Cytochrome c Is Released from Mitochondria in a Reactive Oxygen Species (ROS)-dependent Fashion and Can Operate as a ROS Scavenger and as a Respiratory Substrate in Cerebellar Neurons Undergoing Excitotoxic Death

TLDR

In rat cerebellar granule cells, glutamate toxicity induces both reactive oxygen species production and cytochrome c release, the latter driven by ROS generated either by glutamate or by an exogenous xanthine/xanthine oxidase system. The study aimed to determine the relationship between ROS production and cytochrome c release and to clarify whether released cytochrome c functions as a defense against oxidative stress during early neurotoxicity. During the early 30‑min phase of glutamate toxicity, ~40 % of cytochrome c is released from coupled, intact mitochondria with low ROS, whereas after 3 h mitochondria become uncoupled and produce high ROS; the released cytochrome c can transfer electrons from superoxide to oxygen via the respiratory chain and partially prevent glutamate toxicity when added externally.

Abstract

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome <i>c</i> take place during glutamate toxicity. This investigation was aimed (<i>i</i>) to ascertain whether and how these two processes are related and (<i>ii</i>) to gain insight into the role played by the released cytochrome <i>c</i> in the onset of neurotoxicity. Cytochrome <i>c</i> release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome <i>c</i> release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome <i>c</i> was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochrome<i>c</i> release can be part of a cellular and mitochondrial defense mechanism against oxidative stress.