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