Mouse Models of Alexander Disease

描述（由申请人提供）：亚历山大病（AxD）患者在GFAP编码区内携带杂合突变。为了促进AxD发病机制的机制研究，并提供适合测试潜在疗法的动物模型，我们的长期目标是产生这种疾病的小鼠模型。我们现在已经产生了携带在人类AxD中发现的几种最常见的GFAP突变（相当于R79 H、R239 H、R239 C和R416 W）的小鼠的基因敲入系，并发现突变GFAP的表达诱导罗森塔尔纤维（标志性病理特征），但小鼠是存活的。此外，在适当的遗传修饰剂（例如升高野生型GFAP）的情况下表达突变体GFAP导致致死表型。此外，通过简单的过度表达或突变GFAP的产生来改变GFAP表达导致诱导多个应激途径（aB-晶状体蛋白，Nrf 2），其提示关于发病机制的特定假设，并最终提示治疗策略。为了更准确地理解星形胶质细胞中异常GFAP表达的后果，以及产生反映人类AxD关键表型特征的小鼠模型，我们提出了以下具体目标：在目标1中，我们将测试表达突变GFAP蛋白和/或GFAP过表达的后果，以与人类亚历山大表型的已知特征进行比较。这些研究将包括使用小鼠星形胶质细胞原代培养物的体外模型，以及携带内源性小鼠GFAP基因点突变的体内敲入模型。一系列的属性将被检查，包括生化，功能和行为。还将评估可逆性。在目标2和3中，我们将通过将GFAP小鼠与敲除或新产生的过表达这两种基因的转基因小鼠杂交，分别测试小应激蛋白aB-晶状体蛋白和转录因子Nrf 2作为修饰剂的作用。将评价过量aB-晶状体蛋白或Nrf 2可能实现拯救的具体机制。这些研究有望提供星形胶质细胞中突变中间丝表达的病理学意义的新信息，将提示原发性星形胶质细胞功能障碍导致全身性CMS疾病的机制，并将确定关键的应激途径，这些途径最终可以作为治疗干预的基础，以减轻这种疾病的破坏性影响。

DESCRIPTION (provided by applicant): Alexander disease (AxD) patients carry heterozygous mutations within the coding region of GFAP. To facilitate mechanistic studies on the pathogenesis of AxD, and provide animal models suitable for testing potential therapies, our long-term goal has been to generate mouse models for this disorder. We have now generated knock-in lines of mice carrying several of the most common GFAP mu-tations found in human AxD (equivalent to R79H, R239H, R239C, and R416W), and found that expression of the mutant GFAPs induces Rosenthal fibers (the hallmark pathologic feature), but the mice are viable. Moreover, expressing the mutant GFAPs in the context of appropriate genetic modifiers (such as elevating wild type GFAP) results in a lethal phenotype. In addition, altering GFAP expression either by simple over-expression or production of mutant GFAPs leads to induction of multiple stress pathways (aB-crystallin, Nrf2) that suggest specific hypotheses about pathogenesis and, ultimately, strategies for therapy. With the goals of understanding more precisely the consequences of abnormal GFAP expression in astrocytes, and of generating mouse models that reflect key phenotypic features of AxD in humans, we propose the following specific aims: In Aim 1 we will test the consequences of expressing mutant GFAP protein and/or GFAP over-expression, for comparison with known features of the Alexander phenotype in humans. These studies will include both in vitro models using primary cultures of mouse astrocytes, as well as the in vivo knock-in models carrying point mutations in the endogenous mouse GFAP gene. A range of properties will be examined, including biochemical, functional, and behavioral. Reversibility will also be assessed. In Aims 2 and 3 we will test the roles of the small stress protein aB-crystallin, and the transcription factor Nrf2, respectively, as modifiers, by crossing the GFAP mice with knockouts or with newly generated transgenics that over-express the two genes. Specific mechanisms by which excess aB-crystallin or Nrf2 might achieve rescue will be evaluated. These studies promise novel information on the pathological significance of mutant intermediate filament expression in astrocytes, will suggest mechanisms by which primary astrocyte dysfunction leads to generalized CMS disease, and will identify critical stress pathways that could ultimately serve as the basis for therapeutic interventions to mitigate the devastating effects of this disease.