Subcellular localization and function analysis of PINK1 mitron in PD progression: Mitron modulates mitochondrial morphology to regulate neuronal death

Figures

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  Figure 1. Expressions and subcellular localization of miR-7019-3p in MPTP/MPP+-induced PD mouse neuron cell model. A, the location of miR-7019-3p gene within mouse Pink1 gene. B, expression levels of Pink1 and miR-7019 in main organs of mouse. C, MiR-7019-3p expression levels in mesencephalon of MPTP-induced PD mice model and control group. D, Pink1 mRNA and miR-7019-3p expression levels in MN9D cells and N2a cells. E, subcellular location of miR-7019-3p. Green probe: FISH staining of miR-7019-3p. Red probe: mitotracker staining of mitochondria. The scale bar represents 50 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydrodropyridine; MPP+, 1-methyl-4-phenylpyridiniumion; PD, Parkinson’s disease.

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  Figure 2. The function of miR-7019-3p in neuron cells. A, electron microscope images of mitochondrial in MN9D and N2a cells overexpressed with miR-7019-3p and control mimics. The scale bar represents 500 nm. B, confocal microscopy images of mitochondria, the number of point-like mitochondria increased, and the reticular structure of mitochondria was destroyed. The scale bar represents 2 μm. C, the activity of mitochondria complex I in MN9D and N2a cells overexpressed with miR-7019-3p is evaluated. D, detection of ATP content in MN9D cells overexpressed with miR-7019-3p. MiR-7019-3p expression level in N2a cells treated with MPP+. E, copy number fold change of the mitochondrial DNA. F, MTT experiment results of cells overexpressed and inhibition with miR-7019-3p. G, flow cytometry results to detect the apoptosis of cells transfected with miR-7019-3p mimics or inhibitors. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. MPP+, 1-methyl-4-phenylpyridiniumion; MTT, methyl thiazolyl tetrazolium.

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  Figure 3. Prediction and verification of miR-7019-3p′s target genes. A, expression level of miR-7019-3p–targeting genes in cells overexpressed with miR-7019-3p. B, expression level of OPA1 protein in MN9D cells. C, expression level of OPA1 protein in N2a cells. D, binding region of miR-7019-3p in mRNA 3′ UTR of opa1. Luciferase activity detection of opa1 WT and MUT groups. E, the mitochondria encoded RNA 12s rRNA, 16s rRNA, and polycistronic RNA were predicted as target genes of miR-7019-3p. Potential binding sites between miR-7019-3p and targeting RNAs above. F, expression level of 12s and 16s rRNAs and copy number of mitochondria DNA after treated with MPP+ in N2a cells. Pearson correlation coefficients between miR-7019-3p and 12s rRNA and 16s rRNA. G, after cotransfection of miR-7019-3p and pmirGLO plasmid carried with kinds of target mRNA groups. H, RIP experiment of mitochondrial components including Ago2 protein, to detect Ago2 binding miR-7019-3p and mitochondrial long RNAs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. Opa 1, optic atrophy 1. MPP+, 1-methyl-4-phenylpyridiniumion; RIP, RNA binding protein immunoprecipitation.

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  Figure 4. Prediction and verification of miR-7019-3p′s transcriptional regulatory protein. A, Sp1 mRNA expression level in N2a cells treated with MPP+. B, expression level of miR-7019-3p and pink1 in N2a cells overexpressed with sp1 protein. C, expression level of miR-7019-3p and pink1 mRNA in N2a cells treated with siRNA of sp1. D, expression level of Sp1 mRNA in N2a cells treated with siRNA of sp1 mRNA and different concentration of MPP+. E, expression level of miR-7019-3p in N2a cells treated with siRNA of sp1 mRNA and different concentration of MPP+. F, five kinds of chimeric plasmids based on PGL3 basic plasmid, which carried with different types of sheared binding region sequences of SP1 on upstream of miR-7019 encoded region. The activities of luciferase reporter gene product were detected among six kinds of PGL3 basic plasmids, to show the activated region of SP1. G and H, ChIP result to verify the binding of Sp1 protein and miR-7019-3p upstream DNA sequence. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001. ChIP, chromatin immunoprecipitation; MPP+, 1-methyl-4-phenylpyridiniumion.