Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153

Abstract

Genetic and biochemical studies have established a central role for alpha-synuclein accumulation in the pathogenesis of Parkinson disease. Here, two microRNAs, namely mir-7 and mir-153, have been identified to regulate alpha-synuclein levels post-transcriptionally. These microRNAs bind specifically to the 3'-untranslated region of alpha-synuclein and down-regulate its mRNA and protein levels, with their effect being additive. They are expressed predominantly in the brain with a pattern that mirrors synuclein expression in different tissues as well as during neuronal development, indicating that they play a tuning role in the amount of alpha-synuclein produced. Overexpression of mir-7 and mir-153 significantly reduces endogenous alpha-synuclein levels, whereas inhibition of mir-7 and mir-153 enhances translation of a luciferase construct bearing the alpha-synuclein 3'-untranslated region in primary neurons. These findings reveal a significant additional mechanism by which alpha-synuclein is regulated and point toward new therapeutic regimes for lowering endogenous alpha-synuclein levels in patients with familial or sporadic Parkinson disease.

FIGURE 1.

Diagram of the miRNA binding sites within the SNCA 3′-UTR. A, two conserved sites, one for mir-7 and another for mir-153, are found in the SNCA 3′-UTR. B, bioinformatics prediction of miRNA and murine SNCA transcript interactions. C, predicted hybridization of miRNAs (gray) and SNCA (black) transcript using the RNAhybrid algorithm (36). The minimum free energy required for the hybridization is indicated.

FIGURE 2.

Mir-7 and mir-153 directly reduce SNCA protein expression. A, nonneuronal cell line HEK293 expresses low levels of mir-7 and mir-153. Representative gel shows the products of RT-PCRs amplified with primers specific for mir-7 and mir-153 in postnatal day (P1) murine hippocampus and HEK293 cells transfected with scramble or mir-153/7 vectors. The amount of starting template for each condition was equilibrated relative to U6 RNA. B, luciferase activity assays demonstrated that mir-7 and mir-153 directly suppress SNCA expression by targeting the identified seed regions in the SNCA 3′-UTR. HEK293 cells were co-transfected with both the reporter gene (either wild-type or mutated) and miRNA expression vectors, and luciferase activity was measured 48 h later. For control 1 and control 1/2, the average value of three (scramble1, mir-181, mir-218) and two (mir-181/128, mir-218/377) miRNA constructs expressing miRNA predicted not to bind SNCA 3′-UTR were used, respectively. Data show the mean ± S.E. (error bars) from six independent transfections (***, p < 0.001). C and D, representative Western blot analysis of SNCA protein demonstrating that mir-7 and mir-153 reduce SNCA protein levels through interaction with the 3′-UTR. HEK293 cells were co-transfected with either a full-length (coding region plus 3′-UTR) or a shorter version (coding region alone) SNCA construct and miRNA expression vectors. Cells were harvested 48 h later, and 15 μg of whole cell lysate was loaded in each lane. GAPDH is used as an internal control for loading. Data show the mean ± S.E. (error bars) from four independent experiments (*, p < 0.05; **, p < 0.01). E, representative RT-PCR analysis of SNCA mRNA demonstrating that both mir-7 and mir-153 regulate SNCA mRNA levels. HEK293 cells were co-transfected with a full-length SNCA construct and miRNA expression vectors. Cells were harvested 48 h later, RNA was purified, and RT-PCR for SNCA mRNA was carried out. GAPDH and U6 were used as an internal control for loading. Data show the mean ± S.E. (error bars) from four independent experiments (*, p < 0.05; ***, p < 0.001).

FIGURE 3.

Mir-7 and mir-153 show similar distribution to SNCA mRNA and protein. A, representative gels of the PCR amplification products of mir-7, mir-153, and SNCA mRNA and Western blot analysis of SNCA levels. Different murine tissues of postnatal day 1 (P1) animals are shown. SCG, superior cervical ganglion; TG, trigeminal ganglion. B, midbrain of different ages. C, hippocampus of different ages. D, cortex of different ages. E, representative gels of the PCR amplification products of mir-7, mir-153, and SNCA mRNA in E17 cortical tissue, 3-day neuronal, and 15-day astrocytic cultures. The amount of starting template for each condition was normalized to U6 RNA and GAPDH mRNA or protein.

FIGURE 4.

Mir-153/7 reduces endogenous SNCA protein levels in neurons. A, representative images of transfected cortical neurons stained with SNCA antibody and DAPI. Merged images are shown in the fourth panel. Embryonic day 16 rat cortical neurons were lipofected with scramble1 or mir-7/153 at 5 days after plating by using Lipofectamine 2000. Immunocytochemistry was carried out 40 h after transfection. B, average decrease in SNCA protein levels. 80 neurons were analyzed per experiment. Data show the mean ± S.E. (error bars) from four independent experiments (***, p < 0.001). C, hippocampal neurons were infected with a lentivirus expressing GFP-mir-7/153 (multiplicity of infection, 10) at 4 days after plating. Neurons were harvested 4 days later, and 30 μg of whole cell lysate was loaded in each lane. GAPDH is used as an internal control for loading. D, freshly dissociated cortical neurons were transfected with SNCA luciferase vector and antisense 2′-O-methyl oligonucleotides of mir-7 and mir-153 and assayed 48 h later. Data show the mean ± S.E. (error bars) from four independent experiments (*, p < 0.05).