Mouse Models of Alexander Disease

描述(申请人提供)：Alexander病(AxD)患者携带GFAP编码区的杂合突变。为了促进AxD发病机制的研究，并提供适合测试潜在治疗方法的动物模型，我们的长期目标一直是建立这种疾病的小鼠模型。我们现在已经建立了一些在人类AxD中发现的最常见的GFAP突变的小鼠敲入系(相当于R79H、R239H、R239C和R416W)，并发现突变的GFAP的表达诱导了Rosenthal纤维(这是标志性的病理特征)，但小鼠是存活的。此外，在适当的遗传修饰物(如提升野生型GFAP)的背景下表达突变的GFAP会导致致命的表型。此外，通过简单的过度表达或产生突变的GFAP来改变GFAP的表达会导致多条应激途径(AB-晶体蛋白，Nrf2)的诱导，这些途径提出了关于发病机制的特定假设，并最终提出了治疗策略。为了更准确地了解星形胶质细胞中GFAP异常表达的后果，并建立反映人类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型GFAP蛋白和/或GFAP过表达的后果，以与人类Alexander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的体外模型，以及体内携带内源性小鼠GFAP基因点突变的敲入模型。将检查一系列属性，包括生化、功能和行为。还将评估可逆性。在目标2和目标3中，我们将通过将GFAP小鼠与过量表达这两个基因的转基因小鼠杂交，分别测试小应激蛋白AB-晶体蛋白和转录因子Nrf2作为修饰物的作用。过量的AB-晶体蛋白或Nrf2可能实现救援的具体机制将被评估。这些研究有望提供关于星形胶质细胞突变中间丝表达的病理学意义的新信息，将提示星形胶质细胞功能障碍导致全身性CMS疾病的机制，并将识别关键的应激途径，最终可能作为治疗干预措施的基础，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.