Mechanisms of cyanide neurotoxicity: A role for Bcl -2 proteins

Yan Shou, Purdue University

Abstract

The purpose of the present study was to explore the mechanisms underlying cyanide cytotoxicity in primary cultured cortical neurons. Previous in vivo studies suggested that cyanide induced different modes of cell death in different brain area: necrosis was observed in substantia nigra while cells died mainly through apoptosis in cortex. Based upon morphological and biochemical criteria, cell death induced by cyanide (100–300 μM) in cortical neurons was characterized as apoptosis or programmed cell death. Reactive oxygen species (ROS) and cytosolic free calcium were key mediators in cyanide neurotoxicity. Immediately after cyanide treatment, ROS were generated accompanied by an elevation of intracellular calcium. Increase in cellular stress induced by cyanide further activated cellular redox-sensitive factors including NF-κB and p38 mitogen activated protein (MAP) kinase. Cytochrome c release from mitochondria was a critical event in apoptotic cell death. Once in cytosol, cytochrome c bound to apoptotic protease activating factor 1 (Apaf-1) and further activated caspase cascade. Using western blot analysis and immunohistochemical staining, we established that cytochrome c was released from mitochondria in cyanide-induced apoptosis. Bcl-2 protein family played an important role in regulating cytochrome c release. Both transcriptional regulation and subcellular translocation of Bcl-2 proteins were observed in cyanide-induced apoptosis. Upregulation of two pro-apoptotic members of Bcl-2 protein family: Bcl-Xs and Bax occurred as a result of NF-κB activation. Cytosol-to-mitochondria translocation of Bax and Bad were induced by cyanide. Activation of p38 MAPK was responsible for Bax translocation while calcium-activated-calcineurin contributed to Bad translocation. As a result of transcriptional regulation and subcellular redistribution of Bcl-2 proteins, pro-apoptotic Bcl-2 protein levels were increased in mitochondria which further facilitated cytochrome c release by mechanisms that are still under investigation. Specific inhibitors for NF-κB, p38 MAPK and calcineurin were employed to block the increased levels of pro-apoptotic Bcl-2 proteins in mitochondria and our results suggested that these inhibitors all partly inhibited cytochrome c release and apoptotic cell death induced by cyanide.

Degree

Ph.D.

Advisors

Isom, Purdue University.

Subject Area

Toxicology|Pharmacology

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