Seven studies demonstrated the impact of miR-7 in α-synuclein expression (Junn et al., 2009; Doxakis, 2010; Choi et al., 2014; Fragkouli and Doxakis, 2014; Latreille et al., 2014; Fan et al., 2015; Zhou et al., 2016). From these studies, a total of three confirmed the direct binding of miR-7 to α-synuclein 3′-UTR sequence using luciferase reporter assays in three different in vitro models (SH-SY5Y, HEK293T and primary neurons; Supplementary Table 2). The specificity of the binding site was confirmed by introducing mutations in the α-synuclein 3′-UTR sequence that blocked the effect of miR-7 in the luciferase activity assay. The predicted binding site of miR-7 within the α-synuclein gene required to repress its expression is located at bases 119–217 of the α-synuclein 3′-UTR (Junn et al., 2009; Doxakis, 2010).

The direct effect of miR-7 in α-synuclein expression was first reported by Junn et al. (2009). In particular, transfection with 40 nM of premiR-7-2 in HEK293T cells resulted in a reduction of α-synuclein expression both at protein and mRNA levels. On the other hand, treatment with miR-7 inhibitors significantly increased the levels of α-synuclein protein in SH-SY5Y cells. The direct impact of miR-7 in α-synuclein expression was reproduced by Doxakis (2010) using both HEK293T cells and murine primary neurons.

One of the studies was focused on the role of miR-7 in pancreatic β-cell function (Latreille et al., 2014) and generated a miR-7 conditional knockout mice using Cre/Lox system (miR7a2^fl/fl mice) which developed diabetes due to impaired insulin secretion and β cell differentiation. The direct impact of miR-7 in α-synuclein expression was confirmed in MIN6 cells and pancreatic islets obtained from miR7a2^fl/fl mice. In particular, adenovirus-miR7a-mediated overexpression in MIN6 cells resulted in a reduction of α-synuclein transcript levels, while exposure to miR-7a inhibitors increased α-synuclein mRNA and protein levels. In addition, α-synuclein levels were increased in miR7a2^fl/fl pancreatic islets. Interestingly, miR-7 played a role in insulin secretion by repressing the expression of α-synuclein which in turn modulated the granule fusion with the plasma membrane. These results are in line with the previous observation that α-synuclein, whose exact function still remains unknown, plays a role in neurotransmitter release via regulating the pool of vesicles available in the synaptic bouton and its fusion with the plasma membrane (Murphy et al., 2000; Cabin et al., 2002; Fernández-Chacón et al., 2004; Chandra et al., 2005; Larsen et al., 2006; Mazzulli et al., 2016).

The neuroprotective effect of miR-7 has been assessed under different conditions (Junn et al., 2009; Choi et al., 2014; Fragkouli and Doxakis, 2014; Fan et al., 2015). One of the studies investigated the protective effect of miR-7 in N20Y cells overexpressing mutant A53T α-synuclein challenged with hydrogen peroxide (H₂O₂). Notably, the presence of miR-7 reduced H₂O₂-induced cell death in A53T α-synuclein mutant cells (Junn et al., 2009). Additional protective effects of miR-7 against the MPTP-active metabolite 1-methyl-4-phenylpyridinium (MPP⁺) in vitro was investigated in two studies (Choi et al., 2014; Fragkouli and Doxakis, 2014). Both of them demonstrated that overexpression of miR-7 significantly increased cell viability after MPP⁺ treatment in SH-SY5Y cells, ReNcell VM cells and mouse primary neurons. One of the studies suggested that the protective effect of miR-7 against MPP⁺ is independent of α-synuclein repression, since knocking down α-synuclein in SH-SY5Y cells did not impact on miR-7-enhanced cell viability. This study rather suggested that miR-7 protected against MPP⁺-induced cell death by directly targeting the expression of RelA, a component of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) consequently relieving NF-κB suppression (Choi et al., 2014). On the other hand, Fragkouli and Doxakis (2014) suggested that miR-7 protects against MPP⁺-induced cell death by activating the mTOR pathway. Relevant to this context, SH-SY5Y cells treated with MPP⁺ and subchronic MPTP administration in mice resulted in a significant reduction of miR-7 expression in both models (Junn et al., 2009; Choi et al., 2014; Fragkouli and Doxakis, 2014). Two studies were focused on the protective effect of miR-7 against A53T mutant α-synuclein-induced toxicity (Fan et al., 2015; Zhou et al., 2016). Both studies concluded that miR-7 protects against PD-like degeneration by directly targeting nod-like receptor protein 3 (Nlrp3) expression and therefore modulating NLRP3 inflammasome activation. The protective effect of miR-7 in vivo was also assessed in the MPTP mice model (Zhou et al., 2016), in which the injection of miR-7 mimics into wild type mice treated with subacute MPTP dose rescued the loss of tyrosine hydroxylase (TH)-positive neuron number in the SN and dramatically inhibited Ionized calcium binding adaptor molecule 1 (Iba1) microglial activation via supressing NLRP3 inflammasome-mediated neuroinflammation. Further supporting the concept that miR-7 regulates α-synuclein expression in vivo, Song et al. (2012) reported that schizophrenia-like transgenic mice overexpressing heme oxygenas-1 (HO-1) protein in astrocytes exhibited decreased levels of miR-7 and increased α-synuclein levels in the SN/ventral tegmental area (VTA) at 48-weeks of age compare to control animals.

A total of four studies investigated the impact of miR-153 in α-synuclein expression and its protective effect (Doxakis, 2010; Kim et al., 2013; Fragkouli and Doxakis, 2014; Lim and Song, 2014). Two studies investigated the combined effects of both miR-7, miR-153 and the combination of miR-7/153 (Doxakis, 2010; Fragkouli and Doxakis, 2014). One study predicted the binding site of miR-153 within the α-synuclein gene in the 442–448 bases of the α-synuclein 3′-UTR (Doxakis, 2010). The specificity of the predicted binding site for miR-153 was confirmed in vitro (Doxakis, 2010; Kim et al., 2013; Lim and Song, 2014) using luciferase assays and introducing mutations in the α-synuclein 3′-UTR. The direct effect of miR-153 in α-synuclein expression has now been studied in HEK293T cells. Cotransfection with an α-synuclein plasmid containing the 3′-UTR and miR-153 significantly reduced α-synuclein levels both at protein and mRNA level (Doxakis, 2010). The protective effect of miR-153 was also studied in embryonic murine neurons treated with MPP⁺. As reported with miR-7, overexpression of miR-153 in primary cortical neurons attenuated MPP⁺-induced neurotoxicity by upregulating the mTOR pathway (Fragkouli and Doxakis, 2014).

One study demonstrated that miR-34b and miR-34c directly targeted α-synuclein expression (Kabaria et al., 2015). Computational algorithms were used to predict two miR-34b and one miR-34c binding sites in the 3′-UTR of α-synuclein mRNA (Kabaria et al., 2015): miR-34b binding site #1: located between bases 528–549; miR-34b binding site #2: between bases 732–754; and miR-34c binding site: between bases 1149–1171. These bindings sites were verified by cotransfecting SH-SY5Y cells with a plasmid construct expressing α-synuclein 3′-UTR with miR-34b or miR-34c. Interestingly, the introduction of a polymorphic variation (rs10024743) which lies within the target site 1 of miR-34b significantly decreased the impact of miR-34b in the luciferase activity. As a consequence of the direct binding between miR-34b/miR-34c and α-synuclein, overexpression of miR-34b or miR-34c in SH-SY5Y cells resulted in significant reduction in α-synuclein mRNA and protein levels. Interestingly, miR-34b and miR-34c did not repress β-synuclein, but rather increased its expression by up to 2.3-fold. Moreover, inhibition of miR-34b and miR-34c increased α-synuclein mRNA and protein level as well as the formation of α-synuclein-containing aggregates in DA neurons.

Only one study has demonstrated the direct impact of miR-214 in α-synuclein expression. Using luciferase assays in SH-SY5Y cells, miR-214 has been shown to directly target the α-synuclein 3′-UTR. In addition, miR-214 overexpression reduced α-synuclein expression both at mRNA and protein levels, while downregulation of miR-214 increased not only α-synuclein expression (mRNA and protein) but also the number of α-synuclein-aggregates in cells (Wang et al., 2015). This work also investigated whether the regulation of α-synuclein by miR-214 was the mechanism underlying the neuroprotective effect of Resveratrol. First, they showed that Resveratrol could ameliorate MPP⁺/MPTP-induced cell death both in vitro and in vivo. Interestingly, miR-214 inhibitors reversed the neuroprotective effect of resveratrol treatment in MPP⁺/MPTP models.

One study found that miR-1643 is a direct regulator of α-synuclein expression (Lim and Song, 2014). Luciferase assay in 293TF cells confirmed the direct binding of miR-1643 to α-synuclein 3′-UTR sequence.

α-Synuclein turnover predominantly involves chaperone-mediated autophagy (CMA). Therefore, alterations in CMA result in pathological α-synuclein accumulation. Four studies have investigated how miRNA regulation of CMA influences α-synuclein accumulation (Alvarez-Erviti et al., 2013; Li et al., 2014; Zhang and Cheng, 2014; Su et al., 2016). Firstly, Alvarez-Erviti et al. (2013) transfected SH-SY5Y cells overexpressing α-synuclein with seven miRNAs that directly bind and negatively regulate two key proteins involved in CMA: Lysosome-associated membrane protein 2 (Lamp2a, hsa-miR-21*; hsa-miR-224; hsa-miR-373*; and hsa-miR-379) and Heat shock protein 70 (Hsc70, hsa-miR-26b: hsa-miR-106a*; and hsa-miR-301b). In addition to the expected reduction in Lamp2a and Hsc70 gene expression, transfection with the seven candidate miRNAs significantly increased α-synuclein protein levels. Notably, only two of them (miR-106a* and miR-301b) caused a significant decrease in α-synuclein mRNA levels. Interestingly, miR-106a* was predicted to target the 3′-UTR of α-synuclein although the direct binding has not yet been confirmed.

The impact of miR-21 on Lamp2a and α-synuclein aggregation was confirmed by a second study using SH-SY5Y cells. Cells transfected with miR-21 mimics exhibited decreased levels of Lamp2a both at protein and mRNA levels, and increased α-synuclein only at the protein level. On the other hand, SH-SY5Y cells treated with miR-21 inhibitors displayed increased levels of Lamp2a (protein and mRNA) and decreased α-synuclein levels. This study also suggested that geniposide had a neuroprotective effect against MPP⁺/MPTP by inhibiting α-synuclein expression through the miR-21/Lamp2a axis (Su et al., 2016). In relation to Hsc70, two studies added miR-320 (Li et al., 2014) and miR-16-1(Zhang and Cheng, 2014) as direct regulators of Hsc70 expression, which negatively downregulated Hsc70 expression promoting α-synuclein aggregation in SH-SY5Y cells overexpressing α-synuclein, without affecting α-synuclein mRNA levels. Interestingly, miR-16 is a member of the miR-15/107 superfamily, a miRNA family highly dysregulated in Alzheimer’s disease (AD; Parsi et al., 2015). In this context, a preclinical study aimed to evaluate members of the superfamily miR-15/107 as potential drugs for AD, discovered that the brainstem of mice treated with a miR-16 mimic exhibited decreased α-synuclein protein levels (Parsi et al., 2015). This result was confirmed in HT22 cells, whereby overexpression of miR-16 downregulated α-synuclein protein levels (Parsi et al., 2015).

Another proteolytic pathway related with α-synuclein induced-toxicity is the autophagy-lysosomal pathway (ALP). The impact of ALP-associated miRNAs in α-synuclein expression was studied by Decressac et al. (2013) using rat midbrain overexpressing human wild-type α-synuclein. First, they demonstrated that α-synuclein toxicity is linked to impairment of the transcription factor EB (TFEB), a master regulator of the ALP controlled by mTOR signaling. In this context, AAV-mediated overexpression of miR-128 (which directly targeted TFEB) increased the formation of α-synuclein oligomers and the number of α-synuclein-positive axonal swellings, which resulted in α-synuclein-induced toxicity as revealed by a significant loss of nigral DA neurons, striatal innervation and DA levels, as well as development of motor deficits at 8 weeks after vector injection.

RhoA is a Rho family member that acts downstream of Rho-associated kinase (ROCK) and is a major regulator of the morphological events during apoptosis and neurite extension (Katoh et al., 1998; Shi and Wei, 2007). The fact that miR-133b was previously shown to promote neurite outgrowth and enhance neural function recovery after spinal cord injury and stroke by targeting RhoA (Liu et al., 2009; Yu et al., 2011; Xin et al., 2013), prompted Niu et al. (2016) to investigate the potential neuroprotective effect of miR-133b in the MPP⁺ model. In this scenario, Niu et al. (2016) reported that MPP⁺ treatment reduced miR-133b levels, increased RhoA expression and reduced neurite length in PC2 cells and rat DA neuron. Overexpression of miR-133b reversed the negative impact of MPP⁺ in neurite length and decreased RhoA protein level, although it had no impact on RhoA mRNA levels. Interestingly, ectopic expression of miR-133b in PC2 cells and primary neurons downregulated α-synuclein mRNA levels, both under baseline and MPP⁺ conditions. The authors attributed α-synuclein downregulation to miR-133 inhibition of RhoA, although this pathway has not been experimentally confirmed. Supporting this idea it has been previously reported that RhoA can directly modulate α-synuclein expression by activating the serum response element (SRF) transcription factor and GATA-2 transcription factor which regulates α-synuclein expression via occupancy at the intron-1 (Scherzer et al., 2008; Zhou et al., 2011).

The discovery that SNPs located within the miR-433 binding sites in FGF20 gene were associated with PD (van der Walt et al., 2004; Haghnejad et al., 2015) triggered two studies to investigate the potential impact of miR-433 in α-synuclein expression (Wang et al., 2008; Schmitt et al., 2012). Wang et al. (2008) first demonstrated that miR-433 directly targets the FGF20 mRNA transcript and negatively regulates FGF20 protein translation. They showed that when SH-SY5Y cells were treated with the miR-433-target FGF20, α-synuclein protein levels were significantly increased compared to control cells. The authors suggest that FGF20 might regulate α-synuclein expression via FGF-receptor 1 (FGFR1), as it was previously demonstrated for FGF2 (Ohmachi et al., 2003; Rideout et al., 2003). Supporting this hypothesis, miR-433 did not bind to the α-synuclein 3′-UTR as reported with luciferase assays in Neuro2A and SK-N-SH cells (Schmitt et al., 2012).

Considering the growing evidence that neuroinflammation plays a key role in the pathogenesis and progression of PD, Thome et al. (2016) investigated the impact of miR-155 expression, one of the key microRNA modulators of neuroinflammation, in the α-synuclein transgenic mice. Interestingly, adenovirus-mediated overexpression of α-synuclein (AAV2-Syn) enhanced the expression of miR-155 in the SN of α-synuclein-overexpressing mice compared to control (30% increment 2 weeks after transduction) and induced a 29.7 ± 6.6% loss of TH positive neurons in the SN 6 months after transduction. Reactive microgliosis markers Major Histocompatibility Complex Class II (MHCII) and CD68 were also increased in the AAV2-Syn transgenic mice. Interestingly, genetic deletion of miR-155 prevented the increments of MHCII and CD68 and markedly attenuated the TH positive neuronal loss in the SN of AAV2-syn transgenic mice. These results were confirmed in vitro using primary microglial murine cells. The authors first showed that microglial cells treated with fibrils of human wild-type α-synuclein exhibited increased levels of MHCII and inducible nitric oxide synthase (iNOS), while monomeric α-synuclein did not activate the inflammatory response. On the other hand, α-synuclein fibrils did not activate the inflammatory process in microglial cells derived from miR-155^−/− mice. However, miR-155 mimic treatment restored the inflammatory activity in miR-155^−/− microglial cells.