Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons

Yulia E Kushnareva,Sandra E Wiley,Manus W Ward,Alexander Y Andreyev,Anne N Murphy

doi:10.1074/jbc.m503090200

Abstract

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.

Highlights

Glutamate excitotoxicity underlies neuronal loss in ischemic and traumatic brain injury [1] and likely contributes to dysfunction in chronic forms of neurodegeneration
Mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2؉ uptake capacity (686 ؎ 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2؉ was a smaller percentage of the maximal Ca2؉ uptake capacity
This study indicated that Ca2؉-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death

Summary

Introduction

Glutamate excitotoxicity underlies neuronal loss in ischemic and traumatic brain injury [1] and likely contributes to dysfunction in chronic forms of neurodegeneration. Even though ionic homeostasis appears to be lost and mitochondrial Ca2ϩ loading is maximal after 10 min of glutamate exposure (Fig. 1), if this treatment is halted by addition of MK-801 for the ensuing 24 h, the injury is no longer sufficient to induce LDH release substantially above control levels (Fig. 3), at least at this time point.

Results

Conclusion