Simultaneous Single-Molecule Fluorescence and Conductivity Studies Reveal Distinct Classes of Aβ Species on Lipid Bilayers

Abstract

The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Aβ peptide. These fibrils have been traditionally believed to feature in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low molecular weight oligomers of Aβ that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single molecule optical detection, using fluorescently labeled human Aβ40, combined with membrane conductivity measurements, to monitor the interaction of single oligomeric peptide structures with model planar black lipid membranes (BLM). In a qualitative study, we show that the binding of Aβ to the membrane can be described by three distinctly different behaviors, depending on the Aβ monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few 100 nM, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of less than 10 pS/pore. At higher concentration, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductance (100's of pS) suggesting major disruption of the membrane. KeywordsAβ; amyloid; single molecule; pore; electrophysiology; permeability One of the hallmarks of Alzheimer's disease (AD) is the formation of insoluble neurofibrillary tangles and senile plaques in brain tissue. The major component of these proteinaceous plaques is the 39-43 amino acid Aβ peptide, derived by proteolytic cleavage of the membrane spanning amyloid precursor protein (APP). The mechanism that underlies the pathogenicity of Aβ towards neuronal tissue is the subject of intense research.In vitro studies of Aβ reveal that in aqueous solution the peptide progressively associates from its monomeric form via low molecular weight species to extended β-sheet fibrils, a process NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 April 13. Published in final edited form as:Biochemistry. 2010 April 13; 49(14): 3031-3039. doi:10.1021/bi901444w. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript that is highly dependent upon experimental conditions (1,2). It was originally assumed that Aβ becomes cytotoxic when it forms these large insoluble fibrillar aggregates (3,4), historically referred to as the amyloid hypothesis. For the purposes of this paper we use the term cytotoxic, to mean that Aβ causes deviations from the cell's ...

Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity

“…We also note that leakage in the antimicrobial, Cecropin A, has been suggested to be rate limited by changes in state and not pore size (24). Further parallels can be seen in Aβ from Alzheimer's disease (34), where, for example, the size distribution of oligomers is reported to have an effect on overall leakage rates (35). Thus, the behavior of IAPP, and likely other peptide amyloids such as Aβ, represents a generic property of the interaction of amphipathic peptides with biological membranes.…”

Oxidative Stress and Cell Membranes in the Pathogenesis of Alzheimer's Disease

“…Although such features are not always observed with A␤ proteins (114, 341), there are many reports suggesting that they are formed by other amyloidogenic proteins such as the insulin-associated polypeptide, ␣-synuclein, and serum amyloid A protein (259). It has been suggested that the channel-forming toxic properties of A␤ proteins on a membrane depends on the extent to which it has aggregated into oligomers and that this extent is concentration dependent (276). Model structures of the purported channels have been patterned after ␤-barrel poreforming toxins (8,93).…”

Biochemistry of Amyloid -Protein and Amyloid Deposits in Alzheimer Disease

“…There has been a longstanding controversy in the field as to whether this re-insertion event leads to the formation of a complete transmembrane pore or whether membrane association and partial insertion can disrupt the bilayer to such an extent that its structural integrity is compromised. MD simulations and in vitro artificial lipid membrane models of this insertional event are plentiful (Friedman et al 2009;Lemkul and Bevan 2009;Miyashita et al 2009;Qiu et al 2009;Song et al 2009;Yang et al 2009a,b;Morita et al 2010;Schauerte et al 2010;Wang et al 2010), but rigorous evidence for a hydrophobic membrane-traversing interaction in vivo is lacking. Using photobleaching Förster resonance energy transfer, there was a loss of signal from the hydrophobic carboxyl terminus of Ab as it interacts with the plasma membrane of PC12 cells, which may indicate its sequestration within the lipid bilayer (Bateman and Chakrabartty 2009).…”