Miyuki Murayama

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.

Lithium inhibits amyloid secretion in COS7 cells transfected with amyloid precursor protein C100

To examine the regulation of amyloid secretion in more detail, Abeta sandwich ELISAs with high sensitivity and specificity were developed. Using this technique, we measured Abeta secreted from COS7 cells transiently transfected with APP C100 in the presence of LiCl, a potent glycogen synthase kinase (GSK)-3beta inhibitor. We found that both Abetax-40 and Abetax-42 secretion were reduced by LiCl treatment in a dose-dependent manner. Diminished amyloid secretion was associated with GSK-3beta activity. These results suggest that GSK-3beta might function as a possible mediator for regulating both amyloid deposition and tau pathology in Alzheimers disease (AD), and that lithium should be re-evaluated as a candidate reagent for preventing AD pathology.

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).

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.

Molecular cloning and expression of the rat homologue of presenilin-1☆

The rat homologue of the presenilin-1 (PS-1) gene, which is responsible for early-onset familial Alzheimers disease linked to chromosome 14, was cloned and sequenced. The predicted amino acid sequence showed quite high homology among rat, mouse, and human PS-1. Especially, the amino acid sequences of the putative transmembrane domains were highly conserved among the three species. The expression ĺevel of the PS-1 gene increased during brain development and the number of transcripts of the PS-1 gene changed during brain development. We found one transcript of the PS-1 gene in embryonic day 12 (E12)-E15 rat brain and two transcripts in E18-adult rat brain. Therefore, PS-1 may play a role in neurogenesis.

Cloning of cDNA and expression of the gene encoding rat presenilin-2

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimers disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimers disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

Amphiphilic helix is essential for the activity of brain injury-derived neurotrophic peptide (BINP)

To study the structure‐activity relationships of brain injury‐derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an α‐helical conformation in TFE solution which mimics the membrane environment. In the α‐helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.