Dissipation of potassium and proton gradients inhibits mitochondrial hyperpolarization and cytochrome c release during neural apoptosis.

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.

Up-regulation of Bcl-xL in response to subtoxic beta-amyloid: role in neuronal resistance against apoptotic and oxidative injury.

Neuron death in Alzheimer's disease is believed to be triggered by an increased production of amyloidogenic beta-amyloid peptides, involving both increased oxidative stress and activation of a conserved death program. Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, is expressed at high levels in the adult nervous system. Exposure of neuronal cultures to subtoxic concentrations of beta-amyloid peptide 1-40 (1-10microM) or the fragment 25-35 (1-10microM) up-regulated both bcl-xL mRNA and Bcl-xL protein levels, determined by reverse transcriptase-polymerase chain reaction and western blot analysis. Bcl-xL protein was also up-regulated during oxidative stress induced by exposure to hydrogen peroxide (3-100microM) or ferric ions (1-10microM). In contrast, apoptotic stimuli (exposure to staurosporine or serum withdrawal) actually decreased neuronal Bcl-xL expression. To investigate the role of Bcl-xL in cell death relevant to Alzheimer's disease, we stably overexpressed Bcl-xL in human SH-SY5Y neuroblastoma cells. Cells overexpressing Bcl-xL were significantly protected from beta-amyloid neurotoxicity and staurosporine-induced apoptosis compared to vector-transfected controls. In contrast, Bcl-xL overexpression only conferred a mild protection against oxidative injury induced by hydrogen peroxide. We conclude that up-regulation of Bcl-xL expression in response to subtoxic concentrations of beta-amyloid is a stress response that increases the resistance of neurons to beta-amyloid neurotoxicity primarily by inhibiting apoptotic processes.

Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production.

An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death.

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.

Mitochondrial depolarization is not required for neuronal apoptosis.

Mitochondria are sites of cellular energy production but may also influence life and death decisions by initiating or inhibiting cell death. Mitochondrial depolarization and the subsequent release of pro-apoptotic factors have been suggested to be required for the activation of a cell death program in some forms of neuronal apoptosis. We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (STS) (300 nM). The time course of mitochondrial membrane potential (DeltaPsi(m)) during apoptosis was examined using the probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no decrease in TMRE fluorescence, indicative of mitochondrial depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE fluorescence remained unchanged up to 2 hr and thereafter actually increased significantly. Throughout this time period, the mitochondria could also be depolarized with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 microM), exhibiting the same relative magnitude of fluorescence release (unquenching) as controls. Even after 16 hr of staurosporine treatment, neurons that showed signs of nuclear apoptosis maintained DeltaPsi(m) and could be depolarized with FCCP. In contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine and an antibody to cytochrome c demonstrated at the subcellular level that mitochondrial cytochrome c release also occurred in the absence of mitochondrial depolarization. These data suggest that mitochondrial depolarization is not a decisive event in neuronal apoptosis.

Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector.

We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (30 nM, 24 hr). Treatment with the antioxidant (+/-)-alpha-tocopherol (100 microM) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 microM) significantly reduced staurosporine-induced cell death. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6-8 hr into the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. We then prepared extracts from staurosporine-treated hippocampal neurons and monitored cleavage of acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-Asp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 microM) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragmentation, but failed to prevent the rise in superoxide production and subsequent cell death. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Of note, antioxidants prevented superoxide production, caspase-3-like protease activity, and cell death even when added 4 hr after the onset of the staurosporine exposure. These results suggest a scenario of an early, caspase-1-like activity followed by a delayed intracellular superoxide production that mediates staurosporine-induced cell death of cultured rat hippocampal neurons.