Increasing evidence suggests that formation and propagation of misfolded aggregates of 42-residue human amyloid β (Aβ(1–42)), rather than the more abundant Aβ(1–40), provokes the Alzheimer’s cascade. To date, structural details of misfolded Aβ(1–42) have remained elusive. Here we present the atomic model of Aβ(1–42) amyloid fibril based on solid-state NMR (SSNMR) data. It displays triple parallel-β-sheet segments that are different from reported structures of Aβ(1–40) fibrils. Remarkably, Aβ(1–40) is not compatible with the triple-β motif, as seeding with Aβ(1–42) fibrils does not promote conversion of monomeric Aβ(1–40) into fibrils via cross-replication. SSNMR experiments suggest that the Ala42 carboxyl terminus, absent in Aβ(1–40), forms a salt-bridge with Lys28 as a self-recognition molecular switch that excludes Aβ(1–40). The results provide insight into Aβ(1–42)-selective self-replicating amyloid propagation machinery in early-stage Alzheimer’s disease.

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␤-Amyloid (A␤) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of A␤ aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized A␤1-40 peptide. We found that slow rotation of A␤1-40 solution reproducibly gave self-aggregated A␤1-40 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 A␤1-40 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 A␤1-42 formed ASPD by slow rotation. However, A␤1-42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed Ϸ100-fold-higher toxicity than A␤1-40-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 ␤-sheet interactions, A␤ 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 I͞gly-cogen synthase kinase-3␤ 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 Alzheimer's disease.A 40-to 42-residue peptide named ␤-amyloid (A␤) is a major constituent of senile plaques in Alzheimer's disease (AD) (1). Although multiple pathways have been suggested to lead to AD, recent advances indicate a causal link between A␤ and AD (2), and this idea is supported further by findings that vaccination against A␤ ameliorates behavioral deficits in transgenic mice (3-5). Among various in vivo A␤ species, A␤ 1-42 generally is considered as the primary vehicle of toxicity, whereas A␤ 1-40 , a major species under physiological conditions, is considered less harmful and more resistant to the formation of oligomers than A␤ 1-42 (6). However, it remains controversial which A␤ species contributes predominantly to AD pathogenesis, because both in vitro and in vivo studies have confirmed toxicity of A␤ 1-40 aggregates (7,8).It has been widely accepted that toxicity of A␤ requires aggregation of native A␤ monomers (9-11). Besides fibrils, several types of nonfibrillar aggregates have been reported: A␤ 1-40 oligomers from dimers-hexamers (6, 12-14); a mixture of A␤ 1-42 oligomers named A␤-derived diffusible ligands (ADDLs), ranging from trimers-hexamers up to 24-mers (15); and fibril intermediates named protofibrils (PFs) (16,17). All of these aggregates are mixtures of A␤ oligomers with a variety in oligomer size, and precise morphological analysis of each A␤ a...

Amyloid ␤-protein (A␤) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, A␤ assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10 -15-nm spherical A␤ assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (>100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, A␤-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other A␤ assemblies.Neurodegenerative diseases, such as Alzheimer disease (AD), 2 Parkinson disease, prion diseases, and the polyglutamine diseases, arise from abnormal protein interactions in the central nervous system (1). In these diseases, complex multistep processes of protein conformational change and accretion produce various nonfibrillar assemblies, leading finally to fibrils (1-5). Recent studies have suggested that the early assemblies in this process might be the most toxic, possibly through the exposure of buried moieties and the formation of surface tertiary structures not present in physiologic monomers (6). These surface tertiary structures could mediate abnormal interactions with other cellular components (1).In AD, extensive studies have suggested that accumulation of amyloid ␤-protein (A␤), a physiologic derivative of amyloid precursor protein (APP), plays a primary pathogenic role (7-9). Various forms of assemblies ranging in mass from dimers up to multimers of ϳ1 MDa have been reported as neurotoxins (10 -13) as follows: protofibrils (14); dimers/trimers (natural low-n oligomers) (15); 3-24-mer A␤-(1-42) assemblies termed A␤-derived diffusible ligands (ADDLs) (16); 12-mers termed globulomers (17) or A␤*56 (18); 15-20-mer A␤ assemblies te...

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGFactivated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass ( z 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/ polyacrylamide gel electrophoresis revealed further that the NGF-or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of x 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2 -3 h.The PC12 pheochromocytoma cell line is a useful system for studying the mechanism of action of nerve growth factor (NGF). Exposure of PC12 cells to NGF results in differentiation of the cells, i.e. the conversion from a chromaffinlike phenotype into a sympathetic-neuron-like phenotype [I]. Although the detailed biochemical mechanisms of action of NGF are not known, it is well known that treatment with NGF induces the enhanced phosphorylation of a number of proteins in PC12 cells [2 -81.The mechanisms of mitogenic signal transduction evoked by mitogens such as epidermal growth factor (EGF) has been intensively investigated so far, and a phosphorylation cascade is thought to play an essential role in transduction ofmitogenic signals [9, 101. MAP kinase is one of the kinases involved in the phosphorylation cascade 111 -141. We have previously found that treatment with various mitogens including EGF, platelet-derived growth factor, fibroblast growth factor, insuCorrespondence to E. Nishida,

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Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.NMR | computational modeling | abnormal protein-protein interaction in synapse | hyperexcitotoxicity | protein-protein interaction inhibitors