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Mitochondrial permeability transition can be directly monitored in living neurons
T Gillessen1, C Grasshoff, L Szinicz
1Max-Planck-Institut of Psychiatry, Munich, Germany. gillessen@mpipsykl.mpg.de
Abstract:
Mitochondria have been suggested as key players in apoptotic cell death of neurons and many other tissues, since the release of proapoptotic molecules from mitochondria is implicated in caspase activation. As a potential release mechanism, the occurrence of a large pore opening in the inner membrane (mitochondrial permeability transition pore, PTP) has been proposed, but has not yet been observed directly in neurons. We investigated whether the calcein/Co2+-quenching technique introduced by Petronilli et al. [Biofactors 8 (1998) 263], which allows direct observation of PTP opening, can be applied to neurons. Exposure of calcein-loaded neurons to Co2+ ions resulted in the fading of diffuse cytoplasmic calcein fluorescence, with organelle-restricted fluorescent spots remaining. These spots were colocalized with mitochondrially-entrapped tetramethylrhodamineethylester (TMRE) fluorescence and corresponded to colocalization of calcein and TMRE fluorescence in digitonin-permeabilized neurons. Importantly, extensive neuronal calcium loading, which is assumed to induce PTP opening, resulted in significant fading of mitochondrial fluorescence, suggesting the occurrence of permeability transition. This fluorescence decrease could be completely prevented by the PTP blocker cyclosporin A.
Insights
Researchers directly observed the mitochondrial permeability transition pore (PTP) opening in neurons for the first time. This finding provides direct evidence for PTP
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Mitochondria play a crucial role in neuronal apoptosis via the release of proapoptotic factors.
- The mitochondrial permeability transition pore (PTP) is a proposed mechanism for this release but has not been directly visualized in neurons.
- Understanding PTP dynamics is vital for comprehending neuronal cell death pathways.
Purpose of the Study:
- To adapt and apply the calcein/Co2+-quenching technique for direct observation of PTP opening in neurons.
- To investigate whether neuronal calcium loading induces PTP opening.
- To confirm the role of PTP in neuronal cell death signaling.
Main Methods:
- Utilized the calcein/Co2+-quenching assay to monitor PTP activity in cultured neurons.
- Employed mitochondrially-entrapped tetramethylrhodamineethylester (TMRE) for mitochondrial colocalization.
- Induced PTP opening via extensive neuronal calcium loading and assessed the effect of cyclosporin A.
Main Results:
- The calcein/Co2+-quenching technique successfully visualized PTP opening in neurons, evidenced by changes in calcein fluorescence.
- Mitochondrial fluorescence colocalized with remaining calcein spots, confirming organelle-specific pore activity.
- Neuronal calcium loading led to a significant decrease in mitochondrial fluorescence, indicating PTP opening, which was blocked by cyclosporin A.
Conclusions:
- The calcein/Co2+-quenching technique is effective for directly observing mitochondrial permeability transition pore opening in neurons.
- Neuronal calcium overload triggers PTP opening, supporting its role in neuronal cell death.
- Cyclosporin A effectively inhibits PTP opening in neurons, highlighting its therapeutic potential.