The Rescue on Reperfusion Damage in Cerebral Infarction by Nelonemdaz (RODIN) Trial: Protocol for a Double-Blinded Clinical Trial of Nelonemdaz in Patients with Hyperacute Ischemic Stroke and Endovascular Thrombectomy.

BACKGROUND AND PURPOSE: Nelonemdaz (Neu2000) has both selective antagonism against 2B subunit of N-methyl-D-aspartate receptor and antioxidant activity. This drug provides sufficient evidence of neuroprotection in acute cerebral ischemia/reperfusion models. This phase III trial aims to determine this effect in patients. DESIGN: The Rescue on Reperfusion Damage in Cerebral Infarction by Nelonemdaz is a multicenter, double-blinded clinical trial. A total of 496 patients will be randomly assigned into the nelonemdaz (a total of 5,250 mg divided by 10 times for 5 days) and placebo groups. Patients will be included if they have an acute ischemic stroke (National Institutes of Health Stroke Scale score ≥8) caused by intracranial large vessel occlusion in the anterior circulation (Alberta Stroke Program Early CT Score ≥4), and if they are expected to undergo endovascular thrombectomy within 12 hours after stroke onset. ENDPOINTS: The primary endpoint is a favorable shift in the modified Rankin Scale (mRS) score at 90 days after the first dose of drug. The data will be analyzed by the Cochran-Mantel-Haenszel shift test. The secondary endpoints include functional independence (mRS 0-2) at 35 and 90 days, the favorable shift of mRS at 35 days, the proportion of mRS 0 at 35 and 90 days, and the occurrence rates of symptomatic intracranial hemorrhage within 7 days. CONCLUSION: This trial will clarify the efficacy and safety of nelonemdaz in patients with acute ischemic stroke and endovascular thrombectomy. This study has been registered at ClinicalTrials. gov (NCT05041010).

Nelonemdaz for Patients With Acute Ischemic Stroke Undergoing Endovascular Reperfusion Therapy: A Randomized Phase II Trial.

BACKGROUND: Nelonemdaz is a multitarget neuroprotectant that selectively blocks N-methyl-D-aspartate receptors and scavenges free radicals, as proven in preclinical ischemia-reperfusion studies. We aimed to evaluate the safety and efficacy of nelonemdaz in patients with acute ischemic stroke receiving endovascular reperfusion therapy. METHODS: This phase II randomized trial involved participants with large-artery occlusion in the anterior circulation at baseline who received endovascular reperfusion therapy <8 hours from symptom onset at 7 referral stroke centers in South Korea between October 29, 2016, and June 1, 2020. Two hundred thirteen patients were screened and 209 patients were randomly assigned at a 1:1:1 ratio using a computer-generated randomization system. Patients were divided into 3 groups based on the medication received-placebo, low-dose (2750 mg) nelonemdaz, and high-dose (5250 mg) nelonemdaz. The primary outcome was the proportion of patients with modified Rankin Scale scores of 0-2 at 12 weeks. RESULTS: Two hundred eight patients were assigned to the placebo (n=70), low-dose (n=71), and high-dose (n=67) groups. The groups had similar baseline characteristics. The primary outcome was achieved in 183 patients, and it did not differ among the groups (33/61 [54.1%], 40/65 [61.5%], and 36/57 [63.2%] patients; P=0.5578). The common odds ratio (90% CI) indicating a favorable shift in the modified Rankin Scale scores at 12 weeks was 1.55 (0.92-2.60) between the placebo and low-dose groups and 1.61 (0.94-2.76) between the placebo and high-dose groups. No serious adverse events were reported. CONCLUSIONS: The study arms showed no significant difference in the proportion of patients achieving modified Rankin Scale scores of 0-2 at 12 weeks. Nevertheless, nelonemdaz-treated patients showed a favorable tendency toward achieving these scores at 12 weeks, without serious adverse effects. Thus, a large-scale phase III trial is warranted. REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT02831088.

Excitotoxicity: Still Hammering the Ischemic Brain in 2020.

Interest in excitotoxicity expanded following its implication in the pathogenesis of ischemic brain injury in the 1980s, but waned subsequent to the failure of N-methyl-D-aspartate (NMDA) antagonists in high profile clinical stroke trials. Nonetheless there has been steady progress in elucidating underlying mechanisms. This review will outline the historical path to current understandings of excitotoxicity in the ischemic brain, and suggest that this knowledge should be leveraged now to develop neuroprotective treatments for stroke.

Longitudinal multiparametric MRI study of hydrogen-enriched water with minocycline combination therapy in experimental ischemic stroke in rats.

Free radicals are downstream mediators of several cytotoxic cascades contributing to ischemic brain injury. Molecular hydrogen (H2) is an antioxidant potentially useful in the treatment of stroke. Hydrogen is easy to deliver, biologically non-toxic and diffuses freely through all biological structures including the blood-brain barrier and cellular membranes. This study evaluated the efficacy of hydrogen treatments in a rat stroke model compared to vehicle-treated controls using multiparametric MRI and neurological tests. Additionally, comparison of H2 treatment alone was made with H2 combined with minocycline (H2M) treatment (12 rats per group). The primary findings were: i) H2 therapy reduced infarct volume in both H2 and H2M groups compared to controls at 1 and 7 days after stroke, and ii) both H2 and H2M improved neurologic functional recovery on day 7. The secondary outcomes were: iii) H2M treatment attenuated post-stroke hyperperfusion in the hyperacute phase, and iv) H2M markedly minimized white matter injury. In conclusion, this is the first study to use MRI to longitudinally study H2 and H2M treatment on ischemic stroke and the first study to compare H2 treatment combined with another potential stroke therapeutic (H2M).

Elevated plasma biomarkers of inflammation in acute ischemic stroke patients with underlying dementia.

BACKGROUND: The blood-brain barrier has been a hindrance to developing blood-based diagnostic tests for dementias, as it limits the appearance of brain biomarkers in the blood. Our aim was to see if the natural opening of the blood-brain barrier induced by ischemic stroke would increase serum levels of inflammatory biomarkers known to be elevated in the brains of patients with Alzheimer's disease and other neurodegenerative dementias. METHODS: Forty-three patients with acute ischemic stroke presenting to Stony Brook University Hospital were prospectively enrolled in the study. Eight of these patients were clinically diagnosed as having an underlying neurodegenerative dementia. Blood was drawn acutely within 72 h of stroke symptom onset, and serum levels of the classic inflammatory biomarkers, interleukin-6 (IL-6) and C-reactive protein (CRP) were measured, along with levels of S100B protein (S100B) and complement C3 (CC3). RESULTS: Serum levels of IL-6 and CRP in patients with acute ischemic stroke and underlying dementia (AIS + D) were significantly higher (p = 0.002 and 0.003, respectively) than in patients with acute ischemic stroke alone (AIS). Serum levels of S100B and CC3 did not differ significantly between the groups. CONCLUSIONS: This study supports the possibility that opening of the blood-brain barrier may enhance the blood appearance of brain tissue markers of inflammation associated with neurodegenerative dementia. Further study is warranted to test this possibility, given the recent emergence of methods to open the blood-brain barrier for diagnostic or therapeutic purposes.

Safety and Optimal Neuroprotection of neu2000 in acute Ischemic stroke with reCanalization: study protocol for a randomized, double-blinded, placebo-controlled, phase-II trial.

BACKGROUND: The potential of neuroprotective agents should be revisited in the era of endovascular thrombectomy (EVT) for acute large-artery occlusion because their preclinical effects have been optimized for ischemia and reperfusion injury. Neu2000, a derivative of sulfasalazine, is a multi-target neuroprotectant. It selectively blocks N-methyl-D-aspartate receptors and scavenges for free radicals. This trial aimed to determine whether neuroprotectant administration before EVT is safe and leads to a more favorable outcome. METHODS: This trial is a phase-II, multicenter, three-arm, randomized, double-blinded, placebo-controlled, blinded-endpoint drug trial that enrolled participants aged ≥ 19 years undergoing an EVT attempt less than 8 h from symptom onset, with baseline National Institutes of Health Stroke Scale (NIHSS) score ≥ 8, Alberta Stroke Program Early CT score ≥ 6, evidence of large-artery occlusion, and at least moderate collaterals on computed tomography angiography. EVT-attempted patients are randomized into control, low-dose (2.75 g), and high-dose (5.25 g) Neu2000KWL over 5 days. Seventy participants per group are enrolled for 90% power, assuming that the treatment group has a 28.4% higher proportion of participants with functional independence than the placebo group. The primary outcome, based on intention-to-treat criteria is the improvement of modified Rankin Scale (mRS) scores at 3 months using a dichotomized model. Safety outcomes include symptomatic intracranial hemorrhage within 5 days. Secondary outcomes are distributional change of mRS, mean differences in NIHSS score, proportion of NIHSS score 0-2, and Barthel Index > 90 at 1 and 4 weeks, and 3 months. DISCUSSION: The trial results may provide information on new therapeutic options as multi-target neuroprotection might mitigate reperfusion injury in patients with acute ischemic stroke before EVT. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02831088 . Registered on 13 July 2016.

Medicines for the mind: policy-based "pull" incentives for creating breakthrough CNS drugs.

Several large pharmaceutical companies have selectively downsized their neuroscience research divisions, reflecting a growing view that developing drugs to treat brain diseases is more difficult and often more time-consuming and expensive than developing drugs for other therapeutic areas, and thus represents a weak area for investment. These withdrawals reduce global neuroscience translational capabilities and pose a serious challenge to society's interests in ameliorating the impact of nervous system diseases. While the path forward ultimately lies in improving understandings of disease mechanisms, many promising therapeutic approaches have already been identified, and rebalancing the underlying risk/reward calculus could help keep companies engaged in making CNS drugs. One way to do this that would not require upfront funding is to change the policies that regulate market returns for the most-needed breakthrough drugs. The broader neuroscience community including clinicians and patients should convene to develop and advocate for such policy changes.

nNOS(+) striatal neurons, a subpopulation spared in Huntington's Disease, possess functional NMDA receptors but fail to generate mitochondrial ROS in response to an excitotoxic challenge.

Huntington's disease (HD) is a neurodegenerative condition characterized by severe neuronal loss in the cortex and striatum that leads to motor and behavioral deficits. Excitotoxicity is thought to be involved in HD and several studies have indicated that NMDA receptor (NMDAR) overactivation can play a role in the selective neuronal loss found in HD. Interestingly, a small subset of striatal neurons (less than 1% of the overall population) is found to be spared in post-mortem HD brains. These neurons are medium-sized aspiny interneurons that highly express the neuronal isoform of nitric oxide synthase (nNOS). Intriguingly, neurons expressing large amounts of nNOS [hereafter indicated as nNOS(+) neurons] show reduced vulnerability to NMDAR-mediated excitotoxicity. Mechanisms underlying this reduced vulnerability are still largely unknown and may shed some light on pathogenic mechanisms involved in HD. One untested possibility is that nNOS(+) neurons possess fewer or less functioning NMDARs. Employing single cell calcium imaging we challenged this hypothesis and found that cultured striatal nNOS(+) neurons show NMDAR-evoked responses that are identical to the ones observed in the overall population of neurons that express lower levels of nNOS [nNOS(-) neurons]. NMDAR-dependent deregulation of intraneuronal Ca(2+) is known to generate high levels of reactive oxygen species of mitochondrial origin (mt-ROS), a crucial step in the excitotoxic cascade. With confocal imaging and dihydrorhodamine (DHR; a ROS-sensitive probe) we compared mt-ROS levels generated by NMDAR activation in nNOS(+) and (-) cultured striatal neurons. DHR experiments revealed that nNOS(+) neurons failed to produce significant amounts of mt-ROS in response to NMDA exposure, thereby providing a potential mechanism for their reduced vulnerability to excitotoxicity and decreased vulnerability in HD.

Transient decrease in F-actin may be necessary for translocation of proteins into dendritic spines.

It remains poorly understood as to how newly synthesized proteins that are required to act at specific synapses are translocated into only selected subsets of potentiated dendritic spines. Here, we report that F-actin, a major component of the skeletal structure of dendritic spines, may contribute to the regulation of synaptic specificity of protein translocation. We found that the stabilization of F-actin blocked the translocation of GFP-CaMKII and inhibited the diffusion of 3-kDa dextran into spines (in 2-3 weeks cultures). Neuronal activation in hippocampal slices and cultured neurons led to an increase in the activation (decrease in the phosphorylation) of the actin depolymerization factor, cofilin, and a decrease in F-actin. Furthermore, the induction of long-term potentiation by tetanic stimulation induced local transient depolymerization of F-actin both in vivo and in hippocampal slices (8-10 weeks), and this local F-actin depolymerization was blocked by APV, a N-methyl-D-aspartate (NMDA) receptor antagonist. These results suggest that F-actin may play a role in synaptic specificity by allowing protein translocation into only potentiated spines, gated through its depolymerization, which is probably triggered by the activation of NMDA receptors.

Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia.

The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.

Cu2+ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo.

Wilson's disease results from mutations in the P-type Cu(2+)-ATPase causing Cu(2+) toxicity. We previously demonstrated that exposure of mixed neuronal/glial cultures to 20 microM Cu(2+) induced ATP loss and death that were attenuated by mitochondrial substrates, activators, and cofactors. Here, we show differential cellular sensitivity to Cu(2+) that was equalized to 5 microM in the presence of the copper exchanger/ionophore, disulfiram. Because Cu(2+) facilitates formation of oxygen radicals (ROS) which inhibit pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), we hypothesized that their inhibition contributed to Cu(2+)-induced death. Toxic CU(2+) exposure was accompanied by early inhibition of neuronal and hepatocellular PDH and KGDH activities, followed by reduced mitochondrial transmembrane potential, DeltaPsi(M). Thiamine (1-6 mM), and dihydrolipoic acid (LA, 50 microM), required cofactors for PDH and KGDH, attenuated this enzymatic inhibition and subsequent death in all cell types. Furthermore, liver PDH and KGDH activities were reduced in the Atp7b mouse model of Wilson's disease prior to liver damage, and were partially restored by oral thiamine supplementation. These data support our hypothesis that Cu(2+)-induced ROS may inhibit PDH and KGDH resulting in neuronal and hepatocellular death. Therefore, thiamine or lipoic acid may constitute potential therapeutic agents for Wilson's disease.

Raising intracellular calcium attenuates neuronal apoptosis triggered by staurosporine or oxygen-glucose deprivation in the presence of glutamate receptor blockade.

The relationship between intracellular Ca(2+) ([Ca(2+)](i)) regulation and programmed cell death is not well-defined; both increases and decreases in [Ca(2+)](i) have been observed in cells undergoing apoptosis. We determined [Ca(2+)](i) in cultured murine cortical neurons undergoing apoptosis after exposure to staurosporine or following oxygen-glucose deprivation in the presence of glutamate receptor antagonists. Neuronal [Ca(2+)](i) was decreased 1-4 h after exposure to staurosporine (30 nM). A [Ca(2+)](i) decrease was also observed 1 h after the end of the oxygen-glucose deprivation period when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were added to the bathing medium during the deprivation period. A similar decrease in [Ca(2+)](i) produced by reducing extracellular Ca(2+) or chelating intracellular Ca(2+) was sufficient to induce neuronal apoptosis. Raising [Ca(2+)](i) either by activating voltage-sensitive Ca(2+) channels with (-) Bay K8644 or by application of low concentrations of kainate attenuated both staurosporine and oxygen-glucose deprivation-induced apoptosis.

Potassium attenuates zinc-induced death of cultured cortical astrocytes.

Transient global ischemia induces CA1 hippocampal neuronal death without astrocyte death, perhaps mediated in part by the toxic translocation of zinc from presynaptic terminals to postsynaptic neurons. We tested the hypothesis that cellular depolarization, which occurs in the ischemic brain due to increased extracellular potassium and energy failure, might contribute to astrocyte resistance to zinc-induced death. We previously reported that neurons in mixed cortical neuronal-astrocyte cultures were more vulnerable to a 5-15-min exposure to Zn(2+) than astrocytes in the same cultures. In the present report, we show that (1) neurons in isolation or in conjunction with astrocytes were 2-3-fold more sensitive to a 15-min nondepolarizing Zn(2+) exposure than are glia; (2) KCl-induced depolarization attenuated glial vulnerability to zinc toxicity but potentiated neuronal vulnerability to zinc toxicity; (3) Zn(2+)-induced glial death was attenuated by T-type Ca(2+) channel blockade, as well as compounds that increase NAD(+) levels; and (4) both astrocytic (65)Zn(2+) accumulation and the increase in astrocytic [Zn(2+)](i) induced by Zn(2+) exposure were also attenuated by depolarization or T-type Ca(2+) channel blockers. Zn(2+)-induced cell death in astrocytes was at least in part apoptotic, as caspase-3 was activated, and the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone partially attenuated Zn(2+)-induced death. The levels of peak [Zn(2+)](i) achieved in astrocytes during this toxic nondepolarizing Zn(2+) exposure (250 nM) were substantially greater than those achieved in neurons (40 nM). In glia, exposure to 400 microM Zn(2+) induced a 13-mV depolarization, which can activate T-type Ca(2+) channels. This Zn(2+)-induced astrocyte death, like neuronal death, was attenuated by the addition of pyruvate or niacinamide to the exposure medium.

Involvement of poly ADP ribosyl polymerase-1 in acute but not chronic zinc toxicity.

We have previously suggested that zinc-induced neuronal death may be mediated in part by inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), secondary to depletion of the essential cosubstrate NAD+. Given convergent evidence implicating the NAD+-catabolizing enzyme, poly ADP ribosyl polymerase (PARP) in mediating ATP depletion and neuronal death after excitotoxic and ischemic insults, we tested the specific hypothesis that the neuronal death induced by exposure to toxic levels of extracellular zinc might be partly mediated by PARP. PARP was activated in cultured mouse cortical astrocytes after a toxic acute Zn2+ exposure (350 microm Zn2+ for 15 min), but not in cortical neurons or glia after exposure to a toxic chronic Zn2+ exposure (40 microm Zn2+ for 1-4 h), an exposure sufficient to deplete NAD+ and ATP levels. Furthermore, the neurotoxicity induced by acute, but not chronic, Zn2+ exposure was reduced in mixed neuronal-glial cultures prepared from mutant mice lacking the PARP gene. These data suggest PARP activation may contribute to more fulminant forms of Zn2+-induced neuronal death.

TrkB mediates BDNF-induced potentiation of neuronal necrosis in cortical culture.

In the present study, the signaling mechanisms underlying the effect of brain-derived neurotrophic factor (BDNF) on neuronal necrosis were investigated. Exposure of mature mouse cortical cultures (more than 10 days in vitro (DIV)) to 50-100 ng/ml BDNF for 48 h induced widespread neuronal necrosis that was antioxidant-sensitive. This neuronal necrosis was blocked by the selective NMDA antagonist MK-801, suggesting that prolonged BDNF exposure caused endogenous levels of NMDA receptor activation to become excitotoxic. We examined whether the p75(NTR) played a role in BDNF-induced neuronal death. However, p75(NTR) expression was low in cultured cortical cells, and neutralizing antibodies to p75(NTR) did not attenuate BDNF-triggered neuronal death. In contrast, trkB antisense oligonucleotides and inhibitors of Trk tyrosine kinase blocked BDNF-triggered neuronal death as well as BDNF potentiation of iron-induced oxidative neuronal necrosis, suggesting a critical role for TrkB in this phenomenon. Furthermore, BDNF did not potentiate neuronal necrosis in cortical cultures prepared from embryonic TrkB-null mice. These results suggest that TrkB plays an important role in BDNF-mediated neuronal necrosis.

Membrane-permeant chelators can attenuate Zn2+-induced cortical neuronal death.

Chelating extracellular Zn(2+) with the membrane-impermeant Zn(2+) chelator, CaEDTA, can inhibit toxic Zn(2+) influx and subsequent neuronal death. However, this drug does not cross the blood-brain barrier. In the present study, we explored the ability of two membrane-permeant Zn(2+) chelators to inhibit Zn(2+)-induced death of cultured cortical neurons. Addition of either the high affinity (K(D)=10(-15.6)) Zn(2+) chelator, N, N, N', N', tetrakis (2-pyridylmethyl) etylenediaminepentaethylene (TPEN), or the low affinity (K(D)=10(-6)) Zn(2+) chelator, 1-hydroxypyridine-2-thione (pyrithione), to the culture medium following exposure to extracellular Zn(2+) reduced subsequent neuronal death, even if chelator administration was delayed by up to 1 h. Indeed, some delay was essential for neuroprotection with pyrithione, as co-administration of pyrithione together with extracellular Zn(2+) increased levels of [Zn(2+)](i) and cell death compared to the levels induced by Zn(2+) alone. TPEN, but not pyrithione, was intrinsically toxic at high concentrations, likely due to excessive chelation of [Zn(2+)](i), as this intrinsic toxicity was reduced by prior addition of extracellular Zn(2+). These data point to a potential therapeutic role for membrane-permeant Zn(2+) chelators, perhaps especially possessing low Zn(2+) affinity, in attenuating neuronal death after certain acute insults.

Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress.

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.