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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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Mitochondrial fragmentation following hypoxia-reoxygenation is ameliorated by inactivation of MARCH5 or Drp1.(A) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were cultured in the presence of low oxygen (1%) for 24 hours followed by normoxia for 2 hours. Mitochondrial fragmentation was analyzed following cytochrome c staining in three independent experiments (>200 cell counted/condition). Error bars represent SEM, p-Values for Student's t-test are marked with * (p<0.05) or ** (p<0.01). (B) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were stressed using hypoxia-reoxygenation and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).
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pone-0052637-g004: Mitochondrial fragmentation following hypoxia-reoxygenation is ameliorated by inactivation of MARCH5 or Drp1.(A) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were cultured in the presence of low oxygen (1%) for 24 hours followed by normoxia for 2 hours. Mitochondrial fragmentation was analyzed following cytochrome c staining in three independent experiments (>200 cell counted/condition). Error bars represent SEM, p-Values for Student's t-test are marked with * (p<0.05) or ** (p<0.01). (B) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were stressed using hypoxia-reoxygenation and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).

Mentions: Re-oxygenation following exposure to low oxygen atmosphere mimicking ischemia-reperfusion conditions induces mitochondrial fragmentation in differentiated RGC5 cells (Figure 1 and 4). Re-oxygenation-induced mitochondrial fragmentation was not blocked in cells ectopically expressing MARCH5 or Drp1 when compared to transfected control cells (Figure 4). Interestingly, ectopic expression of MARCH5H43W or Drp1K38A did completely block mitochondrial fragmentation under these conditions (Figure 4A). We confirmed this observation in MARCH5 or MARCH5H43W expressing cells treated with hypoxia-reperfusion using PAGFP diffusion assay (Figure 4B).


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)

Mitochondrial fragmentation following hypoxia-reoxygenation is ameliorated by inactivation of MARCH5 or Drp1.(A) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were cultured in the presence of low oxygen (1%) for 24 hours followed by normoxia for 2 hours. Mitochondrial fragmentation was analyzed following cytochrome c staining in three independent experiments (>200 cell counted/condition). Error bars represent SEM, p-Values for Student's t-test are marked with * (p<0.05) or ** (p<0.01). (B) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were stressed using hypoxia-reoxygenation and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526576&req=5

pone-0052637-g004: Mitochondrial fragmentation following hypoxia-reoxygenation is ameliorated by inactivation of MARCH5 or Drp1.(A) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were cultured in the presence of low oxygen (1%) for 24 hours followed by normoxia for 2 hours. Mitochondrial fragmentation was analyzed following cytochrome c staining in three independent experiments (>200 cell counted/condition). Error bars represent SEM, p-Values for Student's t-test are marked with * (p<0.05) or ** (p<0.01). (B) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were stressed using hypoxia-reoxygenation and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).
Mentions: Re-oxygenation following exposure to low oxygen atmosphere mimicking ischemia-reperfusion conditions induces mitochondrial fragmentation in differentiated RGC5 cells (Figure 1 and 4). Re-oxygenation-induced mitochondrial fragmentation was not blocked in cells ectopically expressing MARCH5 or Drp1 when compared to transfected control cells (Figure 4). Interestingly, ectopic expression of MARCH5H43W or Drp1K38A did completely block mitochondrial fragmentation under these conditions (Figure 4A). We confirmed this observation in MARCH5 or MARCH5H43W expressing cells treated with hypoxia-reperfusion using PAGFP diffusion assay (Figure 4B).

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