Excitotoxic Calcium Overload in a Subpopulation of Mitochondria Triggers Delayed Death in Hippocampal Neurons

Abstract

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons. Beyond transient elevation of cytosolic calcium and perturbation of Na ϩ /K ϩ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca 2ϩ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function.

Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells

“…Therefore, minocycline reduces the electrochemical driving force required for mitochondrial Ca 2+ uptake, resulting in the prevention of [Ca 2+ ] mit overload in excitotoxicity. Accordingly, prevention of mitochondrial Ca 2+ uptake by the mitochondrial Ca 2+ uncoupler FCCP largely abrogated NMDA-induced cell death [9]. Therefore, minocycline inhibit mitochondrial Ca 2+ overload induced by NMDA acting both at the plasma membrane level and at the mitochondrial level.…”

“…It also reduced [Ca 2+ ] mit overload, a pivotal role in excitotoxicity and other models of cell death [9,12,15]. By using bioluminescence imaging with mitochondria-targeted aequorin, we directly observed the effects of minocycline on NMDA-induced mitochondrial Ca 2+ overload.…”

“…Thus, small changes in m may contribute to the cytoprotection mechanisms. Mitochondrial depolarization, caused by low concentrations of mitochondrial uncouplers, has been shown to be protective against NMDA-induced neuronal death [9].…”

“…In addition, mitochondrial Ca 2+ overload plays a pivotal role in many cell death models including excitotoxicity [11]. Specifically, it has been reported that glutamate-induced neurotoxicity depends on mitochondrial Ca 2+ uptake ([Ca 2+ ] m ) [9,12]. Under stress conditions, there is an early increase in [Ca 2+ ] m that may contribute to mitochondrial dysfunction [13,14] and MPTP opening, a point-of-no-return step in the intrinsic pathway of apoptotic cell death [15,16].…”

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

“…Qualitative analyses revealed that more mitochondria were present in the peripheral region of PC somata, in line with the morphometric analysis of EM pictures. Since under pathological conditions such as excitotoxicity, a swelling and rounding up of mitochondria is observed (Bendotti et al, 2001;Pivovarova et al, 2004), the morphology of the peripheral mitochondria was investigated on high-resolution EM images (Fig. 4).…”