YOD1 regulates oxidative damage of dopamine neurons in Parkinson's disease by deubiquitinating PKM2

Abstract

Background: Parkinson's disease (PD) is a common neurodegenerative movement disorder, mainly characterized by the degeneration and loss of dopaminergic neurons in the substantia nigra. Oxidative stress is considered to be a key contributor to dopaminergic neuronal degeneration, triggering a series of downstream events such as mitochondrial dysfunction, neuroinflammation and misfolded protein aggregation, which ultimately exacerbate the development of PD. Deubiquitinating enzymes (DUBs) regulate oxidative stress, but their roles in PD remain unclear.

Methods: GEO database analysis and western blotting were used to analyze the expression of YOD1in PD patients and PD mouse models. Genetic knockout (KO) of YOD1 was performed to assess its effects in PD pathogenesis. The substance of YOD1 was measured via co-immunoprecipitation (Co-IP) coupled with LC-MS/MS analysis. Then the effect of YOD1-mediated motor deficits and oxidative damage were investigated using open field test, swimming test, pole test, immunofluorescence (IF) and cellular analyses.

Results: YOD1 was highly expressed in PD patients and 6-OHDA-induced PD model mice and mediated reactive oxygen species (ROS) production. YOD1 KO ameliorated motor impairments and oxidative stress in PD model mice. YOD1 directly bound PKM2 and reduces its ubiquitination level by removing the K63-linked ubiquitin chain of PKM2, thereby increasing the tetramer level and reducing the dimer level of PKM2. It then inhibited dimerized PKM2 entry into the nucleus and regulated Nrf2-mediated antioxidant responses, but YOD1 does not change the stability of PKM2 protein.

Conclusions: Our study identifies YOD1 as a oxidative-sensitive regulator of PD progression, operating via the YOD1-PKM2-Nrf2 axis. Targeting YOD1 may offer a novel therapeutic strategy for PD.

Key points: YOD1 is highly elevated in different PD model mice and patients with PD. YOD1 is a key regulator in oxidative stress and PD pathology. YOD1-deficient exhibit a protective effect on neuronal oxidative injury. YOD1 targets PKM2-Nrf2 axis in response to oxidative stress.

Keywords: PKM2; Parkinson's disease; YOD1; oxidative stress.

FIGURE 1

YOD1 is significantly elevated in PD animal models and patients with PD. (A, B) GEO database search showing relative YOD1 mRNA expression levels in the substantia nigra of PD patients and non‐PD patients. (C) Expression levels of YOD1 protein in the substantia nigra of C57BL/6 mice injected with 6‐OHDA (Model group) and saline (WT group) into the striatum. (D) Quantification analysis of YOD1 in C. (E) Immunofluorescence co‐staining shows YOD1 (red) expression in dopaminergic neurons (TH+), neurons (NeuN+), astrocytes (GFAP+) and microglia (IBA1+) (all green). (F) Expression level of YOD1 protein in PC12 cells after administration of gradient concentrations of 6‐OHDA. (G) Quantification analysis of YOD1 in G. (H) Expression of YOD1 in PC12 cells treated with different time of 6‐OHDA. (I) Quantification analysis of YOD1 in H. (J) ICC was used to detect the expression of YOD1 in PC12 cells treated with different concentration of 6‐OHDA. Data represent mean ± SEM; *p < .05; **p < .01; ***p < .001 versus control or 6‐OHDA‐treated control.

FIGURE 2

YOD1 knockdown attenuates 6‐OHDA‐induced oxidative stress and apoptosis in PC12 cells. (A) Determination of cell viability after administration of different concentrations of 6‐OHDA(25‐200 µM). (B) Western blot validation of YOD1 knockdown efficiency. (C) YOD1 knockdown improved cell viability induced by 6‐OHDA. (D) YOD1 knockdown reduced LDH levels in cell supernatants after 6‐OHDA stimulation. (E) JC‐1 staining demonstrating that YOD1 knockdown preserves mitochondrial membrane potential. (F) JC‐1 fluorescence quantitative analysis. (G) DCFH‐DA staining showing YOD1 deficiency decreases intracellular ROS accumulation. (H) ROS fluorescence quantitative analysis. (I) Annexin V‐FITC/PI flow cytometry revealing reduced apoptosis in YOD1‐deficient cells. (J) Quantitative analysis of flow cytometry. (K) Western blot analysis of apoptosis‐related proteins (Bax, Bcl‐2 and cleaved caspase‐3) in substantia nigra tissues. (L) Quantitative analysis of western blot results in K by image J. Data represent mean ± SEM of three independent experiments; *p < .05; **p < .01; ***p < .001 versus scrambled control or 6‐OHDA‐treated control.

FIGURE 3

YOD1 knockout significantly improves the motor ability and coordination ability of PD model mice. (A) Experimental timeline schematic. (B) Representative trajectory diagrams of mice in each group in the open field experiment. (C) The total distance travelled by mice in each group in the open field experiment. (D) The number of times mice in each group entered the central area in the open field experiment. (E) The average movement speed of mice in each group in the open field experiment. (F) Representative trajectory diagram of swimming experiment. (G) The total distance travelled by mice in each group during the swimming experiment. (H) The average movement speed of mice in each group during the swimming experiment. (I) Time spent in rod climbing experiment for mice in each group. Data are presented as mean ± SEM (n = 10/group); *p < .05; **p < .01; ***p < .001 versus wild‐type controls or Model group.

FIGURE 4

YOD1 deficiency protects against dopaminergic neuron degeneration and oxidative stress in PD model mice. (A) Representative immunofluorescence images of TH‐positive neurons in substantia nigra sections. (B) Quantification of TH+ neuron density (cells/mm²). (C−E) Oxidative stress markers in substantia nigra lysates: (C) glutathione (GSH) levels, (D) superoxide dismutase (SOD) activity, and (E) malondialdehyde (MDA) content. (F) Western blot analysis of apoptosis‐related proteins (Bax, Bcl‐2 and cleaved caspase‐3) in substantia nigra tissues. (G) Densitometric quantification of protein expression levels normalized to GAPDH in F. Data represent mean ± SEM; *p < .05; **p < .01; ***p < .001 versus wild‐type controls or Model group.

FIGURE 5

YOD1 knockdown ameliorates motor dysfunction and dopaminergic neuron degeneration in A53T α‐synuclein transgenic mice. (A) Schematic overview of the experimental timeline. (B−D) Motor coordination assessments showing: (B) latency to fall in the rotarod test, (C) turning time and (D) total duration in the pole test. (E−G) Locomotor activity analysis from open field testing: (E) representative movement trajectories, (F) total distance travelled and (G) average velocity. (H−J) Swim test performance: (H) representative swimming paths, (I) total swim distance and (J) average swim speed. (K) Representative immunofluorescence images of tyrosine hydroxylase (TH)‐positive neurons in substantia nigra sections. (L) Quantitative analysis of TH+ neuron density. (M) Dopamine (DA) levels measured in substantia nigra tissue lysates. Data are presented as mean ± SEM; *p < .05; **p < .01; ***p < .001 versus control or 6‐OHDA‐treated control.

FIGURE 6

YOD1 directly interacts with PKM2. (A) Experimental workflow for identifying YOD1‐interacting proteins by LC‐MS/MS. (B) MS/MS spectrum from PKM2 peptide. (C) Co‐IP analysis of YOD1 and PKM2 interaction in the substantia nigra of mice infused with or without 100 µM 6‐OHDA. The protein level of PKM2 was detected by western blot. (D) Co‐IP analysis of YOD1 and PKM2 interaction in PC12 cells induced with or without 100 µM 6‐OHDA. (E) Co‐IP analysis of YOD1 and PKM2 interaction in NIH3T3 cells. (F) Schematic diagram of YOD1 structure and design of three different truncated segment plasmids tagged with FLAG (UBX‐like, OTU, Znf‐C2H2). (G) Overexpression of YOD1 and PKM2 by transfer Flag‐YOD1 and His‐PKM2, Flag‐YOD1ΔUBX‐Like and His‐PKM2, Flag‐YOD1ΔOTU and His‐PKM2, Flag‐YOD1ΔZnf‐C2H2 and His‐PKM2 plasmids to NIH3T3 cells. Co‐IP analysis to detect the binding region of YOD1 to PKM2.

FIGURE 7

YOD1 regulates PKM2 ubiquitination by removing its K63 ubiquitin chain. (A) Co‐transfection of His‐PKM2, HA‐UB, Flag‐Yod1 or Flag‐CN overexpression plasmids into PC12 cells. PKM2 ubiquitination level was detected by western blot with His‐specific antibodies. (B) Co‐transfection of His‐PKM2, HA‐UB, Flag‐YOD1 or Flag‐CN overexpression plasmids into NIH3T3 cells. PKM2 ubiquitination level was detected by western blot with His‐specific antibodies. (C) Endogenous PKM2 ubiquitination analysis in YOD1 knockout versus wild‐type cells by co‐immunoprecipitation (Co‐IP)/western blot. (D) Co‐transfection of His‐PKM2, HA‐UB, HA‐K48, HA‐K63 and Flag‐YOD1 overexpression plasmids into NIH/3T3 cells. YOD1‐regulated PKM2 ubiquitination pattern was detected by western blot with His‐specific antibodies. (E) Functional validation using K63R ubiquitin mutant confirming the essential role of K63 linkage in YOD1‐mediated PKM2 deubiquitination.

FIGURE 8

YOD1 modulates PKM2 dimerization and nuclear translocation. (A) PC12 cells were transfected with YOD1 siRNA for 24 h and then stimulated with 100 µM 6‐OHDA for another 24 h. Western blot was used to detect the protein levels of YOD1, PKM2 and YOD1. (B) Comparative assessment of YOD1 and PKM2 protein levels in substantia nigra tissues from wild‐type (WT), 6‐OHDA model (Model), YOD1 knockout (YOD1KO) and YOD1KO+Model groups. (C) PC12 cells were transfected with YOD1 siRNA for 24 h and then stimulated with 100 µM 6‐OHDA for 24 h. Use ICC to detect the nuclear entry of PKM2. (D) PC12 cells were transfected with YOD1 siRNA and stimulated with 100 µM 6‐OHDA for 24 h. Western blot detected the protein expression of Nrf2 in nucleus. (E) RT‐qPCR detection of Nrf2, HO‐1, NQO1 mRNA levels in PC12 cells. YOD1 modulates PKM2 dimerization and nuclear translocation. Data represent mean ± SEM of three independent experiments; *p < .05; **p < .01 versus control siRNA group or 6‐OHDA‐treated control group.