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Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial
Dean D Kindler1, Christine Thiffault, Nina J Solenski
1Center for the Study of Neurodegenerative Diseases and Department of Neurology, University of Virginia, Charlottesville, VA 22908, USA.
Abstract:
Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester (TMRM). Calcein/TMRM-visualized mitochondrial depolarization was prevented by cyclosporin A (CsA) or approximately two-fold increased levels of BclXL protein. Dynamic imaging of mitochondrial potential (Deltapsi M) with TMRM showed that DETA-NO induced cycles of mitochondrial depolarization/repolarization ("flickering"). Fifteen-30 min of DETA-NO exposure caused high-frequency flickering with small peak size; 2 h of DETA-NO produced large peaks with prolonged depolarization. NO-induced flickering but not that from Bax was blocked by the calcium uniporter antagonist Ru360. Our findings show rapid-onset, dynamic regulation of Deltapsi M by NO, implying that neuroprotective therapies for brain ischemia target cell death processes downstream of effects of NO on mitochondria.
Insights
Nitric oxide (NO) rapidly disrupts mitochondrial function in neuroblastoma cells, leading to cell death. Therapies for brain ischemia should target cell death pathways beyond NO
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Nitric oxide (NO) plays complex roles in neuronal function and death.
- Mitochondrial dysfunction is a key event in neurodegenerative diseases and brain ischemia.
Purpose of the Study:
- To investigate the rapid effects of nitric oxide on mitochondrial membrane potential in neuronal cells.
- To elucidate the role of NO in initiating cell death pathways.
Main Methods:
- Utilized SH-SY5Y neuroblastoma and rat cortical neurons.
- Measured mitochondrial membrane potential (Deltapsi M) using JC-1, calcein-AM, and tetramethylrhodamine methyl ester (TMRM).
- Assessed cell death and activation of caspase 3.
- Investigated the effects of cyclosporin A (CsA), BclXL, and Ru360.
Main Results:
- Diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria in neuronal cells.
- DETA-NO triggered caspase 3 activation and cell death in SH-SY5Y cells.
- Mitochondrial depolarization exhibited dynamic "flickering" patterns regulated by NO and calcium influx.
- Cyclosporin A and increased BclXL levels protected against NO-induced depolarization.
Conclusions:
- NO rapidly and dynamically regulates mitochondrial membrane potential in neurons.
- Mitochondrial depolarization induced by NO is an early event in the cascade leading to cell death.
- Neuroprotective strategies for brain ischemia may need to focus on targets downstream of NO's mitochondrial effects.