[Parkin deletion affects PINK1/Parkin-mediated mitochondrial autophagy to exacerbate neuroinflammation and accelerate progression of Parkinson's disease in mice]

Abstract

Objectives: To investigate the role of mitochondrial autophagy disorder caused by deletion of E3 ubiquitin ligase Parkin in neuroinflammation in a mouse model of MPTP-induced Parkinson's disease (PD).

Methods: Wild-type (WT) male C57BL/6 mice and Parkin^-/- mice were given intraperitoneal injections with MPTP or PBS for 5 consecutive days, and the changes in motor behaviors of the mice were observed using open field test. The effects of Parkin deletion on PD development and neuroinflammation were evaluated using immunofluorescence and Western blotting. The changes of the PINK 1/Parkin signaling pathway in the midbrain substantia nigra of the mice were examined to explore the molecular mechanism of Parkin-mediated regulation of mitochondrial autophagy and its effect on neuroinflammation in PD mice.

Results: Compared with their WT counterparts, the Parkin^-/- mice with MPTP injections exhibited significant impairment of motor function with decreased TH⁺ neurons, increased α-synuclein (α-syn) accumulation, and increased numbers of GFAP⁺ and I-ba1⁺ cells in the midbrain substantia nigra. Parkin deletion obviously affected PINK1/Parkin-mediated mitochondrial autophagy to result in significantly increased mtDNA and upregulated expressions of STING and NLRP3 inflammatosomes in the midbrain substantia nigra of MPTP-treated transgenic mice.

Conclusions: Parkin deletion causes mitochondrial autophagy disorder to accelerate PD progression and exacerbates neuroinflammation in mice by affecting the PINK1/Parkin signaling pathway, suggesting the important role of Parkin in early pathogenesis of PD.

Keywords: Parkin; Parkinson's disease; mitochondrial autophagy; neuroinflammation.

图1

WT及Parkin^-/-小鼠注射MPTP后运动行为测试 Fig.1 Behavioral test of wild-type (WT) and Parkin^-/- mice after MPTP injection. A: Flow chart of MPTP-PD modeling in the mice. B: Heat map of movement of the mice in open field tests. C: Motion trajectory graph of the mice. D, E: Statistics of total movement distance and the distance in the central area of the mice in open field test. **P<0.01, ***P<0.001.

图2

WT和Parkin^-/-小鼠MPTP诱导的帕金森病变 Fig.2 MPTP-induced Parkinson's disease in WT and Parkin^-/- mice. A: Immunofluorescence staining of dopaminergic neuron markers TH (red) and DAPI (blue). B: Protein expression level of TH in the midbrain substantia nigra. C: Analysis of TH protein expression. D: Immunofluorescence staining of α-syn (red) and DAPI (blue) in the mouse midbrain. **P<0.01, ***P<0.001.

图3

Parkin^-/-小鼠MPTP诱导神经炎症加剧 Fig.3 Parkin deletion increased neuroinflammation induced by MPTP in mice. A: Immunofluorescence staining of astrocyte markers GFAP (red) and DAPI (blue). B: Co-staining of microglial marker I-ba1 (red) and NLRP3 (green) inflammasomes. C: Analysis of GFAP⁺ cell count (%). D: Analysis of I-ba1⁺ cell count (%).**P<0.01, ***P<0.001, ****P<0.0001.

图4

Parkin调控线粒体自噬的分子机制 Fig.4 Molecular mechanism of Parkin-mediated regulation of mitochondrial autophagy in midbrain substantia nigra. A: Immunofluorescence assay showing abnormal mitochondrial autophagy (white arrows) in Parkin^-/- mice with MPTP injection. B: Western blotting for detecting protein changes in Parkin-mediated regulation of mitochondrial autophagy and neuroinflammation. C: Scanning densitometry for semi-quantitative analysis of the protein expression levels. *P<0.05, **P<0.01 vs WT-MPTP.