Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death

Abstract

As pathogenic Parkin mutations result in the defective clearance of damaged mitochondria, Parkin-dependent mitophagy is thought to be protective against the dopaminergic neurodegeneration observed in Parkinson's disease. Recent studies, however, have demonstrated that Parkin can promote cell death in the context of severe mitochondrial damage by degrading the pro-survival Bcl-2 family member, Mcl-1. Therefore, Parkin may act as a 'switch' that can shift the balance between protective or pro-death pathways depending on the degree of mitochondrial damage. Here, we report that the Parkin interacting protein, Bcl-2-associated athanogene 5 (BAG5), impairs mitophagy by suppressing Parkin recruitment to damaged mitochondria and reducing the movement of damaged mitochondria into the lysosomes. BAG5 also enhanced Parkin-mediated Mcl-1 degradation and cell death following severe mitochondrial insult. These results suggest that BAG5 may regulate the bi-modal activity of Parkin, promoting cell death by suppressing Parkin-dependent mitophagy and enhancing Parkin-mediated Mcl-1 degradation.

Fig. 1. BAG5 regulates Parkin recruitment to mitochondria.

a Representative confocal micrographs of U2OS GFP-Parkin cells transfected with dsRed or FlagBAG5 and treated with 20 μm CCCP for 60 min. Scale bar is 20 μm. b Quantification of the percentage of transfected cells displaying GFP-Parkin recruitment onto the mitochondria by assessing colocalization of GFP-Parkin with TOM20. A minimum of 150 cells per condition were counted in three independent experiments, and statistical significance was determined by student’s t test (*p < 0.05). c Western blot confirming reduction of endogenous BAG5 protein levels in transfected U2OS GFP-Parkin cells. d Representative confocal micrographs of 2 h live cell time-lapse imaging of U2OS GFP-Parkin cells treated with 20 μm CCCP and transfected with non-targeting control or BAG5 siRNA 48 h prior to imaging (see Video 1). e Quantification of GFP-Parkin recruitment to mitochondria by evaluating the percentage of cells displaying colocalization between GFP-Parkin and mito-RFP. At least 200 cells were scored per condition by an individual blinded to the treatment. The vertical bars represent the mean ± SEM for three independent experiments. Two-way ANOVA followed by Bonferroni post hoc test revealed siBAG5 transfected cells had a significantly greater percentage of cells displaying GFP-Parkin recruitment to the mitochondria at timepoints between 30 and 60 min inclusive (*p < 0.05, **p < 0.01,***p < 0.001). Scale bar is 50 μm. f. Representative confocal micrographs of U2OS GFP-Parkin cells transfected with human BAG5 siRNA and rat FlagBAG5 treated with CCCP for 60 min. Scale bar is 20 μm g. Quantification of the percentage of transfected cells displaying GFP-Parkin recruitment onto the mitochondria by assessing colocalization of GFP-Parkin with TOM20. A minimum of 50 Flag negative and Flag positive cells per condition were counted in three independent experiments, and statistical significance was determined by student’s t test (*p < 0.05). h Representative Western blot demonstrating knockdown of human FlagBAG5 but not rat FlagBAG5 by human BAG5 siRNA.

Fig. 2. BAG5 knockdown enhances mitophagy.

a Representative flow cytometry data of U2OS mito-mKeima cell lines also expressing GFP-Parkin or GFP-Parkin^C431S that were untreated or treated with 20 μm CCCP for 4 h after transfection with non-targeting, BAG5, or PINK1 siRNA. Mitophagy was assessed as the percentage of cells displaying an elevated 561/405 nm ratio as captured in the upper gate as we have previously described^,. b Quantification of average percentage of mitophagy from three independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s post hoc test (**p < 0.005, ****p < 0.0001).

Fig. 3. BAG5 enhances cell death and causes increased PARP and Caspase-3 cleavage following CCCP treatment.

a Dose–response curve for CCCP in the GFP and GFP-BAG5 stable cell lines using PrestoBlue viability assay. Experiment performed three times in triplicate. Data back normalized to 0 treatment condition, and statistical analysis was done using two-way ANOVA followed by Bonferroni post hoc testing (***p < 0.001). b Fold change in cell death elicited by 50 μm CCCP treatment (CCCP/DMSO) in the GFP and GFP-BAG5 SH-SY5Y stable cell lines as measured by the DRAQ7 cell death assay using flow cytometry. GFP and GFP-BAG5 expression induced for 24 h using 2 μg/mL doxycycline prior to treatment. Experiment performed two times in triplicate. Statistical significance determined by unpaired t test (****p < 0.0001). Columns represent mean ± SEM. c Same as b comparing the effect of BAG5 KD (siBAG5) to control (siNTC). d Same as b comparing the effect of Parkin KD (siParkin) to control (siNTC) in SH-SY5Y GFP-BAG5 cells. e Western blot demonstrating Parkin knockout (KO) in Parkin KO HEK293T cells compared with the wild-type (WT) HEK293T cells and relative levels of transfected GFP and GFP-BAG5. f Relative percentage of cell death elicited by 50 μm CCCP in Parkin KO HEK293T cells relative to WT HEK293T cells transfected with GFP-BAG5 or GFP alone as a control. Experiment performed two times in triplicate. Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test (**p < 0.01, ***p < 0.001, ****p < 0.0001). Columns represent mean ± SEM. g Western blot illustrating the changes in endogenous PARP, cleaved PARP (cPARP), Caspase-3 (Casp3), and cleaved Caspase-3 (cCasp3) in the GFP and GFP-BAG5 SH-SY5Y stable cell lines following treatment with 50 μm CCCP. Representative blot from three independent experiments. h Quantification of the western blot presented in g. Data obtained from three independent experiments and statistical analysis done using one-way ANOVA followed by Bonferroni post hoc testing (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Columns represent mean ± SEM.

Fig. 4. Parkin and BAG5 modulate Mcl-1 degradation following CCCP treatment.

a Representative western blot illustrating the changes in endogenous Mcl-1 in the GFP and GFP-BAG5 SH-SY5Y cell lines transfected with either pcDNA3.1 vector control or myc-Parkin, and treated with either 1 μm MG132, 50 μm CCCP, or a combination of the two. Representative image obtained from three independent experiments. b Quantification of Mcl-1 from western blots in three independent experiments in the absence of MG132. Data were obtained from three independent experiments and normalized to the GFP alone control condition in each experiment. c Quantification of Mcl-1 from western blots in independent experiments in the presence of MG132. Data were obtained from three independent experiments and normalized to the GFP alone control condition in each experiment. Statistical analysis in b and c was done using one-way ANOVA followed by Bonferroni post hoc testing (*p < 0.05, **p < 0.01, ***p < 0.001). Columns represent mean ± SEM.

Fig. 5. BAG5 knockdown partially rescues Mcl-1 degradation.

a Western blot illustrating the changes in endogenous Mcl-1, BAG5, and exogenous myc-Parkin elicited by siRNA-mediated BAG5 knockdown (siBAG5) vs. Control (siNTC) with or without CCCP treatment in SH-SY5Y cells. b Quantification of Mcl-1 presence in the western blot. Mcl-1 intensity normalized to Actin and expressed as a ratio of the intensity found in the siBAG5 lane vs. siNTC lane. Data obtained from three independent experiments, normalized to siNTC condition and compared using an unpaired t test (*p < 0.05). Columns represent mean ± SEM. c SH-SY5Y cells overexpressing GFP-BAG5 or GFP were transfected with HA-ubiquitin plus siRNA targeting Parkin (siParkin) or control siRNA and then treated with 50 μm CCCP for 18 h. Ubiquitinated proteins were immunoprecipitated with anti-HA antibodies and analyzed by western blot. *Indicates previously probed band from panel below. d Quantification of the level of immunoprecipitated HA-ubiquitinated Mcl-1 (HA-Ub Mcl-1) relative to Mcl-1 protein levels in the inputs. Data obtained from three independent experiments and statistical analysis was done using one-way ANOVA followed by Tukey post hoc testing. Columns represent mean ± SEM.

Fig. 6. Proposed model for the role of BAG5 in regulating Parkin’s functions in mitophagy and cell death.

At baseline conditions, Parkin exists in an inactive state and when activated by PINK1 following mitochondrial depolarization, BAG5 inhibits its recruitment and action in mitophagy while enhancing degradation of Mcl-1, which in turn promotes apoptosis. An additional network of “stress sensor” chaperone proteins is likely required for full activation of Parkin’s cytotoxic action. For example BAG2, BAG4, and CHIP are co-chaperones involved in context-dependent regulation of PINK1 and Parkin.