Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.

Genetic, neuropathological, and biochemical studies have provided strong evidence for a central role of amyloid in the pathogenesis of Alzheimer's disease (AD). We have proposed previously that peptides designed as beta-sheet breakers may be useful in preventing the formation of amyloid plaques. In this study, we describe a modified beta-sheet breaker peptide with improved pharmacological properties, a high rate of penetration across the blood-brain barrier, and the ability to induce a dramatic reduction in amyloid deposition in two different transgenic AD models. In addition, we report for the first time a significant increase in neuronal survival and a decrease in brain inflammation associated with the reduction of amyloid plaques. These results demonstrate that the process of amyloid deposition is one of the causes of neurodegeneration in AD. Moreover, our findings indicate that beta-sheet breaker peptides provide a valuable tool for evaluating further the importance of amyloid in the etiology of AD and suggest that these peptides or some of their derivatives might be good candidates for AD treatment.

The inheritance of the apolipoprotein E (apoE) epsilon4 allele is a prevailing risk factor for sporadic and familial Alzheimer's disease (AD). ApoE isoforms bind directly to Alzheimer's amyloid beta (Abeta) peptides both in vitro and in vivo. Recent studies suggest that association of apoE with lipids may modulate its interaction with Abeta. We examined the binding of lipid-associated and delipidated apoE3 and apoE4 isoforms to Abeta utilizing a solid-phase binding assay and estimated the dissociation constants for the interaction of various apoE and Abeta species. Using native apoE isoforms from stably transfected RAW 264 and human embryonic kidney 293 cells, apoE3 had greater affinity than apoE4 for both Abeta1-40 and Abeta1-42. Delipidation of apoE decreased its affinity for Abeta peptides by 5-10-fold and abolished the isoform-specificity. Conversely, incorporation of apoE isoforms produced by baculovirus-infected Sf9 cells into reconstituted human high-density-lipoprotein lipoparticles restored the affinity values for Abeta peptides and resulted in preferential binding of apoE3. The data demonstrate that native lipid-associated apoE3 binds to Abeta peptides with 2-3-fold higher affinity than lipid-associated apoE4. Since the isoforms' binding efficiency correlate inversely with the risk of developing late-onset AD, the results suggest a possible involvement of apoE3 in the clearance or routing out of Abeta from the central nervous system as one of the mechanisms underlying the pathology of the disease.

Cerebral amyloid-beta (Abeta) deposition is central to the neuropathological definition of Alzheimer disease (AD) with Abeta related toxicity being linked to its beta-sheet conformation and/or aggregation. We show that a beta-sheet breaker peptide (iAbeta5) dose-dependently and reproducibly induced in vivo disassembly of fibrillar amyloid deposits, with control peptides having no effect. The iAbeta5-induced disassembly prevented and/or reversed neuronal shrinkage caused by Abeta and reduced the extent of interleukin-1beta positive microglia-like cells that surround the Abeta deposits. These findings suggest that beta-sheet breakers, such as iAbeta5 or similar peptidomimetic compounds, may be useful for reducing the size and/or number of cerebral amyloid plaques in AD, and subsequently diminishing Abeta-related histopathology.