Neurodegeneration in an Aβ-induced model of Alzheimer's disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Aβ exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Aβ_1–40 synthetic peptide. In mice icv-injected with Aβ, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Aβ-injected mice were prevented by blocking calpain activation with MDL28170 , which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Aβ. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Aβ peptides in vivo , acting as a link between diverse neurotoxic pathways of AD.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

β-Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late-onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to β-amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ_1–42, Aβ_1–40, and Aβ_25–35, increase significantly apoE-liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ_25–35, was further shown to increase significantly the internalization of both apoE3- and apoE4-liposomes, without affecting apoE degradation. Conversely, Aβ_1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE-liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.     

Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo-lytic fragment of the membrane-associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial "plaques" created from aggregated synthetic Aβ peptides. These correspond to full-length Aβ residues 1–40 (Aβ1–40), an internal β-sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ-neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly-peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.     

Rapid Communication: Ca2+ Channel Blockers Attenuate β-Amyloid Peptide Toxicity to Cortical Neurons in Culture

Abstract: Deposit of β-amyloid protein (Aβ) in Alzheimer's disease brain may contribute to the associated neurodegeneration. We have studied the neurotoxicity of Aβ in primary cultures of murine cortical neurons, with the aim of identifying pharmacologic ways of attenuating the injury. Exposure of cultures to Aβ (25–35 fragment; 3–25 4mU M ) generally triggers slow, concentration-dependent neurodegeneration (over 24–72 h). With submaximal Aβ- (25–35) exposure (10 μ M ), substantial (>40% within 48 h) degeneration often occurs and is markedly attenuated by the presence of the Ca²⁺ channel blockers nimodipine (1–20 μ M ) and Co²⁺ (100 μ M ) during the Aβ exposure. However, Aβ neurotoxicity is not affected by the presence of glutamate receptor antagonists. We suggest that Ca²⁺ influx through voltage-gated Ca²⁺ channels may contribute to Aβ-induced neuronal injury and that nimodipine and Co²⁺, by attenuating such influx, are able to attenuate Aβ neurotoxicity.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Calpains mediate p53 activation and neuronal death evoked by DNA damage

DNA damage is an initiator of neuronal death implicated in neuropathological conditions such as stroke. Previous evidence has shown that apoptotic death of embryonic cortical neurons treated with the DNA damaging agent camptothecin is dependent upon the tumor suppressor p53, an upstream death mediator, and more distal death effectors such as caspases. We show here that the calcium-regulated cysteine proteases, calpains, are activated during DNA damage induced by camptothecin treatment. Moreover, calpain deficiency, calpastatin expression, or pharmacological calpain inhibitors prevent the death of embryonic cortical neurons, indicating the important role of calpain in DNA damage-induced death. Calpain inhibition also significantly reduced and delayed the induction of p53. Consistent with the actions of calpains upstream of p53 and the proximal nature of p53 death signaling, calpain inhibition inhibited cytochrome c release and DEVD-AFC cleavage activity. Taken together, our results indicate that calpains are a key mediator of p53 induction and consequent caspase-dependent neuronal death due to DNA damage.     

Role of Cyclin-Dependent Kinase 5 in the Neurodegenerative Process Triggered by Amyloid-Beta and Prion Peptides: Implications for Alzheimer’s Disease and Prion-Related Encephalopathies

Tau hyperphosphorylation, amyloid plaques, and neuronal death are major neuropathological features of Alzheimer’s disease (AD) and Prion-related encephalopathies (PRE). Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, active in post-mitotic neurons, where it regulates survival and death pathways. Overactivation of Cdk5 is conferred by p25, a truncated fragment of the p35 activator formed upon calpain activation. Cdk5 deregulation causes abnormal phosphorylation of microtubule-associated protein tau, leading to neurodegeneration. In this work we investigated the involvement of Cdk5 in the neurodegeneration triggered by amyloid-beta (Aβ) and prion (PrP) peptides, the culprit agents of AD and PRE. As a work model, we used cultured rat cortical neurons treated with Aβ_1–40 and PrP_106–126 synthetic peptides. The obtained data show that apoptotic neuronal death caused by both the peptides was in part due to Cdk5 deregulation. After peptide treatment, p25 levels were significantly enhanced in a pattern consistent with the augment in calpain activity. Moreover, Aβ_1–40 and PrP_106–126 increased the levels of tau protein phosphorylated at Ser202/Thr205. Cdk5 (roscovitine) and calpain (MDL28170) inhibitors reverted tau hyperphosphorylation and prevented neuronal death caused by Aβ_1–40 and PrP_106–126. This study demonstrates, for the first time, that Cdk5 is involved in PrP-neurotoxicity. Altogether, our data suggests that Cdk5 plays an active role in the pathogenesis of AD and PRE.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Complement component C1q inhibits beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms

Alzheimer's disease is a neurodegenerative disorder characterized by neuronal loss, β-amyloid (Aβ) plaques, and neurofibrillary tangles. Complement protein C1q has been found associated with fibrillar Aβ deposits, however the exact contributions of C1q to Alzheimer's disease is still unknown. There is evidence that C1q, as an initiator of the inflammatory complement cascade, may accelerate disease progression. However, neuronal C1q synthesis is induced after injury/infection suggesting that it may be a beneficial response to injury. In this study, we report that C1q enhances the viability of neurons in culture and protects neurons against Aβ- and serum amyloid P (SAP)-induced neurotoxicity. Investigation of potential signaling pathways indicates that caspase and calpain are activated by Aβ, but C1q had no effect on either of these pathways. Interestingly, SAP did not induce caspase and calpain activation, suggesting that C1q neuroprotection is in distinct from caspase and calpain pathways. In contrast to Aβ- and SAP-induced neurotoxicity, neurotoxicity induced by etoposide or FCCP was unaffected by the addition of C1q, indicating pathway selectivity for C1q neuroprotection. These data support a neuroprotective role for C1q which should be further investigated to uncover mechanisms which may be therapeutically targeted to slow neurodegeneration via direct inhibition of neuronal loss.     

Calpain inhibitors confer biochemical, but not electrophysiological, protection against anoxia in rat optic nerves

Calpains are ubiquitous Ca(2+)-activated neutral proteases that have been implicated in ischemic and traumatic CNS injury. Ischemia and trauma of central white matter are dependent on Ca2+ accumulation, and calpain overactivation likely plays a significant role in the pathogenesis. Adult rat optic nerves, representative central white matter tracts, were studied in an in vitro anoxic model. Functional recovery following 60 min of anoxia and reoxygenation was measured electrophysiologically. Calpain activation was assessed using western blots with antibodies against calpain-cleaved spectrin breakdown products. Sixty minutes of in vitro anoxia increased the amount of spectrin breakdown approximately 20-fold over control, with a further increase after reoxygenation to >70 times control, almost as much as 2 h of continuous anoxia. Blocking voltage-gated Na+ channels with tetrodotoxin or removing bath Ca2+ was highly neuroprotective electrophysiologically and resulted in a marked reduction of spectrin degradation. The membrane-permeable calpain inhibitors MDL 28,170 and calpain inhibitor-I (10-100 microM) were effective at reducing spectrin breakdown in anoxic and reoxygenated optic nerves, but no electrophysiological improvement was observed. We conclude that calpain activation is an important step in anoxic white matter injury, but inhibition of this Ca(2+)-dependent process in isolation does not improve functional outcome, probably because other deleterious Ca(2+)-activated pathways proceed unchecked.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Protection of Flupirtine on β-Amyloid-Induced Apoptosis in Neuronal Cells In Vitro: Prevention of Amyloid-Induced Glutathione Depletion

Abstract: Effective drugs are not available to protect against β-amyloid peptide (Aβ)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment Aβ25-35 at 1 µ M in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 µ M Aβ25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 µg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with Aβ25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like DNA fragmentation pattern and by the TUNEL technique. Aβ25-35-induced DNA fragmentation could be abolished by preincubation of the cells with 1 µg/ml flupirtine. Incubation with Aβ25-35 reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/10⁶ cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where Aβ accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.