Pharmacological strategies to block rod photoreceptor apoptosis caused by calcium overload: a mechanistic target-site approach to neuroprotection.

PURPOSE: Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis. METHODS: Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis. RESULTS: Ca2+ overload preceded rod apoptosis in mice and rats, although the extent and kinetics in each differed significantly. The isolated rat model of rod Ca2+ overload/apoptosis showed that blockade of Ca2+ entry through rod cGMP-activated channels with L-cis diltiazem was partially neuroprotective, whereas blockade of Ca2+ entry into rods through L-type Ca2+ channels with D-cis diltiazem or verapamil provided no protection. Inhibition of the mitochondrial Na+/Ca2+ exchanger with D-cis diltiazem provided no protection. CsA and NIM811, mitochondrial permeability transition pore (mPTP) inhibitors, blocked all Ca(2+)-induced apoptosis, whereas the caspase-3 inhibitor DEVD-fmk only blocked the downstream cytochrome c-induced apoptosis. CONCLUSIONS: The successful pharmacological neuroprotective strategies for rod Ca2+ overload/apoptosis targeted the rod cGMP-activated channels or mPTP, but not the rod L-type Ca2+ channels.

Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore.

Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca(2+)- and/or Pb(2+)-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca(2+)] and/or [Pb(2+)] were localized to photoreceptors and produced rod-selective apoptosis. Ca(2+) and/or Pb(2+) induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca(2+) and Pb(2+) were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH(2)F and carbobenzoxy-Asp-Glu-Val-Asp-CH(2)F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH(2)F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca(2+) and Pb(2+). Moreover, they suggest that Ca(2+) and Pb(2+) bind to the internal metal (Me(2+)) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.

Lead-induced alterations in retinal cGMP phosphodiesterase trigger calcium overload, mitochondrial dysfunction and rod photoreceptor apoptosis.

Lead exposure results in the selective apoptotic loss of rods and bipolar cells. During and following developmental lead exposure rod/retinal cGMP phosphodiesterase expression and activity are delayed in onset and decreased, [Ca2+] is elevated, and mitochondrial ATP synthesis is decreased. In vitro studies, using retinas incubated in Ca2+ and/or Pb2+, demonstrate that rods selectively die by apoptosis, retinal mitochondrial ATP synthesis is decreased, mitochondrial cytochrome c is released and caspase activity is increased. These results suggest that lead-induced rod and bipolar cell apoptosis is triggered by Ca2+ and Pb2+ overload due to altered cGMP phosphodiesterase activity and that mitochondrial alterations play a central role in this process.