Resilience to Endoplasmic Reticulum Stress Mitigates Calcium-Dependent Membrane Hyperexcitability Underlying Late Disease Onset in Spinocerebellar Ataxia Type 6

An enduring puzzle in many inherited neurological disorders is the late onset of symptoms despite expression of function-impairing mutant protein early in life. We examined the basis for onset of impairment in Spinocerebellar ataxia type 6 (SCA6), a polyglutamine ataxia with late-onset cerebellar neurodegeneration. In a mouse model of SCA6, we identified a homeostatic response that engages the unfolded protein response early in disease. This protective response provided insight into endoplasmic reticulum (ER) stress-mediated cerebellar Purkinje neuron membrane hyperexcitability as a driver of disease. Age-dependent impairment of chaperone-mediated compensation for ER stress increased calcium-dependent Purkinje neuron membrane excitability. Redundant pathways of the unfolded protein response mediate this resilience to ER stress. ER stress-related decompensation applies also to other late-onset human cerebellar ataxia. These studies elucidate a mechanism of resilience connecting aberrant proteostasis and calcium-dependent intrinsic membrane hyperexcitability to explain delayed disease onset more widely in age-dependent neurodegenerative disease. mRNA sequencing was performed on a NovaSeq instrument (Illumina, Inc.) with ~155 million reads and 150 bp paired-end reads. 5 cerebellum samples from each genotype from each age were used. Samples were prepared individually and pooled together to reduce batch effects. The samples were then demultiplexed into fastq files and statistics were collected using FastQC (Babraham Bioinformatics). The fastq files were trimmed using Trimmomatic (Bolger et al., 2014). 150 bp reads were aligned using STAR (Dobin et al., 2013), and the percent of unique reads were calculated. Raw read counts per gene per sample were calculated using HTSeq (Anders et al., 2015). Outliers were removed based on principal component analysis and hierarchical clustering. Differential gene expression analysis was performed using DESeq2 (Love et al., 2014).多种遗传性神经疾病中存在一个长期存在的未解之谜：尽管功能受损的突变蛋白在生命早期即已表达，但相关疾病症状却呈现晚发性特征。我们针对6型脊髓小脑共济失调（Spinocerebellar ataxia type 6, SCA6）——一种以晚发性小脑神经退变为特征的多聚谷氨酰胺共济失调（polyglutamine ataxia）——的发病机制开展了研究。 在SCA6的小鼠模型中，我们发现疾病早期即激活了一种涉及未折叠蛋白反应（unfolded protein response, UPR）的稳态代偿应答。这一保护性应答揭示，内质网应激（endoplasmic reticulum stress, ER stress）介导的小脑浦肯野神经元膜过度兴奋是疾病发生的驱动因素。年龄依赖性的、针对ER应激的分子伴侣介导代偿功能受损，会进一步增强钙依赖性浦肯野神经元膜兴奋性。未折叠蛋白反应的冗余通路介导了机体对ER应激的抵抗能力。ER应激相关的代偿失调同样可见于其他晚发性人类小脑共济失调。 本研究阐明了一种连接异常蛋白稳态与钙依赖性内在膜过度兴奋的抵抗机制，可更广泛地解释年龄依赖性神经退行性疾病中延迟发病的现象。 本研究使用Illumina公司的NovaSeq仪器开展mRNA测序，共获得约1.55亿条读段，采用150 bp双端测序模式。每个基因型、每个年龄组均取5份小脑样本。样本分别独立制备后进行混合，以降低批次效应。随后使用FastQC（Babraham生物信息学研究所）对样本进行解复用并采集统计数据，利用Trimmomatic（Bolger等，2014）对fastq格式文件进行序列修剪。使用STAR（Dobin等，2013）比对150 bp读段，并计算唯一读段占比。利用HTSeq（Anders等，2015）计算每个样本中每个基因的原始读段计数。基于主成分分析（principal component analysis）与层次聚类（hierarchical clustering）剔除异常样本。使用DESeq2（Love等，2014）完成差异基因表达分析（differential gene expression analysis）。