Abstracts

RATIONALE: Mitochondrial dysfunction and damage have been proposed as key proponents in the pathogenesis of diseases resulting from excitotoxicity. The objective of this study was to investigate whether mitochondrial dysfunction precedes the tissue damage observed after administration of kainic acid. Thus, we performed biochemical studies to examine mitochondrial function in forebrain mitochondria isolated from strains previously identified as resistant (C57BL/6) and vulnerable (FVB/N) to kainate-induced cell death.
METHODS: Non-synaptic mitochondria were isolated from either intact C57BL/6 and FVB/N mice or 24 hours following kainate administration using a previously defined method (Sims, 1993). Induction of the mitochondrial permeability transition pore was estimated from the loss of absorbance at 540 nm measured in energized mitochondrial suspensions. Extramitochondrial free Ca2+ was measured in isolated mitochondria using the hexapotassium salt of Calcium Green-5N. Mitochondrial membrane potential was measured using a cell-permeant dye that accumulates in the mitochondrial matrix (JC-1). Energized mitochondria were incubated at 37[degree]C in the presence of 5 [mu]M JC-1 for 15 minutes and fluorescence was measured with a multi-well plate reader.
RESULTS: Induction of the mitochondrial permeability transition pore in isolated forebrain mitochondria from both strains was observed following the addition of CaCl[sub]2[/sub] (10-100nmol/mg). However, mitochondria from C57BL/6 mice were less sensitive to the effects of increasing Ca2+ as compared to mitochondria from FVB/N mice. Exposure to high calcium levels or kainate appeared to trigger substantial mitochondrial calcium accumulation in FVB/N mice as compared to C57BL/6 mice. In accordance with these results, the addition of Ca2+ induced a significant strain-dependent difference in mitochondrial membrane potential. Only FVB/N mice showed a significant decrease in membrane potential as compared to C57BL/6 mice following calcium addition or after kainate administration.
CONCLUSIONS: While the reason for the selective resistance of C57BL/6 mice to kainate-induced cell death is primarily unknown, results from the present study suggest that mitochondrial dysfunction may not be observed in C57BL/6 mice following exposure to high levels of calcium or following kainate administration. These results suggest that mitochondria from C57BL/6 mice may be able to buffer rises in [Ca2+][sub]i[/sub] such that ATP synthesis is not impaired and mitochondrial membrane homeostasis is maintained. In conclusion, the present study demonstrates that early changes in mitochondrial function relate to genetically determined differences in susceptibility to kainate-induced cell death and may help identify critical molecular events involved in the sequence from mitochondrial dysfunction to cell death.
Support: The James D. and Delia B. Baxter Foundation and NIH grant NS38696-01.