Efficient PHB2 (prohibitin 2) exposure during mitophagy depends on VDAC1 (voltage dependent anion channel 1)

Abstract

Exposure of inner mitochondrial membrane resident protein PHB2 (prohibitin 2) during autophagic removal of depolarized mitochondria (mitophagy) depends on the ubiquitin-proteasome system. This uncovering facilitates the PHB2 interaction with phagophore membrane-associated protein MAP1LC3/LC3. It is unclear whether PHB2 is exposed randomly at mitochondrial rupture sites. Prior knowledge and initial screening indicated that VDAC1 (voltage dependent anion channel 1) might play a role in this phenomenon. Through in vitro biochemical assays and imaging, we have found that VDAC1-PHB2 interaction increases during mitochondrial depolarization. Subsequently, this interaction enhances the efficiency of PHB2 exposure and mitophagy. To investigate the relevance in vivo, we utilized porin (equivalent to VDAC1) knockout Drosophila line. Our findings demonstrate that during mitochondrial stress, porin is essential for Phb2 exposure, Phb2-Atg8 interaction and mitophagy. This study highlights that VDAC1 predominantly synchronizes efficient PHB2 exposure through mitochondrial rupture sites during mitophagy. These findings may provide insights to understand progressive neurodegeneration.

Keywords: Neurodegeneration; PHB2-LC3 interaction; PINK1-PRKN; parkinson disease; porin; ubiquitin-proteasome system.

Figure 1.

VDAC1-PHB2 complex formation during CCCP induced depolarization. (A) isolated rat brain mitochondria are treated with CCCP (10 µm) for 20 min, crosslinked (CS) with EGS and subjected to immunoblotting. Non-crosslinked samples from the same mitochondrial pool were incubated with β mercaptoethanol (βME) and immunoblotted after SDS PAGE. ~72 (appeared between 72 and 55 kDa), ~130 and ≥ 180 kDa band intensity from the crosslinked samples were normalized by ~ 34 kDa band intensity and represented as bar graphs. Arrowheads demarcate ~ 72, ~130 kDa bands and (}) indicate ≥ 180 kDa bands. The experiment was repeated for six times. Experiments with non-crosslinked samples were repeated three times. (B) SH-SY5Y cells are treated with CCCP (10 µm) for 2 h and isolated EGS CS mitochondria are processed for immunoblotting. ~72, ~130 and ≥ 180 kDa band intensity were normalized by ~ 34 kDa band intensity and represented as bar graphs. The experiment was repeated for 4 times. (C) isolated rat brain mitochondria are treated with CCCP (20 min), or isolated mitochondria from 2-h CCCP treated SH-SY5Y cells are immunoprecipitated for either VDAC1 or PHB2. After SDS-PAGE, immunoblots are probed with the indicated antibodies. The experiment was repeated 3 times. (D) VDAC1 KO HEK 293T cells expressing VDAC1-GFP (1-10), GFP (11)-PHB2 and mitoBFP are treated with vehicle or CCCP (10 µm, 20 min). 3D rendered z stack images are represented to demonstrate increased GFP signal after CCCP treatment. The experiment was repeated for 3 times and at least 45 cells were considered. Scale bar: 10 µm. GFP intensity was normalized by MitoBFP intensity and mean values are represented as the bar graph. (E) rat brain mitochondria are treated as mentioned in a and crosslinked with DSP or BMH. Immunoblots represent VDAC1 and PHB2 localization after SDS-PAGE. (*) indicate the missing signal of VDAC1 in the BMH crosslinked samples. (F) mitochondria isolated from control or VDAC1 knockout (KO) HEK 293T cells are treated as mentioned in C and crosslinked as mentioned in figure. Immunoblots are probed with VDAC1 and PHB2 antibodies. Arrowhead demarcates ~ 130 kDa band. Bar graphs represent mean ± SEM. Student’s t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 when compared to control group.

Figure 2.

VDAC1 is required for depolarization induced PHB2 exposure and mitophagy. (A) immunoblots represent protease protection assay for PHB2, mitochondrial inner membrane (ATP5F1A) and matrix (HSPD1) markers in the cell lines after the mentioned treatments. Cells were incubated with CCCP (10 µm)/dids (100 µm) for 4 h. Bar graphs represent mean fold change (± SEM). *p ≤ 0.05, ***p ≤ 0.001 when compared to control group. N = 3, Student’s t test. (B) Representative images of duo link proximity ligation assay for PHB2 and LC3 in SH-SY5Y cells after the mentioned treatments. Cells are treated with DIDS (100 µm) and/or CCCP (10 µm) for 4 h. Scale bar: 10 µm. Bar graphs represent mean number of PLA puncta (± SEM)/cell. ***p ≤ 0.001 when compared to control group. ^##p ≤0.01/^###p ≤0.001 when compared to the only CCCP treated group. The experiment is repeated three times. One-way ANOVA followed by Tukey’s multiple comparisons test. (C) SH-SY5Y or HEK 293T cells are treated with CCCP for 8 h. DIDS is co-treated with CCCP in the mentioned group. Immunoblots for the mentioned proteins are representative of at least three different experiments. Bar graphs represent mean fold change ± SEM. n = 3, *p ≤ 0.05, ***p ≤ 0.001, Student’s t test. (D) quantification of mitophagic cells after transfecting mitoKeima is performed by FACS analysis. Bar graphs represent mean fold change (± SEM) in number of high mitophagic cells after 8 h of CCCP (10 µm) treatment in the mentioned cell lines. The experiment is repeated 3 times. **p ≤ 0.01, ***p ≤ 0.001, Student’s t test.

Figure 3.

Rotenone treatment induces mitophagy and increases porin-Phb2 complex formation in Drosophila. (A) images showing mitoKeima signal from live fly wing (Act5C-GAL4:UAS mitoKeima) after 1 d or 2 d rotenone (1 mm). For wing, images are taken from the indicated region in bright field (scale bar: 10 µm). Images for mitoKeima were also taken from live thoracic muscle after 2 d of rotenone treatment. For thoracic muscle mitochondria, we dissected the thorax and isolated muscles are imaged after submerging in phosphate buffer saline, pH 7.0 (scale bar: 20 µm). White arrowheads indicated mitophagic structures. (B) Drosophila expressing Atg8a-mCherry (Act5C-GAL4:UAS-mCherry-Atg8a) are anaesthetized after 1 d or 2 d of rotenone treatment. Fixed fly thorax is immunostained for blw, mCherry and Atg8a. Images are captured from the indicated portion of thorax as mentioned in bright field or fluorescent image of the whole body (scale: 100 µm) and the representative merged images are provided. White arrowheads indicate colocalization of mCherry, blw and Atg8a signals, which appeared as bright white puncta (scale bar: 20 µm). (C) fixed unstained thorax samples are processed for transmission electron microscope imaging and the selected region from the rotenone treated group demonstrate presence of clear mitophagic structures after 2 d of rotenone treatment (1 mm). Scale bar is as indicated in the images. (D) isolated mitochondria from control and rotenone treated Drosophila are crosslinked with EGS and immunoblotted for porin and Phb2 as mentioned previously. Arrowheads indicate the ~ 70 and ~ 130 kDa signals. (}) indicate signals which appeared above 180 kDa molecular mass marker. Immunoblots are representative of three different experiments. (E) Drosophila treated with rotenone (1 d) are anaesthetized and mitochondrial protein samples are processed for immunoprecipitation. At least 20 flies are processed for individual experiment. Representative blots demonstrate signal for the mentioned proteins after co- immunoprecipitation (IP) for either porin or Phb2. Immunoblots are representative of three different experiments.

Figure 4.

Vdac/porin is necessary for rotenone induced mitophagy in Drosophila. (A) blots demonstrate protease protection assay of Phb2 in mitochondria isolated from WT, porin KO (porin A2/A2) and porin revertant (porin RV) Drosophila lines, after 1 d of rotenone treatment. Bar graphs represent mean ± SEM. *p ≤ 0.05. n = 3-4, Student’s t test. (B) Representative images of duo link proximity ligation assay (PLA) for Phb2 and Atg8a (red) in control or rotenone (2 d) treated fly thorax of the mentioned lines are provided. Phase contrast and DAPI images are merged with the PLA signals. Bar graphs represent mean number of PLA puncta (± SEM). *p ≤ 0.05, **p ≤ 0.01. n = 4-6. Student’s t test. (C) fixed thoracic samples of mentioned fly lines after 2 d of rotenone treatment are processed for immunostaining or for transmission electron microscopy. Confocal microscope images represent colocalization of blw (red) and Atg8a (green). Scale bar: 10 µm. Arrowheads in transmission electron microscopic images indicate mitophagic structures. Scale bar as indicated in the image. At least 5 fly thoraces are imaged. (D) mitochondrial marker proteins (blw and Hsp60) from Drosophila lines are quantified by immunoblotting after 1 d and 2 d of rotenone treatment. Bar graphs represent normalized mean intensity (± SEM) for blw and Hsp60. *p ≤ 0.05, **p ≤ 0.01. n = 3-4. One-way ANOVA followed by Dunnett’s multiple comparisons test.