Molecular mechanisms underlying cell death are a major focus of current biomedical research as aberrant cell death is involved in the pathogenesis of a vast number of diseases including the CNS disorders. Apoptosis, also referred to as programmed cell death, is a process in which a cell dies by activation of an intrinsic genetic program. Apoptosis plays an important role in the development and aging processes in the central nervous system (CNS). Abnormality in apoptosis has been linked to pathogenesis of neurodegenerative diseases and implicated in neuropsychiatric disorders. We have used primary cultures of rat CNS neurons and neurally related cell lines as a model to study molecular mechanisms underlying neuronal apoptosis. We found that glyceraldehyde-3-phospahate dehydrogenase (GAPDH), a glycolytic enzyme, is over- expressed during apoptosis induced by aging of rat cerebellar granule cells (CGCs) in culture. Antisense oligonucleotides to GAPDH block GAPDH over-expression and effectively delay age-induced apoptosis of these cerebellar neurons. These results provide the first evidence for a role of GAPDH in neuronal apoptosis. By the same criteria, we found that GAPDH is involved in age-induced apoptosis of rat cerebral cortical neurons and apoptosis of CGC induced by extracellular potassium deprivation and exposure to cytosine arabinoside (AraC). We found that the AraC-induced apoptosis is robustly protected by the neurotrophins brain-derived neurotrophic factor (BDNF) and NT 4/ 5, but not NT-3. The apoptosis is also associated with an increased expression of two death genes, p53 and Bax. An antisense oligonucleotide to p53 reduces GAPDH induction and protects against AraC-induced apoptosis, suggesting that GAPDH over-expression depends on p53 expression. Moreover, tranfection of the p53 gene into PC12 cells induces GAPDH over-expression and causes ultimate cell death, suggesting that GAPDH is a novel target gene regulated by p53. AraC-induced GAPDH over-expression is predominantly accumulated in the nucleus assessed by subcellular fractionation study and electromicroscopic immunohistochemistry. Translocation of GAPDH to the nucleus occurs in parallel with a loss of GAPDH glycolytic and uracil glycosylase activities, suggesting an alteration in the structure and function of nuclear GAPDH. We also demonstrated that at least six GAPDH isoforms can be detected in the nucleus of cerebellar granule cells. These nuclear isoforms differ in their abundance and are translocated to the nucleus according to a distinct time-course following AraC treatment. Evidence is also available that nuclear GAPDH accumulation precedes activation of caspase-3 and cleavage of its nuclear substrate lamin B1. In CGCs, we studied the role of GAPDH in the excitotoxicity induced by SYM 2081 (2S,4R,-4-methylglutamate), an inhibitor of excitatory amino acid transporter and an agonist of low affinity kainate receptors. We found that SYM-induced excitotoxicity is N-methyl-D-aspartate (NMDA) receptor-mediated and is associated with nuclear translocation of GAPDH. Pretreatment of neurons with valproate, a mood stabilizing drug, suppresses GAPDH nuclear accumulation with a concomitant neuroprotective effect. Chromatin immunoprecipitation revealed that GAPDH is co-present with acetylated histone H3 and that valproate treatment caused a time-dependent decrease in levels of nuclear GAPDH with a concomitant increase in acetylated histone in the chromatin immunoprecipitation complex. Our results strongly suggest that valproate protects neurons from excitotoxicity through inhibition of histone deacetylase and that this neuroprotection involves suppression of excitotoxicity-induced accumulation of GAPDH in the nucleus. We have studied GAPDH abnormalities in a transgenic mouse model of Huntington's disease in which the disease protein (huntingtin) gene expresses expanded (89) CAG repeats. We found that GAPDH is overexpressed in neurons of discrete brain areas such as the hippocampal formation, caudate-putamen and globus pallidus, compared with wild type control. Confocal microscopic analysis revealed a predominant increase of GAPDH in the nucleus. Thus, mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of neurons in selective brain areas. Our results suggest that over-expression of GAPDH in CNS neurons could induce neuronal apoptosis and ultimate neurodegeneration. Moreover, the ability of GAPDH antisense oligonucleotides to rescue neurons from undergoing apoptotic cell death also suggests that such oligonucleotides may be useful for treating neurodegenerative diseases. In an attempt to further elucidate molecular mechanisms underlying apoptosis, we have used CGCs treated with AIDS-related neurotoxins, such as 3-OH- kynurenine (3-HK) and quinolinic acid. We found that 3-HK induces apoptosis in the range of 50-1000 micro-M, while quinolinic acid is ineffective. The 3-HK neurotoxicity is potentiated by superoxide dismutase (SOD) and a cell-permeable SOD mimetic, MnTBAP. Both 3-HK-induced and SOD-potentiated neurotoxicities are blocked by catalase, suggestive of hydrogen peroxide formation. We also found that 3-HK induces apoptotic death of PC12 pheochromocytoma cells and hypothalamic GT1-7 cells. In both cell types, 3-HK-induced apoptosis is robustly protected by dantrolene, a drug that inhibits Ca-2+ efflux from the endoplasmic reticulum to the mitochondria. Moreover, overexpression of Bcl-2, an anti-apoptotic gene product, in GT1-7 cells arrests 3-HK-induced neurotoxicity. Thus, pharmacological manipulation with dantrolene and/or gene therapy with Bcl-2 are potentially useful for the treatment of 3-HK-related neurodegeneration and other forms of neurodegenerative diseases. Glutamate excitotoxicity has been implicated in a variety of neurodegenerative diseases. Using CGCs as a model system, we have studied mechanisms underlying glutamate-induced apoptosis in these cell types. We found that glutamate excitotoxicity is associated with increased expression of apoptotic proteins, p53 and Bax, but with decreased expression of the cytoprotective protein, Bcl-2. The excitotoxicity is also concurrent with inhibition of the cell survival factors, Akt-1 and p-CREB (cyclic AMP-responsive element binding protein) due to activation of protein phosphatase PP2A and PP1, respectively. Additionally, glutamate-induced apoptosis is preceded by activation of p38 kinase and JNK (c-Jun N-terminal kinase), and suppression of these kinase activities results in neuroprotection. Activation of these kinases results in phosphorylation and activation of p53. Moreover, glutamate excitotoxicity mediated through NMDA receptors involves nuclear accumulation of GAPDH. In an attempt to elucidate the role of numerous proteins in apoptosis and neuroprotection, we have succeeded in developing high transfection efficiency technology of siRNA in primary cultures of neurons. Using rat cortical neurons transfected with siRNA for glycogen synthase kinase-3 (GSK-3), we have demonstrated that glutamate-induced apoptotic death involves activation of GSK-3alpha and beta isoforms and that silencing either isoform with its specific siRNA provides full protection against excitotoxicity. This conclusion is corroborated by using inhibitors of GSK-3 such as lithium, SB216763, SB415280, inhibitor I and inhibitor VII. Additionally, our experiments using siRNA silencing of GSK-3alpha and GSK-3beta provide evidence that these two isoforms can have distinct roles in the regulation of transcription factors such as CREB, NF-kB, EGR-1 and Smad3/4.