Akihiko Takashima

Activation of tau protein kinase I/glycogen synthase kinase-3β by amyloid β peptide (25–35) enhances phosphorylation of tau in hippocampal neurons

According to the amyloid hypothesis for the pathogenesis of Alzheimer’s disease (AD), amyloid β peptide (Aβ) directly affects neurons, leading to neurodegeneration and tau phosphorylation, followed by the production of paired helical filaments (PHF) in neurofibrillary tangles (NFT). To analyze the relationship between the phosphorylation sites of tau and the activation of kinases in response to Aβ, we treated cultured rat hippocampal neurons with a peptide fragment of Aβ, Aβ(25–35). Aβ(25–35) treatment activated tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β) but not glycogen synthase kinase-3α (GSK-3α) or mitogen activated protein kinase (MAP kinase) in primary culture of hippocampal neurons. Using antibodies that recognize phosphorylated sites of tau, we showed that tau phosphorylation was enhanced in at least five sites (Ser199, Ser202, Ser396, Ser404, and Ser413 numbered according to the human tau isoform containing 441 amino acid residues), to an extent that depended on the level of TPK I/GSK-3β. Treatment with TPK I/GSK-3β antisense oligonucleotide inhibited the enhancement of tau phosphorylation induced by Aβ(25–35) exposure. Thus, TPK I/GSK-3β activation by Aβ(25–35) may lead to extensive tau phosphorylation.

Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3β and cyclin-dependent kinase 5, a component of TPK II

Abstract Using immunohistochemistry, we examined the localization of four types of proline-directed kinases in the brains of control rats and in the brains of non-demented aged human subjects, subjects with Alzheimer’s disease and those with Down’s syndrome. The four kinases were: cyclin-dependent kinase (cdk) 5, a component of tau protein kinase (TPK) II; TPK I/glycogen synthase kinase (GSK)-3β; GSK-3α; and mitogen-activated protein kinase (MAPK/ERK2). Each of these kinases has been reported to promote the hyperphosphorylation of tau protein in vitro. The kinases were located essentially in neurons, although the intensity and distribution of labeling varied. Antiserum for cdk5 showed the most preferential and consistent labeling of intraneuronal neurofibrillary tangles (NFT). Antiserum for TPK I/GSK-3β also labeled intraneuronal NFT. Double immunolabeling for TPK I/GSK-3β and tau1 showed that TPK I/GSK-3β was closely associated with NFT. Antiserum for GSK-3α labeled neurons weakly, and the intensity of labeling did not differ between neurons with and without NFT. Antiserum for MAPK labeled neurons in superficial cortical layers, but NFT appeared in both superficial and deep cortical layers. These findings suggest that cdk5 and TPK I/GSK-3β are the critically important kinases for the generation in vivo of hyperphosphorylated tau, the main component of the paired helical filaments in NFT.

Characterization of human presenilin 1 using N‐terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing

The majority of cases of early‐onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane‐spanning domain. We produced 7 monoclonal antibodies that react with 3 non‐overlapping epitopes on the N‐terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full‐size PS1 at M r 47 000 and a more abundant M r 28 000 product in membrane extracts from human brain and human cell lines. PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild‐type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.

Nontoxic Amyloid β Peptide1-42 Suppresses Acetylcholine Synthesis POSSIBLE ROLE IN CHOLINERGIC DYSFUNCTION IN ALZHEIMER'S DISEASE

We show here that amyloid β peptide1-42 (Aβ1-42) may play a key role in the pathogenesis of the cholinergic dysfunction seen in Alzheimers disease (AD), in addition to its putative role in amyloid plaque formation. Aβ1-42 freshly solubilized in water (non-aged Aβ1-42), which was not neurotoxic without preaggregation, suppressed acetylcholine (ACh) synthesis in cholinergic neurons at very low concentrations (10-100 nM), although non-aged Aβ1-40 was ineffective. Non-aged Aβ1-42 impaired pyruvate dehydrogenase (PDH) activity by activating mitochondrial τ protein kinase I/glycogen synthase kinase-3β, as we have already shown in hippocampal neurons (Hoshi, M., Takashima, A., Noguchi, K., Murayama, M., Sato, M., Kondo, S., Saitoh, Y., Ishiguro, K., Hoshino, T., and Imahori, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2719-2723). Neither choline acetyltransferase activity nor choline metabolism was affected. Therefore, the major cause of reduced ACh synthesis was considered to be an inadequate supply of acetyl-CoA owing to PDH impairment. Soluble Aβ1-42 increases specifically in AD brain (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem. 271, 4077-4081). This increase in soluble Aβ1-42 may disturb cholinergic function, leading to the deterioration of memory and cognitive function that is characteristic of AD.

Characterization of tau phosphorylation in glycogen synthase kinase-3β and cyclin dependent kinase-5 activator (p23) transfected cells

One of the histopathological markers in Alzheimers disease is the accumulation of hyperphosphorylated tau in neurons called neurofibrillary tangles (NFT) composing paired helical filaments (PHF). Combined tau protein kinase II (TPK II), which consists of CDK5 and its activator (p23), and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate tau to the PHF-form in vitro. To investigate tau phosphorylation by these kinases in intact cells, the phosphorylation sites were examined in detail using well-characterized phosphorylation-dependent anti-tau antibodies after overexpressing the kinases in COS-7 cells with a human tau isoform. The overexpression of tau in COS-7 cells showed extensive phosphorylation at Ser-202 and Ser-404. The p23 overexpression induced a mobility shift of tau, but most of the phosphorylation sites overlapped the endogenous phosphorylation sites. GSK-3beta transfection showed the phosphorylation at Ser-199, Thr-231, Ser-396, and Ser-413. Triplicated transfection resulted in phosphorylation of tau at 8 observed sites (Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-413).

Amyloid β peptide induces cytoplasmic accumulation of amyloid protein precursor via tau protein kinase I/glycogen synthase kinase-3β in rat hippocampal neurons ☆

Abstract Exogenous application of synthetic amyloid β protein (Aβ) is known to induce neurotoxic effects in rat hippocampal culture. We report here that Aβ (25–35) induces accumulation of amyloid precursor protein (APP) derivatives in the cytoplasm of neurons. At the same time, the level of the secreted form of APP released into the culture medium decreases. Tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β) antisense oligonucleotide blocked APP accumulation and prevented neuronal death. These results provide evidence that APP accumulation after Aβ treatment is regulated by TPK I/GSK-3β. Aβ neurotoxicity is probably mediated via phosphorylation of tau by TPK I/GSK-3β, resulting in an impairment of axonal transport, and cytoplasmic accumulation of APP.

Possible Role of Tau Protein Kinases in Pathogenesis of Alzheimer’s Disease

Tau protein kinases (TPK) I and II were isolated as candidate enzymes responsible for the hyperphosphorylation observed in PHF-tau. Four phosphorylation sites of tau were identified for each kinase, accounting for most, but not all, of the major phosphorylation sites of PHF-tau. Immunostaining with anti-TPKI antibody indicated that this kinase is up-regulated in AD brain. Such up-regulation of TPKI and phosphorylatioin of tau were reproduced by treating cultured hippocampal cells with amyloid beta (Abeta) protein. In addition, we found that TPKI can phosphorylate and inactivate pyruvate dehydrogenase (PDH), which is expected to result in depletion of acetyl-CoA, a key substrate of acetyl choline synthesis. Indeed, when septum cells were treated with Abeta, the level of acetyl choline decreased dramatically.

Different effects of Alzheimer-associated mutations of presenilin 1 on its processing

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimers disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site. By transient expression in PC12 cells and rat glioma cell line, C6, we examined their influence on the processing of PS 1. Cys410Tyr inhibited proteolytic processing of PS 1, while Gly384Ala and Leu392Val did not. Thus, the Alzheimer related mutations can be divided into two groups in terms of their effect on the proteolytic cleavage of PS 1.

Immunohistochemical analysis of COOH-termini of amyloid beta protein (Aβ) using end-specific antisera for Aβ40 and Aβ42 in Alzheimer's disease and normal aging

We examined by immunohistochemicistry the carboxyl (C)-terminus extent of the amyloid beta protein (Aβ) that constitutes senile plaques and amyloid angiopathy in the brains of non-demented and Alzheimers disease (AD) subjects. We developed two antisera, which selectively recognized free C-termini of Aβ: BC40 for Aβ40; and BC42 for Aβ42. BC42 labeled various types of senile plaques as well as reference Aβ antiserum, whereas only some parts of the senile plaques were positive with BC40: i.e., all of the plaque cores and some diffuse and primitive plaques. In the brains of non-demented middle-aged subjects, a majority of BC42-positive diffuse plaques were also positive with BC40, but with less intensity than that shown with BC42. The ratio of BC40-negative plaques increased with increasing plaque density. Amyloid in the meningeal vessels showed much greater immnnoreactivity with BC40 than with BC42. Some extracellular neurofibrillary tangles were positive with both BC40 and BC42, although most of them and a...

5-Azacytidine induces toxicity in PC12 cells by apoptosis

5-Azacytidine (5 Az)is a potent inhibitor of DNA methylation, and it may allow inactive genes to become expressed. In a previous study, we demonstrated that 5 Az administered to the dam induced apoptosis in the brains of fetal mice. In this study, the 5 Az-induced apoptosis was further characterized in differentiated PC 12 cells as a model for neuronal apoptosis. Cell death, determined by the activity of released lactate dehydrogenase (LDH) into the medium, occurred from 24 to 48 hrs after 5 Az treatment. Toxicity for differentiated PC 12 cells was observed on treatment with more than 10(-1) micrograms/ml of 5 Az, and it reached the maximal level at 10 micrograms/ml. Cycloheximide, an inhibitor of protein synthesis, prevented 5 Az toxicity, suggesting that this cell death required protein synthesis which could be related to the activation of a dormant gene(s). Electrophoresis of DNA from 5 Az-treated cells evoked ladder formation, indicating the cleavage of DNA into nucleosomes. Scanning electron microscopy demonstrated bleb formation, the so-called apoptotic bodies on the cell surface. The biochemical and morphological findings indicated that 5 Az-induced cell death occurred in the form of apoptosis. 5 Az-induced cell death was prevented by treatment with cAMP but not by treatment with high K+ or deoxycytidine. These results suggest that a cAMP-sensitive mechanism is involved in 5 Az-induced cell death. PC 12 cells should be of value in elucidating the molecular mechanism of 5 Az-induced neuronal apoptosis.