Ronald Wetzel

Polyglutamine (polyGln) aggregation is implicated in the disease progression of Huntington's disease and other expanded CAG repeat diseases. PolyGln aggregationin vitrofollows a simple nucleated growth polymerization pathway without apparent complications such as populated intermediates, alternative assembly pathways, or secondary nucleation phenomena. Previous analysis of the aggregation of simple polyGln peptides revealed that the critical nucleus (the number of monomeric units involved in the formation of an energetically unfavorable aggregation nucleus) is equal to one, suggesting that polyGln nucleation can be viewed as an unfavorable protein folding reaction. We provide here a method for experimentally determining the number of elongation growth sites per unit weight for any polyGln aggregate preparation, a key parameter required for completion of the nucleation kinetics analysis and determination of the thermodynamics of nucleation. We find that, for the polyGln peptide Q₄₇, the second-order rate constant for fibril elongation is 11,400 liters/mol per s, whereasK_n^*, the equilibrium constant for nucleation of aggregation, is remarkably small, equal to 2.6 × 10^-9. The latter value corresponds to a free energy of nucleus formation of +12.2 kcal/mol, a value consistent with a highly unfavorable folding reaction. The methods introduced here should allow further analysis of the energetics of polyGln nucleus formation and accurate comparisons of the seeding capabilities of different fibril preparations, a task of increasing importance in the amyloid field.

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Aβ protofibrils. Library screening yielded several molecules that stimulate Aβ aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts Aβ(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37°C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-Aβ aggregates exhibit a low thioflavin T response. Like Aβ fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-Aβ aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous Aβ fibril formation reaction: ≈12 of the 39 Aβ(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the Aβ molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in Aβ conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester Aβ in a form and/or location that cannot engage the toxic pathway.

The formation of amyloid-like aggregates by expanded polyglutamine (polyGln) sequences is suspected to play a critical role in the neuropathology of Huntington's disease and other expanded CAG-repeat diseases. To probe the folding of the polyGln sequence in the aggregate, we replaced Gln–Gln pairs at different sequence intervals with Pro–Gly pairs, elements that are compatible with β-turn formation and incompatible with β-extended chain. We find that PGQ ₉ and PGQ ₁₀ , peptides consisting of four Q ₉ or Q ₁₀ elements interspersed with PG elements, undergo spontaneous aggregation as efficiently as a Q ₄₅ sequence, whereas the corresponding PGQ ₇ and PGQ ₈ peptides aggregate much less readily. Furthermore, a P _D GQ ₉ sequence containing d -prolines aggregates more efficiently than the peptide with l -prolines, consistent with β-turn formation in aggregate structure. Introduction of one additional Pro residue in the center of a Q ₉ element within PGQ ₉ completely blocks the peptide's ability to aggregate. This strongly suggests that the Q ₉ elements are required to be in extended chain for efficient aggregation to occur. We determined the critical nucleus for aggregation nucleation of the PGQ ₉ peptide to be one, a result identical to that for unbroken polyGln sequences. The PGQ _N peptide aggregates are structurally quite similar to Q ₄₅ aggregates, as judged by heterologous seeding aggregation kinetics, recognition by an anti-polyGln aggregate antibody, and electron microscopy. The results suggest that polyGln aggregate structure consists of alternating elements of extended chain and turn. In the future it should be possible to conduct detailed and interpretable mutational studies in the PGQ ₉ background.

A number of observations point to the aggregation of expanded polyglutamine [poly(Q)]-containing proteins as playing a central role in the etiology of Huntington's disease (HD) and other expanded CAG-repeat diseases. Transfected cell and transgenic animal models provide some of this support, but irrefutable data on the cytotoxicity of poly(Q) aggregates is lacking. This may be due in part to difficulties in observing all aggregated states in these models, and in part to the inability to conclusively rule out the role of monomeric states of the poly(Q) protein. To address these questions, we produced aggregates of simple poly(Q) peptides in vitro and introduced them to mammalian cells in culture. We find that Cos-7 and PC-12 cells in culture readily take up aggregates of chemically synthesized poly(Q) peptides. Simple poly(Q) aggregates are localized to the cytoplasm and have little impact on cell viability. Aggregates of poly(Q) peptides containing a nuclear localization signal, however, are localized to nuclei and lead to dramatic cell death. Amyloid fibrils of a non-poly(Q) peptide are non-toxic, whether localized to the cytoplasm or nucleus. Nuclear localization of an aggregate of a short, Q₂₀, poly(Q) peptide is just as toxic as that of a long poly(Q) peptide, supporting the notion that the influence of poly(Q) repeat length on disease risk and age of onset is at the level of aggregation efficiency. The results support a direct role for poly(Q) aggregates in HD-related neurotoxicity.

In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2–3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37°C undergo a random coil to β-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, β-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal/mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.

Disease-related amyloid fibrils appear to share a common, but poorly understood, structure. We describe here the generation and preliminary characterization of two conformation-specific mAbs, WO1 and WO2, that bind to the amyloid fibril state of the Alzheimer's peptide Aβ(1–40) but not to its soluble, monomeric state. Surprisingly, these Abs also bind to other disease-related amyloid fibrils and amyloid-like aggregates derived from other proteins of unrelated sequence, such as transthyretin, islet amyloid polypeptide, β₂-microglobulin, and polyglutamine. At the same time, WO1 and WO2 do not bind to the native protein precursors of these amyloids, nor do they bind to other kinds of protein aggregates. This new class of Abs associated with a fundamental amyloid-folding motif appear to recognize a common conformational epitope with little apparent dependence on amino acid side chain information. These Abs should contribute to the understanding of amyloid structure, assembly, and toxicity and also may benefit the development of diagnostic and therapeutic agents for amyloid diseases.

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q₂₀ have been reported to be insoluble in water, but dissolution in – and evaporation from ‐ a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q₄₄ to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies—including in vitro aggregation experiments—on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.

We describe here experiments designed to characterize the secondary structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide Aβ, using hydrogen-deuterium exchange measurements evaluated by mass spectrometry. The results show that ≈50% of the amide protons of the polypeptide backbone of Aβ(1–40) resist exchange in aqueous, neutral pH buffer even after more than 1,000 h of incubation at room temperature. We attribute this extensive, strong protection to H-bonding by residues in core regions of β-sheet structure within the fibril. The backbone amide hydrogens exchange at variable rates, suggesting different degrees of protection within the fibril. These data suggest that it is unlikely that the entire Aβ sequence is involved in H-bonded secondary structure within the amyloid fibril. Future studies using the methods described here should reveal further details of Aβ fibril structure and assembly. These methods also should be amenable to studies of other amyloid fibrils and protein aggregates.