Trace detection of the conformational transition of beta-amyloid peptide (Abeta) from a predominantly alpha-helical structure to beta-sheet could have a large impact in understanding and diagnosing Alzheimer's disease. We demonstrate how a novel nanofluidic biosensor using a controlled, reproducible surface enhanced Raman spectroscopy active site was developed to observe Abeta in different conformational states during the Abeta self-assembly process as well as to distinguish Abeta from confounder proteins commonly found in cerebral spinal fluid.

b-amyloid peptide (Ab) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Ab readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Ab aggregates or oligomers is still under investigation. In this article, we show that different Ab incubation conditions in vitro can affect the rate of Ab fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Ab aggregates faster than Ab prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Ab aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Ab fibrils formed with agitation. In addition, Ab fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Ab aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Ab aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Ab oligomers.

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␤-Amyloid (A␤) is the primary protein component of senile plaques associated with Alzheimer's disease and has been implicated in the neurotoxicity associated with the disease. A variety of evidence points to the importance of A␤-membrane interactions in the mechanism of A␤ neurotoxicity and indicates that cholesterol and gangliosides are particularly important for A␤ aggregation and binding to membranes. We investigated the effects of cholesterol and sialic acid depletion on A␤-induced GTPase activity in cells, a step implicated in the mechanism of A␤ toxicity, and A␤-induced cell toxicity. Cholesterol reduction and depletion of membraneassociated sialic acid residues both significantly reduced the A␤-induced GTPase activity. In addition, cholesterol and membrane-associated sialic acid residue depletion or inhibition of cholesterol and ganglioside synthesis protected PC12 cells from A␤-induced toxicity. These results indicate the importance of A␤-membrane interactions in the mechanism of A␤ toxicity. In addition, these results suggest that control of cellular cholesterol and/or ganglioside content may prove useful in the prevention or treatment of Alzheimer's disease.An important pathological hallmark of Alzheimer's disease (AD) 1 is the formation and progressive deposition of insoluble amyloid fibrils within the cerebral cortex (1). The key constituent of these amyloid deposits has been identified as a 39 -43-amino acid long polypeptide, ␤-amyloid peptide (A␤), which is derived primarily from the proteolytic cleavage of a much larger amyloid precursor protein (2). Presenilin 1 and 2 are believed to be involved in the proteolytic processing of the A␤ fragment (3-6). Evidence for the causative role of A␤ in the pathogenesis of AD partly comes from genetic studies, which linked mutations in the amyloid precursor protein and in the presenilins to inheritable forms of AD (7-9). Further evidence originates from in vitro toxicity studies with synthetic A␤ peptides, which have shown that A␤, in an aggregated state (fibril, protofibril, low molecular weight oligomer, or diffusible, nonfibrillar ligand), is toxic to neurons in culture (10 -17). Although it seems certain that A␤ plays a role in neurotoxicity associated with AD, the molecular mechanism of A␤ neurotoxicity remains unclear.Increasing evidence indicates that the neuronal cell membrane is important in the mechanism of A␤ toxicity. Studies (18 -21) have indicated that membrane components such as cholesterol and gangliosides alter the affinity of A␤ for phospholipid membranes. Once associated with the membranes, negatively charged phospholipids, cholesterol, and gangliosides have been shown to increase the ␤-sheet content and/or rate of aggregation of A␤ (19,(21)(22)(23)(24). Both in vivo and in vitro, alterations in soluble cholesterol and/or cholesterol biosynthesis have also been shown to affect the normal processing of amyloid precursor protein (25)(26)(27). In these studies, the inhibition of cholesterol synthesis led to decreased A␤ formation (25,27). In...

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beta-Amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. A beta has proven to be toxic only when aggregated; however, the structure of the aggregated species associated with toxicity is unknown. In the present study, we use hydrogen-deuterium isotope exchange (HX)-electrospray ionization mass spectrometry (MS) along with enzymatic digestion as a tool to examine at near residue level, the changes in A beta structure associated with aggregation to a fibril form. Our results show that the structure of A beta intermediate species formed early in the course of fibrillogenesis is dependent upon solvent conditions. Additionally, the HX-MS data of peptic A beta fragments suggest that the C-terminal segment of the peptide is approximately 35% protected from exchange in fibril-containing samples, relative to monomeric A beta species prepared in DMSO/H(2)O. The N-terminus (residues 1-4) is completely unprotected from exchange, and the fragment containing residues 5-19 is over 50% protected from exchange in the fibril-containing samples. This work contributes to our understanding of A beta structure associated with aggregation and toxicity and further application of this approach may aid in the design of agents that intervene in the A beta aggregation processes associated with neurotoxicity.

Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Aβ) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Aβ and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Aβ(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Aβ aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Aβ toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multicomponent oligomer distributions of aggregating peptides and proteins.