Abstract
The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.
Highlights
The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries
Triggered by Calpain and Bid—To examine the ability of calpain I to cleave and release endogenous AIF from mitochondria, we initially used isolated mouse liver mitochondria as a model system because, in contrast to brain mitochondria, these mitochondria represent a homogenous population that is highly sensitive to mitochondrial outer membrane permeabilization by Bid (Fig. 1)
When mitochondria were treated with the same amount of active calpain I (2.5 units/ml) in the presence of Bid (100 nM), a dramatic enhancement of AIF release occurred compared with calpain treatment alone
Summary
Is an approach frequently employed to examine the mechanistic underpinnings of the release of toxic proteins normally confined to the intermembrane space [23]. In this system, mitochondrial permeabilization by the pro-apoptotic Bcl-2 family members Bid and Bax failed to reconstitute the AIF redistribution observed in cell and tissue models, apoptogenic cytochrome c, Smac/DIABLO, and Htra2/Omi were faithfully released [21]. Pure lipid vesicles of mitochondrial inner membrane composition were employed to directly assess the effect of calpain on the ability of AIF to associate with phospholipids
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