Ryoichi Ishitani

Evidence that Glyceraldehyde‐3‐Phosphate Dehydrogenase Is Involved in Age‐Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: 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 Alzheimers 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 Alzheimers disease.

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNCs). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.

Pro-apoptotic protein glyceraldehyde-3-phosphate dehydrogenase promotes the formation of Lewy body-like inclusions.

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has long been recognized as a classical glycolytic protein; however, previous studies by our group and others have demonstrated that GAPDH is a general mediator initiating one or more apoptotic cascades. Our most recent findings have elucidated that an expression of a pro‐apoptotic protein GAPDH is critically regulated at the promoter region of the gene. Apoptotic signals for its subsequent aggregate formation and nuclear translocation are controlled by the respective functional domains harboured within its cDNA component. In this study, coexpression of GAPDH with either wild‐type or mutant (A53T) α‐synuclein and less likely with β‐synuclein in transfected COS‐7 cells was found to induce Lewy body‐like cytoplasmic inclusions. Unlike its full‐length construct, the deleted mutant GAPDH construct (C66) abolished these apoptotic signals, disfavouring the formation of inclusions. The generated inclusions were ubiquitin‐ and thioflavin S‐positive appearing fibrils. Furthermore, GAPDH coimmunoprecipitated with wild‐type α‐synuclein in this paradigm. Importantly, immunohistochemical examinations of post mortem materials from patients with sporadic Parkinsons disease revealed the colocalized profiles immunoreactive against these two proteins in the peripheral zone of Lewy bodies from the affected brain regions (i.e. locus coeruleus). Moreover, a quantitative assessment showed that about 20% of Lewy bodies displayed both antigenicities. These results suggest that pro‐apoptotic protein GAPDH may be involved in the Lewy body formation in vivo, probably associated with the apoptotic death pathway.

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 μM, 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.

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 Alzheimers disease.

5-Hydroxytryptamine binding to butanol extracts from myelin fragments

The myelin fraction of rat brain stem was treated with butanol-water mixtures, and the extracted proteolipids were separated by Sephadex LH20 column chromatography. 2 peaks of proteolipids eluted in chloroform-methanol 4/1 showed the binding capacity for C14·5-HT. This finding suggests the necessity of the more careful investigations for the probability of proteolipids as receptor proteins in the central nervous system.

Implication of acidic lipids in 5-hydroxytryptamine receptor mechanisms

To establish the possible involvement of acidic lipids in 5-HT receptor mechanisms, we subjected whole rat brain synaptic plasma membranes to treatment with several kinds of lipid-modifying reagents and examined the [3H]5-HT and [3H]spiperone binding properties of the membranes. [3H]5-HT binding was decreased by treatment with Azure A, while [3H]spiperone binding was not altered. Similarly, prior treatment with arylsulphatase reduced the former binding, but had no effect on the latter binding. On the other hand, neither [3H]ligand binding was sensitive to phospholipases C and D. In contrast, prior treatment with phospholipase A2 (unheated) drastically decreased the [3H]5-HT binding and also affected the [3H]spiperone binding to some extent. Chelation of Ca2+ by EGTA (5 mM) prior to incubation of membranes with the unheated phospholipase A2 did not completely prevent the inhibitory effect of this enzyme on [3H]5-HT binding, while in the heated enzyme (at 100 degrees C for 10 min) EGTA exhibited this preventive effect perfectly. Furthermore, it was an interesting find that at least a low concentration of the heated phospholipase A2 (0.01 U) had no effect on the [3H]spiperone binding, as contrasted with the case of [3H]5-HT binding. In addition, the reduction of [3H]5-HT binding capacity in membranes treated with phospholipase A2 (heated and unheated) was restored only slightly by treatment with BSA (1%). Scatchard analysis of the [3H]5-HT binding showed that Azure A and phospholipase A2 (heated) decreased the Bmax values with no significant alteration in the KD values, whereas arylsulphatase increased only the KD value. All these observations infer that certain acidic lipids may play a role as the recognition site(s) or modulator(s) of 5-HT1 receptor molecules.

Butanol extracts from myelin fragments. VI. Lack of specificity of 5-hydroxytryptamine binding to recombinate fraction with sulphatides and phosphatidylinositol

Abstract To clarify the participation of sulphatides and PI for the binding of 5-HT to myelin butanol extracts, binding experiments of 14C·5-HT to the recombinate fraction of these two acidic lipids were performed. The recombinate fraction was incubated with 5 × 10−7 M of 14C·5-HT, and elution profile of 14C·5-HT, sulphatides and PI was examined by Sephadex LH20 column chromatography. Three components were eluted with CM 4:1 and elution areas of those relatively corresponded to each other. On the other hand, when 3 fold excess volumes of solvents were used 14C·5-HT was also eluted with CM 4:1 but the clear difference was observed in the elution pattern of sulphatides and PI. In the myelin butanol extracts, we have reported that the 5-HT binding macromolecules present in the myelin extracts would be composed of saturable and non-saturable components, and ACh, DA and tryptamine specifically inhibited the saturable 5-HT binding. Therefore, on the recombinate system of two acidic lipids the specificity of 14C·5-HT binding was investigated by displacement experiments. The results indicated that only ACh slightly inhibited the 14C·5-HT binding but 5-HT, DA and tryptamine had no effect. All these observations suggest that other component(s) besides sulphatides and PI may be implicated in the binding of 5-HT to myelin butanol extracts.

Butanol extracts from myelin fragments. III. SDS-urea gel electrophoretic studies

The butanol extracts from myelin fragments showing 5-HT binding capacity were studied by SDS-urea gel electrophoresis. 5 main bands were observed and their molecular weights were determined by the method of Ferguson relationship, and revealed that these extracts contained proteolipid protein, DM-20 and basic proteins. Furthermore, the reconstituted fraction with crude basic proteins and lipids showed the saturable binding capacity for C14·5-HT.