Alexander Disease: Cellular and Molecular Mechanisms

描述(申请人提供)：Alexander病是一种罕见的、典型的致命的神经退行性疾病，由编码III型中间丝蛋白GFAP的基因杂合突变引起。这种疾病的病理特征是罗森塔尔纤维，这是一种胞浆内包涵体，包含中间细丝和小的热休克蛋白，在CMS期间聚集在星形胶质细胞中。我们小组之前的研究表明，GFAP突变是几乎所有Alexander病的原因，并将这一信息迅速转化为临床实践，作为诊断的标准。然而，GFAP突变导致星形胶质细胞功能障碍和疾病的机制尚不清楚。基于前一次资助期间的结果，我们提出了一个假说来指导实验，强调GFAP水平的升高与细胞应激反应的诱导和包涵体的形成是发病的关键因素。该项目的目标是研究应激反应的分子机制和功能后果，探索包涵体形成的生化组成和下游影响，并识别和表征疾病表型的遗传修饰因素。我们的研究跨越了遗传学、生化、细胞学、生理学和形态学的方法来解决这些问题。该计划将连接五个实验室；其中三个实验室继续前一个赠款时期，并加入两个新的小组，带来新的方法和技术(生理学和果蝇)。该计划将通过促进五个实验室之间的试剂、动物和结果的共享，通过思想的交流，以及实验室成员之间的定期交流和会议，来促进关于神经胶质细丝功能障碍在疾病中的作用的重点努力。这些研究有望对胶质细丝在星形胶质细胞细胞生物学中的作用以及星形胶质细胞在大脑功能和疾病中的作用提供新的见解。 项目1 主要调查者：迈克尔·布伦纳 标题：亚历山大病的生物化学和遗传学 描述(申请人提供)：胶质纤维酸性蛋白(GFAP)是一种结构蛋白，几乎只在星形胶质细胞中发现。我们的实验室最近发现，GFAP基因编码区的突变会导致亚历山大病(AxD)，这是一种罕见但通常是致命的中枢神经系统疾病。这种疾病的特点是存在以GFAP为主要成分的蛋白质聚集体。这项建议的目的是开发更多关于GFAP突变导致AxD的机制的信息。在目标1中，我们将确定RFS的组成，以获得疾病机制的线索，这一方法已被证明对其他蛋白质聚集性疾病有效。聚集体将被部分提纯，并通过质谱学鉴定其蛋白质成分。在目标2中，我们将确定GFAP是否被异常脱亚胺或磷酸化，已知的两种GFAP修饰会影响其聚合，并存在于其他神经退行性疾病中。在目标3中，我们还将确定突变形式是否特定地积累在聚集体中。这将检验这样一种假设，即突变的GFAP本身无毒，但通过导致GFAP积累而导致疾病。

DESCRIPTION (provided by applicant): Alexander disease is a rare and typically fatal neurodegenerative disease that results from heterozygous mutations in the gene encoding the type III intermediate filament protein GFAP. The pathological signature of the disorder is the Rosenthal fiber, a cytoplasmic inclusion containing intermediate filaments and small heat shock proteins that accumulates in astrocytes throughout the CMS. Prior investigations by our groups have let to the acceptance of GFAP mutations as the cause for nearly all cases of Alexander disease, and the rapid translation of this information to clinical practice as the standard for diagnosis. However, the mechanisms by which GFAP mutations cause astrocyte dysfunction and disease remain unclear. Based on results obtained during the previous grant period we have developed a hypothesis to guide experiments that emphasizes expression of elevated levels of GFAP in concert with induction of a cellular stress response and formation of inclusions as key elements of pathogenesis. The goals of this Program Project are to investigate molecular mechanisms and functional consequences of the stress response, to explore biochemical composition and downstream effects of inclusion body formation, and to identify and characterize genetic modifiers of disease phenotype. Our studies span genetic, biochemical, cellular, physiological, and morphological approaches to these questions. The Program will link five laboratories; three of these continue from the previous grant period, and two new groups join that bring novel approaches and techniques (physiology and Drosophila). The Program will promote a focussed effort on the role of glial filament dysfunction in disease, by fostering sharing of reagents, animals, and results among the five labs, through cross-fertilization of ideas, and by regular communication and meetings among laboratory members. These studies promise novel insights into the role of glial filaments in the cell biology of astrocytes, and the role of astrocytes in brain function and disease. PROJECT 1 Principal Investigator: Michael Brenner Title: Biochemistry and Genetics of Alexander Disease Description (provided by applicant): Glial fibrillary acidic protein (GFAP) is a structural protein found almost exclusively in astrocytes. Our laboratory recently found that mutations in the coding region of the GFAP gene cause Alexander disease (AxD), a rare but usually fatal disorder of the central nervous system. This disease is characterized by the presence of protein aggregates which have GFAP as a primary constituent. The purpose of this proposal is to develop additional information about the mechanism by which the GFAP mutations cause AxD. In Aim 1 we will determine the composition of the RFs to obtain clues for the disease mechanism, an approach that has proved fruitful for other protein aggregate disorders. The aggregates will be partially purified, and their protein components identified by mass spectrometry. In Aim 2 we will determine if the GFAP is abnormally deiminated or phosphorylated, two modifications of GFAP known to affect its polymerization and to be present in other neurodegenerative disorders. In Aim 3 we will also determine whether the mutant form specifically accumulates in the aggregates. This will test the hypothesis that mutant GFAP is not toxic per se, but produces disease by causing GFAP accumulation.