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Inactivation of MARCH5 prevents mitochondrial fragmentation and interferes with cell death in a neuronal cell model.

Fang L, Hemion C, Goldblum D, Meyer P, Orgül S, Frank S, Flammer J, Neutzner A - PLoS ONE (2012)

Bottom Line: Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay.Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5.Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University Basel, Basel, Switzerland.

ABSTRACT

Purpose: To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods: RGC5 cells transfected with expression constructs for MARCH5, MARCH5(H43W), Dpr1(K38A) or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results: Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5(H43W) or inactive Drp(K38A), mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5(H43W) or Drp1(K38A) did significantly delay cell death.

Conclusion: Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.

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Related in: MedlinePlus

Inactivation of MARCH5 or Drp1 delays induction of apoptosis and cell death.RGC5 cells expressing (A) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (B) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 100 mmHg for 24 hours, 1 µM rotenone for 6 hours or combined 100 mmHg pressure and 1 µM rotenone in the presence of the pan-caspase inhibitor zVAD-fmk. Following treatment, cells were fixed and cytochrome c release from mitochondria into the cytosol was assessed by fluorescence microscopy (>200 cell counted/condition). The bar graphs represent four independent experiments with * marking p<0.05 and ** marking p<0.01 (Student's t-test). RGC5 cells expressing (C) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (D) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 1 µM rotenone for 0, 24 or 48 hours and the amount of dead cells was measured by flow cytometry following 7-AAD staining of DNA.
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pone-0052637-g005: Inactivation of MARCH5 or Drp1 delays induction of apoptosis and cell death.RGC5 cells expressing (A) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (B) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 100 mmHg for 24 hours, 1 µM rotenone for 6 hours or combined 100 mmHg pressure and 1 µM rotenone in the presence of the pan-caspase inhibitor zVAD-fmk. Following treatment, cells were fixed and cytochrome c release from mitochondria into the cytosol was assessed by fluorescence microscopy (>200 cell counted/condition). The bar graphs represent four independent experiments with * marking p<0.05 and ** marking p<0.01 (Student's t-test). RGC5 cells expressing (C) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (D) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 1 µM rotenone for 0, 24 or 48 hours and the amount of dead cells was measured by flow cytometry following 7-AAD staining of DNA.

Mentions: Furthermore, we assessed whether modulation of mitochondrial morphology through expression of Drp1K38A or MARCH5H43W altered the sensitivity of differentiated RGC5 cells to apoptotic stimuli. To this end, RGC5 cells were exposed either to 100 mmHg elevated pressure for one day, rotenone alone, or a combination of elevated pressure and rotenone, and apoptotic induction in the presence of pan-caspase inhibitor was assessed by counting the release of cytochrome c from mitochondria. As shown in Figure 5, exposing RGC5 to these stress conditions leads to the induction of apoptotic cell death in control cells and in cells expressing wildtype MARCH5 (Figure 5A) or Drp1 (Figure 5B). Interestingly, expression of MARCH5H43W (Figure 5A) or Dpr1K38A (Figure 5B) resulted in significant suppression of apoptotic cell death induction.


Inactivation of MARCH5 prevents mitochondrial fragmentation and interferes with cell death in a neuronal cell model.

Fang L, Hemion C, Goldblum D, Meyer P, Orgül S, Frank S, Flammer J, Neutzner A - PLoS ONE (2012)

Inactivation of MARCH5 or Drp1 delays induction of apoptosis and cell death.RGC5 cells expressing (A) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (B) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 100 mmHg for 24 hours, 1 µM rotenone for 6 hours or combined 100 mmHg pressure and 1 µM rotenone in the presence of the pan-caspase inhibitor zVAD-fmk. Following treatment, cells were fixed and cytochrome c release from mitochondria into the cytosol was assessed by fluorescence microscopy (>200 cell counted/condition). The bar graphs represent four independent experiments with * marking p<0.05 and ** marking p<0.01 (Student's t-test). RGC5 cells expressing (C) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (D) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 1 µM rotenone for 0, 24 or 48 hours and the amount of dead cells was measured by flow cytometry following 7-AAD staining of DNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526576&req=5

pone-0052637-g005: Inactivation of MARCH5 or Drp1 delays induction of apoptosis and cell death.RGC5 cells expressing (A) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (B) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 100 mmHg for 24 hours, 1 µM rotenone for 6 hours or combined 100 mmHg pressure and 1 µM rotenone in the presence of the pan-caspase inhibitor zVAD-fmk. Following treatment, cells were fixed and cytochrome c release from mitochondria into the cytosol was assessed by fluorescence microscopy (>200 cell counted/condition). The bar graphs represent four independent experiments with * marking p<0.05 and ** marking p<0.01 (Student's t-test). RGC5 cells expressing (C) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (D) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 1 µM rotenone for 0, 24 or 48 hours and the amount of dead cells was measured by flow cytometry following 7-AAD staining of DNA.
Mentions: Furthermore, we assessed whether modulation of mitochondrial morphology through expression of Drp1K38A or MARCH5H43W altered the sensitivity of differentiated RGC5 cells to apoptotic stimuli. To this end, RGC5 cells were exposed either to 100 mmHg elevated pressure for one day, rotenone alone, or a combination of elevated pressure and rotenone, and apoptotic induction in the presence of pan-caspase inhibitor was assessed by counting the release of cytochrome c from mitochondria. As shown in Figure 5, exposing RGC5 to these stress conditions leads to the induction of apoptotic cell death in control cells and in cells expressing wildtype MARCH5 (Figure 5A) or Drp1 (Figure 5B). Interestingly, expression of MARCH5H43W (Figure 5A) or Dpr1K38A (Figure 5B) resulted in significant suppression of apoptotic cell death induction.

Bottom Line: Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay.Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5.Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University Basel, Basel, Switzerland.

ABSTRACT

Purpose: To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods: RGC5 cells transfected with expression constructs for MARCH5, MARCH5(H43W), Dpr1(K38A) or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results: Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5(H43W) or inactive Drp(K38A), mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5(H43W) or Drp1(K38A) did significantly delay cell death.

Conclusion: Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.

Show MeSH
Related in: MedlinePlus