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.

Familial hypercholesterolaemia (FH) is characterized by increased circulating low-density lipoprotein (LDL) cholesterol leading to premature atherosclerosis and coronary heart disease. Although FH is usually caused by mutations in LDLR, mutations in APOB and PCSK9 also cause FH but only a few mutations have been reported, APOB p.R3527Q being the most common. However, 30-80% of clinical FH patients do not present an identifiable mutation in any of the described genes. To identify the genetic cause of the hypercholesterolaemia in 65 patients without mutations in LDLR, PCSK9 or in fragments of exon 26 and 29 of APOB currently analysed, we performed whole sequencing of APOB by pyrosequencing. A total of 10 putative mutations in APOB were identified. Flow cytometry with fluorescently labelled LDL from patients and relatives showed that p.Arg1164Thr (exon 22) and p.Gln4494del (exon 29) presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB3527. The proliferation assays with U937 cells showed reduced growth for both cases. The variant p.Tyr1247Cys was found to be neutral and other three alterations were considered polymorphisms. Our results emphasize the need to study the whole APOB in routine protocols to improve patient identification and cardiovascular risk assessment.

p.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.