have developed a neuronal cell culture model to test these and related hypotheses. Preliminary studies demonstrate that treatment of undifferentiated neuronal cells with astroglial conditioned medium (CM) or purified APP induces neuritogenesis and no toxicity. By contrast, CM- or APP-treatment of long-term differentiated ("aged") neuronal cells, provided the differentiating agent (dbcAMP) is reduced or eliminated, induces abortive sprouting and degeneration. CM and APP are benign in these aged cultures when sprouting is blocked by maintaining dbcAMP at high concentrations. Our previous studies demonstrate that hyperactivation of calpain (calcium-activated protease) and protein kinase C (PKC) in these cells induces ALZ-5O immunoreactivity. We will determine whether CM- and APP-induced neurotoxicity is accompanied by ALZ-50 induction, and whether beta-amyloid generation by neurons involves calpain and PKC hyperactivation by the intracellular delivery of specific calpain and PKC activator and inhibitors. The potential role of aberrantly phosphorylated tau (generating ALZ-50) in neurodegeneration will be examined by antisense oligonucleotide-mediated suppression of tau synthesis prior to CM- and APP-treatment, and determining whether neurodegeneration is lessened; these analyses will provide information on whether tau contributes to, or is a by-product of, neurodegeneration. The hypothesis that neurodegeneration is a consequence of recapitulation of sprouting will be further explored by antisense oligonucleotide- mediated down-regulation of the growth associated protein, GAP-43, which is required for sprouting in these cells. Potentially crucial interactions with extracellular matrix components will be examined by inclusion of laminin in alternate cultures. The studies contained within this proposal are not designed to be comprehensive in terms of including all glial-derived growth factors expressed either constituitively or during development and regeneration. Neither do they attempt to elucidate the initiating event(s) in AD. However, by exploring the possibility that glial cells exacerbate AD neuropathology, it is hoped that novel insights towards therapeutic approaches may evolve.