Deficiency of parkin causes neurodegeneration and accumulation of pathological α-synuclein in monkey models
Rui Han, Qi Wang, Xin Xiong, Xiusheng Chen, Zhuchi Tu, Bang Li, Fei Zhang, Chunyu Chen, Mingtian Pan, Ting Xu, Laiqiang Chen, Zhifu Wang, Yanting Liu, Dajian He, Xiangyu Guo, Feng He, Peng Wu, Peng Yin, Yunbo Liu, Xiaoxin Yan, Shihua Li, Xiao-Jiang Li, Weili Yang
Rui Han, Qi Wang, Xin Xiong, Xiusheng Chen, Zhuchi Tu, Bang Li, Fei Zhang, Chunyu Chen, Mingtian Pan, Ting Xu, Laiqiang Chen, Zhifu Wang, Yanting Liu, Dajian He, Xiangyu Guo, Feng He, Peng Wu, Peng Yin, Yunbo Liu, Xiaoxin Yan, Shihua Li, Xiao-Jiang Li, Weili Yang
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Research Article Aging Neuroscience

Deficiency of parkin causes neurodegeneration and accumulation of pathological α-synuclein in monkey models

Abstract

Parkinson’s disease (PD) is characterized by age-dependent neurodegeneration and the accumulation of toxic phosphorylated α-synuclein (pS129-α-syn). The mechanisms underlying these crucial pathological changes remain unclear. Mutations in parkin RBR E3 ubiquitin protein ligase (PARK2), the gene encoding parkin that is phosphorylated by PTEN-induced putative kinase 1 (PINK1) to participate in mitophagy, cause early onset PD. However, current parkin-KO mouse and pig models do not exhibit neurodegeneration. In the current study, we utilized CRISPR/Cas9 technology to establish parkin-deficient monkey models at different ages. We found that parkin deficiency leads to substantia nigra neurodegeneration in adult monkey brains and that parkin phosphorylation decreases with aging, primarily due to increased insolubility of parkin. Phosphorylated parkin is important for neuroprotection and the reduction of pS129-α-syn. Consistently, overexpression of WT parkin, but not a mutant form that cannot be phosphorylated by PINK1, reduced the accumulation of pS129-α-syn. These findings identify parkin phosphorylation as a key factor in PD pathogenesis and suggest it as a promising target for therapeutic interventions.

Authors

Rui Han, Qi Wang, Xin Xiong, Xiusheng Chen, Zhuchi Tu, Bang Li, Fei Zhang, Chunyu Chen, Mingtian Pan, Ting Xu, Laiqiang Chen, Zhifu Wang, Yanting Liu, Dajian He, Xiangyu Guo, Feng He, Peng Wu, Peng Yin, Yunbo Liu, Xiaoxin Yan, Shihua Li, Xiao-Jiang Li, Weili Yang

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Figure 4

The phosphorylation of parkin at S65 relies on PINK1 expression in the primates.

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The phosphorylation of parkin at S65 relies on PINK1 expression in the p...
(A) The structure schematic of human full-length PINK1 that contains a mitochondrial targeting sequence (MTS), a transmembrane domain (TM), and a kinase domain (KD), and a mutant PINK1 (M1A) that was generated by replacing methionine with alanine at amino acid position 1. (B) The plasmids of full-length PINK1 and PINK1 (M1A) were cotransfected with WT human parkin plasmid in 293 cells for 48 hours, and the cells were collected for Western blotting analysis. Two different anti-pS65-parkin (AF3500 and CST-36866S) antibodies were used to detect parkin phosphorylation. Western blotting results show that parkin phosphorylation at S65 occurred only in the presence of PINK1. (C) Phos-tag gel analysis also shows that PINK1 deficiency in the M6 monkey brain cortex reduces phosphorylation of pS65-parkin. (D) Knocking down PINK1 via siRNA in cultured monkey astrocytes reduced phosphorylation of parkin (pS65-parkin). (E) Brain stem (BS) lysates from a WT monkey at the age of 8 years were treated with alkaline phosphatase (1 U/μL) for 0.5 and 1 hour at 37°C to dephosphorylate endogenous proteins. Western blotting analysis revealed that dephosphorylation eliminated the immunoreactivity of pS65-parkin to the 2 antibodies (AF3500 and CST) specific to phosphorylated parkin. Phosphorylated S112-BAD served as a control. The PINK1 mutant (M6) monkey BS tissues were included to show that endogenous pS65-parkin (red arrows) is reduced by knocking down PINK1. The ratios of phosphorylated proteins to total proteins from 4 independent experiments are presented beneath the blot (n = 2 animals). (F) Western blotting analysis of parkin, PINK1, and pS65-parkin expression in different brain regions in monkey (3 years old) and human (68 years old) who had passed away due to cancer. The level of pS65-parkin (arrows) recognized by 2 antibodies is dependent on the level of PINK1, but not the total parkin. Hippo, hippocampus. (G) Reduced parkin and pS65-parkin in different brain regions of a 3-year-old parkin mutant monkey (Park-4) when compared with a 3-year-old WT monkey. However, there was no specific alteration of pS65-parkin signals in parkin KO mouse brain tissues. Three independent experiments were conducted. Vin, vinculin. (H) Representative images of immunostaining of pS65-parkin in different brain regions (cortex, SN, striatum) of an 8-year-old monkey also showing that pS65-parkin is more abundant in the SN compared with other regions of monkey brains. The adult brain regions (cortex, striatum, and SN) injected with AAV9 parkin gRNA/Cas9 (parkin KD) show reduced levels of pS65-parkin. Representative Western blotting results and immunostaining images are from at least 3 independent experiments of 2 biological replicates.