Aiming to explore the functional role of the deregulated miRNAs both in neuronal function and homeostasis, as well as in neuropathological states, pathway enrichment analyses were conducted with experimentally validated targets of the deregulated miRNAs identified in the DE analyses. Using validated targets exclusively obviously restricts the
84 number of potential miRNA-mRNA pairs offered by computational target prediction (usually based on prediction by nucleotide complementarity), but offers the highest level of evidence for enriched biological processes and excludes the bias of non-canonical miRNA pairing or imperfect seed match that would not lead to any relevant biological consequence to the cell or organism (Agarwal et al., 2015; Friedman et al., 2008; Garcia et al., 2011). The enrichment analyses were conducted for targets of up- and down-regulated miRNAs individually.
For up-regulated targets, the significantly enriched biological processes were characterized as neuron-related (32%), nucleus/chromosome related (30%) and cytoskeleton/cell growth-related (20%) (as the most representative GO categories). These findings indicate an important involvement of the up-regulated candidates in neuronal processes and also suggest that several of these miRNAs are likely originated in neurons, fitting well with the RNA samples of midbrain source analyzed here. Moreover, midbrain tissue biopsies contain a variety of neuronal and glial cell types (La Manno et al., 2016), which are likely accounting to the different categories annotated for the regulated miRNAs (depicted in figure 10). The top 5 most significant biological processes are related to regulation of cell proliferation, differentiation, cell cycle and neurogenesis, well in line with the results retrieved by the literature screening (Basak et al., 2016; Oh et al., 2018; Pieczora et al., 2017; Pons-Espinal et al., 2017; Qu et al., 2019). Remarkably, cell proliferation- and cell cycle-related proteins are known to play an important role also in the survival and maintenance of mature neurons (Herrup and Yang, 2007; Omais et al., 2018). Aberrant cell cycle was also reported to be intimately linked to apoptosis of post-mitotic neurons and neurodegeneration overall (Feddersen et al., 1992; Heintz, 1993). Interestingly, the regulation of targets related to cell proliferation and cell death seems to happen in both directions, being enriched for both up- and down-regulated miRNAs. That might indicate that the enrichment on the aforementioned categories might be related both to a response to the neurodegenerative processes occurring in the PD-affected brains, as well as reflect an impairment of mechanisms related to cell maintenance and survival leading to the neurodegeneration in PD. More to that, regulation of cell death and apoptotic process also figure among the enriched GO terms for the validated targets of up-regulated miRNAs,
85 providing further evidence for the involvement of these candidates in (or as a response to) the cell death occurring in PD midbrains (Kim et al., 2016; Li et al., 2013a; Xie et al., 2017).
On the other hand, for the target of PD down-regulated miRNAs, the majority of the enriched GO terms belong to nucleus/chromosome-related processes (26%), cytoskeleton/cell growth-related processes (24%) and vesicular-related processes (19%).
Interestingly, no enriched processes seem to be directly related to neuronal processes for the validates targets of PD down-regulated miRNAs. It’s important to account, though, that the number of down-regulated miRNAs in the PD condition is almost 3 times smaller than the up-regulated category, which consequently decreases the number of analyzed targets substantially. Some of the enriched biological processes seem to be related to mitochondrial mechanisms (7%), which also have fundamental implications in PD pathophysiology (Bose and Beal, 2016; Liu et al., 2017; Rocha et al., 2018). When analyzed individually, the biological processes enriched for the targets of down-regulated miRNAs are mostly related to the regulation of gene expression, transcription / RNA metabolism and regulation of biosynthetic processes, in line to what was observed for the results from the up-regulated miRNAs. Interestingly, GO terms related to apoptotic process and cellular response to stress figure among the enriched categories for the targets of down-regulated miRNAs, indicating that a possible insufficient miRNA regulation of stress- and apoptotic- related genes might be taking place for some of the identified candidates.
In general, for the functional annotation of miRNA targets, it is important to consider that RNA material from a diversity of cells populating the analyzed midbrains was used for the sequencing. Thus, the enriched biological processes annotated for miRNA-targets are reflecting cellular changes not only of neurons but also of glial and immune cells present at the analyzed tissue. This is especially important since a massive activation of the immune response was observed when analyzing the transcriptomics results for these samples (as discussed in section 4.4). As many of the most significant pathways enriched for targets of the deregulated miRNAs were related to cell proliferation and cell cycle and positive regulation of cellular biosynthetic processes, it is likely that several of the enriched pathways are related not only to the apoptosis of dopaminergic cells but also to an immune cell proliferation taking place in the PD-affected brains, for example(Hirsch and Hunot, 2009; Hunot and Hirsch, 2003; Kempuraj et al., 2016; McGeer et al., 1988a; Nazmi et al.,
86 2019; Rea et al., 2018; Viviani, 2004). To a certain extent, the aforementioned findings indicate a disturbance in the homeostatic state of the cell populations present in the analyzed midbrains and might reflect possible cellular responses to dopaminergic insults and the neurodegenerative events taking place at the PD-affected midbrains. The exact events happening at the transcriptomic and proteomic levels and detailed miRNA-mRNA interaction pairs were further explored in subsequent steps of this work.
4.4 Transcriptomic profiling documents dopaminergic depletion and indicates an