Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress

Abstract

Prohibitin (PHB) is a ubiquitously expressed and evolutionarily conserved mitochondrial protein with multiple functions. We have recently shown that PHB up-regulation offers robust protection against neuronal injury in models of cerebral ischemia in vitro and in vivo, but the mechanism by which PHB affords neuroprotection remains to be elucidated. Here, we manipulated PHB expression in PC12 neural cells to investigate its impact on mitochondrial function and the mechanisms whereby it protects cells exposed to oxidative stress. PHB over-expression promoted cell survival, whereas PHB down-regulation diminished cell viability. Functionally, manipulation of PHB levels did not affect basal mitochondrial respiration, but it increased spare respiratory capacity. Moreover, PHB over-expression preserved mitochondrial respiratory function of cells exposed to oxidative stress. Preserved respiratory capacity in differentiated PHB over-expressing cells exposed to oxidative stress was associated with an elongated mitochondrial morphology, whereas PHB down-regulation enhanced fragmentation. Mitochondrial complex I oxidative degradation was attenuated by PHB over-expression and increased in PHB knockdown cells. Changes in complex I degradation were associated with alterations of respiratory chain supercomplexes. Furthermore, we showed that PHB directly interacts with cardiolipin and that down-regulation of PHB results in loss of cardiolipin in mitochondria, which may contribute to destabilizing respiratory chain supercomplexes. Taken together, these data demonstrate that PHB modulates mitochondrial integrity and bioenergetics under oxidative stress, and suggest that the protective effect of PHB is mediated by stabilization of the mitochondrial respiratory machinery and its functional capacity, by the regulation of cardiolipin content. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Keywords: PC12 cells; mitochondria; oxidative stress; prohibitin; respiratory chain complexes.

Figure 1. PHB levels in PC12 cells affect cell viability in oxidative stress conditions

(A) Western blots analyses for PHB. PC12 cells stably transfected with PHB overexpression, shRNA silencing, and vector control plasmids and grown in normal growth medium were harvested and lysed. Each lane was loaded with 20μg of cell lysate. β-actin was used as loading control. (B) Protein band intensity quantification. Mean band intensity was normalized to β-actin levels and expressed as a proportion of control lines. **p<0.01 compared to vector or shSCR lines, n=8 independent experiments. (C) Cell viability analyses. Cell viability was determined using MTS assay after cells were treated for 2hrs with H₂O₂ (0.2mM) or vehicle. Viability was calculated as percentage of vehicle treated cells for each line. ***p<0.001 compared to vector or shSCR, n=6 independent experiments. The data are shown as mean±SEM.

Figure 2. PHB levels modulate mitochondrial respiration

PC12 cells treated with H₂O₂ (0.2 mM, 2 hrs) or vehicle were analyzed by oxygraph. (A) Basal respiration. (B) Proton leakage measurement after oligomycin (1μM) addition. (C) Maximum respiration induced by the protonophore FCCP (1μM). (D) Spare respiratory capacity obtained by subtracting basal respiration from maximum respiration. *p<0.05 compared to vehicle treated cells, #p<0.05 compared to corresponding shSCR group. Each bar represents between 6 and 9 samples from 3 independent experiments. (E) Relative mitochondrial DNA copy number measured by real-time RT-qPCR and normalized to 28sRNA nuclear DNA. The data are shown as mean±SEM.

Figure 3. PHB levels modulate mitochondrial length in differentiated PC12 cells

PC12 cells on glass bottom dishes were transfected with mito-RFP and induced to differentiate with NGF for 6 days. (A) Mitochondrial length distribution in normal (upper panels) and in oxidative stress conditions (lower panels). (B) Box-and-whisker diagrams showing mitochondrial length distribution derived from the Kruskal-Wallis test for the four cell lines in basal conditions (untreated) and under oxidative stress (H₂O₂ treatment). The box is interquartile range from the first to the third (midspread 50%); the data range (max and min values) is represented by the lines above and below the box; the bar inside the box represents the median value. Significant differences among groups revealed by the Dunn’s test for multiple comparisons are indicated (*p<0.05, total n=359–763 mitochondria per group in normal conditions and n=211–1096 mitochondria per group in H₂O₂ treated cells). (C) Representative images of mitochondrial network in PHB expressing and PHB knockdown cells in basal conditions and after oxidative stress. Data were obtained from three independent experiments measuring individual mitochondria.

Figure 4. PHB knockdown in primary neurons causes mitochondrial fragmentation

Primary neurons co-transfected with PHB siRNA and mt-dsRed were cultured for 5 days before PHB protein level and mitochondrial morphology analyses. (A) Western blot analysis of PHB protein level in si-PHB and si-Ctrl transfected neurons and β-actin as loading control. (B) Quantitative measurement of PHB protein band intensity normalized to β-actin. (C) Representative confocal images of mitochondrial morphology in the neurites of neurons transfected with mt-dsRed and either si-PHB or si-Ctrl as indicated. Images were taken with 63X oil objective. (D) Quantitative analyses of siRNA transfected neurons containing elongated tubular or fragmented mitochondria. Neurons containing a mix of elongated and fragmented mitochondria were grouped separately. *p<0.05, n=3 separate experiments totaling 224 cells for si-PHB group and 254 for si-Ctrl group. Data are shown as mean±SEM.

Figure 5. PHB expression modulates mitochondrial complex I subunit levels and RSC formation

Mitochondria from PHB overexpressing or knockdown cells and respective controls were partially purified and lysed with either RIPA buffer for SDS-PAGE or by digitonin for BN-PAGE analyses. (A, B) Mitochondrial complex CI level assessment with CI subunit NDUFA9 specific antibody and quantitative protein band analysis. HSP60 was used as loading control. (C, D) CI level assessment with a different antibody specific for CI subunit NDUFVI and protein band intensity measurements. (E, F) CIII level assessment with antibody specific for CIII subunit core 2 and protein band intensity measurements. (G) Supercomplex assembly assessment with antibodies for CI subunit A9. Mitochondrial preparations were solubilized using digitonin and separated by BN-PAGE. (H) Supercomplex assembly assessed by antibody specific for CIII core 2 subunit. Shown in each panel is one representative blot image from 3–4 separate experiments. *p<0.05, **p<0.01. The data are shown as mean±SEM.

Figure 6. PHB expression prevents oxidative breakdown of CI

Oxidative breakdown of CI was assayed in mitochondrial preparations from WT PC12 cells. Intact CI was solubilized using maltoside from mitochondria of cells that were treated with increasing concentrations of H₂O₂ for 2 hrs (A) or with 0.5mM H₂O₂ for 0, 30, 60, 90, 120, or 180 minutes (B). Solubilized intact CI was resolved by BN-PAGE and probed with antibodies to CI subunit NDUFA9. To assess the effect of PHB on CI stability, PHB overexpressing or knockdown cells and a vector control line were treated with 0.5mM H₂O₂ for 4 hrs. Mitochondrial preparations were lysed and content of representative CI subunit NDUFA9 was assayed by SDS-PAGE and western blotting (C). CI NDUFA9 band intensity was normalized using TIM23 (D). *p<0.05, n=4 separate experiments; ns, not significant. The data are shown as mean±SEM.

Figure 7. PHB binds cardiolipin and modulates its content in mitochondria

(A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.