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。包括阿尔茨海默病在内的许多神经退行性疾病的发病机制都被认为是在不同的亚细胞室中积累的未折叠蛋白中间产物。这项研究的一个关键组成部分是在小鼠身上获得患者数据和明确定义的动物模型，以便充分利用可用于遗传操作的广泛工具，例如通过同源重组去除基因和引入异源转基因。在特定的目标1中，将在体外更详细地检验一种假说，即神经退化源于内质网中错误折叠的蛋白质的积累。这些数据将与患者的疾病严重程度相关，这是通过详细的临床评估和体外转基因试验确定的。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。在特定的目标2中，将从分子和形态两个方面详细描述突变小鼠的发病机制，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层次的理解。在具体目标3中，我们将在小鼠神经退行性疾病模型中从RNA、蛋白质和免疫细胞化学水平检测最近发现的一些重要的基因和蛋白质，这些基因和蛋白质是由内质网中的蛋白质积累激活的，以确定蛋白质错误折叠是否激活了与其他研究人员在体外系统中研究不同细胞类型的信号通路相似的信号通路。此外，使用微阵列筛选识别下游靶基因将寻求识别和表征由未折叠蛋白质激活的信号通路，并最终确定细胞是存活还是死亡。总之，这些目标有望确定源于蛋白质积累的重要病理过程，并可能导致减轻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，这些疾病的原因是蛋白质错误折叠，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.