Midbrain dopamine oxidation links ubiquitination of glutathione peroxidase 4 to ferroptosis of dopaminergic neurons
Jie Sun, … , Li Zhang, Rong-Rong He
Jie Sun, … , Li Zhang, Rong-Rong He
Published May 15, 2023
Citation Information: J Clin Invest. 2023;133(10):e165228. https://doi.org/10.1172/JCI165228.
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Midbrain dopamine oxidation links ubiquitination of glutathione peroxidase 4 to ferroptosis of dopaminergic neurons

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the gradual loss of midbrain dopaminergic neurons in association with aggregation of α-synuclein. Oxidative damage has been widely implicated in this disease, though the mechanisms involved remain elusive. Here, we demonstrated that preferential accumulation of peroxidized phospholipids and loss of the antioxidant enzyme glutathione peroxidase 4 (GPX4) were responsible for vulnerability of midbrain dopaminergic neurons and progressive motor dysfunctions in a mouse model of PD. We also established a mechanism wherein iron-induced dopamine oxidation modified GPX4, thereby rendering it amenable to degradation via the ubiquitin-proteasome pathway. In conclusion, this study unraveled what we believe to be a novel pathway for dopaminergic neuron degeneration during PD pathogenesis, driven by dopamine-induced loss of antioxidant GPX4 activity.

Authors

Jie Sun, Xiao-Min Lin, Dan-Hua Lu, Meng Wang, Kun Li, Sheng-Rong Li, Zheng-Qiu Li, Cheng-Jun Zhu, Zhi-Min Zhang, Chang-Yu Yan, Ming-Hai Pan, Hai-Biao Gong, Jing-Cheng Feng, Yun-Feng Cao, Feng Huang, Wan-Yang Sun, Hiroshi Kurihara, Yi-Fang Li, Wen-Jun Duan, Gen-Long Jiao, Li Zhang, Rong-Rong He

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Figure 4

DAQ and α-synuclein contribute to NEDD4-mediated GPX4 ubiquitin proteasome degradation.

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DAQ and α-synuclein contribute to NEDD4-mediated GPX4 ubiquitin proteaso...
(A) Binding of DAQ to recombinant GPX4 protein was quantitated by BLI. (B) The interaction between DAQ (150 μM) and GPX4 in PC12 cells transfected with GPX4-expressing plasmid was detected by pull-down assay. (C) Proteomic analysis of target proteins pulled down with a DAQ probe and whole cell lysates from PC12 cells. Volcano plots showed the fold changes of DAQ versus control with significance calculated by t test. (D) LC-MS/MS analysis identified GPX4-Cys102 as the binding site for DAQ. (E) GPX4 degradation was assessed by cycloheximide (CHX, 100 μM) chase at indicated time points (left) and the degradation curve was plotted (right) using PC12 cells (n = 3 by 2-way repeated measures ANOVA). (F) Western blotting showed that MG132 (10 μM) reversed DAQ-induced GPX4 degradation compared with 3-methyladenine (3-MA, 10 mM) in PC12 cells. (G) Ubiquitination of GPX4 was determined by coIP using PC12 cells transfected with FLAG-tagged GPX4 plasmid. (H) Diagram illustrating the experimental design for identifying GPX4-binding proteins by coIP-MS. (I) Ubiquitination of GPX4 and the expression of NEDD4 were examined by coIP using HEK293 cells transfected with plasmids of FLAG-tagged GPX4 and MYC-tagged ubiquitin. (J and K) The role of NEDD4 in GPX4 degradation was verified in HEK293 cells transfected with NEDD4 siRNA and plasmids of overexpression (HA-tagged) or mutation (C1286S). Enzymatic activities of GPX4 in PC12 cells (L) or A53T mice (M) as analyzed by LC-MS/MS. All data represent mean ± SEM (for cells, n = 3, for mice, n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group, ##P < 0.01 versus the DAQ-treated cells, by 1-way ANOVA with Bonferroni’s posthoc test (for F), or independent-samples t tests (for C, H, L, and M).