Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice [human-miRNA]

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, have injected vehicle, CTL-sRNA (obtained from non-affected individuals) or HD-sRNA (obtained from patients with HD) from different brain areas (putamen, PT; cortex, CTX and cerebellum, CB) into the striatum of wild type (WT) mice and demonstrate that sRNA produced in the putamen and cortex of HD patients are sufficient to induce HD pathology in vivo. We have performed small RNA (sRNA) sequencing of the human samples used for injection. Here we describe the procedure to obtain and sequence human sRNA isolated from the different brain areas. Overall design: Human dissected brain areas (putamen, cortex and cerebellum) of non-affected individuals and patients with Huntington's disease were placed in QIAzol solution (QIAGEN; 79306), followed by RNA extraction with the miRNeasy mini kit (QIAGEN; 217004) as indicated by the manufacturer. Determinations of RNA quality and quantity were made with a 2100 Bioanalyzer (Agilent Technologies) and an ND-1000 spectrophotometer (Thermo Fisher Scientific), respectively. All RNA samples showed an RNA integrity number (RIN) of 7 or higher. ). Equivalent amounts of total RNA were mixed to obtain a representative pool of CTL-RNAs (n=10-14) and HD-RNAs (n=11-14) from each brain area. Then, small RNA (sRNA) fractions were purified from total RNA with the RNA Clean & Concentrator-5 Kit (Zymo Research; R1015) according to the manufacturer's instructions.

亨廷顿病（Huntington's disease, HD）是一类因亨廷顿（huntingtin, HTT）基因编码区CAG三核苷酸重复扩增引发的神经退行性疾病，其核心病理标志为进行性运动改变与纹状体中等多棘神经元（striatal medium spiny neurons, MSNs）选择性丢失。既往多数研究聚焦于其编码蛋白产物的致病效应，但越来越多证据表明，突变HTT mRNA内的扩增CAG重复序列及其衍生的小CAG重复RNA（small CAG repeat RNAs, sCAG）参与了HD的病理生理过程。目前，蛋白毒性与RNA毒性各自对HD病理生理的具体贡献仍未明确，且HD中显著失调的其他类别小RNA（small RNAs, sRNA）的功能也尚未明晰。本研究将来自不同脑区（壳核（putamen, PT）、大脑皮层（cortex, CTX）与小脑（cerebellum, CB））的溶剂对照、对照小RNA（CTL-sRNA，取自健康个体）或HD相关小RNA（HD-sRNA，取自HD患者）注射至野生型（wild type, WT）小鼠的纹状体中，证实HD患者壳核与皮层来源的小RNA足以在体内诱导HD病理改变。本研究已对用于注射的人类样本开展了小RNA（sRNA）测序，下文将详述从不同脑区中分离获取人类小RNA并进行测序的实验流程。
实验整体设计：分别将健康个体与HD患者的解剖后脑区（壳核、大脑皮层及小脑）置于QIAzol裂解液（QIAGEN；货号79306）中，随后按照制造商说明书使用miRNeasy迷你试剂盒（QIAGEN；货号217004）完成RNA提取。分别采用2100生物分析仪（Agilent Technologies）与ND-1000分光光度计（赛默飞世尔科技）检测RNA的质量与浓度。所有RNA样本的RNA完整性指数（RNA integrity number, RIN）均≥7。取等量总RNA进行混合，以构建每个脑区中对照RNA池（CTL-RNAs，n=10~14）与HD相关RNA池（HD-RNAs，n=11~14）的代表性混合样本。随后按照制造商说明书，使用RNA Clean & Concentrator-5试剂盒（Zymo Research；货号R1015）从总RNA中纯化小RNA组分。