Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate Apoptosis, one form of programmed cell death, is a normal physiological process that occurs during development and prunes various cell populations to maintain a homeostasis (1, 2). Abnormal apoptosis has been linked to pathogenesis of a number of human diseases including cancer, viral infections, autoimmune diseases, and neurodegenerative disorders (3, 4). Apoptosis can be induced by various extrinsic and intrinsic factors such as activation of cell-surface receptors, growth factor withdrawal, oxidative stress, hormonal stimulation, and cell cycle perturbation (4). In this regard, it has been shown that cytosine arabinonucleoside (AraC) kills postmitotic rat sympathetic neurons in a manner similar to that caused by deprivation of nerve growth factor (5). Moreover, Dessi et al. (6) have reported that treatment of immature cerebellar granule cells (CGC) in culture with a high concentration of AraC induces a cascade of events including de novo synthesis of RNA and protein, leading to apoptosis of these neurons, as indicated by chromatin condensation and internucleosomal DNA cleavage. However, neither the underlying mechanism nor the protein(s) involved in AraC-induced apoptosis is clear.CGC are the most abundant neurons in the cerebellum and mature postnatally, in contrast to other types of cerebellar neurons (7). Therefore, cultured CGC represent the most enriched neuronal model available for studying the molecular mechanisms by which apoptosis is involved. For example, it has been shown that apoptosis results when CGC are exposed to nondepolarizing concentrations of KCl (8-10) or 13-amyloid peptide containing residues 25-35 (11). In a previous study (12), we found that CGC undergo age-induced apoptosis in culture, which is associated with the over-expression of a particulate 38-kDa protein that we identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Antisense oligodeoxyribonucleotides directed against GAPDH mRNA block this GAPDH over-expression and significantly delay age-induced apoptosis of cultured CGC. In an initial effort to explore the generality of the involvement of GAPDH in apoptosis, we undertook the present study. Here, we show that AraC-induced apoptotic death of immature CGC in culture is robustly inhibited by pretreatment with two antisense, but not sense, oligonucleotides to GAPDH. AraCinduced apoptosis is preceded by an increase in GAPDH mRNA which can be suppressed by these GAPDH antisense oligonucleotides, while the corresponding sense oligonucleotides are totally inactive in this regard. MATERIALS AND METHODSCell Culture. CGC were prepared from 8-day-old SpragueDawley rats and cultured as described (13). The dissociated cells were suspe...

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.

We recently reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is directly involved in cytosine arabinonucleoside (ara-C)- and low K+-induced neuronal death of cultured cerebellar granule cells. The former is entirely due to apoptosis, whereas the latter involves both apoptosis and necrosis. We examined the subcellular distribution of the overexpressed GAPDH occurring during apoptosis by using both subcellular fractionation and immunocytochemistry with a monoclonal antibody directed against this overexpressed protein. When immature cerebellar neurons were exposed to ara-C, an overexpression of GAPDH was observed, primarily in the nuclear fraction. In contrast, low K+ exposure of mature cerebellar neurons induced the overexpression of GAPDH not only in the nuclear fraction but also in the mitochondrial fraction. In both paradigms, no significant change of GAPDH levels occurred in the microsomal and cytosolic fractions. Moreover, pretreatment with GAPDH antisense oligonucleotide or classic apoptotic inhibitors clearly suppressed the accumulation of GAPDH protein in these subcellular loci. This discrete nuclear localization of GAPDH during apoptosis was supported further by immunoelectron microscopy. Quantitative assessment of GAPDH immunogold labeling revealed that a approximately 5-fold increase in the intensity of gold particles was observed within the nucleus of apoptotic cells. Thus, the current results raise the possibility that neuronal apoptosis may be triggered by GAPDH accumulation in the nucleus, resulting in perturbation of nuclear function and ultimate cell death.