Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of A␤42 are believed to be the main cause of AD; therefore, disruption of A␤ oligomerization is a promising approach for developing therapeutics for AD. Formation of A␤42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of A␤42 may get incorporated into oligomers of A␤42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using A␤ fragments with the general formula A␤(x؊42) (x ‫؍‬ 28 -39). A cell viability screen identified A␤(31-42) as the most potent inhibitor. In addition, the shortest peptide, A␤(39 -42), also had high activity. Both A␤(31-42) and A␤(39 -42) inhibited A␤-induced cell death and rescued disruption of synaptic activity by A␤42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of A␤42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with A␤42 to form heterooligomers. Thus, A␤(31-42) and A␤(39 -42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.Alzheimer's disease ͉ amyloid ␤-protein ͉ inhibitor design A lzheimer's disease (AD) is the predominant cause of dementia and one of the leading causes of death among elderly people. It is estimated that there are currently Ϸ27 million people suffering from AD worldwide (1). Because the world population is aging rapidly, if no cure is found in the near future AD will become an epidemic (2).The amyloid cascade hypothesis proposed that amyloid ␤-protein (A␤) fibrils-an aggregated form of A␤ found in amyloid plaques in the brains of patients with AD-were the neurotoxic agents causing AD (3). However, in recent years, multiple lines of evidence have led to a revision of this view, and today the primary toxins causing AD are believed to be early-forming A␤ oligomers rather than A␤ fibrils (4, 5). This paradigm shift suggests that efforts toward development of therapeutic agents targeting A␤ assembly should be directed at A␤ oligomers rather than fibrils. In particular, genetic, physiologic, and biochemical data indicate that oligomers of the 42-aa form of A␤, A␤42, are most strongly linked to the etiology of AD (6-9) and therefore are a particularly attractive target for inhibitor design.Several groups have reported small-molecule inhibitors of A␤ oligomerization (10-13). The importance of understanding the mechanism of inhibition recently has been highlighted (14) after findings that many small-molecule inhibitors of fibrillogenesis may act nonspecifically, likely making them unsuitable for treating amyloid-related disorders (15). In addition, inhibition of fibril formation may actually lead to stabilization of toxic oligomers (16). Interestingly, when oligomers are stabiliz...

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Understanding the structural and assembly dynamics of the amyloid ␤-protein (A␤) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the A␤ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for A␤40) or 42 (for A␤42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil 3 ␣/␤ 3 ␤ transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr 1 -substituted homologues of A␤40 and A␤42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr 1 ]A␤40 fibrils. Substitution of A␤40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both A␤40 and A␤42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr 1 ) to alter A␤ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that A␤ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD) 4 is the most common cause of latelife dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid ␤-protein (A␤) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Neurotoxicity studies have shown that A␤ assemblies are potent neurotoxins (5,(13)(14)(15)(16)(17)(18)(19)(20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble A␤ oligomers inhibit hippocampal long term potentiation (4,5,13,15,17,18,22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of A␤ (24,25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of A␤ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 ϫ g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activati...

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)...

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.