Crystallization and preliminary X-ray analysis of the archaeosine tRNA-guanine transglycosylase from Pyrococcus horikoshii.

The archaeosine tRNA-guanine transglycosylase from the hyperthermophilic archaeon Pyrococcus horikoshii was crystallized and preliminary X-ray characterization was performed. Single crystals were grown by the hanging-drop vapour-diffusion method, using sodium/potassium phosphate and sodium acetate as precipitants. The space group is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 99.28 (14), c = 363.74 (56) A. The cryocooled crystals diffracted X-rays beyond 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8 (Harima). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.

GAPDH knockdown rescues mesencephalic dopaminergic neurons from MPP+ -induced apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) has a number of diverse functions apart from glycolytic function. We explored the possible involvement of GAPDH in 1-methyl-4-phenylpyridinium (MPP+)-induced death of mesencephalic dopaminergic neurons (MDNs) in culture. MPP+ (10 and 20 microM, 24 h) exposure selectively decreased the survival of tyrosine hydroxylase positive (TH+) MDNs, which manifested apoptotic features including shrinkage of the cell body, chromatin condensation and nuclear fragmentation. Two types of GAPDH antisense oligonucleotides almost completely rescued MDNs from MPP+ toxicity. GAPDH was strongly expressed in apoptotic TH+ neurons, and MPP+ exposure significantly increased the percentage of TH+ neurons in which GAPDH is over-expressed. Confocal microscopic analysis demonstrated the nuclear accumulation of GAPDH in neurons undergoing MPP+-induced apoptosis. These results suggest that MPP+ causes apoptosis of MDNs, concomitant with the over-expression and nuclear accumulation of GAPDH.

Over-expression of GAPDH induces apoptosis in COS-7 cells transfected with cloned GAPDH cDNAs.

The cDNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) were cloned from cerebellar neurons undergoing age-induced apoptosis and/or healthy cells. COS-7 cells were transfected with the isolated GAPDH cDNAs using to the Lipofectamine method. Assessment of cell death in this paradigm was performed by monitoring the co-transfected luciferase activities and the characterization of cell death was examined by the DNA fragmentation assay and Hoechst dye nuclear staining. These observations show that over-expression of GAPDH occurring from both cDNAs robustly induces apoptotic death in the transfected COS-7 cell cultures. Confocal-immunocytochemical studies using this GAPDH-specific monoclonal antibody revealed that nuclear translocation of overexpressed GAPDH is a primary apoptotic event. Our results directly demonstrate that over-expressed GAPDH functions as a 'killing protein' in apoptosis.

ONO-1603, a potential antidementia drug, delays age-induced apoptosis and suppresses overexpression of glyceraldehyde-3-phosphate dehydrogenase in cultured central nervous system neurons.

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 microM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 microM in contrast to a narrow effective range of 3 to 10 microM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 microM, whereas THA elicits severe neurotoxicity at a dose of >/=30 microM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.

Nuclear localization of overexpressed glyceraldehyde-3-phosphate dehydrogenase in cultured cerebellar neurons undergoing apoptosis.

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.

Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells.

We have reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is involved in age-induced apoptosis of the cultured cerebellar granule cells that grow in a depolarizing concentration (25 mM) of KCI. The present study was undertaken to investigate whether GAPDH overexpression also occurs and participates in apoptosis of the cerebellar granule cells that result from switching the culturing conditions from high (25 mM) to low (5 mM) concentrations of KCl. We found that exposure of granule cells to low potassium (K+) for 24 hr induces not only apoptosis but also necrotic damage. The latter is supported by the morphological observations that a subpopulation of neurons showed cell swelling, extensive cytoplasmic vacuolization, damaged mitochondria, and apparently intact nuclei. Treatments with two antisense but not sense oligodeoxyribonucleotides directed against GAPDH attenuated low K+-induced neuronal death by approximately 50%. Morphological inspection revealed that GAPDH antisense oligonucleotides preferentially blocked low K+-induced apoptosis with little or no effect on necrotic damage. Similar to antisense oligonucleotides, actinomycin-D partially inhibited low K+-induced death of granule cells with a predominant effect on apoptosis. In contrast, cycloheximide almost completely blocked low K+-induced neuronal death and seemed to prevent both apoptotic and necrotic damage. The levels of GAPDH mRNA and protein were markedly increased in a time-dependent manner after low K+ exposure. The overexpression of GAPDH mRNA and protein was completely blocked by cycloheximide, actinomycin-D, and its antisense but not sense oligonucleotides. Taken together, these results lend credence to the view that exposure of cerebellar granule cells to low K+ induces both apoptosis and necrosis and that only the apoptotic component involves overexpression of GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

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 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells.

ONO-1603, a potential antidementia drug, shows neuroprotective effects and increases m3-muscarinic receptor mRNA levels in differentiating rat cerebellar granule neurons.

We have reported that the antidementia drug tetrahydroaminoacridine (THA; 30 microM) is neuroprotective and neurotrophic and selectively increases m3-muscarinic acetylcholine receptor (mAChR) mRNA levels in differentiating cerebellar granule cells. Here, we examined whether novel prolyl endopeptidase inhibitor ONO-1603, a potential antidementia drug, induces similar effects in these cerebellar neurons. Supplement of ONO-1603 (0.03 microM) to cultures grown in 15 mM KCl-containing media was found to markedly promote neuronal survival and neurite outgrowth and enhance [3H]N-methylscopolamine binding to mAChRs. Moreover, ONO-1603 increased the level of m3-mAChR mRNA and stimulated mAChR-mediated phosphoinositide turnover. The common actions of ONO-1603 and THA suggest that these properties could be related to their putative antidementia activities and that this model system may be used to screen for drugs effective in the treatment for Alzheimer's disease.

An antisense oligodeoxynucleotide to glyceraldehyde-3-phosphate dehydrogenase blocks age-induced apoptosis of mature cerebrocortical neurons in culture.

We recently reported that the age-induced apoptotic death of cultured cerebellar neurons is correlated with an increased expression of a particulate-bound 38-kDa protein that we identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To determine whether this phenomenon of GAPDH overexpression occurs in other cell types, we selected primary cultures of cerebrocortical cells for testing, because under normal culture conditions, cortical neurons die progressively after 15 days in vitro. As with cerebellar neurons, this age-induced neuronal death involves ultrastructural changes and internucleosomal DNA fragmentation characteristic of apoptosis and is effectively prevented by actinomycin-D and cycloheximide. Moreover, a GAPDH antisense oligodeoxyribonucleotide arrested this cortical neuronal death for about 4 to 5 days and thus was more effective than cycloheximide. By contrast, its corresponding sense oligonucleotide had no effect. Additionally, the age-induced apoptosis of cortical neuronal cultures is effectively protected by aurintricarboxylic acid and tetrahy-droaminoacridine (an antidementia drug). Before cell death, GAPDH mRNA levels increased by about 2-fold and the increase was blocked by the above-mentioned neuroprotective agents and the GAPDH antisense, but not sense, oligonucleotide. The effects of antisense oligonucleotide are more robust in the present case than those found with cerebellar neurons, and they indicate a significant, though at present not defined, role of GAPDH in the apoptotic process occurring in these two types of neurons.

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.

Glyceraldehyde-3-phosphate dehydrogenase is over-expressed during apoptotic death of neuronal cultures and is recognized by a monoclonal antibody against amyloid plaques from Alzheimer's brain.

The age-induced apoptotic death of cerebellar neurons in culture is associated with over-expression of a 38-kDa particulate protein identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both the age-induced apoptosis and the 38-kDa protein overexpression were effectively suppressed by the presence of tetrahydroaminoacridine, an antidementia drug, or aurintricarboxylic acid. This over-expressed 38-kDa protein and purified GAPDH were found to react with a monoclonal antibody (mAb), Am-3, which was raised against amyloid plaques from Alzheimer's brain, but not with mAb, AmT-1, which was produced using synthetic amyloid beta peptide. These results raise the possibility that GAPDH is also involved in the neurodegeneration during the development of Alzheimer's disease.

Tryptamine induces phosphoinositide turnover and modulates adrenergic and muscarinic cholinergic receptor function in cultured cerebellar granule cells.

Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC50 = 56 microM). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca2+ and Na+. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4 beta-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC50 values of approximately 0.4 and approximately 2.5 mM, respectively. Pretreatment of cells with tryptamine (0.5 mM) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca(2+)- and Na(+)-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.

Tetrahydroaminoacridine increases m3-, but not m2-, muscarinic acetylcholine receptor mRNA levels in differentiating cerebellar granule cells.

We used Northern blot hybridization to determine whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a potential antidementia drug, selectively altered the levels of muscarinic acetylcholine receptor (mAChR) mRNA in differentiating cerebellar granule cells. Granule cells were cultured for 8 days in media containing 15 mM K+, 25 mM K+ or 15 mM K+ plus 30 microM THA. High K+ markedly increased the levels of m2- and m3-mAChR mRNA in the surviving cells. In contrast, THA increased the levels of m3-mAChR mRNA, but had little or no effect on m2-mAChR mRNA levels. These results suggest that THA selectively up-regulates the synthesis of m3-mAChR mRNA.

Autoradiographic demonstration of an increase in muscarinic cholinergic receptors in cerebellar granule cells treated with tetrahydroaminoacridine.

The neurotrophic and neurosurviving effects of 9-amino-1,2,3,4-tetrahydroacridine (THA), a putative antidementia agent, were studied in cultured granule cells using biochemical and morphological methods. The addition of 30 microM THA to cultures grown in 15 mM K(+)-containing media markedly increased cell survival and enhanced [3H]N-methylscopolamine binding to muscarinic cholinergic receptors (mAChRs). Furthermore, receptor autoradiographic studies revealed that neuronal cells were labelled over both cell bodies and fibers by the [3H]receptor ligand. These observations provide direct evidence that THA promotes the expression of mAChR binding sites in differentiating cerebellar granule cells.

Tetrahydroaminoacridine is neurotrophic and promotes the expression of muscarinic receptor-coupled phosphoinositide turnover in differentiating cerebellar granule cells.

We have investigated whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug with potential antidementia activity, has a trophic action on differentiating cerebellar granule cells by using the method of [3H]inositol incorporation into inositol-containing phospholipid. Addition of THA (30-50 microM) prevented the extensive neuronal degeneration which occurred in the growth medium containing "low" K+ (15 mM). These effects were similar to the neuroprotective action caused by the presence of 100 microM N-methyl-D-aspartate (NMDA). Neurotrophic effects of THA and NMDA on cells grown in low K+ were also demonstrated by direct microscopic examination of cellular morphology. Measurement of phosphoinositide (PI) response in the rescued cells indicated that NMDA modestly promoted the PI response to carbachol and norepinephrine but markedly stimulated the activity induced by glutamate. In contrast, although THA had little or no influence on the maturation of the norepinephrine- and glutamate-induced PI response, it selectively enhanced the activity stimulated by carbachol. Furthermore, the THA treatment drastically increased the Vmax value of carbachol-induced PI turnover with no significant alteration in the EC50 value. Scatchard analysis of the binding of N-[3H]methylscopolamine to intact granule cells indicated a selective increase in the maximum binding value in cells grown in THA-supplementing medium. These observations suggest that THA seems to selectively up-regulate muscarinic cholinergic receptors.

Effect of phospholipase A2 on temperature-induced high-affinity [3H]tryptamine binding sites in rat brain.

To investigate a link between membrane phospholipids and tryptamine binding molecules, we examined the effects of phospholipases A2 and D on the temperature-sensitive high-affinity [3H]tryptamine binding sites in rat brain. When the phospholipase A2-treated membranes were exposed to 1% bovine serum albumin (BSA) before assaying for [3H]tryptamine binding, a complete dose-dependent inhibition curve was observed. At a concentration of 0.03 U, the action of phospholipase A2 resulted in the splitting of phosphatidylserine (PS), choline phosphatides (PC) and ethanolamine phosphatides (PE) by about 32, 34 and 65%, respectively, and reduced [3H]ligand binding by about 32%. On the contrary, in the case of phospholipase D (500 U), PS and PC decreased by about 8% and 33% and PE by about 29% with no significant alteration in the binding capacity. Moreover, Scatchard analysis of the [3H]tryptamine binding showed that phospholipase A2 drastically increased only the KD value of the high affinity sites, and this was accompanied by a decrement of the Bmax values of both the high and low affinity binding sites. From these results, it is inferred that certain lipids (PS) may be a modulator for the function of the temperature-induced high-affinity [3H]tryptamine binding molecules.

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts.

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.

Effects of protein-modifying reagents on brain tryptamine binding sites: possible involvement of a thiol group in temperature-induced high-affinity [3H]tryptamine binding sites.

To investigate the biochemical nature of temperature-induced high-affinity [3H]tryptamine binding sites, we subjected whole rat brain synaptic membranes to treatment with various protein-modifying reagents and examined the subsequent [3H]tryptamine binding properties of the membranes. Pretreatment of the membrane preparations with NEM, NBS, PCMB, PAPMA and MA, but not with iodoacetamide, DTT, glutathione and cysteine, reduced the [3H]tryptamine binding. In addition, to at least approx. 10(-4) M, the inactivation properties of NEM, PCMB, PAPMA and MA, except for NBS, were temperature-dependent. Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with these thiol reagents conformed to a curved line, as well as in the case of the control membranes. Nonlinear regression analysis of these data showed that NEM decreased the Bmax values of both the high and low affinity binding sites with no significant alteration in the KD values, whereas PCMB, PAPMA and MA increased only the KD value of the high affinity sites, accompanying the decrease of the Bmax values of both sites. These results indicate that the temperature-induced high-affinity [3H]tryptamine binding molecule(s) is a thiol protein.