Molecular Mechanisms in Neurodegeneration

这项研究的长期目标是确定和表征蛋白质错义突变导致神经退行性疾病的分子机制。 未折叠蛋白质中间体在各种亚细胞区室中的积累被认为是许多神经退行性疾病（包括阿尔茨海默病）的发病机制的基础。 这项研究的一个关键组成部分是患者数据和小鼠中定义明确的动物模型的可用性，以便充分利用可用于遗传操作的广泛工具，例如通过同源重组进行基因切除和引入异源转基因。 在具体目标1中，将在体外更详细地测试神经变性源于内质网中错误折叠蛋白质积累的假设。 这些数据将与通过详细的临床评价和体外转染测定确定的患者疾病严重程度相关。此外，尸检标本将在RNA、蛋白质和免疫细胞化学水平上进行表征，以确定人类的发病机制是否与动物模型相似。在特定目标2号突变小鼠的发病机制，其中通常激活的蛋白质积累的信号通路已被破坏，在敲除小鼠，将详细的分子和形态学特征，以提供更深入的了解蛋白质错误折叠在神经退行性疾病的参与。 在特定目标3号中，将在神经退行性疾病小鼠模型的RNA、蛋白质和免疫细胞化学水平上检查最近发现的通过内质网中蛋白质积累而激活的一些重要基因和蛋白质，以确定蛋白质错误折叠是否激活与其他研究者在体外系统中研究不同细胞类型所鉴定的信号通路相似的信号通路。 此外，将寻求使用微阵列筛选来鉴定下游靶基因，以鉴定和表征由未折叠蛋白质激活的信号通路，并最终确定细胞存活还是死亡。 总之，这些目标预计将确定源于蛋白质积累的重要病理过程，并可能导致改善疾病严重程度的策略。 此外，从这些研究中获得的知识可能适用于改善其他神经退行性疾病，这些疾病的原因是蛋白质错误折叠，但遗传或代谢缺陷是未知的。

The long term goals of this research are to define and characterize the molecular mechanisms by which missense mutations in proteins cause neurodegenerative disease. The accumulation of unfolded protein intermediates in various subcellular compartments is thought to underlie pathogenesis for a number of neurodegenerative diseases, including Alzheimers disease. A critical component of this research is the availability of patient data and well-defined animal models in mice so that full use can be made of the extensive tools available for genetic manipulation, such as gene ablation by homologous recombination and the introduction of heterologous transgenes. In Specific Aim number 1 an hypothesis that neurodegeneration stems from the accumulation of misfolded proteins in the endoplasmic reticulum will be tested in greater detail in vitro. These data will be correlated with disease severity in patients as determined by detailed clinical evaluation and in vitro transfection assays. Furthermore, autopsy specimens will be characterized at the levels of RNA, protein and immunocytochemistry to determine if pathogenesis in humans is similar to that in the animal models. In Specific Aim number 2 the pathogenesis of mutant mice, for which signaling pathways normally activated by protein accumulation have been disrupted in knockout mice, will be characterized in detail both molecularly and morphologically to provide a deeper understanding of the involvement of protein misfolding in neurodegenerative disease. In Specific Aim number 3 a number of important genes and proteins recently found to be activated by protein accumulation in the endoplasmic reticulum will be examined at the levels of RNA, protein and immunocytochemistry in mouse models of neurodegenerative disease to determine if protein misfolding activates similar signaling pathways to those identified by other investigators studying different cell types in in vitro systems. Furthermore, the identification of downstream target genes using microarray screens will be sought to identify and characterize the signaling pathways that are activated by unfolded proteins, and ultimately determine if a cell survives or dies. Together, these Aims are expected to identify important pathological processes stemming from protein accumulation and may lead to strategies that ameliorate disease severity. Moreover, the knowledge gained from these studies may be applicable to the amelioration of other neurodegenerative diseases for which protein misfolding is a cause but the genetic or metabolic defects are unknown.