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 and traditionally thought to be a house-keeping gene, is overexpressed during neuronal apoptosis in cultured neurons including cerebellar granule cells (CGCs) and cortical neurons. Using antisense oligonucleotides to GAPDH, we provided the first evidence for a role of GAPDH in neuronal apoptosis such as in CGCs treated with cytosine arabinoside (AraC). We also found that AraC-induced apoptosis of CGCs is robustly protected by the neurotrophins brain-derived neurotrophic factor (BDNF) and NT 4/ 5. The AraC-induced apoptosis is also associated with an increased expression of two death genes, p53 and Bax. Our results suggest that GAPDH over-expression depends on p53 expression, and that GAPDH is a novel target gene regulated by p53. AraC-induced GAPDH over-expression is predominantly accumulated in the nucleus, and this nuclear translocation occurs in parallel with a loss of GAPDH glycolytic and uracil glycosylase activities, but precedes the activation of caspase-3.[unreadable] In CGCs, we studied the role of GAPDH in the neuro-excitotoxicity induced by SYM 2081 (2S,4R,-4-methylglutamate), an inhibitor of excitatory amino acid transporter and an agonist of low affinity kainate receptors. 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 and anti-convulsant, 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 to alter gene expression.[unreadable] 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, and GAPDH is a target of therapeutic intervention.[unreadable] 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. 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 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 also causes phosphorylation and activation of p53. Moreover, glutamate excitotoxicity mediated through NMDA receptors involves nuclear accumulation of GAPDH.[unreadable] 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. During the spontaneous death of cortical neurons in cultures, GSK-3betaSerine9, but not GSK-3alphaSerine21 isoform is dephosphorylated, and is selectively activated. 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.[unreadable] In a collaborative effort with Dr. J-S Hongs section of NIEHS, NIH, we also investigated the apoptosis of microglia derived from the midbrain of rats. We recently reported that valproate pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic neurotoxicity through inhibition of microglia over-activation. We now demonstrated that valproate and other histone deacetylase inhibitors (butyrate and trichostatin A) induce apoptosis of microglia, which shows typical apoptotic hallmarks including loss of mitochondrial membrane potential, phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Our results underscore the potential utility of histone deacetylase inhibitors in preventing inflammation-related neurodegenerative disorders.[unreadable] In conjunction with the group of Dr. Zheng-Hong Qin at Soochow University School of Medicine in China, we have investigated mechanisms underlying apoptotic death of striatal neurons resulting from unilateral infusion of quinolinic acid (QA), an NMDA receptor agonist, into one side of the striata. Cell cycle re-entry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether NF-kB activation promotes cell cycle entry and neuronal apoptosis, we studied the relationship between NF-kB-mediated cyclin induction, BrdU incorporation and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected QA elicits a rise in cyclin D1 mRNA and protein levels. QA-induced NF-kB activation occurs in striatal neurons and non-neuronal cells and partially co-localizes with elevated cyclin D1 immunoreactivity and TUNEL-positive nuclei. QA triggers DNA replication as revealed by BrdU incorporation, and some striatal replicating cells were identified as neurons. Blockade of NF-kB nuclear translocation with the recombinant peptide, NF-kB SN50, attenuates QA-induced elevation in cyclin D1 and cell replication. QA-induced internucleosomal DNA fragmentation is blunted by G1/S phase cell cycle inhibitors. These findings suggest that excitotoxin-induced neuronal apoptosis may result from, at least partially, a failed cell cycle attempt. In another study, we evaluated the potential contribution of Bcl-2, p53 and c-Myc to the differential vulnerability of striatal neurons to QA. We found that p53 and c-Myc are robustly induced in medium-sized neurons, but not in large neurons, which are enriched in Bcl-2. Thus, the selective vulnerability of striatal medium spiny neurons to degeneration correlates with low levels of Bcl-2 and high levels of p53 and c-Myc.