Alexander Disease: Cellular and Molecular Mechanisms

描述（由申请人提供）：许多脑部疾病研究集中在神经元毒性机制上。然而，神经胶质细胞是神经系统中最丰富的一类细胞，对这些细胞在疾病状态中的功能作用知之甚少。编码星形胶质细胞特异性中间丝、胶质细胞酸性蛋白（GFAP）的基因突变导致亚历山大病，这是一种典型的儿童疾病，表现为癫痫发作和严重的白色病变。髓鞘形成障碍伴随着星形胶质细胞中富含GFAP的包涵体的形成，称为罗森塔尔纤维。这些聚集体中存在GFAP，并且观察到小鼠星形胶质细胞中GFAP过表达产生严重的神经系统综合征和罗森塔尔纤维形成，这导致了以下假设：亚历山大病是由功能机制的显性获得产生的，可能与GFAP的异常聚集有关。为了建立亚历山大病模型，我们在果蝇中表达了正常和亚历山大病相关的GFAP突变体。我们发现，GFAP在果蝇胶质细胞中的过度表达导致形成大量含有GFAP的Rosenthal-fiber样包涵体，癫痫发作频率增加，以及非细胞自主神经变性。初步的遗传分析表明，蛋白质错误折叠和氧化应激在介导GFAP毒性中起关键作用。此外，我们在我们的转基因动物中看到了自噬和JNK通路的强烈上调。蛋白质错误折叠、氧化应激、JNK途径激活和自噬与亚历山大病和小鼠和哺乳动物细胞培养系统中的亚历山大病模型有关。因此，我们的模型似乎可以识别GFAP毒性的相关途径。我们还开发了一种适合于在我们的模型中进行正向遗传分析的筛选测定。我们现在提出了一个大规模的GFAP细胞毒性的正向遗传分析。我们的实验将利用新创建的全基因组范围的转基因RNAi集合。从我们的工作中出现的新见解可以在与该计划项目的其他成员合作的生化，细胞和转基因小鼠实验中进一步评估。 公共卫生相关性：亚历山大病是一种破坏性的疾病，它会攻击患者大脑中的神经胶质和神经元，导致严重残疾和早死。我们的研究将开发和利用强大的疾病模型，可用于了解发生的精确细胞和生化异常，从而为有效的治疗铺平道路。

DESCRIPTION (provided by applicant): Much brain disease research focuses on neuronal mechanisms of toxicity. However, glia comprise the most abundant class of cells in the nervous system and little is known about the functional role of these cells in disease states. Mutations in the gene encoding the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), cause Alexander disease, a typically childhood disorder that manifests with seizures and severe white matter pathology. Dysmyelination is accompanied by the formation of GFAP-rich inclusions in astrocytes termed Rosenthal fibers. The presence of GFAP in these aggregates and the observation that overexpression of GFAP in mouse astrocytes produces a severe neurological syndrome and Rosenthal fiber formation has led to the hypothesis that Alexander disease is produced by a dominant gain of function mechanism, perhaps related to abnormal aggregation of GFAP. To create a model of Alexander disease, we have expressed normal and Alexander disease-linked mutant versions of GFAP in Drosophila. We find that overexpression of GFAP in Drosophila glia leads to formation of numerous GFAPcontaining, Rosenthal-fiber like inclusion bodies, increased seizure frequency, and non-cell autonomous neurodegeneration. Preliminary genetic analysis suggests that protein misfolding and oxidative stress play key roles in mediating toxicity of GFAP in our model. Further, we see robust upregulation of autophagy and the JNK pathway in our transgenic animals. Protein misfolding, oxidative stress, JNK pathway activation and autophagy have been implicated in Alexander disease and Alexander disease models in mice and mammalian cell culture systems by other members of the Program Project. Thus, our model appears to identify relevant pathways of GFAP toxicity. We have also developed a screening assay suitable for forward genetic analysis in our model. We now propose a large-scale forward genetic analysis of GFAP cellular toxicity. Our experiments will utilize a newly created whole genome wide transgenic RNAi collection. Novel insights emerging from our work can be further evaluated in biochemical, cellular, and transgenic mouse experiments working with other members of the Program Project. PUBLIC HEALTH RELEVANCE: Alexander disease is a devastating disorder that attacks glia and neurons in the brains of patients, leading to severe disability and early death. Our studies will develop and utilize powerful models of the disease that can be used to understand the precise cellular and biochemical abnormalities that occur, and thus pave the way for effective therapies.