Inactivation of MARCH5 prevents mitochondrial fragmentation and interferes with cell death in a neuronal cell model
Lei Fang1, Charles Hemion, David Goldblum
1Department of Biomedicine, University Basel, Basel, Switzerland.
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.
Insights
The mitochondrial ligase MARCH5 impacts neuronal cell death. Inhibiting mitochondrial fragmentation protects cells from glaucoma-related stress, suggesting therapeutic potential.
Area of Science:
- Cell Biology
- Neuroscience
- Mitochondrial Dynamics
Background:
- Mitochondrial morphology is crucial for neuronal health.
- Dysfunctional mitochondria are implicated in neurodegenerative diseases like glaucoma.
- The role of mitochondrial ubiquitin ligase MARCH5 in neuronal stress response is not fully understood.
Purpose of the Study:
- To investigate the role of MARCH5 in regulating mitochondrial morphology and apoptosis in neuronal precursor cells.
- To assess the impact of MARCH5 on neuronal cell survival under glaucoma-relevant stress conditions.
Main Methods:
- RGC5 cells were transfected with MARCH5 or its mutants and exposed to elevated pressure, oxidative stress, or hypoxia-reoxygenation.
- Mitochondrial morphology was analyzed using cytochrome c staining and PAGFP diffusion assays.
- Apoptosis was quantified by measuring cytochrome c release and using flow cytometry.
Main Results:
- Elevated pressure and oxidative stress induced mitochondrial fragmentation in control and MARCH5-expressing cells.
- Inactive MARCH5 and Drp1 mutants significantly blocked mitochondrial fragmentation.
- Both MARCH5 expression and stress conditions promoted apoptotic cell death, which was delayed by dominant-negative MARCH5 or Drp1 mutants.
Conclusions:
- Mitochondrial fragmentation is a key event in stress-induced neuronal cell death.
- Interfering with mitochondrial fission machinery protects neuronal cells from apoptosis.
- Targeting mitochondrial dynamics may offer a therapeutic strategy for glaucoma.
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