Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice [Mouse RNA-seq]

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 (VEH), CTL-sRNA-PT (obtained from the putamen of non-affected individuals) or HD-sRNA-PT (obtained from the putamen of patients with HD) 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, including transcriptional alterations. To reveal the transcriptional changes produced by sCAG, we co-injected human sRNAs with a locked nucleic acid (LNA) modified anti-sense oligonucleotide complementary to sCAG (HD-sRNA-PT + LNA-CTG) or an analogous scrambled LNA (HD-sRNA-PT + LNA-SCB). We have performed mRNA-seq in the different experimental groups: Vehicle, CTL-sRNA-PT, HD-sRNA-PT, HD-sRNA-PT + LNA-CTG, and HD-sRNA-PT + LNA-SCB. Here we describe the procedure to obtain and sequence human sRNA dataset isolated from the different brain areas. Overall design: Human dissected putamen 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-PT (n=10-14) and HD-RNAs-PT (n=11-14). 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. Each sRNA pool was injected into the mouse striatum, using stainless steel bilateral cannulas (26-gauge; Bilaney Consultants Ltd) that were implanted at the following coordinates: anteroposterior (AP) +0.6 mm, mediolateral (ML) ±2 mm and dorsoventral (DV) -1 mm from bregma. Infusions were performed at 0.25 µl/min by using an infusion pump. Bilateral infusions of 2 µl of vehicle, CTL-sRNA-PT, HD-sRNA-PT, HD-sRNA-PT + LNA-SCB, HD-sRNA-PT + LNA-CTG, CTL-sRNA-PT + LNA-SCB, CTL-sRNA-PT + LNA-CTG (0.24 µg/µl) were carried out. Infusions were administered twice per week, for two weeks, and were preceded by a vehicle infusion. Infusion cannulas were left in place for 5 minutes after the injection to ensure a complete diffusion. Animals were sacrificed 48 hours after the last infusion and the cerebral hemispheres were processed for RNA extraction.