BAG5 Interacts with DJ-1 and Inhibits the Neuroprotective Effects of DJ-1 to Combat Mitochondrial Oxidative Damage

Abstract

Loss-of-function mutations in gene encoding DJ-1 contribute to the pathogenesis of autosomal recessive early-onset familial forms of Parkinson's disease (PD). DJ-1 is a multifunctional protein and plays a protective role against oxidative stress-induced mitochondrial damage and cell death, but the exact mechanism underlying this is not yet clearly understood. Here, using coimmunoprecipitation (Co-IP) and immunofluorescence methods, we prove that Bcl-2-associated athanogene 5 (BAG5), a BAG family member, interacts with DJ-1 in mammalian cells. Moreover, we show that BAG5 could decrease stability of DJ-1 and weaken its role in mitochondrial protection probably by influencing dimerization in stress condition. Our study reveals the relationship of BAG5 and DJ-1 suggesting a potential role for BAG5 in the pathogenesis of PD through its functional interactions with DJ-1.

Figure 1

Interaction of BAG5 with DJ-1. (a) Co-IP of overexpressed DJ-1 and BAG5. HEK293 cells were cotransfected with DJ-1-eGFP and BAG5-Myc or eGFP followed by IP with anti-GFP polyclonal antibody. In Western blot, anti-GFP antibody (Ab1218) was used for examining DJ-1 and anti-Myc antibody (#2276) for BAG5. The results showed that DJ-1 could coimmunoprecipitate BAG5. (b) Conversely, similar results were obtained by anti-Myc antibody IP (#2272). (c) Co-IP was performed using DJ-1 antibody (αDJ-1, #5933) that recognized endogenous DJ-1. In Western blot, anti-DJ-1 antibody (ab11251) was used for examining endogenous DJ-1 and anti-BAG5 antibody (ab56738) for endogenous BAG5 protein. (d) Immunofluorescence colocalization analysis of DJ-1 (green) with BAG5 (red) in HEK293 cells (above) or primary hippocampal neurons of rat transfected with DJ-1-GFP (green) and BAG5-Myc (red) (below).

Figure 2

BAG5 reduces the levels of DJ-1, which are reversed by overexpressing Hsp70. (a) HEK293 cells stably expressing DJ-1-HA alone or with BAG5-Myc. Exogenous DJ-1 levels are obviously reduced in cells expressing BAG5-Myc comparing with cell expressing DJ-1-HA alone. Moreover, the overexpression of Hsp70-flag reversed the BAG5-mediated decrease of DJ-1-HA levels in cells stably expressing exogenous BAG5 and DJ-1. Actin was used as a loading control. (b) Quantitative data from (a) is shown. Data is normalized to DJ-1 group which was set to 1. (c) Knockdown endogenous Hsp70 accelerate reduction of ectopic DJ-1 induced by overexpression of BAG5. (d) Quantitative data from (c) is shown. The results are indicated as mean ± SEM (n = 3, ^∗∗p < 0.01, ^∗p < 0.05).

Figure 3

BAG5 attenuated the stability of DJ-1. (a) HEK293 cells stably expressing DJ-1-HA alone, or with BAG5-Myc. All cells were treated with 1 μM CHX to inhibit total protein synthesis and cell's extracts were analyzed by Western blot using the specified antibodies at 0 hr, 12 hr, and 24 hr, respectively. Actin acted as a loading control. In the presence of CHX, BAG5 decreased the DJ-1 levels. (b) Quantitative data from (a) showed that it reduces half-life from 24 hr to 15 hr. Data is normalized to 0 h group which was set to 100%. The results are indicated as mean ± SEM (n = 3, ^∗∗p < 0.01).

Figure 4

BAG5 attenuated the protective property of DJ-1 on mitochondrial function. HEK293 cells were stably expressed with empty vector or BAG5-Myc together with DJ-1-HA, with or without rotenone treatment. 24 h after treatment, ΔΨm and ROS production were measured by flow cytometry. Quantitative analysis of the ΔΨm and ROS was shown in (a) and (b), respectively. Data is normalized to vector + DJ-1 group which was set to 100%. Results are shown as the mean ± SEM (n = 3, ^∗p < 0.05, ^∗∗p < 0.01).

Figure 5

BAG5 inhibited DJ-1 recruitment to mitochondria under rotenone treatment. HEK293 cells stably expressed DJ-1-HA alone (a, c) or with BAG5-Myc (b, d). DJ-1 and mitochondria were stained by HA (red) and Tom20 (green) antibodies, respectively. Without rotenone treatment, exogenous DJ-1-HA (green) has almost no localization on mitochondria marker Tom20 (red) in cells stably expressing vector (a) or BAG5-Myc (b). After being treated with rotenone, increased mitochondrial localization of DJ-1 was observed in cells stably expressing DJ-1-HA alone (c) comparing with BAG5-Myc + DJ-1-HA (d). The enlarged area is displayed as a white box. Quantitative analysis of colocalization is shown (e). Results are shown as mean ± SEM (^∗p < 0.05).

Figure 6

BAG5 influences the dimerization of DJ-1 in mitochondria. Mitochondria were isolated in HEK293 cells stably expressing DJ-1-flag alone or with BAG5-Myc for examining the levels of dimer and monomer by Western blot. Tom20 acted as control protein in mitochondria. HEK293 cells coexpressing BAG5 and DJ-1 had a lower level of dimer and a higher level of monomer in mitochondria than HEK293 cells expressing DJ-1 alone (a). Quantitative data from (a) is shown (b). Results were shown as the mean ± SEM (n = 3, ^∗p < 0.05).

Figure 7

BAG5 diminished the protective effects of DJ-1 on rotenone-induced cell apoptosis. The stable expression of cells is indicated (a). After 24 h of rotenone treatment, the induction of apoptosis was determined by Annexin V-FITC/PI double staining assay, and the degree of apoptotic cell death was quantified. Quantitative data from (a) is shown in (b). Results are shown as mean ± SEM (n = 3, ^∗p < 0.05, ^∗∗p < 0.01).