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

1. 为了在小鼠中建立SCA6模型，我们在Cacna1a位点产生了三株携带正常、扩增或超扩增CAG重复束的敲入（KI）小鼠。通过对这些动物的分析，我们发现扩大的CAG重复序列本身并不影响通道的固有电生理特性，并且SCA6的发病机制显然与年龄依赖性过程相关，并伴有突变Cav2.1通道蛋白的积累。通过细胞模型、KI小脑和死后患者样本，我们发现CAG重复通道的扩增影响了CACNA1A外显子47位点的剪接位点选择，导致小脑浦肯野神经元中含有多聚谷氨酸的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。我们证实Cav2.1通道在sc6小脑的c端部分有更多的分裂。目前正在研究裂解片段在SCA6发病机制中的作用。通过表达含有扩展聚谷氨酰胺的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，BIPs是一系列细胞保护膜可渗透的五肽，可以显著保护细胞免受聚谷氨酰胺诱导的毒性。因此，Bax可能是多聚谷氨酰胺诱导毒性的关键介质，BIPs可能为开发多聚谷氨酰胺疾病的治疗方法提供新的策略。我们开发了一种新的RNA干扰策略来治疗主要遗传性神经退行性疾病。我们发现这种方法可以选择性地抑制细胞中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，该药物已被证明可增加小脑浦肯野细胞的兴奋性。这些患者在临床症状的几个方面都有显著改善，提示4-AP可能对治疗该疾病有用。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