We previously reported that brain-enriched miRNAs can differentiate healthy controls from genetic and idiopathic PD subjects [14]. Here, we report results of an equally large validation study conducted on twelve miRNAs that were significantly or marginally non-significantly altered in our original study. With the exception of two miRNAs, miR-139-5p and miR-433-3p, all other miRNAs displayed very similar expression patterns and differences between healthy control and PD subjects in both studies. Combining the data, it was revealed that miR-22-3p, miR-124-3p, miR-136-3p, miR-154-5p and miR-323a-3p are differentially expressed between healthy controls and PD subjects. Of these, miR-22-3p [20–22], miR-124-3p [23], and miR-136-3p [21, 24] have been identified as deregulated in other PD biomarker studies, further validating current findings and their replicability at different neurological centers.
There is considerable information regarding the biological role of some of these deregulated miRNAs. miR-22-3p targets GBA, which encodes for β-glucocerebrosidase, a lysosomal enzyme involved in sphingolipid degradation; mutations in GBA are the single largest risk factor for PD [25]. In addition, miR-22 is neuroprotective in multiple neurodegeneration and traumatic brain injury models, has regenerative capabilities, and is involved in several aspects of neuronal development including cell cycle length, polarization of migrating neurons, and long-term synaptic plasticity [26–28]. miR-124-3p is the most abundant neuronal miRNA in the nervous system, and is considered indispensable for neuronal fate determination, differentiation, and plasticity [29, 30]. It is anti-inflammatory and protects dopaminergic neurons from MPTP- and 6-OHDA -induced toxicity via multiple pathways [31]. Less is known about miR-136-3p, miR-154-5p, and miR-323a-3p. miR-136-3p expression is upregulated in synaptoneurosomes at preclinical stage of prion disease and its overexpression protects cells from neuroinflammation following ischemic insults [32, 33]. miR-154-5p is differentially regulated during morphine self-administration and is predicted to have an important role in dopaminergic neuron differentiation and mu-opioid receptor regulation [34]. miR-323a-3p is upregulated in mild cognitive impairment, and differentially regulated by 6-OHDA and ischemia/reperfusion injury [35, 36]. Importantly, we found that all five deregulated miRNAs are significantly increased in PD; the fact that most of these miRNAs appear to have strong neuroprotective properties at multiple settings, may suggest that they are regulated as a compensatory response to brain impairment.
Men have fifty percent higher incidence of PD [37] and, interestingly, we found that the expression of three brain-enriched miRNAs, miR-330-5p, miR-433-3p and miR-495-3p was significantly higher in male subjects. Considering that the ratio of males to women was different in our two studies, we think that this partly explains the inconsistency in miR-433-3p expression between them. Little is known of their biological roles. miR-330-5p targets mRNAs involved in activity-dependent synaptic plasticity in the hippocampus [38]. miR-433-3p targets follicle-stimulating hormone (FSH) expression in the anterior pituitary, and HIF1α levels in the brain during hypoxia inhibiting neuron proliferation and migration [39, 40]. miR-495-3p targets are enriched for addiction and pro-survival genes, including BDNF, CAMK2 and ARC [41]. Furthermore, miR-495-3p expression is upregulated following deep brain stimulation [42]. Collectively, it is documented that these three miRNAs have rather negative impact on neuronal processes affected in PD, however, further work is required to better delineate their roles, and if/how their higher expression in males affects vulnerability to PD.
Examining the chromosomal coordinates of the differentially-expressed brain miRNAs, we found that miR-136-3p, miR-154-5p, miR-323a-3p, miR-433-3p, and miR-495-3p are located on Chr14q32, suggesting that they are co-deregulated by transcription factors or methylation. This area is inherently unstable and a 1.1 Mb microdeletion (14q32.2.q32.3, coordinates Chr14: ~100,400,000 -101,500,000) has been reported in a number of patients displaying motor delay, hypotonia, and feeding problems [43, 44]. This genomic rearrangement is thought to be generated after the 500 bp expanded repeats flanking the deletion boundaries undergo either non-allelic homologous recombination (NAHR) or form secondary structures that interfere with normal DNA replication and chromosome condensation [43]. It should be noted that the particular location also hosts fifteen protein coding genes, some of which are imprinted [43, 44]. More recently, miRNAs of this cluster were found to be among the most longitudinally stable miRNAs, indicating that they are ideal biomarkers to monitor the progression pathophysiological states including PD, reiterating the importance of the current findings [45]. Moreover, we have used the TransmiR v2 experimental, manually curated, database to probe the TFs that regulate the expression of these deregulated brain miRNAs. Transcription binding sites for CREB1, CEBPB and MAZ were found in all miRNAs located at Chr14q32. In addition, a CREB1 site was found at miR-22 gene locus. CREB1 is essential for neuronal survival and axonal growth via both transcription of neurotrophins and neurotrophin-dependent CREB1-mediated transcription of pro-survival genes [46]. CREB1 is also required for adult neurogenesis, synaptic plasticity and memory formation [47, 48]. Similarly to CREB1, CEBPB has been implicated in the control of neuronal development and survival, including processes such as cell fate determination, synthesis and response to trophic factors, learning, and memory [49, 50]. MAZ on the other hand, has been linked to neural stem cell differentiation towards the glial cell lineage [51] and the induction of the NMDA receptor subunit type 1 (NR1) gene after neuronal differentiation [52]. Overall, these data indicate that epigenetic deregulation at Chr14q32 locus maybe responsible or contribute to neuronal miRNA differential expression in PD.
To explore the molecular pathways controlled by the deregulated miRNAs, in silico analysis of KEGG pathways and GO terms was performed. KEGG categories such as ‘Long-term depression’, ‘TGF-beta signaling pathway’, ‘FoxO signaling pathway’, ‘Estrogen signaling pathway’, ‘ErbB signaling pathway’, and ‘Neurotrophin signaling pathway’, that are implicated in PD- associated processes, particularly dopaminergic neuron development and survival, were over-represented [53–60]. ‘Cellular protein modification’, ‘Nucleic acid binding transcription factor activity’, ‘Cell death’, ‘Catabolic process’, were overrepresented among the biological processes affected. Cellular protein modifications such as phosphorylation, ubiquitination, truncation, acetylation, nitration and sumoylation of PD-linked proteins have emerged as important modulators of pathogenic mechanisms in PD [61, 62]. Transcription factor changes are also of particular interest, as they indicate that there is not only misexpression at the mRNA translation level by miRNA deregulation, but that there exists a second wave of en masse deregulation involving transcription-wide changes. Recently, we have integrated data across twenty-four PD biomarker studies and identified 25 miRNAs reported deregulated in at least two studies [13]. Five of the currently reported deregulated miRNAs are found in that list. Interestingly, following stringent bioinformatic analysis and different bioinformatic tools to the ones used in the current manuscript [TargetScan 7.2 for miRNA target prediction in conjunction with DAVID v6.8 for pathway analysis as opposed to DIANA mirPath v.3 used here], revealed that all top four GO categories controlled by the 25 deregulated miRNAs were likewise associated with transcription factors. Hence, it appears that transcription factor misexpression is an important component of miRNA deregulation in PD.