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...

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

Nonfibrillar assemblies of amyloid ␤-protein (A␤) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using A␤ labeled at the N terminus or Lys 16 , we uncovered two distinct assembly pathways. One leads to highly toxic 10 -15-nm spherical A␤ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ϳ330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ϳ128 kDa (ϳ32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys 16 -labeled peptide disturbed fibril formation because the A␤ 16 -20 region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying A␤ assembly steps at which inhibition may be beneficial.

Edited by Miguel De la RosaKeywords: Miraculin Taste-modifying protein Signal sequence Apoplast Kunitz-type soybean protease inhibitor a b s t r a c t Miraculin, a glycoprotein that modifies sour tastes into sweet ones, belongs to the Kunitz-type soybean trypsin inhibitor (STI) family. To clarify the functional relation of miraculin with Kunitz-type STIs, we investigated its subcellular localization and trypsin inhibitory activity. In transgenic Arabidopsis thaliana, miraculin, fused to yellow fluorescent protein, localized to and outside the plasma membrane depending on the putative secretion signal peptide. When transgenic seedlings were cultured in liquid medium, miraculin was present in the supernatant only after cellulase treatment. No trypsin inhibitory activity was detected in native or recombinant miraculin. In conclusion, miraculin is secreted outside the plasma membrane through the function of a signal peptide, conserved in Kunitz-type STIs, whereas its trypsin inhibitory activity may be lost during its evolution.