The Parkinson's disease gene product DJ-1 modulates miR-221 to promote neuronal survival against oxidative stress

Abstract

DJ-1 is a highly conserved protein that protects neurons against oxidative stress and whose loss of function mutations are linked to recessively inherited Parkinson's disease (PD). While a number of signaling pathways have been shown to be regulated by DJ-1, its role in controlling cell survival through non-coding RNAs remains poorly understood. Here, using a microarray screen, we found that knocking down DJ-1 in human neuroblastoma cells results in down-regulation of microRNA-221 (miR-221). This is one of the most abundant miRNAs in the human brain and promotes neurite outgrowth and neuronal differentiation. Yet the molecular mechanism linking miR-221 to genetic forms of PD has not been studied. Consistent with the microarray data, miR-221 expression is also decreased in DJ-1^-/- mouse brains. Re-introduction of wild-type DJ-1, but not its PD-linked pathogenic M26I mutant, restores miR-221 expression. Notably, over-expression of miR-221 is protective against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death, while inhibition of endogenous miR-221 sensitizes cells to this toxin. Additionally, miR-221 down-regulates the expression of several pro-apoptotic proteins at basal conditions and prevents oxidative stress-induced up-regulation of bcl-2-like protein 11 (BIM). Accordingly, miR-221 protects differentiated DJ-1 knock-down ReNcell VM human dopaminergic neuronal cells from MPP⁺-induced neurite retraction and cell death. DJ-1 is a known activator of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway and may modulate miR-221 levels in part through this pathway. We found that inhibiting ERK1/2 decreases miR-221 levels, whereas over-expressing ERK1 in DJ-1 knock-down cells increases miR-221 levels. These findings point to a new cytoprotective mechanism by which DJ-1 may increase miR-221 expression through the MAPK/ERK pathway, subsequently leading to repression of apoptotic molecules. The inability of a pathogenic DJ-1 mutant to modulate miR-221 further supports the relevance of this mechanism in neuronal health and its failure in DJ-1-linked PD.

Keywords: Autosomal recessive; DJ-1; Oxidative stress; PARK7; Parkinson's disease; miR-221; microRNA (miRNA).

Graphical abstract

Fig. 1

miR-221 expression is modulated by wild-type DJ-1 but not its pathogenic mutant. SH-SY5Y cells were transfected with either pooled DJ-1 siRNA (si-DJ-1) or non-targeting control siRNA (si-NT). (A) Immunoblotting shows that DJ-1 is knocked down effectively by pooled DJ-1 siRNA. (B) DJ-1 knock-down (KD) decreases mature miR-221 and (C) precursor pre-miR-221. (D) DJ-1 knockout mouse brains have lower levels of mature miR-221 compared to wild-type mice. (E) DJ-1 knockout mice lack DJ-1 expression. (F) SH-SY5Y cells were transfected with either si-NT or si-DJ-1 for 24 h, followed by transfection with either empty control vector, FLAG-tagged wild-type DJ-1, or FLAG-tagged pathogenic M26I mutant DJ-1. Immunoblotting shows expression of FLAG-tagged wild-type and mutant DJ-1 protein, and the downregulation of endogenous DJ-1 protein in DJ-1 KD cells. (G) Transfection of wild-type DJ-1 but not its pathogenic M26I mutant in DJ-1 KD cells rescued mature miR-221 expression and (H) precursor pre-mir-221 levels. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) relative to control. (B–D) analyzed using two-tailed student's t-test. (G, H) analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig. 2

miR-221 is cytoprotective under MPP⁺insult. (A) SH-SY5Y cells were transfected with either pre-miR negative control (pre-miR-NC) or pre-miR-221. Transfected cells were treated with indicated concentrations of MPP⁺ for 24 h before assessing cell survival using MTS assay. Over-expression of miR-221 resulted in significantly higher cell viability following MPP⁺ treatment. (B) Transfected cells showed robust increase in mature miR-221 levels and (C) down-regulation of known miR-221 mRNA target, fragile X mental retardation 1 transcript (FMR1). (D) SH-SY5Y cells were transfected with either anti-miR negative control (anti-miR-NC) or anti-miR-221 that binds to endogenous mature miR-221. Cell survival of transfected cells was assessed after MPP⁺ treatment at the indicated concentrations for 24 h, and showed that inhibition of endogenous miR-221 resulted in a significant decrease of cell viability. (E) Anti-miR-221 transfected cells showed a decrease in mature miR-221 levels and (F) up-regulation of FMR1. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. (B, C, E, F) analyzed using two-tailed student's t-test. (A, D) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig. 3

miR-221 Down-Regulates transcripts involved in apoptosis, including Pro-Apoptotic protein BIM, and protects DJ-1 knock-down cells from cell death. (A) Compared to cells transfected with negative control pre-miR (pre-miR-NC), SH-SY5Y cells transfected with pre-miR-221 showed a decrease of mRNA transcripts implicated in apoptosis, including BIM, BMF, BNIP3L, and FOXO3A. (B) All three isoforms of BIM protein (BIM_EL, BIM_L, BIM_S) were decreased by over-expression of miR-221 and (C) increased by the inhibition of miR-221 using anti-miR-221. (D) Pre-miR-221 transfected cells were treated with 250 uM H₂O₂ for 24 h. Compared to control, over-expression of miR-221 prevented the oxidative stress mediated induction of pro-apoptotic BIM transcript. (E) Cells were made to stably express non-targeting control (Ctrl) short hairpin RNA (shRNA) or pooled DJ-1 shRNA. Cells were then transduced with either lentiviral control miR (Ctrl miR) or miR-221 for 24 h. Stable DJ-1 KD decreases the levels of mature miR-221, while transduction with lentiviral miR-221 robustly increases its levels. (F) Cells were then treated with the indicated concentrations of MPP⁺ for 24 h, and cell death was assessed using LDH assay. Compared to control KD cells, stable DJ-1 KD leads to increased cell death, which is rescued when miR-221 is over-expressed. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (A, D, F) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test. (B, C) analyzed using two-tailed student's t-test. (E) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig. 4

miR-221 mitigates the loss of neuron morphology in terminally differentiated, human ReNcell VM dopaminergic neurons treated with MPP⁺. To investigate the role of miR-221 in terminally differentiated human neuronal cells, (A) neural progenitor cells (NPCs) derived from the ventral mesencephalon (ReNcell VM) were terminally differentiated to dopaminergic neurons using an established pre-aggregation protocol. (B) Differentiated ReNcell VM cells were transduced with lentivirus containing control (lenti-Ctrl miR) or miR-221 (lenti-miR-221). Cells transduced with lenti-miR-221 showed robust up-regulation of mature miR-221. (C) These cells were treated with 0.5 mM MPP⁺ or vehicle (DMEM) for 24 h. Differentiated ReNcell VM cells exhibited GFP expression from viral transduction, stained positively for the dopaminergic neuron marker tyrosine hydroxylase (TH). Following MPP⁺ exposure, cells exhibited loss of neurite morphology, which was mitigated by miR-221 over-expression (Scale bar: 100 µm). Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (****p ≤ 0.0001) relative to control. (B) analyzed using two-tailed student's t-test.

Fig. 5

miR-221 prevents MPP⁺-induced neurite retraction and cell loss in DJ-1 KD ReNcell VM dopaminergic neurons. ReNcell NPCs were made to stably express either control non-targeting shRNA or pooled DJ-1 shRNA, then differentiated into post-mitotic dopaminergic neurons. (A) Immunoblotting shows that DJ-1 is knocked down effectively by DJ-1 shRNA. (B) Quantification of (A). (C) As seen previously in SH-SY5Y cells, DJ-1 KD decreases miR-221 levels in differentiated ReNcell VM neurons. (D) Control KD (Ctrl KD) or DJ-1 KD cells were transduced with either lenti-Ctrl miR or lenti-miR-221 and exposed to 0.5 mM MPP⁺ or vehicle (DMEM) for 24 h. MPP⁺ exposure resulted in cell loss and neuritic retraction (Scale bar: 100 µm). (E) Measurements of neurite length indicated that miR-221 protects DJ-1 KD neurites from MPP⁺. (F) Compared to control KD cells, long neurites (≥ 80 µm, longer than 75th percentile of all neurites) were less preserved among DJ-1 KD cells treated with MPP⁺. This effect was reversed when DJ-1 KD cells were made to over-express miR-221. (G) MPP⁺ treatment significantly decreased the cell count in Ctrl miR transduced neurons, whereas miR-221 transduced neurons showed a smaller, non-significant decrease in cell count. For (E, G), n = 450 neurites per group, representative of length measurements taken from 15 microscopic fields per experimental group across three biological replicates. For (F), mean values of percent neurites ≥ 80 µm long were generated with bootstrap resampling, where each sample run contained n = 100 neurites per group. Resampling runs were repeated 500 times to generate confidence intervals for mean values of percent neurites ≥ 80 µm. For (G), Average cell count was assessed by counting DAPI-positive nuclei in these fields. For (B, C, E, G), data are presented as means ± S.E.M. For (F), data are presented as means ± Confidence Interval. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (B, C) analyzed using two-tailed student's t-test. (E, F, G) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig. 6

DJ-1 modulates miR-221 expression through the MAPK/ERK1/2 pathway. To investigate whether DJ-1 could modulate miR-221 levels through its effect on the MAPK/ERK pathway, (A) SH-SY5Y cells were treated with MEK1/2 inhibitor, U0126 (50 µM), or vehicle (DMSO) for 24 h. This led to a decrease in levels of active, phosphorylated ERK1/2 without impacting the levels of total ERK1/2 or DJ-1 protein. (B) The inhibition of ERK1/2 phosphorylation led to down-regulation of miR-221 levels. (C) DJ-1 KD cells were transfected with FLAG-tagged hERK1 or vector-only control for 48 h. hERK1 overexpression increased phosphorylation of ERK1 in DJ-1 KD cells (D) and partially rescued the decrease in miR-221 levels. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (**p ≤ 0.01, ****p ≤ 0.0001) relative to control. (B) analyzed using two-tailed student's t-test. (D) analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test. Veh = vehicle. Vec = vector-only control.