Elucidation of pathomechanism and development of treatment of spinocerebellar ataxia type 6

1.为了在小鼠中建立SCA6模型，我们建立了三个品系的KI小鼠，分别携带正常、扩增和超扩增的CAG重复序列。基于对这些动物的分析，我们发现扩展的CAG重复本身并不影响通道的内在电生理特性，并且SCA6的发病显然与伴随突变的Cav2.1通道蛋白的积聚的年龄相关的过程有关。利用细胞模型、Ki小脑和身体标本，我们发现CAG重复序列的扩张影响了CACNA1A47外显子的剪接位点选择，导致小脑浦肯野神经元中含多聚谷氨酰胺的Cav2.1通道的相对丰度增加。因此，SCA6突变不仅产生编码谷氨酰胺扩展的通道蛋白的转录本，而且可能通过调节剪接事件上调其表达。我们确认了…下的CAV2.1频道在SCA6小脑中，更多的OES在其C-末端部分被切割。目前正在研究裂解片段在SCA6发病机制中的作用。利用表达含有膨化聚谷氨酰胺的ataxin-3 C端片段的细胞模型，我们证明了BIPs(Bax抑制多肽)对Bax的抑制作用，这种抑制Bax的多肽是一系列细胞保护膜通透性的五肽，可显著保护细胞免受多聚谷氨酰胺的毒性。因此，Bax可能是聚谷氨酰胺毒性的关键介质，BIPs可能为开发治疗聚谷氨酰胺疾病的药物提供了新的策略。我们开发了一种新的RNA干扰策略，用于治疗以遗传为主的神经退行性疾病。我们发现用这种方法可以在细胞中选择性地抑制SCA6等位基因。我们用钾通道阻滞剂4-二氨基吡啶(4-AP)治疗SCA6患者，它已被证明能增加小脑浦肯野细胞的兴奋性。这些患者的临床症状在几个方面都有显著改善，表明4-AP可能对治疗该病有用。较少

1. In order to model SCA6 in mice, we generated three strains of knock-in (KI) mice carrying normal, expanded, or hyper-expanded CAG repeat tracts in Cacna1a locus. Based on the analysis of these animals, we found that the expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channel and the pathogenesis of SCA6 was apparently linked to an age-dependent process accompanied with accumulation of mutant Cav2.1 channel protein.2. Using cellular models, KI cerebella, and postmortem patients' samples, we revealed that expansion of the CAG repeat tract affected splice-site selection at CACNA1A exon 47 locus, leading to the increased relative abundance of polyglutamine-containing Cav2.1 channels in cerebellar Purkinje neurons. Thus, the SCA6 mutation not only gives rise to transcripts encoding glutamine-expanded channel proteins, but also upregulates their expression, possibly by regulating splicing events.3. We confirmed that the Cav2.1 channel underg … More oes cleavage at its C-terminal portion in SCA6 cerebella. The role of the cleaved fragment in SCA6 pathogenesis is now being examined.4. Using cellular models expressing C-terminal fragment of ataxin-3 containing expanded polyglutamine, we demonstrated that the inhibition of Bax by BIPs (Bax-inhibiting polypepdides), which were developed as a series of cytoprotective membrane -permeable pentapeprides, markedly protected cells from polyglutamine-induced toxicity. Thus, Bax may be a key mediator of polyglutamine-induced toxicity and BIPs may provide a new strategy for developing therapeutics for polyglutamine diseases.5. We developed a new RNA interference strategy for treating dominantly inherited neurodegenerative diseases. We found that SCA6 allele could be selectively suppressed with this method in cells.6. We treated SCA6 patients with a potassium channel blocker 4-diaminopyridine (4-AP), which has been shown to increase the excitability of cerebellar Purkinje cells. These patients showed significant improvement in several aspects of clinical symptoms, suggesting that 4-AP may be useful for treating the disease. Less