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...

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (␣APF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in ␣APF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric ␣-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a ␤ barrel structural motif.Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2). The genetic linkage between the mutant allele and disease is evidence of the causal relationship of amyloid accumulation to pathogenesis, and many of the mutations either destabilize the natively folded state, produce more amyloidogenic protein, or they increase its propensity to aggregate (3). Although fibrillar amyloid deposits are among the most obvious pathognomonic features of disease, their role in pathogenesis is not clear. The extent of fibrillar amyloid plaque deposition does not correlate well with Alzheimer's disease pathogenesis, and there are a significant number of non-demented individuals that have equivalent amounts of amyloid plaques as disease patients (4). Pathological changes are observed in transgenic animals before the onset of amyloid plaque accumulation (5, 6), and it has been reported that soluble A␤ oligomers correlate better with dementia than insoluble, fibrillar deposits (7, 8), suggesting that oligomeric forms of A␤ may represent the primary toxic species. Soluble oligomers have been implicated as the primary ...

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current–voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area compressibility of the lipid has a very large effect. Membranes with a large area compressibility modulus are almost insensitive to amyloid oligomers, but membranes formed from soft, highly compressible lipids are highly susceptible to amyloid oligomer-induced conductance changes. Furthermore, membranes formed using the solvent decane (instead of squalane) are completely insensitive to the presence of oligomers. One simple explanation for these effects on bilayer conductance is that amyloid oligomers increase the area per molecule of the membrane-forming lipids, thus thinning the membrane, lowering the dielectric barrier, and increasing the conductance of any mechanism sensitive to the dielectric barrier.

It is well established that Alzheimer's amyloid beta-peptides reduce the membrane barrier to ion transport. The prevailing model ascribes the resulting interference with ion homeostasis to the formation of peptide pores across the bilayer. In this work, we examine the interaction of soluble prefibrillar amyloid beta (Abeta(1-42))-oligomers with bilayer models, observing also dramatic increases in ion current at micromolar peptide concentrations. We demonstrate that the Abeta-induced ion conductances across free-standing membranes and across substrate-supported "tethered" bilayers are quantitatively similar and depend on membrane composition. However, characteristic signatures of the molecular transport mechanism were distinctly different from ion transfer through water-filled pores, as shown by a quantitative comparison of the membrane response to Abeta-oligomers and to the bacterial toxin alpha-hemolysin. Neutron reflection from tethered membranes showed that Abeta-oligomers insert into the bilayer, affecting both membrane leaflets. By measuring the capacitance of peptide-free membranes, as well as their geometrical thicknesses, the dielectric constants in the aliphatic cores of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers were determined to be epsilon = 2.8 and 2.2, respectively. The magnitude of the Abeta-induced increase in epsilon indicates that Abeta-oligomers affect membranes by inducing lateral heterogeneity in the bilayers, but an increase in the water content of the bilayers was not observed. The activation energy for Abeta-induced ion transport across the membrane is at least three times higher than that measured for membranes reconstituted with alpha-hemolysin pores, E(a) = 36.8 vs. 9.9 kJ/mol, indicating that the molecular mechanisms underlying both transport processes are fundamentally different. The Abeta-induced membrane conductance shows a nonlinear dependence on the peptide concentration in the membrane. Moreover, E(a) depends on peptide concentration. These observations suggest that cooperativity and/or conformational changes of the Abeta-oligomer particles upon transfer from the aqueous to the hydrocarbon environment play a prominent role in the interaction of the peptide with the membrane. A model in which Abeta-oligomers insert into the hydrophobic core of the membrane-where they lead to a local increase in epsilon and a concomitant reduction of the membrane barrier-describes the experimental data quantitatively.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable /8-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. (J. Clin. Invest.