Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization

Background: The pathogenesis of bilirubin encephalopathy and Alzheimer's di sease appears to result from accumulation of unconjugated bilirubin (UCB) a nd amyloid-beta (A beta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembra ne proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) a nd tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and no nhepatic cells. In this study, we further characterize UCB- and A beta -ind uced cytotoxicty in isolated neural cells, and investigate membrane perturb ation during incubation of isolated mitochondria with both agents. In addit ion, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. Materials and Methods: Primary rat neuron and astrocyte cultures were incub ated with UCB or A beta peptide, either alone or in the presence of UDC. Ap optosis was assessed by DNA fragmentation and nuclear morphological changes . Isolated mitochondria were treated with each toxic, either alone or in co mbination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was mea sured spectrophotometrically and cytochrome c protein levels determined by Western blot. Results: Incubation of neural cells with both UCB and A beta induced apopto sis (p < 0.01). Coincubation with UDC reduced apoptosis by >50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by A<beta> (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and A<beta>, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swe lling and cytochrome c efflux mediated by UCB (p < 0.05). Conclusion: UCB and A beta peptide activate the apoptotic machinery in neur al cells. Toxicity occurs through a mitochondrial-dependent pathway which i n part involves opening of the permeability transition pore. Furthermore, m embrane permeabilization is required for cytochrome c release from mitochon dria and can be prevented by UDC or TUDC. These data suggest that the mitoc hondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC ma y be potential therapeutic agents.