A long-lived Aß oligomer resistant to fibrillization.

The hydrophobic Aß peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of Aß increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of Aß is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that Aß peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this Aß dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel Aß oligomer that resists conversion to fibrils and remains stable for more than one year.

Structural heterogeneity and intersubject variability of Aß in familial and sporadic Alzheimer's disease.

Point mutations in the amyloid-ß (Aß) coding region produce a combination of mutant and WT Aß isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aß determines WT Aß conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aß deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aß, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aß40 fibrils into transgenic mice expressing only WT Aß induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aß40 prions induce a conformation of WT Aß similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aß prion conformations, which kinetically dominate the spread of prions in the brain.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Structural Polymorphism of Alzheimer's ß-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study.

The amyloid-ß (Aß) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aß sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aß. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aß40 fibrils. (13)C, (15)N-labeled synthetic E22G Aß40 peptides are studied and compared with wild-type and Osaka E22Δ Aß40 fibrils. Under the same fibrillization conditions, Arctic Aß40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aß40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aß40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aß42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aß40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aß peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

Specific aromatic foldamers potently inhibit spontaneous and seeded Aß42 and Aß43 fibril assembly.

Amyloid fibrils are self-propagating entities that spread pathology in several devastating disorders including Alzheimer's disease (AD). In AD, amyloid-ß (Aß) peptides form extracellular plaques that contribute to cognitive decline. One potential therapeutic strategy is to develop inhibitors that prevent Aß misfolding into proteotoxic conformers. Here, we design specific aromatic foldamers, synthetic polymers with an aromatic salicylamide (Sal) or 3-amino benzoic acid (Benz) backbone, short length (four repetitive units), basic arginine (Arg), lysine (Lys) or citrulline (Cit) side chains, and various N- and C-terminal groups that prevent spontaneous and seeded Aß fibrillization. Ac-Sal-(Lys-Sal)3-CONH2 and Sal-(Lys-Sal)3-CONH2 selectively inhibited Aß42 fibrillization, but were ineffective against Aß43, an overlooked species that is highly neurotoxic and frequently deposited in AD brains. By contrast, (Arg-Benz)4-CONH2 and (Arg-Sal)3-(Cit-Sal)-CONH2 prevented spontaneous and seeded Aß42 and Aß43 fibrillization. Importantly, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibited formation of toxic Aß42 and Aß43 oligomers and proteotoxicity. None of these foldamers inhibited Sup35 prionogenesis, but Sal-(Lys-Sal)3-CONH2 delayed aggregation of fused in sarcoma (FUS), an RNA-binding protein with a prion-like domain connected with amyotrophic lateral sclerosis and frontotemporal dementia. We establish that inhibitors of Aß42 fibrillization do not necessarily inhibit Aß43 fibrillization. Moreover, (Arg-Sal)3-(Cit-Sal)-CONH2 inhibits formation of toxic Aß conformers and seeding activity, properties that could have therapeutic utility.

Distinct synthetic Aß prion strains producing different amyloid deposits in bigenic mice.

An increasing number of studies continue to show that the amyloid ß (Aß) peptide adopts an alternative conformation and acquires transmissibility; hence, it becomes a prion. Here, we report on the attributes of two strains of Aß prions formed from synthetic Aß peptides composed of either 40 or 42 residues. Modifying the conditions for Aß polymerization increased both the protease resistance and prion infectivity compared with an earlier study. Approximately 150 d after intracerebral inoculation, both synthetic Aß40 and Aß42 prions produced a sustained rise in the bioluminescence imaging signal in the brains of bigenic Tg(APP23:Gfap-luc) mice, indicative of astrocytic gliosis. Pathological investigations showed that synthetic Aß40 prions produced amyloid plaques containing both Aß40 and Aß42 in the brains of inoculated bigenic mice, whereas synthetic Aß42 prions stimulated the formation of smaller, more numerous plaques composed predominantly of Aß42. Synthetic Aß40 preparations consisted of long straight fibrils; in contrast, the Aß42 fibrils were much shorter. Addition of 3.47 mM (0.1%) SDS to the polymerization reaction produced Aß42 fibrils that were indistinguishable from Aß40 fibrils produced in the absence or presence of SDS. Moreover, the Aß amyloid plaques in the brains of bigenic mice inoculated with Aß42 prions prepared in the presence of SDS were similar to those found in mice that received Aß40 prions. From these results, we conclude that the composition of Aß plaques depends on the conformation of the inoculated Aß polymers, and thus, these inocula represent distinct synthetic Aß prion strains.

Hsp104 suppresses polyglutamine-induced degeneration post onset in a drosophila MJD/SCA3 model.

There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.

Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients.

It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures, including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate noncooperatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits cooperatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates, but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable "cross-ß" cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and nonamyloid clients, which impose distinct mechanical demands.