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ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoproteins and regulates the generation of high density lipoproteins. Previously, we have shown that lack of Abca1 significantly increases amyloid deposition and cognitive deficits in Alzheimer's disease model mice expressing human amyloid-β protein precursor (APP). The goal of this study was to determine if ABCA1 plays a role in memory deficits caused by amyloid-β (Aβ) oligomers and examine neurite architecture of pyramidal hippocampal neurons. Our results confirm previous findings that Abca1 deficiency significantly impairs spatial memory acquisition and retention in the Morris water maze and long-term memory in novel object recognition of APP transgenic mice at a stage of early amyloid pathology. Neither test demonstrated a significant difference between Abca1ko and wild-type (WT) mice. We also examined the effect of intra-hippocampal infused Aβ oligomers on cognitive performance of Abca1ko mice, compared to control infusion of scrambled Aβ peptide. Age-matched WT mice undergoing the same infusions were also used as controls. In this model system, we found a statistically significant difference between WT and Abca1ko mice infused with scrambled Aβ, suggesting that Abca1ko mice are vulnerable to the effect of mild stresses. Moreover, examination of neurite architecture in the hippocampi revealed a significant decrease in neurite length, number of neurite segments, and branches in Abca1ko mice when compared to WT mice. We conclude that mice lacking ABCA1 have basal cognitive deficits that prevent them from coping with additional stressors, which is in part due to impairment of neurite morphology in the hippocampus.

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells., Huntington's disease is caused by a polyglutamine stretch expansion in the first exon of huntingtin. Here, the authors use infrared spectroscopy and solid-state NMR and show that polymorphic huntingtin exon1 fibres differ in their flanking regions but not their core polyglutamine amyloid structures.

Passive amyloid-β (Aβ) vaccination has shown significant effects on amyloid pathology in pre-depositing amyloid-β protein precursor (AβPP) mice but the results in older mice are inconsistent. A therapeutic effect of LXR and RXR agonists consisting of improved memory deficits and Aβ pathology has been demonstrated in different Alzheimer's disease (AD) mouse models. Here, we report the effect of a combination of N-terminal Aβ antibody and synthetic LXR agonist T0901317 (T0) on AD-like phenotype of APP23 mice. To examine the therapeutic potential of this combination, the treatment of mice started at 11 months of age, when amyloid phenotype in this model is fully developed, and continued for 50 days. We show that Aβ immunization with or without LXR agonist restored the performance of APP23 transgenic mice in two behavior paradigms without affecting the existing amyloid plaques. Importantly, we did not observe an increase of brain microhemorrhage which is considered a significant side effect of Aβ vaccination. Target engagement was confirmed by increased Abca1 and ApoE protein level as well as increased ApoE lipidation in soluble brain extract. In interstitial fluid obtained by microdialysis, we demonstrate that immunization and T0 significantly reduced Aβ levels, indicating an increased Aβ clearance. We found no interaction between the immunotherapy and T0, suggesting no synergism, at least with these doses. The results of our study demonstrate that anti-Aβ treatments can ameliorate cognitive deficits in AβPP mice with advanced AD-like phenotype in conjunction with a decrease of Aβ in brain interstitium and increase of ApoE lipidation without affecting the existing amyloid plaques.

Protein aggregation and/or amyloid formation is an important intrinsic constraint on protein sequence evolution1–4, the focus of a variety of cellular housekeeping operations5, a significant challenge in biotechnology6–9 and a component of the molecular mechanisms of a number of important human neurodegenerative diseases10,11. Significant effort has been expended in developing an understanding of the molecular determinants of protein aggregation, and in particular in devising algorithms that might be predictive for the propensity of amino acid sequences to aggregate12–20. The fundamental approaches taken in both the development of these algorithms and their validation differ considerably. Empirical assessments of these algorithms have been limited, however, and, where attempted, have in some cases been restricted to amino acid compositions that are highly favorable in promoting aggregation. Recently, we defined several contributions of the short, 17-amino acid peptide segment called httNT, which is located at the N-terminus of the Huntington's disease protein huntingtin (htt), to spontaneous amyloid formation by N-terminal fragments of htt such as htt exon121–26. For example, we found that the httNT segment, as part of exon1-like fragments, greatly enhances nucleation of amyloid growth by the adjacent polyglutamine (polyQ) segment, without itself becoming incorporated into amyloid21–23. We also found that the httNT sequence alone, as a separate peptide, is an effective, if transient, inhibitor of amyloid nucleation by exon1-like fragments25. As part of an effort to determine the molecular basis for this inhibition, we generated a series of scrambled variants of the httNTQ sequence25. Most of these sequences were well-behaved under our experimental conditions and could be evaluated as inhibitors in our experiments. Some of them could not be evaluated, however, because they themselves undergo efficient spontaneous growth of β-aggregates under our experimental conditions. This was especially intriguing because the WT httNT sequence by itself aggregates very slowly and only at high concentrations, and then only to form α-helix rich oligomers22,23. It struck us that the behavior of this set of scrambled peptides might serve as a good test for how well previously described amyloid prediction algorithms can predict facile amyloid formation in a novel series of sequences not designed for that purpose. In this paper we report the spontaneous aggregation tendencies of the full set of scrambled sequences and evaluate how well a number of recently described predictive algorithms are able to distinguish aggregating peptides from peptides that remain soluble under physiological conditions. The results show that the available algorithms are only modestly successful at predicting amyloid propensity under conditions approaching physiological. In fact, it is not clear that any of these algorithms is capable of evaluating kinetics constraints on amyloid formation, even among sequences that might be capable of achieving snug steric zipper27, amyloid-like packing arrangements.

Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to "misfolded" monomers. One way to vet these candidates is to develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary β-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called "βHP," that exhibits greatly improved amyloid nucleation without measurably enhancing β-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within βHP. Modifications predicted to allow formation of a fully H-bonded β-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-β-hairpin H-bonding within βHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong β-hairpin encouraging motifs only rarely form β-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.

Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aβ aggregation mechanism that uses Aβ-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aβ-polyQ hybrids, and by extension for full-length Aβ peptides, in which a modular Aβ C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a β-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an engineered Aβ C-terminal fragment delays aggregation onset by Aβ-polyglutamine peptides and redirects assembly of Aβ42 fibrils., The elucidation of amyloid nucleation mechanisms remains challenging as early oligomeric intermediates are transient and difficult to distinguish. Here the authors use Aβ- polyglutamine hybrid peptides designed to slow and limit amyloid maturation to provide insights into the structures of Aβ self-assembly intermediates.

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.

The 17-amino-acid N-terminal segment (htt(NT)) 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 htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) 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 htt(NT) 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 htt(NT)Q(N) 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.

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in “domain-swapping” fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.

The aggregation of polyglutamine containing protein sequences is implicated in a family of familial neurodegenerative diseases, the expanded CAG repeat diseases. While the cellular aggregation process undoubtedly depends on the flux and local environment of these proteins, their intrinsic physical properties and folding/aggregation propensities must also contribute to their cellular behavior. Here we describe a series of methods for determining mechanistic details of the spontaneous aggregation of polyQ-containing sequences, including the identification and structural examination of aggregation intermediates.

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ(40) aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ(42) and decreases Aβ(42) toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I(KO) mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I(KO) mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I(KO) mice, insoluble Aβ(40) is increased more than 10-fold, and Aβ(42) is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I(KO) mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.

The aggregated, β-sheet rich amyloid structure represents a stable, alternatively folded state of polypeptides. Amyloid fibrils are associated with several important neurodegenerative diseases, such as Alzheimer’s and Huntington’s diseases 1, as well as a number of peripheral diseases of organ failure 2. Amyloid fibrils can be produced in vitro from many proteins, consistent with the polymeric structure of proteins and the relationship of amyloid fibrils to synthetic polymers 3. The fundamental unit of amyloid fibrils is the cross-β structure, in which β-sheet extended chains and sheet-sheet stacking interactions are perpendicular to the fibril axis and β-sheet H-bonds are parallel to the fibril axis 4. Details of the three-dimensional structures of amyloid fibrils are still being elucidated 5; 6; 7; 8; 9; 10; 11; 12 One striking feature of amyloid fibrils that sets them apart from most globular proteins is the ability of a single polypeptide chain to grow into more than one stable structure 13. The existence of multiple protein aggregate conformations, each of which can propagate with retention of structure, has long been speculated to be the basis for strain and species barrier effects in mammalian and yeast prion biology 14; 15. Polymorphism at the electron microscopy level, for example in Aβ amyloid fibrils 16; 17, has been known for some time, but it has not been clear whether these shape differences were due merely to different modes of super-assembly of a common protofilament structure, or to more substantial internal structural differences, such as β-sheet formation and side-chain packing. Previous solid state NMR and electron microscopy analyses suggest that the folded structures of Aβ(1–40) in two polymorphic amyloid fibrils are only modestly different, while the major structural differences are in how the folded peptides pack within the fibril cross-section 9; 18. Analysis of amyloid polymorphs of other protein sequences, however, suggests the possibility of polymorphic structures differing more extensively in the details of segmental folding, H-bonding and packing within the fibril 13; 19, and this is further suggested by the different manners in which sequence-related fragments from amyloid proteins pack within “β-spine” crystal structures 6. Polymorphism in amyloid fibrils may have profound biological consequences. It has been demonstrated that different polymorphic yeast prion fibrils generated in vitro produce different prion strain behavior when these are introduced into yeast 20; 21. Two structurally and functionally different polymorphic fibrils have been generated by exposing Aβ(1–40) to different growth conditions in vitro 22, and the identification of a third polymorphic form produced by elongation of Aβ peptides in vitro using fibrils extracted from AD brain 23 supports the idea that fibril polymorphism may contribute to variations within human diseases. In this paper we describe the creation of five self-propagating amyloid fibril structures by subjecting wild type Aβ(1–40) to different trial growth conditions. These Aβ(1–40) polymorphic fibrils vary considerably in structural properties. In particular, we find that amyloid polymorphs exhibit significant differences in the extent and locations of stable β-sheet, as probed by the number of backbone amides highly protected from hydrogen-deuterium exchange. We find that fibril stabilities assessed by their free energies of elongation 24 correlate extremely well with these β-sheet contents, consistent with the central role of β-sheet in fibril structure. The results illustrate the facility with which some peptides produce fibril polymorphs and suggest that β-sheet content contributes significantly to fibril properties.

Spinocerebellar ataxia type 1 (SCA1) is one of a group of nine expanded CAG repeat diseases, in which polyglutamine (polyQ) expansion above a threshold is associated with increased disease risk and aggregation. SCA1 is unique in which the polyQ in the disease protein, ataxin1, often contains a few His residues that appear to block toxicity. Here, we ask how His insertions affect aggregation by comparing a Q(30) peptide with and without a centrally inserted His-Gln-His sequence. We found that at pH 7.5-8.5, His interruptions decrease polyQ aggregation rates but do not change the spontaneous growth mechanism: nucleated growth polymerization with a critical nucleus of one without non-fibrillar intermediates. The decreased aggregation rates are because of reductions in nucleation equilibrium constants. At pH 6, however, the His-interrupted peptide aggregates by a different mechanism that involves a low ThT-binding intermediate and produces a polymorphic amyloid product. In aggregates grown at pH 7.5, the His residues are solvent-accessible. Aggregates of His-inserted polyQ are good seeds for Q(30) elongation, suggesting the potential to recruit polyQ proteins in the cell. Our data are therefore most consistent with His insertions blocking toxicity by suppressing rates and/or altering pathways of spontaneous aggregation.

Amyloid formation reactions exhibit two classes of polymorphisms: the metastable intermediates commonly observed during amyloid formation and the range of conformationally distinct mature fibrils often seen at the reaction endpoint. Although recent data suggest that spherical oligomers and protofibrils in most cases are not obligate intermediates of amyloid assembly, oligomeric states might sometimes serve as on-pathway intermediates. Mature amyloid polymorphs self-propagate as a result of the normally very high fidelity of amyloid elongation, giving rise to strain behavior and species barriers in prion phenomena. Oligomers, protofibrils and various polymorphic forms of mature amyloid fibrils seem to be distinguished by differences in atomic structure that give rise to differences in observed morphologies.

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington’s disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

More than 20 human diseases are associated with amyloid fibril formation, one of which is Huntington's Disease (HD). HD, which poses a risk for ∼200,000 Americans, is a neurodegenerative disease that degrades cognitive and motor functions and is one of ten disorders to feature polyglutamine (polyQ) domain expansion. The self-aggregation of expanded polyQ segments in the huntingtin protein (htt) leads to fibril formation. Flanking regions of the polyQ domain affect the kinetics of this aggregation. As protein aggregation appears to correlate to disease onset, we aim to understand the structure of the mature fibrils, a critical step toward resolving structural changes along the fibril formation pathway.Fibrils of site-specific isotopically labeled peptides mimicking polyQ-containing N-terminal fragments of the htt protein were studied by magic-angle-spinning (MAS) solid-state NMR (ssNMR). We probed the conformation of both the polyQ amyloid core and its flanking regions by multidimensional MAS ssNMR, finding an a-helical conformation for residues in the very N-terminus (httNT), which is known to initiate the aggregation process. Structural models of the aggregation pathway and fibril were assessed based on structural data, relaxation times, and order parameters from MAS ssNMR measurements. Fibrils featuring mutations in the linker region connecting the httNT helix to the amyloid core were also investigated. Earlier animal studies have shown these mutations to reduce toxicity, and EM shows significant changes in fibril morphology. The site-specific effects of these and other mutations, on both the httNT and the polyQ core, were explored through structural and dynamics measurements. The characterization of the mutants, which mimic post-translational Ser phosphorylation, suggests biophysical connections between the stability and molecular structure of species along the aggregation pathway and the observed changes in toxicity.

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington's disease. Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

Aggregation of expanded polyglutamine repeat-containing fragments of the huntingtin (htt) protein may play a key role in Huntington's disease. Consistent with this hypothesis, two Ser-to-Asp mutations in the 17-amino-acid N-terminal htt(NT) segment abrogate both visible brain aggregates and disease symptoms in a full-length Q(97) htt mouse model while compromising aggregation kinetics and aggregate morphology in an htt fragment in vitro [Gu et al. (2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron64, 828-840]. The htt(NT) 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 htt(NT) 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 posttranslational 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 that they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser-to-Asp mutants.

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

Summary The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntington's disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.

The N-terminal 17 amino acid sequence in huntingtin (httNT) plays a crucial role in the aggregation of htt N-terminal fragment peptides (htt NTFs). In the current mechanistic model, httNT segments pack into α-helical bundles to form oligomers that create high local concentrations of appended polyglutamine (polyQ) segments, favoring nucleation of polyQ amyloid. Consistent with this model, we found previously that isolated httNT peptide added in trans inhibits the aggregation of htt NTF peptides, presumably by co-assembling with htt NTFs into mixed oligomers with decreased local polyQ concentrations. In a further test of this model, we found that the aggregation inhibitory activities of a set of ten scrambled httNT sequences correlate with their α-helical potential. We have now selected three of these scrambled sequences - (a) one that inhibits htt NTF aggregation (schttNTASSQ), (b) one that does not inhibit (schttNTFAKF), and (c) one that readily forms amyloid fibrils itself (unlike httNT or the other sequences) but does not inhibit (schttNTSAFM) - and used them in place of httNT to make synthetic analogs of htt NTF by adding a Q37P10K2 sequence. Consistent with expectations, we found that the peptide with the inhibitory, high α-helical potential, leader sequence (schttNTASSQ) exhibits an aggregation profile similar to that of the httNT-containing control peptide. In contrast, despite their identical amino acid compositions, htt NTF analogs containing the other two scrambled sequences (low aggregation inhibition, low α-helix) exhibit aggregation behavior more typical of simple polyQ sequences, hence deriving no kinetic benefit from their N-terminal sequences. Our results support a strong role for α-helix formation within httNT in greatly enhancing the kinetics of formation of the polyQ core of htt NTF amyloid.