DECREASES IN ROD PHOTORECEPTOR MITOCHONDRIAL RESPIRATION OCCUR UPSTREAM OF CASPASE ACTIVATION IN DIVALENT CATION–INDUCED ROD–SELECTIVE APOPTOSIS.

Abstract

Abstract: : Purpose:Rod–selective apoptosis produced by developmental lead exposure in mice is mediated by the translocation of cytosolic Bax to Pb²⁺ and Ca²⁺ overloaded rod mitochondria, loss of the mitochondrial transmembrane potential (Δψm) and activation of the cytochrome c (cyt c)–caspase cascade (He et al., PNAS 2003). Ricci et al. (JCB 2003) suggest that caspase–3 mediates the loss of Δψm in permeabilized cells by disrupting complex I and II, but not complex III, of the electron transport chain (ETC). Therefore we investigated whether this latter mechanism occurs in rods during divalent cation overload. Methods:Isolated retinas from adult wild–type (controls) and transgenic mice overexpressing Bcl–x_L only in photoreceptors were briefly exposed to Ca²⁺ and/or Pb²⁺. Isolated adult rat retinal mitochondria, in the presence of different site–specific substrates, were exposed to nanomolar concentrations of Pb²⁺ with or without Ca²⁺. Results:In isolated mouse retinas, Pb²⁺ and/or Ca²⁺ induced rod–selective apoptosis characterized by genomic DNA fragmentation, Bax translocation to the mitochondria, decreases in rod mitochondrial respiration and Δψm, cyt c release and caspase activation. Bcl–x_L overexpression or the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A (CsA) completely blocked all these divalent cation–induced apoptotic effects, whereas the pan–caspase inhibitor z–VAD–fmk only blocked caspase activation and DNA fragmentation. In isolated rod mitochondria, Pb²⁺ with or without Ca²⁺ produced mitochondrial swelling, cyt c release and concentration–dependent decreases in mitochondrial respiration at complexes I, II and III of the ETC. CsA, but not z–VAD–fmk, completely blocked the divalent cation–induced mitochondrial swelling, cyt c release and inhibition of respiration at all three ETC sites. Conclusions:These results show that Bcl–x_L and CsA prevent the loss of Δψm, inhibition of ETC and rod–selective apoptosis in Bax permeabilized and divalent cation–overloaded rod mitochondria by blocking the release of cyt c through the PTP. Thus, the caspases were activated downstream of, and not prior to, mitochondrial cyt c release. Therefore the caspases do not appear to play a role in the inhibition of rod mitochondrial respiration. These findings have relevance for therapies in progressive human and animal retinal and neuronal degenerations where divalent cation overload and mitochondrial dysfunction occur.

Keywords: apoptosis/cell death • mitochondria • photoreceptors