Apoptosis In Alzheimer's Disease Research Articles

Gender and age-dependent differences in the mitochondrial apoptogenic pathway in Alzheimer's disease

Age-related mitochondrial oxidative stress is highly gender dependent. The aim of this study was to determine the role of gender in the mitochondrial contribution to neuronal apoptosis in Alzheimer's disease (AD). We used mitochondria isolated from brains of Wistar rats to study the toxicity of ß-amyloid peptide (Aß), and found that it increases mitochondrial peroxide production, nitration and oxidation of proteins, and release of cytochrome c. The toxic effects occurred in young males and in old females but not in young females, indicating their resistance to Aß. This resistance was abolished with age. These toxic effects of Aß were prevented by heme. Our findings provide a molecular mechanism for the contribution of Aβ to the mitochondrial dysfunction and oxidative stress seen in AD, as well as for the mitochondria-dependent pathway of apoptosis in AD. Gender and age-related differences seen in the development of AD can also be partially explained.

Bile acids and apoptosis modulation: an emerging role in experimental Alzheimer's disease

The potential role of apoptosis in Alzheimer's disease (AD) has been an area of intense research in recent years. Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA) are endogenous bile acids that act as potent inhibitors of apoptosis. Their therapeutic effects have been tested in many experimental pathological conditions, including neurological disorders, such as AD. TUDCA regulates precise transcriptional and post-transcriptional events that impact mitochondrial function in neurons. TUDCA not only stabilizes the mitochondrial membrane and prevents Bax translocation, inhibiting the release of cytochrome c and the activation of caspases, but also interferes with upstream factors, including cell cycle-related proteins. In addition, TUDCA is capable of inducing survival pathways. Here, we review the role of apoptosis in AD and discuss the therapeutic potential of TUDCA in treating this disease.

Lovastatin protects human neurons against Aβ-induced toxicity and causes activation of β-catenin–TCF/LEF signaling

Alzheimer's disease (AD) is characterized by cognitive decline due to excess amyloid beta peptide (Aβ), neurofibrillary tangles, and neuronal loss. Aβ promotes neuronal apoptosis in AD by activating glycogen synthase kinase-3β (GSK-3β), leading to degradation of β-catenin and inactivation of Wnt signaling. β-Catenin interacts with the T-cell factor (TCF)/Lymphoid enhancer factor (LEF)-nuclear complex to mediate Wnt signaling and cell survival. Statins are associated with decreased prevalence of AD. Lovastatin has been shown to decrease the production of Aβ and to promote neuronal survival. The mechanisms of how statins promote neuronal survival are unclear. We propose that the neuroprotective effect of lovastatin may be due to inactivation of GSK-3β activity, resulting in induction of Wnt signaling. Here, we report that lovastatin prevented Aβ-induced apoptosis in human SK-NSH cells. This was accompanied by reduction in active GSK-3β, and increased nuclear translocation of β-catenin, TCF-3, and LEF-1. Lovastatin treatment induced an increase in TCF/LEF–chloramphenicol acetyl transferase (CAT) gene reporter activity. More importantly, β-catenin and TCF were required for the neuroprotective function of lovastatin. Our results suggest that lovastatin protects neuronal cells from Aβ-induced apoptosis and causes reduction in GSK-3β activity, resulting in activation of Wnt signaling.

The modular systems biology approach to investigate the control of apoptosis in Alzheimer's disease neurodegeneration

Apoptosis is a programmed cell death that plays a critical role during the development of the nervous system and in many chronic neurodegenerative diseases, including Alzheimer's disease (AD). This pathology, characterized by a progressive degeneration of cholinergic function resulting in a remarkable cognitive decline, is the most common form of dementia with high social and economic impact. Current therapies of AD are only symptomatic, therefore the need to elucidate the mechanisms underlying the onset and progression of the disease is surely needed in order to develop effective pharmacological therapies. Because of its pivotal role in neuronal cell death, apoptosis has been considered one of the most appealing therapeutic targets, however, due to the complexity of the molecular mechanisms involving the various triggering events and the many signaling cascades leading to cell death, a comprehensive understanding of this process is still lacking.Modular systems biology is a very effective strategy in organizing information about complex biological processes and deriving modular and mathematical models that greatly simplify the identification of key steps of a given process.This review aims at describing the main steps underlying the strategy of modular systems biology and briefly summarizes how this approach has been successfully applied for cell cycle studies. Moreover, after giving an overview of the many molecular mechanisms underlying apoptosis in AD, we present both a modular and a molecular model of neuronal apoptosis that suggest new insights on neuroprotection for this disease.

Molecular Insights into Mechanisms of the Cell Death Program:Role in the Progression of Neurodegenerative Disorders

Synaptic degeneration and death of neurons in limbic and cortical brain regions are the fundamental processes responsible for the manifestation of cognitive dysfunction and behavioural abnormalities in Alzheimer's disease (AD). Despite the various genetic and environmental factors, and the aging process itself that may lead to the manifestation of AD, multiple evidence from studies in experimental models and in AD brain tissue demonstrate that the underlying neurodegeneration is associated with morphological and biochemical features of apoptosis. At the cellular level, neuronal apoptosis in AD may be initiated by oxidative stress and related DNA damage, disruption of cellular calcium homeostasis, or endoplasmic reticulum (ER) stress. The molecular mechanisms of the biochemical cascades of apoptosis are beginning to be understood and involve upstream effectors such as Par-4, p53, and pro-apoptotic Bcl-2 family members, which mediate mitochondrial dysfunction and subsequent release of pro-apoptotic proteins, such as cytochrome c or apoptosis inducing factor (AIF), and subsequent caspase-dependent and -independent pathways which finally result in degradation of proteins and nuclear DNA. The regulation of apoptotic cascades is complex and involves transcriptional control as well as posttranscriptional protein modifications, such as protease-mediated cleavage, ubiquitination or poly(ADP-ribosylation). More recently, the regulation of protein phosphorylation by kinases and phosphatases is emerging as a prerequisite mechanism in the control of the apoptotic cell death program. A better understanding of the molecular underpinnings of neuronal apoptosis will lead to novel preventive and therapeutic approaches to the neurodegenerative processes in Alzheimer's disease and other neurological disorders where programmed cell death is prominent.

Pro-NGF from Alzheimer's Disease and Normal Human Brain Displays Distinctive Abilities to Induce Processing and Nuclear Translocation of Intracellular Domain of p75NTR and Apoptosis

The pro form of neurotrophic growth factor (pro-NGF), purified by chromatography from human Alzheimer's disease (AD)-affected brains (ADhbi-pro-NGF), has been shown to induce apoptotic cell death in neuronal cell cultures through its interaction with the p75 neurotrophin receptor (p75NTR). In the present work, we report that ADhbi-pro-NGF stimulates processing of p75NTR with alpha- and gamma-secretases, yielding a 20-kd intracellular domain (p75(ICD)) that translocates to the nucleus. This process was accompanied by delayed apoptosis. In AD, p75(ICD) was significantly increased in human entorhinal cortex. Although human frontal cortex has been described as showing a higher pro-NGF increase in AD, the increase in the entorhinal cortex paralleled p75NTR processing in its intracellular domain. In addition, pro-NGF isolated from AD-affected brains differed functionally from pro-NGF isolated from comparably aged control brains, with pro-NGF isolated from control brains being unstable and undergoing degradation to NGF when added to cell culture. As p75(ICD) and pro-NGF are both mediators of apoptosis and are both found in increased levels in the cerebral cortex in AD, the present data have implications for understanding neuronal degeneration in AD.

Novel Dimeric Acetylcholinesterase Inhibitor Bis(7)-tacrine, but Not Donepezil, Prevents Glutamate-induced Neuronal Apoptosis by Blocking N-Methyl-d-aspartate Receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Biomarkers for apoptosis in Alzheimer's disease

Alzheimer's disease (AD) affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. Today there is no cure for this devastating and always lethal disease and therefore it is of great interest for patients, relatives and societies to find new drugs that can hinder the disease process. During the progression of AD a substantial amount of neurons degenerate in the brain. The mechanisms of cell death involved in AD have not been fully elucidated. However, there are several reports showing that neurons die partly by apoptosis in the AD brain. Drugs blocking apoptosis could therefore be potentially useful for early prevention of neuronal cell death. Biomarkers for apoptosis should be important tools in the evaluation of drug effects and in the diagnostics of AD. Here we review the current knowledge in the field and discuss potential biomarkers for apoptosis in AD.

Rosuvastatin reduces caspase-3 activity and up-regulates α-secretase in human neuroblastoma SH-SY5Y cells exposed to Aβ

An increasing number of studies suggest that disturbances in cholesterol homeostasis may promote the formation and deposition of beta-amyloid (Aβ) and the progression of Alzheimer's disease (AD). In this paper, we have analyzed the effect of the lipid-lowering compound rosuvastatin on apoptosis and caspase-3 activity in human neuroblastoma SH-SY5Y cells. Exposure of SH-SY5Y cells to Aβ 1–42 alone resulted in a significantly increased caspase-3 activity approximately by 35% (135 ± 15%, p < 0.05), and decreased α-secretase activity by 34% (67.4 ± 2.7%, p < 0.001) compared to the controls (100 ± 18.1%). Rosuvastatin alone decreased caspase-3 activity by 15% (85.3 ± 1.5%, p < 0.0005) compared to the controls and by 50% to cells exposed to Aβ alone ( p < 0.00005). Cells exposed to rosuvastatin alone had a higher α-secretase activity compared to cells exposed to Aβ (76.4 ± 23.8%, n.s.) but a slightly lower activity compared to the controls (n.s.). Pre-treatment of SY-SY5Y cells with rosuvastatin prior to incubation with Aβ 1–42 resulted in decreased caspase-3 activity by ∼15% compared to the controls and by ∼48% (86.8 ± 16.9%, p < 0.05) compared to cells treated with Aβ 1–42 alone. Also, α-secretase activity was increased by approximately 50% compared to the controls and by 84% (151.3 ± 10.1%, p < 0.05), compared to cells treated with Aβ 1–42 alone. Mevalonate abrogated the effect of rosuvastatin in vitro. To our knowledge, this is the first study demonstrating that the hydrophilic compound rosuvastatin decreases caspase-3 activity and increases α-secretase activity in human neuroblastoma SH-SY5Y cells exposed to Aβ in vitro. These effects are essential for modulation of the amyloidogenic pathway and mediators of apoptosis in AD.

Prospects for antiapoptotic drug therapy of neurodegenerative diseases

The evidence for a role of apoptosis in the neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), and in the more acute conditions of cerebral ischemia, traumatic brain injury (TBI), and spinal cord injury (SCI) is reviewed with regard to potential intervention by means of small antiapoptotic molecules. In addition, the available animal models for these diseases are discussed with respect to their relevance for testing small antiapoptotic molecules in the context of what is known about the apoptotic pathways involved in the diseases and the models. The principal issues related to pharmacotherapy by apoptosis inhibition, i.e., functionality of rescued neurons and potential interference with physiologically occurring apoptosis, are pointed out. Finally, the properties of a number of small antiapoptotic molecules currently under clinical investigation are summarized. It is concluded that the evidence for a role of apoptosis at present is more convincing for PD and ALS than for AD. In PD, damage to dopaminergic neurons may occur through oxidative stress and/or mitochondrial impairment and culminate in activation of an apoptotic, presumably p53-dependent cascade; some neurons experiencing energy failure may not be able to complete apoptosis, end up in necrosis and give rise to inflammatory processes. These events are reasonably well reflected in some of the PD animal models, notably those involving 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone. In sporadic ALS, an involvement of pathways involving p53 and Bcl-2 family members appears possible if not likely, but is not established. The issue is important for the development of antiapoptotic compounds for the treatment of this disease because of differential involvement of p53 in different mutant superoxide dismutase (SOD) mice. Most debated is the role of apoptosis in AD; this implies that little is known about potentially involved pathways. Moreover, there is a lack of suitable animal models for compound evaluation. Apoptosis or related phenomena are likely involved in secondary cell death in cerebral ischemia, TBI, and SCI. Most of the pertinent information comes from animal experiments, which have provided some evidence for prevention of cell death by antiapoptotic treatments, but little for functional benefit. Much remains to be done in this area to explore the potential of antiapoptotic drugs. There is a small number of antiapoptotic compounds in clinical development. With some of them, evidence for maintenance of functionality of the rescued neurons has been obtained in some animal models, and the fact that they made it to phase II studies in patients suggests that interference with physiological apoptosis is not an obligatory problem. The prospect that small antiapoptotic molecules will have an impact on the therapy of neurodegenerative diseases, and perhaps also of ischemia and trauma, is therefore judged cautiously positively.

Increased expression of intranuclear AChE involved in apoptosis of SK-N-SH cells.

Programmed cell death plays an integral role in neurodegenerative diseases such as Alzheimer's disease (AD). Acetylcholinesterase (AChE) was suggested to be neurotoxic in vivo and in vitro and accelerate assembly of amyloid peptide into Alzheimer's fibrils. In our experiments, we found increased AChE expression in apoptotic neuroblastoma SK-N-SH cells after long-term culture. Our results first showed that in apoptotic SK-N-SH cells, AChE aggregated in the nucleus and suppression of AChE expression with antisense oligonucleotide could save the cells from apoptosis. It was also found that caspase-3 activity was parallel with AChE activation in apoptotic SK-N-SH cells. These results suggest that AChE plays an important role in the procession of neuroblastoma cell apoptosis and favor the association between AChE and neuronal apoptosis in AD.

Intron-Exon Swapping of Transglutaminase mRNA and Neuronal Tau Aggregation in Alzheimer's Disease

In order to understand the mechanism for insoluble neurotoxic protein polymerization in Alzheimer's disease (AD) brain neurons, we examined protein and gene expression for transglutaminase (TGase 2; tissue transglutaminase (tTG)) in hippocampus and isocortex. We found co-localization of tTG protein and activity with tau-positive neurofibrillary tangles, whereas mRNA and sequence analysis indicated an absolute increase in tTG synthesized. Although apoptosis in AD hippocampus is now an established mode of neuronal cell death, no definite underlying mechanism(s) is known. Since TGase-mediated protein aggregation is implicated in polyglutamine ((CAG)(n)/Q(n) expansion) disorder apoptosis, and expanded Q(n) repeats are excellent TGase substrates, a role for TGase in AD is possible. However, despite such suggestions almost 20 years ago, the molecular mechanism remained elusive. We now present one possible molecular mechanism for tTG-mediated, neurotoxic protein polymerization leading to neuronal apoptosis in AD that involves not its substrates (like Q(n) repeats) but rather the unique presence of alternative transcripts of tTG mRNA. In addition to a full-length (L) isoform in aged non-demented brains, we found a short isoform (S) lacking a binding domain in all AD brains. Our current results identify intron-exon "switching" between L and S isoforms, implicating G-protein-coupled signaling pathways associated with tTG that may help to determine the dual roles of this enzyme in neuronal life and death processes.

Alteration of Caspases and Apoptosis-Related Proteins in Brains of Patients with Alzheimer's Disease

Dysregulated programmed cell death or apoptosis is suggested to be involved in the pathogenesis of Alzheimer's disease (AD). Caspases, the major effectors of apoptosis, are cysteine proteases that cleave crucial substrate proteins exclusively after aspartate residues. The activity of caspases are delicately regulated by a variety of proteins that possess distinct domains for protein–protein interaction. To further substantiate the role of apoptosis in AD, we investigated the levels of nine different proteins involved in apoptosis by Western blot technique in frontal cortex and cerebellum of control and AD subjects. The protein levels of caspase-3, -8, and -9, DFF45 (DNA fragmentation factor 45), and FLIP (Fas associated death domain (FADD)-like interleukin-1β-converting enzyme inhibitory proteins) were decreased, whereas those of ARC (apoptosis repressor with caspase recruitment domain) and RICK (Receptor interacting protein (RIP)-like interacting CLARP kinase) increased in AD. In contrast, cytochrome c and Apaf-1 (apoptosis protease activating factor-1) were unchanged. Regression analysis revealed no correlation between levels of protein and postmortem interval. However, inconsistent correlation was found between age and levels of proteins as well as among the levels of individual proteins. The current findings showed that dysregulation of apoptotic proteins indeed exists in AD brain and support the notion that it may contribute to neuropathology of AD. The study further hints that apoptosis in AD may occur via the death receptor pathway independent of cytochrome c. Hence, therapeutic strategies that ablate caspase activation may be of some benefit for AD sufferers.

Apoptosis and Alzheimer's disease.

Cell death by apoptosis comprises a sequence of events leading to the activation of caspases. Caspases execute the fragmentation of the cellular protein and DNA, ultimately, leading to disintegration of the cell. Apoptosis is a tightly regulated physiological mechanism that is crucial during development and thereafter for the maintenance of the balance between cell division and cell death. In contrast to the rather smoothly operating cell death machinery of apoptosis, necrosis is caused by insults leading to the rapid disruption of cellular metabolism and the non-physiological disintegration of the cells. Frequently, toxic events or traumatic challenges trigger the rapid necrotic cell death. Apoptosis and necrosis can be discriminated by a number of morphological and biochemical characteristics. To describe the specific mechanisms of cell death occurring during neurodegenerative disorders, such as Alzheimer's disease (AD), many investigations, both in vivo and in vitro, have attempted to label the particular pathway of cell death either as apoptosis or as necrosis. The elucidation of the mechanism of cell death promises to identify novel pharmaceutical targets for the prevention and therapy of AD. Apoptotic and necrotic cells can be found in AD tissue, and both pathways can be mimicked employing a variety of models systems of AD-associated nerve cell degeneration. Certain genes that are linked to familial AD may render neurons more vulnerable to apoptosis, but it has to be stressed that the vast majority of AD cases are sporadic and not strictly genetically determined. Apoptosis and necrosis may overlap, may sequentially occur under certain conditions, and may not be detected unequivocally. In conclusion, on the basis of the presently available data it has to be stated that although many studies in vivo and in vitro favor apoptosis in AD, there is considerable evidence that a mixture of both events may contribute to neurodegeneration in AD and to its final pathology.

Avoidance of Apoptosis in Alzheimer's Disease.

Internucleosomal DNA fragmentation, which is a well-known feature of apoptosis but not an absolute criterion for identifying apoptosis (1), has often been observed in the brain tissue of Alzheimer's disease (AD). However, classical apoptotic morphology such as nuclear condensation, membrane blebbing and apoptotic bodies are seldom seen in AD brain. In this issue of Journal of Alzheimer's Disease, Velez-Pardo et al. have reported the DNA fragmentation using terminal dUTP labeling (TUNEL) in postmortem brains of familial AD with presenilin-1 [E280A] mutation. Importantly, no classical apoptotic morphology has been observed also in the brains of presenilin-1 familial AD. Furthermore, Velez-Pardo et al. have shown that there is no obvious correlation between DNA fragmentation and the severity of amyloid deposition as well as between DNA fragmentation and the severity of neurofibrillary tangle formation. An apoptotic pathway only takes several hours or at most a few days for completion. In the development of the lateral motor column of the chick embryo, 8,000 cells out of 20,000 cells die, i.e. loss of 40% of the population occurs within 3 days (2). The fact that only 5% of the population in the lateral motor column is undergoing apoptosis at any particular time in this period (2) indicates that apoptotic pathway requires about 10 hours for completion. In a striking contrast with the physiologically programmed cell death, loss of 40% of the population occurs (3,000 neurons out of 7,000 neurons per 50 micron-thick section are lost) within 10 years in the temporal cortex neurons of AD (3). If we suppose that 20-40% of neurons of the temporal cortex are undergoing degeneration at any given time in the course of AD, an individual neuron in the temporal cortex of AD requires 5-10 years to die. Indeed, we can observe neurons displaying many of the features of apoptosis in AD. This fact argues that neurons in AD have mounted an effective defense to apoptotic death (an avoidance of apoptosis) rather than actual completion of apoptosis (4,5). It is noteworthy that nucleic acid oxidation occurs widely in vulnerable neuronal populations in AD (6) and oxidative damage can directly cause DNA fragmentation (7). Therefore, DNA damage possibly resulting from oxidative stress involves vulnerable neurons in AD beyond the distribution of amyloid deposition or neurofibrillary tangles, which may be related to neuronal cell death occurring independently of the classical AD pathology (3).

Apoptosis in Alzheimer's disease--an update.

Alzheimer's disease (AD) is the most common human neurodegenerative disorder characterized by the progressive deterioration of cognition and memory in association with the presence of senile plaques, neurofibrillary tangles, and massive loss of neurons. Most cases of AD are late-onset and sporadic, but in some cases the disease is inherited as an autosomal dominant trait. Four different genes, the amyloid precursor protein, apolipoprotein E, and presenilins 1 and 2 have been implicated in the etiology of familial AD. It is now generally accepted that massive neuronal death due to apoptosis is a commmon characteristic in the brains of patients suffering from neurodegenerative diseases, and apoptotic cell death has been found in neurons and glial cells in AD. This review summarizes the current findings regarding the evidence for apoptosis in AD and discusses the possible involvement of apoptosis-regulating factors in the pathology of AD. Modification of the apoptotic cascade could be considered as a primary therapeutic strategy for the disease.

Depletion of intracellular zinc induces protein synthesis-dependent neuronal apoptosis in mouse cortical culture.

The central nervous system (CNS) contains a large amount of zinc; a substantial fraction of it is located inside synaptic vesicles of glutamatergic terminals in chelatable forms and released in a calcium-dependent manner with intense neuronal activity. Recently, it has been shown that excessive zinc influx can kill neurons in rats subjected to transient forebrain ischemia. On the other hand, severe depletion of zinc has been also reported to induced cell death in certain nonneuronal cells. Since decreases in tissue zinc have been associated with Alzheimer's disease (AD) and senile macular degeneration, we examined whether depletion of intracellular zinc with a zinc chelator can directly induce neuronal death in mouse cortical cultures. Exposure of cortical cultures to a cell-permeant zinc-chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 0.5-3.0 microM) induced gradually developing neuronal degeneration accompanied by various features of apoptosis: cell body shrinkage, nuclear condensation and fragmentation, and internucleosomal DNA breakage. At higher concentrations, TPEN induced additional glial cell death. TPEN-induced cell death was completely blocked by coaddition of zinc. Addition of a protein synthesis inhibitor cycloheximide as well as a caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-fluoromethyl ketone (zVAD-fmk) markedly attenuated TPEN-induced neuronal death. On the other hand, brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), phorbol 12-myristate 13-acetate (PMA), high K+, or an antioxidant, trolox, did not show any protective effect. The present results demonstrated that depletion of intracellular zinc induces protein synthesis-dependent neuronal apoptosis in cortical culture. Combined with the findings that extracellular zinc may promote extracellular beta-amyloid (A beta) aggregation and that total tissue zinc is reduced in AD, present results suggest a possibility that redistribution of zinc from intracellular to extracellular space may synergistically contribute to neuronal apoptosis in AD.