Molecular Mechanisms in Neurodegeneration

这项研究的长期目标是定义和描述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）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的可用性和在小鼠中定义良好的动物模型，以便充分利用可用于基因操作的广泛工具，例如同源重组的基因消融和引入异源转基因。在特异性目标1中，神经变性源于内质网错误折叠蛋白积累的假设将在体外进行更详细的测试。这些数据将与患者的疾病严重程度相关联，由详细的临床评估和体外转染测定确定。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。在特异性目标2中，突变小鼠的发病机制，通常由蛋白质积累激活的信号通路在敲除小鼠中被破坏，将从分子和形态学上详细描述，以提供对蛋白质错误折叠在神经退行性疾病中的参与的更深入的理解。在《特异性目标3》中，我们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研究最近发现的由内质网蛋白质积累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了类似于其他研究人员在体外系统中研究不同细胞类型时发现的信号通路。此外，使用微阵列筛选下游靶基因的鉴定将寻求鉴定和表征未折叠蛋白激活的信号通路，并最终确定细胞是否存活或死亡。总之，这些目标有望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其他神经退行性疾病的改善，这些疾病是蛋白质错误折叠的原因，但遗传或代谢缺陷尚不清楚。

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.