Novel knock-in mouse models of spinocerebellar ataxia type 12

项目摘要 脊髓小脑性共济失调12型（SCA 12）是一种罕见的神经退行性疾病，由CAG/CTG引起 PPP 2 R2 B的外显子7中的扩增，PPP 2 R2 B是编码蛋白磷酸酶2A的调节单元的基因。SCA 12是 其特征在于震颤、步态异常和精神综合征。神经病理学上，单个SCA 12 大脑已经变得可用，显示大脑皮层和小脑明显萎缩， 在多个家系中，浦肯野细胞的丢失与MRI结果一致。PPP 2 R2 B转录物或者是 与至少八个变体剪接，每个变体具有不同的N-末端区域。外显子7中的重复序列本身包括 在至少三种剪接变体中。在两个变体中，重复福尔斯落入预测编码多聚丝氨酸的ORF中 （polySer）片段，产生来自正常等位基因的短polySer片段和来自正常等位基因的长polySer片段。 扩展等位基因我们从细胞系统获得的初步数据表明，即使在没有AUG起始密码子的情况下， 长的polySer可以通过重复相关的非AUG（RAN）翻译产生，并且具有长的polySer的蛋白质可以通过重复相关的非AUG（RAN）翻译产生。 聚丝氨酸，而不是短聚丝氨酸，具有毒性。 通过CRISPR辅助的同源重组，我们成功地产生了人源化的SCA 12。 敲入（KI-10或80）小鼠模型，其中小鼠外显子2被其人对应外显子7替换 有10到80个CAG三胞胎我们假设SCA 12 KI-80小鼠模型将重现一些特征， 多聚氨基酸片段，特别是长聚丝氨酸，可以在人SCA 12疾病中表达。 SCA 12 KI-80，触发毒性，因此SCA 12发病机制，重复扩增可能改变PPP 2 R2 B 剪接和表达。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 表征新型SCA 12 KI-80小鼠模型沿着对照SCA 12 KI-10模型和WT。在Aim中 2、我们将确定预测的含polySer蛋白在SCA 12中的表达和潜在的毒性 通过将polySer表达和聚集与小鼠表型的进展相关联， 行为异常和脑萎缩我们将通过RT-PCR检测PPP 2 R2 B的剪接和表达 KI-80与KI-10和WT的qPCR比较。拟议的实验提供了一些初步步骤， 确定SCA 12的潜在治疗靶点，以及用于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.