Soluble oligomers are common to most amyloids and may represent the primary toxic species of amyloids, like the Abeta peptide in Alzheimer's disease (AD). Here we show that all of the soluble oligomers tested display a common conformation-dependent structure that is unique to soluble oligomers regardless of sequence. The in vitro toxicity of soluble oligomers is inhibited by oligomer-specific antibody. Soluble oligomers have a unique distribution in human AD brain that is distinct from fibrillar amyloid. These results indicate that different types of soluble amyloid oligomers have a common structure and suggest they share a common mechanism of toxicity.

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca 2؉ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of diseaserelated amyloids (A␤, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6 -6 g ml ؊1 ) rapidly (ϳ5 s) elevated intracellular Ca 2؉ , whereas equivalent amounts of monomers and fibrils did not. Ca 2؉ signals evoked by A␤42 oligomers persisted after depletion of intracellular Ca 2؉ stores, and small signals remained in Ca 2؉ -free medium, indicating contributions from both extracellular and intracellular Ca 2؉ sources. The increased membrane permeability to Ca 2؉ cannot be attributed to activation of endogenous Ca 2؉ channels, because responses were unaffected by the potent Ca 2؉ -channel blocker cobalt (20 m). Instead, observations that A␤42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca 2؉ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.Alzheimer disease (AD) 1 is characterized by the appearance in the brain of plaques, containing extracellular deposits of amyloid ␤-peptide (A␤) that result from altered proteolytic processing of amyloid precursor protein, together with intracellular neurofibrillary tangles containing misfolded tau (1).Brain regions with plaques and tangles exhibit reduced numbers of synapses, and neurites associated with plaques and tangles are often damaged, suggesting a pivotal role for A␤ in the neuropathology of AD (2-5). Moreover, numerous other neurodegenerative disorders (including Huntington, Parkinson, and prion diseases) are also associated with the formation and accumulation of amyloid fibrils in specific brain areas (6, 7). These commonalities suggest a general mechanism of action for the more than 100 human amyloid-related diseases, whereby normally soluble peptides and proteins undergo aberrant folding (8).Aggregation of A␤ proceeds through several conformational states, including dimers, spherical oligomers composed of 10 -24 monomers, and strings of oligomers (protofibrils), before finally assuming an insoluble fibrillar conformation (9). The initial formulation of the "amyloid hypothesis" of AD specifically implicated fibrillar amyloid deposits (10). However, more recent evidence suggests that soluble oligomers may be the principal neurotoxic agent (11-15). Soluble A␤ oligomers are found in the cerebrospinal fluid of ...

Amyloid fibrillization is multistep process involving soluble oligomeric intermediates, including spherical oligomers and protofibrils. Amyloid oligomers have a common, generic structure, and they are intrinsically toxic to cells, even when formed from non-disease related proteins, which implies they also share a common mechanism of pathogenesis and toxicity. Here we report that soluble oligomers from several types of amyloids specifically increase lipid bilayer conductance regardless of the sequence, while fibrils and soluble low molecular weight species have no effect. The increase in membrane conductance occurs without any evidence of discrete channel or pore formation or ion selectivity. The conductance is dependent on the concentration of oligomers and can be reversed by anti-oligomer antibody. These results indicate that soluble oligomers from many types of amyloidogenic proteins and peptides increase membrane conductance in a conformation-specific fashion and suggest that this may represent the common primary mechanism of pathogenesis in amyloid-related degenerative diseases.Soluble amyloid oligomers are a common intermediate in the pathway for amyloid fibril formation and have been implicated as the primary toxic species of amyloids related to neurodegenerative disease (1-6). More recent reports indicate that soluble amyloid oligomers are intrinsically toxic even when they are formed from proteins that are not normally related to degenerative disease (3), and the toxic activity of soluble oligomers may be related to a common generic structure that they share (6). Although the primary mechanism of amyloid toxicity is not clear, the fact that different amyloids reside in either the cytosolic or extracellular compartments and the observation that cytosolic amyloid aggregates are toxic when applied externally to cells (6, 7) points to the cell plasma membrane as a potential primary target of amyloid pathogenesis. Indeed, there are many reports of membrane perturbations caused by amyloids like A␤ (8), but it isn't clear whether these effects are specific to soluble oligomers nor whether they are common to other types of amyloids. Here we report that homogeneous populations of spherical amyloid oligomers and protofibrils increase the conductivity of membranes by a non-channel mechanism. This effect is observed for all soluble oligomers tested regardless of protein sequence and is not observed for amyloid fibrils or soluble low molecular weight species, suggesting that the increase in membrane conductivity may be a primary common mechanism of amyloid oligomer pathogenesis. MATERIALS AND METHODSPeptide Synthesis-Peptide synthesis: A␤ peptides, prion 106 -126, and IAPP 1 were synthesized by fluoren-9-ylmethoxy carbonyl chemistry using a continuous flow semiautomatic instrument as described previously (9). The purity was checked by analytical reverse phase-high performance liquid chromatography and by electrospray mass spectrometry. Polyglutamine KKQ40KK was a gift from Dr. Ronald Wetzel, and ␣-synuclein was a gif...

Alzheimer disease is characterized by the abnormal aggregation of amyloid ␤ peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid ␤ oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid ␤ aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid ␤ monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.Protein aggregation into amyloid fibrils is a pathological hallmark of many neurodegenerative diseases, including Alzheimer disease (AD). 2 AD is characterized, in part, by the aggregation of amyloid ␤ protein (A␤) into fibrillar amyloid plaques in select areas of the brain (1). Compelling evidence indicates that A␤ aggregation is critical for neurodegeneration, suggesting that preventing this process may be an effective therapeutic approach for the treatment of AD (2-4).A number of small molecules have been reported to inhibit A␤ fibrillogenesis. Since it was initially presumed that toxicity is associated with mature fibers (5-8), the majority of inhibitor screens have been directed toward identifying modulators of A␤ fibrillization. These fibril inhibitor screens have resulted in the discovery of multiple inhibitor molecules (9 -24). Some compounds have also been shown to inhibit A␤-mediated cellular toxicity, and this activity was correlated with modulation of fibrillization (13,15,16,21,25).However, A␤ aggregation is a complicated process and appears to involve more than a simple conversion of soluble monomer to fiber. More recent evidence has pointed to the role of soluble amyloid oligomers or prefibrillar aggregation intermediates as the primary toxic species in degenerative amyloid diseases (2, 3). Electron microscopy and atomic force microscopy have identified spherical particles of ϳ3-10 nm that appear at early times of incubation and disappear as mature fibrils appear (26). These spherical oligomers a...