␤-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...

In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh tnscripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.In mice, germ cell precursors, termed primordial germ cells (PGCs), are generated in the epiblast. They consist of putative pluripotential cells that can be first identified as a small cluster of alkaline phosphatase-positive cells in the extraembryonic mesoderm at 7.25 days postcoitum (dpc). Then they move down to the embryonic mesoderm at the posterior end of the primitive streak (8.0 dpc), migrate through the hindgut endoderm, and colonize the developing genital ridges (10.5-11.5 dpc), in which they are destined to form functional germ cells (1, 2). Despite these detailed morphological observations, molecular mechanisms regulating this developmental pathway still remain unclear.Unlike mammals, it has been well established that several maternal factors are involved in germ cell determination of many animal species. In particular, in Drosophila it has been shown that the pole plasm localized at the posterior pole ofthe oocyte contains determining substances for the abdomen and the germ lineage. After fertilization, only the nuclei that migrate into the pole plasm are destined to form the germ-line progenitors (pole cells). Genetic identification ofgenes whose function is required for pole cell formation has revealed at least eight maternally active genes, cappucino, spire, staufen, oskar, vasa, valois, mago-nashi, and tudor (3, 4). Among these genes, vasa (vas) is one of the best characterized. Homozygous mutant vasa females produce no eggs. In ectopic formation of pole cells induced by mislocalization of oskar mRNA to the anterior pole of the oocyte, only vasa and tudor were required for ectopic pole cell formation (5-10). The gene vas encodes a DEAD (Asp-Glu-Ala-Asp)-family protein of putative RNA helicases, which is found to be present as a component of both the polar granules at the posterior end of the oocyte and the nuage structure in the germ cells, and zygotic expression is also restricted to the ge...

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...

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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