OTUD5 Protects Dopaminergic Neurons by Promoting the Degradation of α-Synuclein in Parkinson's Disease Model

Abstract

Defective clearance and accumulation of α-synuclein (α-Syn) is the key pathogenic factor in Parkinson's disease (PD). Recent studies emphasize the importance of E3 ligases in regulating the degradation of α-Syn. However, the molecular mechanisms by which deubiquitinases regulate α-Syn degradation are scarcely studied. In this study, it is found that the protein levels of α-Syn are negatively regulated by ovarian tumor protease deubiquitinase 5 (OTUD5) which protects dopaminergic (DA) neurons in the PD model. Mechanistically, OTUD5 promotes K63-linked polyubiquitination of α-Syn independent of its deubiquitinating enzyme activity and mediates its endolysosomal degradation by recruiting the E3 ligase neural precursor cell expressed developmentally downregulated 4 (NEDD4). Furthermore, OTUD5 conditional knockout in DA neurons results in more severe α-Syn related pathology and dyskinesia after injection of α-Syn preformed fibrils (PFF). Overall, the data unveil a novel mechanism to regulate the degradation of α-Syn and provide a new therapeutic strategy to alleviate DA neurodegeneration.

Keywords: deubiquitinase; endolysosomal pathway; nedd4; otud5; parkinson's disease; α‐synuclein.

Figure 1

OTUD5 facilitated the degradation of a‐Syn. A) Gradient amount of Flag‐OTUD5 was transfected in SH‐SY5Y cells, the protein levels of α‐Syn were detected by western blot. Quantification of α‐Syn protein levels in SH‐SY5Y cells was normalized to GAPDH (n = 3 biologically independent). (B) RT‐PCR analysis of SNCA mRNA levels in SH‐SY5Y cells transfected with plasmid expressing OTUD5. Quantification of SNCA mRNA levels in SH‐SY5Y cells was normalized to β‐actin (n = 3 biologically independent). (C) Immunoblot analysis of α‐Syn protein level in SH‐SY5Y cells transfected with siRNA target for OTUD5. Quantification of α‐Syn protein levels in SH‐SY5Y cells was normalized to GAPDH (n = 5 biologically independent). (D) RT‐PCR analysis of SNCA mRNA levels in SH‐SY5Y cells transfected with siRNA targeting OTUD5. Quantification of SNCA mRNA levels in SH‐SY5Y cells was normalized to β‐actin (n = 3 biologically independent). (E) Immunoblot analysis and quantification of lysates from primary cultured midbrain neurons of WT or OTUD5 CKO mice. Quantification of α‐Syn protein levels was normalized to GAPDH (n = 6 biologically independent). (F) Immunofluorescence staining for α‐Syn (green) was performed in SN regions of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice. Quantification of fluorescence intensity of α‐Syn was performed by ImageJ software (n = 6 mice per group). Scale bar, 20 µm. (G) Immunoblot analysis of extracts from SH‐SY5Y cells overexpressed OTUD5 treated with cycloheximide (CHX, 100 µg mL⁻¹) (n = 3 biologically independent). The interaction between Flag‐OTUD5 and CHX treated time was statistically significant (P = 0.018), which was analyzed by repeated measure ANOVA. (H) Immunoblot analysis of extracts from SH‐SY5Y cells transfected with OTUD5 sgRNAs or scrambled sgRNA (sg‐Ctrl). (I) Immunoblot analysis of extracts from OTUD5 knockout SH‐SY5Y cells treated with CHX (n = 3 biologically independent). The interaction of sg‐OTUD5 and CHX treated time was statistically significant (P = 0.049), which was analyzed by repeated measure ANOVA. Data were expressed as the mean ± SEM. One‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (A). Two‐tailed Student's t‐tests were used for statistical analysis in (B–F). Two‐way ANOVA followed by Tukey's post hoc tests were used for statistical analysis in (G) and (I). ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

OTUD5 promoted endolysosomal degradation of α‐Syn. (A) Immunoblot analysis of extracts from SH‐SY5Y cells transfected with Flag‐OTUD5 then treated with MG132 (10 µM), chloroquine (CQ, 10 µM) or 3‐MA (10 mM) for 6 h. Quantification of α‐Syn levels in SH‐SY5Y cells was normalized to GAPDH (n = 3 biologically independent). (B) Representative confocal images of immunofluorescence staining for α‐Syn (red) and lysosome marker LAMP1 (green) in SH‐SY5Y cells transfected with the plasmids expressing Flag‐OTUD5 and followed by CQ treatment for 6 h. Scale bar, 10 µm. (C) Western blots were performed in lysates of ATG5 knockout (sg‐ATG5) cells and its control (sg‐Ctrl) cells transfected with Flag‐OTUD5. Quantification of α‐Syn protein levels was shown on the right (n = 3 biologically independent). The interaction of sg‐ATG5 and Flag‐OTUD5 was not statistically significant (P = 0.716), which was tested by 2*2 factorial analysis. (D) Confocal microscopic analysis of the co‐localization of endogenous α‐Syn (red) and LC3 (green). Scale bar, 10 µm. (E) Confocal microscopic analysis of the co‐localization of endogenous α‐Syn (red) and Rab7 (green). Scale bar, 10 µm. Co‐localization was quantified by using Pearson's correlation coefficient method and scatter map from ImageJ software in (B), (D), and (E). Data were expressed as the mean ± SEM. One‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (A). Two‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (C). ns, not significant (p > 0.05), *p < 0.05, **p < 0.01.

Figure 3

OTUD5 interacted with α‐Syn and promoted its K63‐linked ubiquitination. (A) Co‐immunoprecipitation (Co‐IP) was performed with lysates from HEK293T cells expressing His‐α‐Syn and Flag‐OTUD5. IP with anti‐His antibody probed with anti‐Flag antibody. (B) Co‐IP was performed with lysates from HEK293T cells expressing His‐α‐Syn and Flag‐OTUD5. IP with anti‐Flag antibody, probed with anti‐His antibody. (C) Confocal microscopy of HEK293T cells transfected with plasmids expressing Flag‐OTUD5 (green) and His‐α‐Syn (red). Scale bar, 10 µm. (D) Co‐IP was performed to analyze the endogenous OTUD5 and α‐Syn in SH‐SY5Y cells treated with α‐Syn PFF. (E) Confocal analysis of the co‐localization of endogenous α‐Syn (red) and OTUD5 (green) in SH‐SY5Y cells stimulated by α‐Syn PFF. Scale bar, 10 µm. (F) SH‐SY5Y cells transfected with Flag‐OTUD5 (WT) or Flag‐OTUD5 (C224S), α‐Syn protein level was detected by immunoblotting. Quantification of α‐Syn levels in SH‐SY5Y cells was normalized to GAPDH (n = 6 biologically independent). (G) Immunoblot analysis of lysates from HEK293T cells transfected with HA‐tagged ubiquitin (HA‐Ub), His‐α‐Syn, and Flag‐OTUD5 (WT) or Flag‐OTUD5 (C224S), followed by 6 h CQ (10 µM) treatment. IP with anti‐His antibody, probed with anti‐HA antibody. (H) Immunoblot analysis of lysates from HEK293T or OTUD5 knockout HEK293T cells transfected with HA‐tagged ubiquitin (HA‐Ub), His‐α‐Syn, followed by IP with anti‐His, probed with anti‐HA. (I) HEK293T cells were transfected with HA‐Ub, HA‐tagged K48‐linked ubiquitin (HA‐K48) or HA‐tagged K63‐linked ubiquitin (HA‐K63), His‐α‐Syn and Flag‐OTUD5 followed by 6 h CQ (10 µM) treatment. Cells were harvested, denatured, and lysed for immunoprecipitation with anti‐His antibody. The ubiquitination level of His‐α‐Syn was assessed by immunoblotting with anti‐HA antibody. (J) Co‐IP and immunoblot analysis of lysates from SN of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice. IP with anti‐α‐Syn antibody, probed with anti‐K63‐Ub antibody. Co‐localization was quantified by using Pearson's correlation coefficient method. Intensity profiles of indicated proteins along the plotted lines were analyzed by ImageJ line scan analysis in (C) and (E). Data were expressed as the mean ± SEM. One‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (F). ns, not significant (p > 0.05), **p < 0.01.

Figure 4

OTUD5 recruited E3 ligase NEDD4 to promote the degradation of α‐Syn. (A) SH‐SY5Y cells were transfected with siRNA for NEDD4 or negative control (NC) and Flag‐OTUD5 or the empty Flag‐vector (EV), the protein levels of α‐Syn were detected by western blot. Quantification of α‐Syn protein levels was shown on the right panel (n = 3 biologically independent). The interaction of si‐NEDD4 and Flag‐OTUD5 was significant (P = 0.012). (B) SH‐SY5Y cells were transfected with EGFP‐NEDD4 or empty EGFP‐vector and Flag‐OTUD5 or the empty Flag‐vector (EV), the protein levels of α‐Syn were detected by western blot. Quantification of intensity of α‐Syn was shown on the right panel (n = 3 biologically independent). The interaction of Flag‐OTUD5 and EGFP‐NEDD4 was not statistically significant (P = 0.509). (C) Overexpressed EGFP‐NEDD4 in SH‐SY5Y cells transfected sgRNA targeting OTUD5 (sg‐OTUD5) or sg‐Ctrl, the protein levels of α‐Syn were detected using an anti‐α‐Syn antibody. Quantification of the intensity of α‐Syn was shown on the right panel (n = 3 biologically independent). The interaction effect of sg‐OTUD5 and EGFP‐NEDD4 was statistically significant (P = 0.013). (D, E) Exogenous interactions between OTUD5 and NEDD4 were analyzed by Co‐IP in HEK293T cells transfected Flag‐OTUD5 and EGFP‐NEDD4. (F) Confocal microscopy of HEK293T cells transfected with EGFP‐NEDD4 (green) and Flag‐OTUD5 (red). Scale bar, 10 µm. (G) Co‐IP and immunoblot analysis of extracts of HEK293T cells transfected with EGFP‐NEDD4, His‐α‐Syn, and Flag‐OTUD5 or Flag‐OTUD5 C224S treated with CQ (10 µM) for 6 h. (H) The interaction between EGFP‐NEDD4 and His‐α‐Syn was attenuated in HEK293T OTUD5 knockout cells. (I) Co‐IP and immunoblot analysis of lysates from WT HEK293T cells or OTUD5 knockout HEK293T cells transfected with HA‐tagged K63‐linked ubiquitin (K63‐Ub), EGFP‐NEDD4 and His‐α‐Syn. IP with anti‐His antibody, probed with anti‐HA antibody. Co‐localization was quantified by Pearson's correlation coefficient method from ImageJ software. Intensity profiles of indicated proteins along the plotted lines were analyzed by ImageJ line scan analysis in (F). Data were expressed as the mean ± SEM. Two‐way ANOVA for 2*2 factorial analysis and Tukey's post hoc tests were used for statistical analysis in (A–C). ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

OTUD5 was downregulated in PD mouse model induced by α‐Syn PFF. Wild type (WT) mice were injected in bilateral striatum (STR) with α‐Syn PFF (5µg each side) or PBS at 1, 2, and 3 months post injection (mpi). (A) Schematic diagram of stereotactic injection of PBS or α‐Syn PFF in the STR. (B) Rotarod test and (C) pole test were used to measure motor coordination (n = 8 mice per group). (D) Immunoblot analysis of protein expression levels in SN from WT mice injected with α‐Syn PFF at 1, 2, and 3 mpi. Quantification of indicated protein levels was normalized to GAPDH (n = 6 mice per group). (E) Photomicrographs and quantification of OTUD5 (green) and TH (red) in SN sections of mice after α‐Syn PFF treatment (n = 6 mice per group). Scale bars, 20 µm. (F) Co‐IP and immunoblot analysis of SN lysates from WT mice injected with α‐Syn PFF at 1, 2, and 3 mpi. IP with anti‐α‐Syn antibody, probed with anti‐K63‐Ub antibody. Quantification of indicated protein levels was normalized to GAPDH (n = 6 mice per group). (G) Photomicrographs and quantification of SN sections stained for p‐α‐Syn (Ser129) (green), TH (red), and DAPI from mice injected with α‐Syn PFF at 3 mpi (n = 6 mice per group). Scale bar, 20 µm. (H) Pearson's correlation analysis showing the correlations between OTUD5 and p‐α‐Syn levels in mice brain tissues (n = 6 mice per group; n = 24 mice in total). (I) Pearson's correlation analysis showing the correlation between OTUD5 and K63‐linked polyubiquitylation of α‐Syn in mice brain tissues (n = 6 mice per group; n = 24 mice in total). Data were expressed as the mean ± SEM. One‐way ANOVA followed by Tukey's test was used for statistical analysis in (B‐D, F). Two‐tailed Student's t‐tests were used for statistical analysis in (E) and (G). ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

OTUD5 conditional deficiency compromised dopaminergic neurodegeneration in PD mouse model induced by α‐Syn PFF. (A) A diagram for the mice generation of OTUD5 specific deletion in dopaminergic (DA) neurons. (B) PCR was performed on genomic DNA extracted from mice tail to confirm mice genotypes. (C) Western blot analysis of lysates from SN of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice. Quantification of indicated protein levels was shown on the right (n = 6 mice per group). (D) Immunofluorescence staining for TH (red) and OTUD5 (green) was performed in SN regions of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice. Quantification of fluorescence intensity of OTUD5 were performed by ImageJ software (n = 6 mice per group). Scale bar, 20 µm. (E) Rotarod test and (F) pole test were used to evaluate the motor coordination on mice injected with α‐Syn PFF at 3 mpi (n = 8 mice per group). The interaction of OTUD5 conditional knockout (CKO) and α‐Syn PFF was statistically significant in (E) (P = 0.046). In (F), the interaction of OTUD5 CKO and α‐Syn PFF was significant (P = 0.033). (G) Micrographs of DA neurons in SN of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF by TH staining. Scale bar, 200 µm. Quantification of TH‐positive neurons in the SN was measured by ImageJ software (n = 6 mice per group). The interaction of OTUD5 CKO and α‐Syn PFF was not statistically significant (P = 0.293). (H) Micrographs of STR sections stained for TH fiber density of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. Scale bar, 200 µm. The average optical density of DA fiber in the STR was measured by ImageJ software and the data were normalized to WT‐PBS group (n = 6 mice per group). The interaction between OTUD5 CKO and α‐Syn PFF was not statistically significant (P = 0.145). (I) Immunoblot analysis of lysates from SN of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. Quantification of indicated protein levels was normalized to GAPDH (n = 6 mice per group). The interaction of OTUD5 CKO and α‐Syn PFF was statistically significant (P = 0.015). Data were expressed as the mean ± SEM. Two‐tailed Student's t‐tests were used for statistical analysis in (C) and (D). Two‐way ANOVA for 2*2 factorial analysis and Tukey's post hoc tests were used for statistical analysis in (E‐I). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

OTUD5 protected dopaminergic neurons in α‐Syn PFF‐induced PD model. (A) Immunoblot analysis of lysates from SN of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. Quantification of indicated protein levels was normalized to GAPDH (n = 6 mice per group). The interaction between OTUD5 CKO and α‐Syn PFF was not statistically significant in regulating α‐Syn level (P = 0.831) while the interaction was significant in regulating p‐α‐Syn (Ser129) level (P = 0.036). (B) Representative micrographs of SN sections stained for p‐α‐Syn (Ser129) (green) and TH (red) of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. Scale bar, 20 µm. Quantification of p‐α‐Syn (Ser129) immunofluorescence intensity was shown on the right panel (n = 6 mice per group). The interaction between OTUD5 CKO and α‐Syn PFF was statistically significant (P = 0.004). (C) Representative micrographs of STR regions stained for p‐α‐Syn (Ser129) (green) of OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. Quantification of p‐α‐Syn (Ser129) immunofluorescence intensity was performed by ImageJ software (n = 6 mice per group). Scale bar, 20 µm. The interaction between OTUD5 CKO and α‐Syn PFF was statistically significant (P = 0.003). (D) Co‐IP and immunoblot analysis of SN lysates from OTUD5^fl/Y/Cre⁻ and OTUD5^fl/Y/Cre⁺ mice treated with PBS or α‐Syn PFF. IP with anti‐α‐Syn antibody, probed with anti‐K63‐Ub antibody. (E) Schematic illustration for OTUD5 negatively regulating α‐Syn protein. Under normal state, the constitutively expressed OTUD5 recruited NEDD4 to α‐Syn and promoted the K63‐linked polyubiquitination of α‐Syn, leading to its degradation through the endolysosomal pathway. Thus, the balance between the generation and degradation of α‐Syn maintained DA neuronal homeostasis (left panel). In the pathological state of PD induced by α‐Syn PFF, the downregulated OTUD5 in DA neurons attenuated the interaction between NEDD4 and α‐Syn and the following endolysosomal degradation of α‐Syn, leading to the elevated protein level of α‐Syn. The upregulated α‐Syn accelerated the conversion into pathological α‐Syn and DA neuron loss induced by α‐Syn PFF (right panel). Data were expressed as the mean ± SEM. Two‐way ANOVA for 2 × 2 factorial analysis and Tukey's post hoc tests were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.