D with the mRNA targets for 5 of the 11 miRNAs. For 4 of the 5 miRNAs with significant target association to the HD canonical pathway, we confirmed their expression by qPCR in the monkey cortical tissues (miR940 did not have commercially available Taqman primers for qPCR) (Figure 1D). For the qPCR verification, we focused on the BLU-554 site monkeys (HD4, HD7, and HD8) that had corresponding pathology. Quantitation by qPCR revealed a significant downregulation of miR-128a in the brains of the HD monkeys (Figure 1D), consistent with our microarray results. Additionally, miR-128a was also downregulated in the brains of pre-symptomatic and post-symptomatic HD patients (Figure 1E) when compared to controls (p < 0.05).miR-128a regulates 3'-UTR activity of genes with known roles in HD canonical pathwayThe HD canonical signaling genes HIP-1, HTT, SP-1, and GRM5 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27741243 are all predicted gene targets of miR-128a. To determine whether miR-128a regulates the 3′-UTRs of these genes, luciferase reporter assays were developed and co-transfection assays were performed to examineKocerha et al. Molecular Brain 2014, 7:46 http://www.molecularbrain.com/content/7/1/Page 4 ofFigure 2 Expression of mHTT in monkey frontal cortex. A) Brain sections of HD monkey (rHD4, rHD7 and rHD8) and GFP monkey (C1 and C3) were immunostained with mEM48. Low magnification (upper panels, 40? and high magnification (lower panels, 100? shows that mHTT forms intranuclear inclusions (arrows) and neuropil aggregates (arrowheads). B) mEM48 immunoblot of frontal cortex showed high-molecular-mass mutant HTT aggregate (arrow) in the stacking gel. – tubulin was used as an internal control. C) Quantification of the mHTT aggregate in the stacking gel showed the relative level of mHTT aggregates in the frontal cortex. All HD monkeys have significantly higher level of mHTT aggregate compared to the controls. D) The percentage of mEM48 positive cells with or without intranuclear inclusions was calculated and compared among HD monkey frontal cortex immunostained with mEM48. HD7 has significantly more nuclei with intranuclear inclusions than HD4 and HD8. The data was presented as mean ?SE. *P < 0.05.the effect of miR-128a mimic on 3'-UTR activation. Transfection of miR-128a mimic significantly reduced the 3'-UTR activation of the WT constructs for HIP-1, HTT, SP-1, and GRM5 in the luciferase reporter assays (Figure 4). Suppression of HIP-1, HTT, and SP-1 by miR-128a mimic (55 , 36 , and 30 respectively) was highly significant compared to the negative control (NC) (p < 0.0001 by One-way ANOVA followed by Tukey's post-hoc multiple comparison). GRM5 3' UTR was suppressed by the miR-128a mimic with a significance of p < 0.001. None of the site-specific mutant (MUT) control reporter constructs for the 4 genes examined were significantly regulated by the miR-128a mimic.Discussion In this study, we examine ncRNA regulation in HD monkeys and identified 11 significant disease-associated miRNAs. This is the first study which analyzes miRNA regulation in a transgenic primate model of a human disease, with a goal of helping to bridge or expand on previous results in HD rodents and human patients. The HD monkeys offer a unique resource to identify pathogenic ncRNA mechanisms that are either conserved from lower vertebrates to humans or are primate specific. For example, miR-451, one of the 11 miRNAs wefound modulated in the HD monkeys, is also upregulated in HD patients [24]. However, miR-451 does not appear to be disrupted.