Novel knock-in mouse models of spinocerebellar ataxia type 12

项目总结 脊髓小脑性共济失调12型(SCA12)是一种罕见的由CAG/CTG引起的神经退行性疾病 PPP2R2B基因外显子7的扩增，PPP2R2B是一种编码蛋白磷酸酶2A调节单位的基因。SCA12是 以震颤、步态异常和精神症状为特征的。神经病理学上，单一的SCA12 已有的大脑显示大脑皮层和小脑明显萎缩，并有明显的 多个家系的浦肯野细胞丢失与MRI表现一致。PPP2R2B文本是另一种选择 至少有8个变异体，每个变异体都有不同的N-末端区域。外显子7的重复序列本身也包括在内 至少有三种剪接变体。在两个变种中，重复序列落入预测编码多丝氨酸的ORF中 (PolySer)区，从正常等位基因产生短的PolySer区，从等位基因产生长的PolySer区 扩展的等位基因。我们来自细胞系统的初步数据表明，即使在没有Aug起始密码子的情况下， 可以通过重复相关非Aug(RAN)翻译产生更长的PolySer，且具有更长的 PolySer，但不是短的PolySer，具有有毒特性。 通过CRISPR辅助的同源重组，我们成功地产生了人源化的SCA12 敲入(Ki-10或80)小鼠模型，其中小鼠外显子2被其人类对应的外显子7取代 有10或80个CAG三胞胎。我们假设SCA12Ki-80小鼠模型将概括一些特征 人类SCA12疾病；多氨基酸区，特别是长的PolySer，可能在 SCA12Ki-80，触发毒性从而导致SCA12的发病机制，以及重复扩增可能改变PPP2R2B 拼接和表达。在目标1中，我们将进行行为测试、神经病理学研究和神经成像 目的：研究SCA12Ki-80小鼠模型及其对照的SCA12Ki-10小鼠模型和WT。在AIM 2，我们将确定预测的含有PolySer的蛋白在SCA12中的表达和潜在的毒性 通过将PolySer的表达和聚集与小鼠表型的进展相关联， 行为异常和脑萎缩。通过RT-PCR检测PPP2R2B的剪接和表达 与Ki-10和WT相比，Ki-80细胞有较高的增殖活性。拟议的实验提供了以下几个初步步骤 确定SCA12的潜在治疗靶点，以及测试合理疗法的适当模型。

PROJECT SUMMARY Spinocerebellar ataxia type 12 (SCA12) is a rare neurodegenerative disease caused by a CAG/CTG expansion in exon 7 of PPP2R2B, a gene encoding regulatory units of the protein phosphatase 2A. SCA12 is characterized by tremor, gait abnormalities, and psychiatric syndromes. Neuropathologically, the single SCA12 brain that has become available revealed prominent atrophy of the cerebral cortex and cerebellum, with a noted loss of Purkinje cells consistent with MRI findings in multiple pedigrees. PPP2R2B transcript is alternatively spliced with at least eight variants, each with a different N-terminal region. The repeat in exon 7 is itself included in at least three splice variants. In two variants, the repeat falls into ORFs predicted to encode the poly serine (polySer) tracts, giving rise to short polySer tracts from the normal allele and long polySer tracts from the expanded allele. Our preliminary data from cell systems suggests that even in the absence of AUG start codons, long polySer may be produced by repeat associated non-AUG (RAN) translation, and that proteins with a long polySer, but not a short polySer, have toxic properties. By CRISPR-assisted homologous recombination, we have successfully generated a humanized SCA12 knock-in (KI-10 or 80) mouse model, in which the mouse exon 2 was replaced by its human counterpart exon 7 with 10 or 80 CAG triplets. We hypothesize that the SCA12 KI-80 mouse model will recapitulate some features of the human SCA12 disease; and that poly amino acid tracts, especially long polySer, may be expressed in SCA12 KI-80, triggering toxicity hence SCA12 pathogenesis, and that the repeat expansion may alter PPP2R2B splicing and expression. In Aim 1, we will perform behavioral testing, neuropathology studies and neuroimaging to characterize the novel SCA12 KI-80 mouse model, along with its control SCA12 KI-10 model and WT. In Aim 2, we will determine the expression and potential toxicity of the predicted polySer containing proteins in SCA12 KI mice by correlating the polySer expression and aggregation with the progression of the mouse phenotype, behavior abnormalities and brain atrophy. We will examine the splicing and expression of PPP2R2B by RT-PCR and qPCR in KI-80, compared with KI-10 and WT. The proposed experiments provide some of the first steps in determining potential therapeutic targets for SCA12, and the appropriate models for testing rational therapeutics.