Repression of alpha-synuclein expression and toxicity by microRNA-7

Abstract

alpha-Synuclein is a key protein in Parkinson's disease (PD) because it accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons. Intraneuronal levels of this protein appear critical in mediating its toxicity, because multiplication of its gene locus leads to autosomal dominant PD, and transgenic animal models overexpressing human alpha-synuclein manifest impaired function or decreased survival of dopaminergic neurons. Here, we show that microRNA-7 (miR-7), which is expressed mainly in neurons, represses alpha-synuclein protein levels through the 3'-untranslated region (UTR) of alpha-synuclein mRNA. Importantly, miR-7-induced down-regulation of alpha-synuclein protects cells against oxidative stress. Further, in the MPTP-induced neurotoxin model of PD in cultured cells and in mice, miR-7 expression decreases, possibly contributing to increased alpha-synuclein expression. These findings provide a mechanism by which alpha-synuclein levels are regulated in neurons, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for PD and other alpha-synucleinopathies.

Fig. 1.

miR-7 regulates endogenous α-Syn levels. (A) Schematic diagram of human α-Syn mRNA containing the predicted conserved target site of miR-7. The seed match is indicated with the box. (B) miR-7 reduces the level of endogenous α-Syn in HEK293T cells. Cells were transfected with miR-7 precursor at the indicated concentrations and harvested at 48 h after transfection. A representative Western blot using SYN-1 antibody is shown. (C) Mean levels of α-Syn protein in B calculated from 3 independent experiments. (D) Quantitative RT-PCR analysis of α-Syn and EGFR mRNA expression in HEK293T cells. Cells were transfected with 40 nM miR-7 precursor or scrambled sequence and harvested 24 h later. Both mRNA expressions are normalized to GAPDH mRNA and are shown as a ratio to control (scrambled miR-7 sequence)-transfected cells. Experiments were done in triplicates. Data are shown as means ± SD. *, P < 0.01 for difference between control and miR-7-treated samples.

Fig. 2.

miR-7 acts on the 3′-UTR of α-Syn mRNA. (A) Schematic diagrams of human α-Syn 3′-UTR luciferase constructs showing wild-type and mutant (α-Syn-3′-mUTR) miR-7 target sequences. (B) HEK293T cells were transfected with luciferase constructs shown in A and 40 nM miR-7 and harvested 24 h later. Luciferase activities normalized to β-galactosidase activity are shown as a percentage of control (scrambled miR-7 sequence). (C) miR-7 expressed from plasmid pri-miR-7-2 inhibits luciferase activity in HEK293T cells. Transfected cells were harvested 24 h later. *, P < 0.05; **, P < 0.01 for difference between control and miR-7-treated samples.

Fig. 3.

miR-7 inhibitor increases α-Syn protein level. (A) SH-SY5Y cells were transfected with the indicated concentrations of anti-miR-7 inhibitor (2′-O-methyl) and harvested 48 h later. A representative Western blot using SYN-1 antibody is shown. (B) Mean amount of α-Syn measured from 3 independent experiments was normalized to β-actin. (C) SH-SY5Y cells transfected with luciferase constructs and 50 nM 2′-O-methyl inhibitor of miR-7 and harvested 24 h later. Luciferase values normalized to β-galactosidase activity are shown as a percentage of control (scrambled miR-7 inhibitor). *, P < 0.05; **, P < 0.01 for difference between control and miR7 inhibitor-treated samples.

Fig. 4.

miR-7-mediated reduction of α-Syn level protects against cell death. (A) Schematic diagram of A53T mutant α-Syn construct without its 3′-UTR, with wild-type 3′-UTR, or miR-7 target site mutant 3′-UTR. (B) Mouse neuroblastoma NS20Y cells were transfected with constructs shown in A along with 50 nM miR-7 or control (scrambled miR sequence). pEGFP-C1 was used as internal control for normalization of transfection efficiency. A representative Western blot using LB509 antibody is shown. The relative levels of α-Syn normalized to EGFP expression were calculated based on band density using NIH Image J software with each scrambled miR transfected sample set to 1.0. (C) NS20Y cells were transfected with α-Syn (A53T) constructs in the presence of scrambled miR (control) or presence of miR-7. pSV-β-Gal plasmid was used as internal control for normalization of transfection efficiency. After transfection, cells were treated with 200 μM H₂O₂ for 16 h. Experiments were performed in triplicates and repeated independently 3 times. *, P < 0.05 for difference between control and miR-7-treated samples.

Fig. 5.

miR-7 expression in the mouse brain and in brain cell types. (A) Total RNA samples isolated from brain regions were used for qRT-PCR analysis of miR-7. Amounts are calculated relative to the level in substantia nigra. (B) Expression levels of miR-7 measured in primary neurons and astrocytes by qRT-PCR. (C) Mice were challenged with MPTP for 5 days, and 2 weeks later ventral midbrain tissue was subjected to qRT-PCR analysis of miR-7 normalized to U6 level. *, P < 0.05 for difference between saline and MPTP-treated mice.