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 and traditionally thought to be a house-keeping gene, is over- expressed during neuronal apoptosis in cultured neurons including cerebellar granule cells (CGCs) and cortical neurons. Using antisense oligonucleotides to GAPDH, we provide the first evidence for a role of GAPDH in neuronal apoptosis. In CGCs treated with cytosine arabinoside (AraC) we found that the drug-induced apoptosis is robustly protected by the neurotrophins brain-derived neurotrophic factor (BDNF) and NT 4/ 5, but not NT-3. 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 assessed by subcellular fractionation study and 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. 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 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. 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. 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 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. However, 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. In collaboration with the group of Dr. Zheng-Hong Qin at Soochow University School of Medicine, 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 evidenced by BrdU incorporation; some striatal BrdU-positive cells were identified as neurons by co-localization with NeuN. Blockade of NF-kB nuclear translocation with the recombinant peptide, NF-kB SN50, attenuates QA-induced elevation in cyclin D1 and BrdU incorporation. QA-induced internucleosomal DNA fragmentation is blunted by G1/S phase cell cycle inhibitors. These findings suggest that NF-kB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin-induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.
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