Oligomeric forms of amyloid β-protein (Aβ) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Aβ42 interfered with assembly of fulllength Aβ42 and inhibited Aβ42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Aβ42 assembly and neurotoxicity, here, we investigated the interaction between Aβ42 and CTFs using photo-induced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Aβ42 assembly and Aβ42-induced toxicity. Inhibition of Aβ42-induced toxicity was found to correlate with stabilization of oligomers with hydrodynamic radius (R H ) = 8-12 nm and attenuation of formation of oligomers with R H = 20-60 nm. In contrast, inhibition of Aβ42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Aβ42-induced toxicity. Our findings provide an important insight into the mechanisms by which different CTFs inhibit the toxic effect of Aβ42 and suggest that stabilization of non-toxic Aβ42 oligomers is a promising strategy for designing inhibitors of Aβ42 neurotoxicity. KeywordsAmyloid β-protein; aggregation; neurotoxicity; inhibitor; dynamic light scattering Alzheimer's Disease (AD) is the most common neurodegenerative disease, affecting over 35 million people worldwide (1). Abundant evidence suggests that oligomeric forms of amyloid * To whom correspondence should be addressed: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Neuroscience Research Building 1, Room 451, 635 Charles E. Young Drive South, Los Angeles, CA 90095-7334, USA. gbitan@mednet.ucla.edu. Tel.: +1-310-206-2082; Fax.: +1-310-206-1700 The sequences of such inhibitors were based on random selection (9), selfrecognition of the central hydrophobic cluster (CHC) of Aβ (10-13), or structural modifications of sequences from the CHC or C-terminal regions (14-17). As the hypothesis of the cause of AD shifted from Aβ fibril formation and deposition to oligomeric Aβ, the design strategy for peptide inhibitors for treatment of AD was adjusted to target Aβ oligomerization. In view of the important role of the C-terminus in Aβ42 assembly and toxicity, we hypothesized that the C-terminal fragments (CTFs) of Aβ42 might disrupt Aβ42 assembly and inhibit its neurotoxicity. In a previous study, we confirmed this hypothesis and found that Aβ42 CTFs, Recently, we reported a systematic characterization of biophysical properties of all the CTFs in the original series (19), to which we added for additional structural insight two Aβ40 CTFs, Aβ(34-40) and , and a fragment derived from the putative folding nucleus of Aβ, Aβ(21-30) (20). We found that most of Aβ42 CTFs longer than 8 residues readily formed β-sheet-rich fibrils, whereas the shorter CTFs did not. The two Aβ40 CTFs were substantially less prone to aggregation than their Aβ42 CTF counterparts (19). Surprisingly, Aβ(30-40)...

BACKGROUND Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome (CFS) and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized micro-neutralization assays as a surrogate marker for detection of anti-MLV serological responses – with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses. STUDY DESIGN AND METHODS We developed a high-throughput micro-neutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not pre-immune serum. 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization. RESULTS 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed. CONCLUSION A micro-neutralization assay was developed for detection of XMRV, and can be applied in a high-throughput format for large scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV/MLV. It is likely that this moderate neutralization is mediated through another, non-specific mechanism.

Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer’s disease. Previously, we found that the C-terminal fragments (CTFs), Aβ(30–42) and Aβ(31–42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x–42) peptides, (x=28–39). Therefore, we chose these peptides as leads for further development. These CTFs are 12/13-amino-acid long, hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N-terminus resulted in toxic peptides. Therefore, next we incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting β-sheet formation. Focusing on Aβ(31–42), we used a two-step N-methyl (N-Me) amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side-chain. This strategy allowed exploring the role of both side-chain structure and N-Me substitution in inhibitory activity. We found that the introduction of N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31–42). In particular, N-Me amino acid substitution at positions 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short-peptides is highly structure-specific, providing basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.