Kaori Yasutake

Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β

β-Amyloid (Aβ) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of Aβ aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized Aβ1–40 peptide. We found that slow rotation of Aβ1–40 solution reproducibly gave self-aggregated Aβ1–40 containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other Aβ1–40 aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10- to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when Aβ1–42 formed ASPD by slow rotation. However, Aβ1–42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed ≈100-fold-higher toxicity than Aβ1–40-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break β-sheet interactions, Aβ may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I/glycogen synthase kinase-3β in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimers disease.

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.

Direct association of presenilin-1 with β-catenin

Families bearing mutations in the presenilin‐1 (PS1) gene develop Alzheimers disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co‐immunoprecipitation with PS1 in transfected COS‐7 cells indicates that PS1 directly interacts with endogenous β‐catenin, and the interaction requires residues 322–450 of PS1 and 445–676 of β‐catenin. Both proteins are co‐localized in the endoplasmic reticulum. Over‐expression of PS1 reduces the level of cytoplasmic β‐catenin, and inhibits β‐catenin‐T cell factor‐regulated transcription. These results indicate that PS1 plays a role as inhibitor of the β‐catenin signal, which may be connected with the AD dysfunction.

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.

Aβ amyloidosis induces the initial stage of tau accumulation in APPSW mice

Abstract To clarify how Aβ deposits induce secondary tauopathy, the presence of phosphorylated tau, glycogen synthase kinase 3α (GSK3α), GSK3β, cyclin-dependent kinase 5 (CDK5), mitogen-activated protein kinase (MAPK) and fyn were examined in the Tg2576 brain showing substantial brain Aβ amyloidosis and behavioral abnormalities. Phosphorylated tau at Ser199, Thr231/Ser235, Ser396 and Ser413 accumulated in the dystrophic neurites of senile plaques. The major kinase for tau phosphorylation was GSK3β. Smaller contributions of GSK3α, CDK5 and MAPK were suggested. Thus, brain Aβ amyloidosis has a potential role in the induction of tauopathy leading to the mental disturbances of Alzheimers disease.

Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimers disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10(-6) M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau.

Dual Roles of Proteasome in the Metabolism of Presenilin 1

Abstract : Presenilin 1 (PS1) has been identified as a causative gene for most early‐onset familial Alzheimers disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild‐type and familial Alzheimers disease‐associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full‐length PS1 was significantly accumulated. The levels of N‐ and C‐terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full‐length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N‐terminal fragment was attenuated by the inhibitor. Finally, 28‐kDa N‐ and 20‐kDa C‐terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.

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.

Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope

A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimers disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.