The alteration of NAD(H) binding to the apoptosis inducing factor (AIF) as a cause of neurodegeneration

Mitochondria fulfil two different functions, representing both the cell power engine and a checkpoint of programmed cell death. The latter task depends on proteins whose release from the organelle trigger apoptosis through several possible pathways. Among such proteins, is the apoptosis inducing factor (AIF), a Janus flavoprotein possessing both pro-death and pro-life activities (1). Indeed, besides its involvement in caspase-independent apoptosis, it has a major role in the maintenance of the mitochondrial respiratory chain. In mammals, AIF gene inactivation is embryonically lethal, while the so-called Harlequin mouse (Hq), where AIF expression is downregulated, display OXPHOS defect, neuromuscular impairment, and is regarded as a model of neurodegeneration. To date three human point-mutations that results in pathogenic AIF variants have been identified (1, 2). Affected subjects display a variety of severe neurological and muscular symptoms, which could stem from increased apoptogenic and/or decreased pro-life activities of mutated AIF forms. The G308E mutation in human AIF causes neurodegeneration associated with OXPHOS impairment (2). Unfortunately, the molecular mechanism of the mitochondrial action of AIF is still essentially unknown. It has be proposed that AIF could act either as a low-turnover NAD(P)H oxidoreductase or as a redox sensor (1). It has been shown that AIF slowly reacts with NAD(P)H forming a stable charge-transfer (CT) complex where reduced FAD is exceptionally resistant to oxidation by O2. CT formation is strictly coupled to protein dimerization. With the aim to dissect the mechanism of CT formation and dimerization, and to determine the effects of the G307E replacement in murine AIF (equivalent to G308E of human AIF), we investigated in vitro the properties of AIF-G307E in comparison to those of the wild-type protein. We found that CT complex formation by reaction of pre-reduced AIF with NAD+ is much faster than by reaction of oxidized AIF with NADH. A conformational transition linked to the FAD redox state induces AIF dimerization independently from ligand binding. The ligand-concentration dependence of the rate of CT formation is consistent with two-step mechanism, where an initial low-affinity complex, which is formed very fast, reorganizes more slowly to yield the final high-affinity CT dimeric ensemble. The G307E replacement has no effect on both the O2 reactivity and the dissociation rate of the dimeric CT complex, but it greatly slows down the rate of its formation. When the CT complex is generated by reaction between AIF and NADH, the mutation increases ca. 35-fold the Kd of the initial complex, while, when it is formed through the binding of NAD+ to reduced AIF, the mutation decreases ca. 40-fold the rate constant of the second step. Our results indicate that, whatever the actual process generating the CT complex of AIF within mitochondria is, its selective slowing down is per se sufficient to impair its protective action towards OXPHOS and to induce a severe progressive neurological deficit. 1. Sevrioukova, I.F. (2011) Antiox. redox signal. 14, 2545-2579. 2. Berger, I, Ben-Neriah, Z., Dor-Wolman, T., et al. (2011) Mol. Genet. Metab. 104, 517-520.