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 we found modulated in the HD monkeys, is also upregulated in HD patients . However, miR-451 does not appear to be disrupted in HD mice models, suggesting the involvement of this miRNA in disease progression may be restricted to primates. On the contrary, we found miR-128a downregulated in the HD monkeys and human patients, and there are published reports also showing its corresponding downregulation in at least 2 distinct HD mice models . Additionally, our human data for miR-128a corresponds with other reported datasets in HD patients . The miR-128a results, however, were not previously highlighted in the previous publications.
Although it is important to identify both primate-specific and conserved molecular mechanisms in disease, we focused on miR-128a in this study due to the compelling list of genes it is predicted to target in HD, including; SP1, HIP1 as well as HTT itself. Our in vitro luciferase reporter assays revealed the binding sites for miR-128a in HIP1, SP1, and HTT are all regulated by miR-128a compared to stringent site-specific mutant controls. Although we experimentally examined a subset of the predicted miR-128a gene targets, there are likely more genes which miR-128a regulates within the HD pathway. Furthermore, a few of the other 11 miRNAs we identified to be associated with HD in this study are also likely to regulate genes within the HD pathway. A future point of interest could examine whether a distinct group of miRNAs work together as a network to govern the HD genes. MiR-940, one of the 11 HD-associated miRNAs in the transgenic monkeys, is also predicted to target HTT. Correspondingly, although we examined miR-128a regulation of the SP1 transcription factor, other miRNAS identified in this study also bioinformatically bind to SP1 (such as miR-320, miR-133, and miR-181). Overall, this report points to a collective miRNA targeting of HTT or genes which regulate HTT (i.e. SP1, HIP1, etc.). Furthermore, several of the dysregulated miRNAs in the HD monkeys are also predicted to target the insulin like growth factor −1 gene (IGF1) or its receptor (IGF1-R), including; miR-128a, miR940, miR-320, and miR133. It has previously been shown that IGF1 signaling is linked to HD and other neurodegenerative diseases [31–33]. Future studies of these other miRNAs and their target genes, along with miR-128a, could help develop a more complete understanding of ncRNA regulation in HD.
miR-128a is neuronally-enriched [34, 35] and one of the most abundantly expressed miRNAs in human and mouse brain . A recent report indicated a principal role for miR-128a in motor activity and neuronal excitability, and its downregulation can provoke epileptic seizures . HD is partly characterized by deficits in motor activity, leading to significant impact on associated movements and possible development of seizures in patients [37, 38]. Tan et al. showed that the seizures in mice with deficient miR-128a expression can be alleviated with treatment of an anti-convulsant drug . Moreover, the authors showed that the ablation of miR-128a specifically in the dopamine 1 receptor-expressing neurons (D1-neurons) leads to juvenile hyperactivity and seizures , consistent with other reports implicating D1-neurons in the symptoms of HD .
Several studies have also linked miR-128a with tumor repression and apoptosis [40, 41]. Interestingly, apoptosis related signaling has consistently been implicated in HD pathogenesis [42–45]. One possibility is that the polyQ expansion in HTT of HD patients signals for apoptosis and neurodegenerative cascades through the regulation of miR-128a. Indeed, we found caspase-3, a component of the apoptosis pathway [44, 46, 47], was dysregulated in HD monkeys (Figure 2). It has also been reported that miR-128a is involved in the regulation of neuronal differentiation and survivability, including through the targeting of transcription factors and neurotrophins [48, 49]. Overexpressing miR-128a represses the levels of the neurotrophin-3 receptor (NTRK3) gene [48, 49] and the transcription factor E2F3A . Moreover, it has also been shown that miR-128a can target genes involved in neuronal differentiation and viability through the regulation of nonsense-mediated decay (NMD) [28, 51, 52]. NMD is an RNA surveillance pathway which can provoke the degradation of a subset of RNA transcripts. It has been suggested that increased expression of miR-128a can modulate the NMD pathway, resulting in elevated levels of important neuronal proteins [28, 51, 52].
Overall, our results suggest that miRNAs, and more specifically miR-128a, may play a pivotal role in HD pathogenesis. Epigenetic regulation of HD by miRNAs could provide a viable target for future therapeutics, particularly when one miRNA, such as miR-128a, regulates multiple genes within the HD signaling pathway.