Molecular Pathogenesis of spinocerebellar ataxia type 12

项目摘要 脊髓小脑性共济失调12型（SCA 12）是一种常染色体显性遗传的神经退行性疾病， 发现和特点是由我们的小组，值得注意的临床震颤，步态异常，和神经精神 综合征和神经病理学上的大脑皮质和小脑萎缩。SCA 12是由 PPP 2 R2 B基因CAG/CTG扩增突变的研究 磷酸酶2A（PP 2A）。正常等位基因携带4至31个三联体，而疾病等位基因携带43至78个三联体。 SCA 12是印度北部第二常见的共济失调，在美国发现了SCA 12病例， 意大利和中国。没有治疗方法。我们的初步数据表明PPP 2 R2 B至少包含16个外显子 多个选择性剪接变体，导致至少8种具有不同N 末端结构域，其中Bβ1和Bβ2是迄今为止最丰富和最好表征的。N-末端结构域有助于PP 2A全酶的定位，因此有助于其相对丰度 可能是建立蛋白质磷酸化模式的关键。该重复序列位于外显子5'端 7，但也包含在可变剪接变体的转录本内。基于 根据大量的初步数据，我们假设SCA 12中CAG重复扩增改变了PPP 2 R2 B 表达和剪接，导致PP 2A靶向的变化，这有助于SCA 12的发病机制， 磷酸化蛋白质组的改变。其次，我们假设由重复序列扩增引起的错误剪接 产生编码毒性多丝氨酸段的转录物，该多丝氨酸段有助于SCA 12发病机制。我们的目标是 每一个都旨在探索SCA 12发病机制的一个特定方面，同时探索更普遍的 神经退行性变的原则与重复扩增和PP 2A的功能，使用遗传 工程小鼠和人类iPSC。在目标1中，我们将确定SCA 12突变对PPP 2 R2 B的影响。 表情在目的2中，我们将确定异常PPP 2 R2 B表达对PP 2A底物靶向的影响。 和磷酸化蛋白质组。在目标3中，我们将确定PP 2A底物的变化如何靶向和异常地 表达的多丝氨酸导致神经毒性。如果我们的假设是正确的，那么SCA 12的发病机制源于 重复扩增诱导PPP 2 R2 B表达的变化，1）改变PP 2A底物靶点， 磷酸化蛋白质组，和2）产生有毒的多丝氨酸束。建立这些途径将建立 SCA 12治疗干预的多个潜在靶点。与此同时，我们的实验将更多地 一般建立了重复扩增对启动子和剪接功能的潜在影响，以及 神经变性中的PP 2A失调，特别令人感兴趣的是给出了其他神经变性中PP 2A失调的证据。 神经退行性疾病，如阿尔茨海默病。

PROJECT SUMMARY Spinocerebellar ataxia type 12 (SCA12) is an autosomal dominant neurodegenerative disease, discovered and characterized by our group, notable clinically for tremor, gait abnormalities, and neuropsychiatric syndromes, and neuropathologically for atrophy in cerebral cortex and cerebellum. SCA12 is caused by a CAG/CTG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A (PP2A). Normal alleles carry 4 to 31 triplets, whereas disease alleles carry 43 to 78 triplets. SCA12 is the second most common ataxia in North India, with cases of SCA12 detected in the United States, Italy, and China. There is no treatment. Our preliminary data indicates that PPP2R2B contains at least 16 exons with multiple alternative splice variants, resulting in at least 8 protein isoforms (termed B1-B8) with different N terminal domains, among which Bβ1 and Bβ2 are by far the most abundant and best characterized. The N-terminal domains contribute to the localization of the PP2A holoenzyme, and hence their relative abundance may be critical in establishing patterns of protein phosphorylation. The repeat is located in the 5’ region of exon 7 in a predicted promoter region, but is also contained within the transcripts of alternative splice variants. Based on extensive preliminary data, we hypothesize that the CAG repeat expansion in SCA12 alters PPP2R2B expression and splicing, leading to changes in PP2A targeting that contribute to SCA12 pathogenesis by alteration of phosphoproteome. Secondarily, we hypothesize that missplicing induced by repeat expansion generates a transcript encoding a toxic polyserine tract that contributes to SCA12 pathogenesis. Our aims are each designed to explore a specific aspect of SCA12 pathogenesis, and simultaneously to explore more general principles of neurodegeneration related to repeat expansion and the function of PP2A, using genetically engineered mice and human iPSCs. In Aim 1, we will determine the effect of the SCA12 mutation on PPP2R2B expression. In Aim 2, we will determine the effect of abnormal PPP2R2B expression on PP2A substrate targeting and the phosphoproteome. In Aim 3, we will determine how changes in PP2A substrate targets and aberrantly expressed polyserine contribute to neurotoxicity. If our hypothesis is correct, SCA12 pathogenesis derives from repeat-expansion-induced changes in PPP2R2B expression that 1) alters PP2A substrates targets and hence the phosphoproteome, and 2) generates toxic polyserine tracts. Establishing these pathways will establish multiple potential targets for therapeutic intervention in SCA12. At the same time, our experiments will more generally establish the potential effect of repeat expansion on promoter and splicing function, and the role of PP2A dysregulation in neurodegeneration, of particular interest given evidence of PP2A dysregulation in other neurodegenerative disorders, such as Alzheimer’s disease.