Spinocerebellar ataxia 12: Mechanisms of Neuronal Injury

描述（由申请人提供）：脊髓小脑共济失调12型（SCA12）是一种神经退行性疾病，最近由作者描述。SCA12与PPP2R2B中的CAG重复扩增突变相关，PPP2R2B是编码PPP2R2B的基因，作为蛋白磷酸酶2A（PP2A）的调节亚基。目的是1）描述这种新疾病的临床表型，2）深入了解SCA12中介导神经元功能障碍和细胞丢失的分子和细胞机制。待检验的主要假设是扩增突变导致PP2A调节亚基表达增加，导致PP2A活性改变，从而导致神经元毒性。目的1探讨SCA12的临床、MRI及病理特点。将在两个不同的时间点对第一个SCA12亲属的受影响成员进行神经学、神经心理学和精神病学评估，以确定SCA12的临床特征及其时间进程。这些发现将与脑磁共振成像（MRI）上观察到的变化进行比较。将对第一个家系先证者的尸检大脑进行大体病理学检查和特定脑区的细胞学变化。目的2将确定SCA12中CAG重复扩增改变基因表达的机制。我们将检验SCA12 CAG重复序列位于磷酸酶亚基基因PPP2R2B的启动子区域内，重复序列的扩增导致PPP2R2B表达增加的假设。将使用生物信息学和实验方法来确认CAG重复序列在开放阅读框外的PPP2R2B的5'区域中的位置。启动子测定将揭示含有重复序列的区域是否是功能性启动子，以及重复序列扩增是否增加启动子活性。如果是这样，将确定增加启动子活性所必需的重复扩增的性质。目的3将使用N2a神经母细胞瘤细胞和原代神经元培养物中PPP2R2B的过表达来分析SCA12的细胞培养模型中的神经元损伤。这将检验PPP2R2B的过表达对细胞有毒的假设。在CMV启动子调控下PPP2R2B过表达后，然后通过PPP2R2B在其自身启动子控制下过表达，分析培养细胞的存活。将通过比较用正常与扩增的CAG重复转染的细胞的损失来测试不同PPP2R2B表达水平的相对毒性。第二个假设是PPP2R2B过表达的毒性是蛋白磷酸酶2A功能改变的结果。为了测试功能，我们将在PPP2R2B过表达后测定PP2A的酶活性、底物特异性和细胞内定位，并确定PP2A调节剂对细胞存活的影响。SCA12的研究可能会提供洞察负责神经元损失的机制，和潜在的治疗，这种和其他三核苷酸重复疾病和SCA，由于基因启动子突变的其他疾病，以及其他条件，导致磷酸化功能失调的调节。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disorder recently described by the authors. SCA12 is linked to a CAG repeat expansion mutation in PPP2R2B, the gene that encodes PPP2R2B, as regulatory subunit of protein phosphatase 2A (PP2A). Goals are 1) to delineate the clinical phenotype of this new disease and 2) to gain insight into the molecular and cellular mechanisms mediating neuronal dysfunction and cell loss in SCA12. The main hypothesis to be tested is that the expansion mutation leads to increased expression of the regulatory subunit of PP2A, resulting in altered PP2A activity that contributes to neuronal toxicity. Aim 1 is to delineate the clinical, MRI and neuropathologic features of SCA12. Neurological, neuropsychological and psychiatric evaluations will be performed on affected members of the first SCA12 kindred at two different times points to define the clinical features of SCA12 and their time course. These findings will be compared to changes seen on brain magnetic resonance images (MRIs). An autopsy brain from the proband in this first kindred will be examined for gross pathology and cytologic changes in specific brain regions. Aim 2 will determine the mechanism by which the CAG repeat expansion in SCA12 alters gene expression. We will test the hypothesis that the SCA12 CAG repeat is within the promoter region of the phosphatase subunit gene PPP2R2B, and the expansion of the repeat causes increased PPP2R2B expression. Bioinformatic and experimental approaches will be used to confirm location of the CAG repeat in a 5' region of PPP2R2B outside of an open reading frame. Promoter assays will reveal if the region containing the repeat is a functional promoter, and if repeat expansion increases promoter activity. If so, the properties of the repeat expansion necessary for increased promoter activity will be determined. Aim 3 will analyze neuronal injury in a cell culture model of SCA12 using over-expression of PPP2R2B in N2a neuroblastoma cells and primary neuronal cultures. This will test the hypothesis that over-expression of PPP2R2B is toxic to cells. Survival of cultured cells will be analyzed following over-expression of PPP2R2B under regulation of a CMV promoter, and then by over-expression of PPP2R2B under control of its own promoter. The relative toxicity of different levels of PPP2R2B expression will be tested by comparing loss of cells transfected with normal vs. expanded CAG repeats. A 2nd hypothesis is that the toxicity of over-expressed PPP2R2B is a consequence of altered protein phosphatase 2A function. To test function, we will assay enzyme activity, substrate specificity, and intracellular localization of PP2A following over-expression of PPP2R2B and determine the effect of PP2A modulators on cell survival. Investigation of SCA12 may provide insight into the mechanisms responsible for neuronal loss, and potential therapy, for this and other trinucleotide repeat diseases and SCAs, additional disorders due to mutations in gene promoters, and other conditions that result from dysfunctional regulation of phoshorylation.