Spinocerebellar ataxia 12: Mechanisms of Neuronal Injury

脊髓小脑共济失调 12：神经元损伤的机制

• 批准号: 6774734
• 负责人: ELIZABETH O'HEARN
• 金额: $ 34.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6774734
• 关键词: Asians; India; brain; cerebellar ataxia /dyskinesia; clinical research; enzyme activity; family genetics; gene expression; gene mutation; genetic disorder; genetic promoter element; human subject; human tissue; laboratory rat; magnetic resonance imaging; molecular pathology; neural degeneration; neurons; neuropathology; nucleic acid structure; pathologic process; patient oriented research; phenotype; phosphomonoesterases; postmortem; psychological tests; tissue /cell culture

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.

描述（由申请人提供）：棘脑脑性共济失调类型12（SCA12）是作者最近描述的神经退行性疾病。 SCA12与编码PPP2R2B的PPP2R2B中的CAG重复扩张突变有关，作为蛋白质磷酸酶2a（PP2A）的调节亚基。目标是1）描述这种新疾病的临床表型，以及2）洞悉SCA12中介导神经元功能障碍和细胞损失的分子和细胞机制。要测试的主要假设是扩张突变导致PP2A调节亚基的表达增加，从而导致PP2A活性改变，这导致神经元毒性。目标1是描述SCA12的临床，MRI和神经病理特征。将对在两个不同时间的第一个sca12的受影响成员进行神经，神经心理学和精神病学评估，以定义SCA12的临床特征及其时间课程。这些发现将与在脑磁共振图像（MRI）上看到的变化进行比较。在第一个亲戚中，将检查来自概率的尸检大脑，以了解特定大脑区域的严重病理和细胞学变化。 AIM 2将确定SCA12中CAG重复扩展改变基因表达的机制。我们将测试以下假设：SCA12 CAG重复重复位于磷酸酶亚基基因PPP2R2B的启动子区域内，并且重复膨胀引起的膨胀增加了PPP2R2B的表达。生物信息学和实验方法将用于确认CAG在开放阅读框架外的PPP2R2B 5'区域中重复的位置。启动子测定将揭示包含重复的区域是否是功能启动子，并且是否重复扩展会增加启动子活性。如果是这样，将确定增加启动子活性所需的重复扩展的特性。 AIM 3将使用N2A神经母细胞瘤细胞和原发性神经元培养物中的PPP2R2B过度表达在SCA12的细胞培养模型中分析神经元损伤。这将检验以下假设：PPP2R2B的过表达对细胞有毒。在CMV启动子调节PPP2R2B之后，将分析培养细胞的存活，然后通过在其自身启动子控制下对PPP2R2B的过表达。不同水平的PPP2R2B表达的相对毒性将通过比较正常与扩展的CAG重复序列的细胞丢失来测试。第二个假设是，过表达PPP2R2B的毒性是蛋白质磷酸酶2a功能改变的结果。为了测试功能，我们将测定PPP2R2B过表达后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。对SCA12的研究可能会深入了解该机制，用于对这种和其他三核苷酸重复疾病和SCAS的神经元丧失和潜在疗法，这是由于基因启动子的突变以及其他phoshorytration的功能障碍调节所带来的其他疾病引起的其他疾病。