The 17-amino-acid N-terminal segment (httNT) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to httNT itself, form α-helix-rich oligomeric intermediates, only peptides with QN of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the httNT sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only httNTQN peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.

Since polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that, over a short repeat length range from Q26 to Q23, the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex anti-parallel β-sheet framework for the nucleus, and further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in aggregation nucleus size may play a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-␤ (A␤) peptide. However, the monomeric state of this aggregationprone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ؎ 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both A␤ 40 and A␤ 42 remain predominantly monomeric up to 3 M, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If A␤ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.Alzheimer disease (AD) 2 is a degenerative brain disorder that is associated with the presence of extracellular aggregates of amyloid-␤ (A␤) (1), which is an ϳ4.5-kDa peptide containing 39 -42 residues. Recent studies indicate that small soluble oligomers are key to A␤ toxicity (2-4). In the AD brain, both A␤ monomers and dimers have been isolated, and the dimers have been shown to impair synaptic plasticity in mouse hippocampal slices (5). In contrast, A␤ monomers have been shown to be devoid of neurotoxicity (5) and have in fact been suggested to be neuroprotective (6, 7). The monomer to oligomer transition is therefore not only the obligatory first event of aggregation, it is also the key event determining the transformation of a benign protein to a neurotoxic one.We address this transition from a thermodynamic viewpoint: an aggregation-capable molecule should have a defined equilibrium between monomers and dimers (or oligomers), such that it is primarily monomeric below a certain concentration. Any oligomer-enriched solution prepared below such a concentration must be thermodynamically unstable and must dissociate to monomers at a given rate. To understand AD in terms of A␤ aggregation, we need to understand how this concentration compares with the in vivo concentrations of A␤ (which is estimated to be Ͻ Ͻ1 M) (8 -11) and what the kinetics of A␤ oligomer dissociation is.However, experiments probing the monomer to oligomer transition have been difficult to perform due to the low concentration at which this transition most likely occurs, and they have yielded rather confusing results. Some studies have ...

Aggregation of expanded polyglutamine (polyQ) repeat containing fragments of the huntingtin (htt) protein may play a key role in Huntington’s disease (HD). Consistent with this hypothesis, two Ser to Asp mutations in the 17 amino acid N-terminal httNT segment abrogate both visible brain aggregates and disease symptoms in a full length Q97 htt mouse model while compromising aggregation kinetics and aggregate morphology in a htt fragment in vitro (Gu et al., Neuron 64, 828–840 (2009)). The httNT segment has been shown to play a critical role in facilitating nucleation of amyloid formation in htt N-terminal exon1 fragments. We show here how these Ser to Asp mutations dramatically affect aggregation kinetics and aggregate structural integrity. First, these negatively charged Ser replacements impair the assembly of the α-helical oligomers that play a critical role in htt amyloid nucleation, thus providing an explanation for reduced amyloid formation rates. Second, these sequence modifications alter aggregate morphology, decrease aggregate stability, and enhance the steric accessibility of the httNT segment within the aggregates. Together these changes make the sequence-modified peptides kinetically and thermodynamically less likely to aggregate and more susceptible, if they do, to post-translational modifications and degradation. These effects also show how phosphorylation of a protein might achieve cellular effects via direct impacts on the protein’s aggregation properties. In fact, preliminary studies on exon1-like molecules containing phosphoryl-Ser residues at positions 13 and 16 show they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser to Asp mutants.