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Bcl-2 family proteins are principal regulators of cell death during normal development as well as in many disease states. Differentiated cerebellar granule neurons are protected from apoptosis by depolarizing concentrations of potassium. Further, these cells acquire resistance to glutamate-mediated excitotoxicity when pre-exposed to subtoxic concentrations of the glutamate receptor agonist, N-methyl-D-aspartate. Here, we report that the expression of bcl-2, bcl-xL, bcl-xS, bax and bad mRNA as well as of Bcl-2, Bax, Bcl-XL, Bcl-XS and Bag-1 proteins is not modulated in these two paradigms of neuronal cell death. However, mitochondrial release of cytochrome c, which is thought to be controlled by Bcl-2 family proteins, is detected 5 h after switching the neurons to low potassium conditions. Thus, there appears to be regulation of Bcl-2 family protein bioactivity in the absence of altered protein expression during potassium deprivation-induced apoptosis of cerebellar granule neurons.     

Metabolic and genetic analyses of apoptosis in potassium/serum-deprived rat cerebellar granule cells

Cerebellar granule cells maintained in medium containing serum and 25 mM potassium undergo an apoptotic death within 96 hr when switched to serum-free medium with 5 mM potassium. Because large numbers of apparently homogeneous neurons can be obtained, this represents a potentially useful model of neuronal programmed cell death (PCD). Analysis of the time course and extent of death after removal of either serum or K+ alone demonstrated that a fast-dying (T(1/2) = 4 hr) population (20%) responded to serum deprivation, whereas a slow-dying (T(1/2) = 25 hr) population (80%) died in response to K+ deprivation. Taking advantage of the complete death after removing both K+ and serum, changes in metabolic events and mRNA levels were analyzed in this model. Glucose uptake, protein synthesis, and RNA synthesis fell to <35% of control by 9 hr after potassium/serum deprivation, a time when 85% of the cells were still viable. The pattern of the fall in these metabolic parameters was similar to that reported for trophic factor-deprived sympathetic neurons. Most mRNAs decreased markedly after K+/serum deprivation. Levels of c-jun mRNA increased fivefold in potassium/serum-deprived granule cells; c-jun is required for cell death of sympathetic neurons. mRNA levels of cyclin D1, c-myb, collagenase, and transin remained relatively constant in potassium/serum-deprived granule cells. These data demonstrate the existence of two populations of granule cells with respect to cell death and define common metabolic and genetic events involved in neuronal PCD.     

Ethanol induces apoptosis in cerebellar granule neurons by inhibiting insulin-like growth factor 1 signaling

The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. IGF-1 protected granule neurons from apoptosis in medium containing 5 mM K+. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 mM K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15-20%) was observed at 1 mM and was half-maximal at 45 mM. The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

TGF-beta-induced apoptosis of cerebellar granule neurons is prevented by depolarization

The regulation of programmed cell death in the developing nervous system involves target-derived survival factors, afferent synaptic activity, and hormone- and cytokine-dependent signaling. Cultured immature cerebellar granule neurons die by apoptosis within several days in vitro unless maintained in depolarizing (high) concentrations of potassium (25 mM K+). Here we report that transforming growth factors (TGF)-beta1, -beta2, and -beta3 accelerate apoptosis of these neurons when maintained in physiological (low) K+ medium (5mM K+) as assessed by measures of viability, quantitative DNA fragmentation, and nuclear morphology. TGF-beta-induced apoptosis of these neurons is not blocked by CNTF and LIF, cytokines that enhance neuronal survival when applied alone, or by IGF-I, which prevents apoptosis upon potassium withdrawal. In contrast, neurons that differentiate in high K+ medium for several days in vitro acquire resistance to TGF-beta-mediated cell death. Granule neurons maintained in either low or high K+ medium produce latent, but not bioactive, TGF-beta1 and -beta2. Because neutralizing TGF-beta antibodies fail to augment survival of low K+ neurons, the cerebellar neurons are apparently unable to activate latent TGF-beta. Thus, apoptosis of low K+ neurons is not attributable to endogenous production of TGF-beta. Taken together, our data suggest that TGF-beta may limit the expansion of postmitotic neuronal precursor populations by promoting their apoptosis but may support survival of those neurons that have maturated, differentiated, and established supportive synaptic connectivity.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

N-Methyl-D-aspartate inhibits apoptosis through activation of phosphatidylinositol 3-kinase in cerebellar granule neurons. A role for insulin receptor substrate-1 in the neurotrophic action of n-methyl-D-aspartate and its inhibition by ethanol

Primary cultured rat cerebellar granule neurons underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. N-methyl-D-aspartate (NMDA) protected granule neurons from apoptosis in medium containing 5 mM K+. Inhibition of apoptosis by NMDA was blocked by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but it was unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. The antiapoptotic action of NMDA was associated with an increase in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an increase in the binding of the regulatory subunit of PI 3-kinase to IRS-1, and a stimulation of PI 3-kinase activity. In the absence of extracellular Ca2+, NMDA was unable to prevent apoptosis or to phosphorylate IRS-1 and activate PI 3-kinase. Significant inhibition of NMDA-mediated neuronal survival by ethanol (10-15%) was observed at 1 mM, and inhibition was half-maximal at 45-50 mM. Inhibition of neuronal survival by ethanol corresponded with a marked reduction in the capacity of NMDA to increase the concentration of intracellular Ca2+, phosphorylate IRS-1, and activate PI 3-kinase. These data demonstrate that the neurotrophic action of NMDA and its inhibition by ethanol are mediated by alterations in the activity of a PI 3-kinase-dependent antiapoptotic signaling pathway.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway

Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1-100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1-2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.     

Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs)

IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrated that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila, Reaper (RPR), through their baculovirus IAP repeat (BIR) region. Expression of IAPs blocked RPR-induced apoptosis and resulted in the accumulation of RPR in punctate perinuclear locations which coincided with IAP localization. When expressed alone, RPR rapidly disappeared from the cells undergoing RPR-induced apoptosis. Expression of P35, a caspase inhibitor, also blocked RPR-induced apoptosis and delayed RPR decline, but RPR remained cytoplasmic in its location. Mutational analysis of RPR demonstrated that caspases were not directly responsible for RPR disappearance. The physical interaction of IAPs with RPR provides a molecular mechanism for IAP inhibition of RPR's apoptotic activity.     

Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation

The inhibitor of apoptosis (IAP) proteins form a highly conserved gene family that prevents cell death in response to a variety of stimuli. Herein we describe a newly defined murine IAP, designated Tiap, that proved to be a murine homologue of human survivin based on sequence comparison. TIAP has one baculovirus IAP repeat and lacks a C-terminal RING finger motif. TIAP interacted with the processed form of caspase 3 and inhibited caspase-induced cell death. Histological examinations revealed that TIAP is expressed in growing tissues such as thymus, testis, and intestine of adult mice and many tissues of embryos. In in vitro studies, TIAP was induced in splenic T cells activated with anti-CD3 antibody or Con A, and the expression of TIAP was up-regulated in synchronized NIH 3T3 cells at S to G2/M phase of the cell cycle. We propose that during cell proliferation, cellular protective activity may be augmented with inducible IAPs such as TIAP.     

The dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce necrotic- and apoptotic-like death of rat cerebellar granule cells

The neurotoxic properties of the dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. Beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of beta-N-methylamino-L-alanine were blocked by D(-)-2-amino-5-phosphonopentanoic acid, and those of beta-N-oxalylamino-L-alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K+ (30 mM)- to a low K+ (10 mM)-containing medium. In cerebellar granule cells exposed to beta-N-methylamino-L-alanine or beta-N-oxalylamino-L-alanine (3 mM), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce death over 12-72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.     

Glia modulate NMDA-mediated signaling in primary cultures of cerebellar granule cells

Excessive activation of N-methyl-D-aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Evidence that glyceraldehyde-3-phosphate dehydrogenase is involved in age-induced apoptosis in mature cerebellar neurons in culture

Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.