INV OF ASTROCYTE CASPASE ACTV & CD40/CD40L SIGNALING INTERACTIONS IN ALZHEIMER?S

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 问题和假设的简要背景： Tau是一种微管相关蛋白，稳定神经元细胞骨架，在囊泡运输和轴突极性中发挥作用。Tau的病理改变被认为在包括阿尔茨海默病(AD)在内的几种神经退行性疾病中起到了致病作用。在AD中，β-淀粉样蛋白(A？)沉积伴随着神经元细胞骨架逐渐被不可溶的NFT取代，这些NFT是在tau的修饰后发展起来的，包括过度磷酸化和蛋白水解性切割。然而，到目前为止，A？沉积和NFTs之间的关系仍然不清楚。A？和NFTs之间的一个可能的联系是caspase的异常激活，caspase是负责正确执行细胞程序性死亡或凋亡的蛋白水解酶。最近的研究确定，caspase的激活可能会将这两种标志性病理联系在一起。因此，在人类AD脑中，我们的实验室以及其他实验室已经证明了tau的caspase-裂解产物(Ccp)的存在，这些ccp与NFT形成的标记相关联。此外，半胱氨酸氨基转移酶裂解的tau在AD脑内与细胞内的A？1-42共定位，并在体外被A？1-42诱导。总之，这些结果表明，缠结和淀粉样蛋白通过共同的途径相互连接，即caspase介导的蛋白分解。为了直接验证这一假设，我们在本申请中建议将AD的新型TG小鼠模型(3xTg-AD小鼠)与过表达抗细胞凋亡蛋白的小鼠(Bcl-2 OE TG小鼠)杂交的效果进行检验。我们的总体假设是，这种杂交的后代将在与3xTg-AD小鼠相关的病理和行为问题中表现出caspase激活的减弱和预防。 这项建议的具体目标是： 目的1：建立高表达抗细胞凋亡蛋白Bcl2的新型AD转基因小鼠模型。初步数据表明，在3xTg-AD小鼠中，tau的caspase裂解是以年龄依赖的方式发生的。BCL-2是一种功能强大的抗细胞凋亡蛋白，它能将多种类型的神经元从损伤或疾病引起的凋亡中拯救出来，并能提高神经元在发育过程中的存活率。为了达到这个目的，我们测试了这样的假设，即这两个转基因小鼠模型杂交产生的后代是可行的，并以年龄相关的方式适当地过表达Aü、tau和Bcl2。由于所有后续的特定目标只能在这个特定目标成功后才能实现，我们将提供初步数据，以证明此类后代成功杂交并存活至少24个月。 目的2：探讨过表达Bcl2是否能阻止caspase激活、tau和APP的裂解，减少AD小鼠的缠结改变。最近的体外和体内数据表明，半胱氨酸氨基转移酶的激活和随后的tau的切割可能是AD中Aβ与NFTs之间的关键事件。为此，我们计划直接验证这一假说，通过确定抗凋亡蛋白Bcl-2的过度表达是否可以阻止AD动物模型中caspase的激活和NFT的形成。 目的：探讨半胱氨酸天冬氨酸氨基转移酶在3xTg-AD小鼠Bcl2过度表达后淀粉样前体蛋白和tau蛋白的转化中是否起作用。有证据表明，淀粉样前体蛋白(APP)和tau都是caspase介导的切割的底物。在3xTg-AD小鼠模型中，Bcl2的过度表达和caspase活性的减弱可能导致APP、Aβ和tau水平的变化。我们推测，在Bcl2过表达后，我们将观察到APP和tau的更高水平。 目的：探讨3xTg-AD小鼠过表达Bcl2是否能改善认知功能。这一建议的一个重要决定因素将不仅是评估病理，还将评估对Bcl-2过表达后的认知。除了使用一种涉及位置识别的新记忆范式外，还将使用标准的莫里斯迷宫或径向臂迷宫。 综上所述，我们相信这个项目的设计和基本原理应该可以让我们回答caspase是否在AD相关的发病机制中发挥积极作用。我们认为，这是一项重要而及时的研究，可以确定治疗这种疾病的新靶点。这一建议的结果可能会为测试药物阻断caspase作为治疗AD的干预措施提供动力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brief Background of the problem and hypothesis: Tau is a microtubule-associated protein that stabilizes the neuronal cytoskeleton and functions in vesicular transport and axonal polarity. Pathological alterations in tau are thought to play a causative role in several neurodegenerative disorders, including Alzheimer's disease (AD). In AD, beta-amyloid (A¿) deposition is accompanied by the gradual replacement of the neuronal cytoskeleton with insoluble NFTs that develop following modifications of tau that include hyperphosphorylation and proteolytic cleavage. However, to date the relationship between A¿ deposition and NFTs remains unknown. One possible link between A¿ and NFTs is the aberrant activation of caspases, proteolytic enzymes responsible for the proper execution of programmed cell death or apoptosis. Recent studies have determined that caspase activation may interconnect these two hallmark pathologies. Thus, in the human AD brain our laboratory as well as others have demonstrated the presence of caspase-cleavage products (CCPs) of tau that are associated with markers of NFT formation. Furthermore, caspase-cleaved tau co-localizes with intracellular A¿1-42 in the AD brain and is induced by A¿1-42, in vitro. Collectively, these results indicate that tangles and amyloid are interconnected through a common pathway, namely caspase-mediated proteolysis. To test this hypothesis directly, we propose in the present application to examine the effects of crossing a novel Tg mouse model of AD (3xTg-AD mice) with mice overexpressing the antiapoptotic protein, Bcl-2 (Bcl-2 OE Tg mice). Our overall hypothesis is that progeny of such a cross will show attenuation in caspase activation and prevention in the pathology and behavioral problems associated with 3xTg-AD mice. The specific Aims of the proposal are: Aim 1: To establish a novel transgenic mouse model of AD that overexpresses the antiapoptotic protein, Bcl-2. Preliminary data suggest that caspase cleavage of tau occurs in an age-dependent manner in 3xTg-AD mice. Bcl-2 is powerful anti-apoptotic protein that rescues many types of neurons from apoptosis caused by injury or disease and can enhance neuronal survival during development. In this aim, we test the hypothesis that progeny resulting from crossing these two Tg mice models are viable and properly overexpress A¿, tau and Bcl-2 in an age-dependent manner. Because all subsequent specific aims can only be carried out following the success of this specific aim, we will provide preliminary data to demonstrate the successful crossbreeding and survival of such progeny for at least 24 months. Aim 2: Assess whether overexpression of Bcl-2 will prevent caspase activation, the cleavage of tau and APP and lead to fewer tangle alterations in AD mice. Recent in vitro and in vivo data suggest that caspase activation and subsequent cleavage of tau may be a key event linking A¿ with NFTs in AD. In this aim, we plan to test this hypothesis directly, by determining whether overexpression of an antiapoptotic protein, Bcl-2, prevents caspase activation and NFT formation in an animal model of AD. Aim 3: Determine whether caspases play a role in the turnover of the amyloid precursor protein and tau following the overexpression of Bcl-2 in 3xTg-AD mice. Evidence suggests that both the amyloid precursor protein (APP) and tau are substrates for caspase-mediated cleavage. The overexpression of Bcl-2 and attenuation of caspase activation may lead to alterations in the levels of APP, A¿ and tau in 3xTg-AD mice. We hypothesize that we will observe higher levels of both APP and tau following overexpression of Bcl-2. Aim 4: To determine whether overexpression of Bcl-2 in 3xTg-AD mice improves cognition. An important determinant for this proposal will be to assess not only the pathology but also cognition following overexpression of Bcl-2. Standard Morris or radial arm mazes will be employed in addition to using a novel memory paradigm involving place recognition. Taken together, we believe the design and rationale for this project should allow us to answer whether caspases play an active role in the pathogenesis associated with AD. We believe this to be both an important and timely study that could identify new targets for the treatment of this disease. Results from this proposal may provide impetus to test pharmacological blockade of caspases as a therapeutic intervention in treating AD.