EXACERBATION OF AD NEUROPATHOLOGY BY ASTROGLIAL FACTORS

许多发展研究都记录了 星形胶质细胞支持神经元迁移和突起 结果这些研究呈现出意想不到的新维度， 近年来，通过观察明显相关的现象， 阿尔茨海默病（AD）的神经退行性事件。一个标志 AD是一种潜在的神经毒性物质的细胞外沉积， 肽，（β-淀粉样蛋白，其通过交替切割 较大的膜相关APP。这种蛋白质与 神经胶质-神经元发育的相互作用是， 分泌型APP与蛋白酶连接蛋白II（PNII）同源， 神经胶质衍生的分泌分子， 并抑制神经元表面蛋白酶，介导神经突生长 在发展过程中。 反应性神经胶质增生和存活神经元的发芽失败都是神经元死亡的原因。 在AD大脑的受影响区域中常规观察到。这些发现促使 考虑到星形胶质细胞的重演 神经突促进因子的分泌，包括PMII/APP和层粘连蛋白， 可能会促使发芽失败，导致额外的神经元周期， 退化 经历失败的发芽尝试的神经元可能 产生大量APP的淀粉样蛋白片段。 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， 未分化神经元细胞与星形胶质细胞条件培养基（CM） 或纯化的APP诱导神经突发生且无毒性。相比之下，CM- 或APP处理长期分化的（“老化的”）神经元细胞， 只要分化剂（dbcAMP）减少或消除， 诱导发芽和退化。CM和APP是良性的， 这些老化的培养物在发芽时通过将dbcAMP维持在 高浓度。我们以前的研究表明， 钙蛋白酶（钙激活蛋白酶）和蛋白质的过度激活 这些细胞中的激酶C（PKC）诱导ALZ-5 O免疫反应性。我们将 确定CM和APP诱导的神经毒性是否伴有 ALZ-50诱导，以及神经元产生β-淀粉样蛋白是否涉及 钙蛋白酶和PKC通过细胞内递送特异性 钙蛋白酶和PKC激活剂和抑制剂。的潜在作用 神经变性中异常磷酸化tau蛋白（产生ALZ-50） 将通过反义寡核苷酸介导的tau抑制来检查 在CM和APP处理之前进行合成，并确定是否 神经退行性变减少;这些分析将提供信息 关于tau蛋白是导致神经退行性变还是其副产品。 假设神经退化是重演的结果 将进一步探索的发芽反义寡核苷酸- 介导的生长相关蛋白GAP-43的下调， 是这些细胞发芽所必需的。潜在的关键 与细胞外基质成分的相互作用将通过 在交替培养物中包含层粘连蛋白。 本建议中所载的研究并非旨在 包括所有胶质源性生长因子 在组成型或发育和再生期间表达。 他们也没有试图阐明AD的起始事件。 然而，通过探索神经胶质细胞加剧AD的可能性， 神经病理学，希望能为治疗提供新的见解 方法可以演变。

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.