Exploding the Repeat Length Paradigm while Exploring Amyloid Toxicity in Huntington's Disease.

Huntington's disease (HD) is a progressive, familial neurodegenerative disease triggered by the expansion of a polyglutamine (polyQ) track in the protein huntingtin (htt). PolyQ sequences up to Q36 in htt are not known to be toxic, while polyQ lengths above Q36 almost invariably lead to increased disease risk and decreased ages of onset. The large number of physical states (monomers, dimers, tetramers, non-ß oligomers, nanofibrils, and clustered amyloid fibrils) on the self-association landscape, with their overlapping kinetics of formation, have greatly complicated identification of the molecular species responsible for HD toxicity, drawing attention to the need for innovative approaches.After reports of HD-associated intraneuronal htt inclusions in 1997, we elucidated aggregation mechanisms of both simple polyQ sequences and the more complex polyQ-containing "exon1" fragment of htt (htt-ex1). Grounded in this work, the more recent results described here were made possible by breakthroughs in the molecular design of diagnostic polyQ derivatives and in fluorescence applications for characterizing amyloid assembly intermediates. Thus, insertion of ß-turn-promoting mutations into relatively short, disordered polyQ sequences created "pro-ß-hairpin" polyQs (ßHPs) that exhibit amyloid formation rates comparable to the enhanced rates seen with expanded polyQ peptides. Introduction of "ß-breaker" mutations into these ßHP polyQ sequences created molecules that are blocked from aggregating into amyloid and also can inhibit amyloid formation by other polyQ proteins. These mutational effects were then successfully transferred into more complex htt-ex1 sequence backgrounds. Insights into the aggregation properties of htt-ex1 derivatives-as well as into the nucleation process itself-were obtained using fluorescence correlation spectroscopy (FCS) and a novel thioflavin-T (ThT) protocol that allows quantitation of htt-ex1 assembly intermediates.Using these tools, we quantified physical states of htt-ex1 at different growth times in mammalian PC12 cells engineered for inducible expression of both normal and expanded polyQ repeat length versions of htt-ex1. For expanded polyQ versions, we found tetramers, oligomers, and fibrils (but no monomers) all populated in these cells at a time when the first indication of toxicity (nuclear DNA damage) was observed. These experiments provided a strong hint that monomeric forms of htt-ex1 are not involved in toxicity, but we were otherwise unable to implicate a specific toxic self-assembled state because of the overlapping kinetics of formation. To gain a more intimate focus and control over the timelines of htt-ex1 self-assembly and the resulting toxic response, we engineered various htt-ex1-ßHP molecules-with and without added ß-breaker mutations-that could be expressed in rat neuronal and Drosophila models of HD. In both models, novel htt-ex1-ßHP analogues exhibiting strong aggregation in spite of their very short polyQ repeat lengths proved to be toxic, dramatically breaking the "repeat length paradigm" and strongly suggesting that the toxic species must be some kind of aggregate. In both models, ß-breaker analogues of htt-ex1-ßHP that are slow to make amyloid-instead favoring accumulation of non-ß oligomers-were nontoxic. In contrast, htt-ex1-ßHP analogues that rapidly progress to amyloid states were toxic, suggesting that an aggregate possessing the fundamental amyloid folding motif is very likely the major toxic species in HD.

RAD52 is required for RNA-templated recombination repair in post-mitotic neurons.

It has been long assumed that post-mitotic neurons only utilize the error-prone non-homologous end-joining pathway to repair double-strand breaks (DSBs) associated with oxidative damage to DNA, given the inability of non-replicating neuronal DNA to utilize a sister chromatid template in the less error-prone homologous recombination (HR) repair pathway. However, we and others have found recently that active transcription triggers a replication-independent recombinational repair mechanism in G0/G1 phase of the cell cycle. Here we observed that the HR repair protein RAD52 is recruited to sites of DNA DSBs in terminally differentiated, post-mitotic neurons. This recruitment is dependent on the presence of a nascent mRNA generated during active transcription, providing evidence that an RNA-templated HR repair mechanism exists in non-dividing, terminally differentiated neurons. This recruitment of RAD52 in neurons is decreased by transcription inhibition. Importantly, we found that high concentrations of amyloid ß, a toxic protein associated with Alzheimer's disease, inhibits the expression and DNA damage response of RAD52, potentially leading to a defect in the error-free, RNA-templated HR repair mechanism. This study shows a novel RNA-dependent repair mechanism of DSBs in post-mitotic neurons and demonstrates that defects in this pathway may contribute to neuronal genomic instability and consequent neurodegenerative phenotypes such as those seen in Alzheimer's disease.

Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of ß-hairpin-containing ß-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical ß-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

Mutational analysis implicates the amyloid fibril as the toxic entity in Huntington's disease.

In Huntington disease (HD), an expanded polyglutamine (polyQ > 37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an α-helix-rich oligomer, or a ß-sheet-rich amyloid fibril. Here we describe an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in our experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. These engineered analogs are novel tools for mapping properties of polyQ self-assembly intermediates and products that should similarly be useful in the analysis of other expanded polyQ diseases. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.

Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency.

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis.

Biophysical underpinnings of the repeat length dependence of polyglutamine amyloid formation.

There are now 10 expanded CAG repeat diseases in which both disease risk and age of onset are strongly dependent on the repeat length of the polyglutamine (polyQ) sequence in the disease protein. Large, polyQ-rich inclusions in patient brains and in cell and animal models are consistent with the involvement of polyQ aggregation in the disease mechanism. This possibility is reinforced by studies showing strong repeat length dependence to the aggregation process, qualitatively mirroring the repeat length dependence of disease risk. Our understanding of the underlying biophysical principles that mediate the repeat length dependence of aggregation, however, is far from complete. A previous study of simple polyQ peptides showed that N*, the size of the critical nucleus that controls onset of aggregation, decreases from unfavorable tetramer to favorable monomer over the range Q23 to Q26. These data, however, do not explain why, for all peptides exhibiting N* ∼ 1, spontaneous aggregation rates continue to increase with increasing repeat length. Here we describe a novel kinetics analyses that maps out the nonlinear dependence with repeat length of a nucleation efficiency term that is likely related to aspects of nucleus structure. This trend accounts for why nucleus size increases to tetrameric at repeat lengths of Q23 or below. Intriguingly, both aggregation and age of onset trend with repeat length in similar ways, exhibiting large changes per added Gln at low repeat lengths and small changes per added Gln at relatively long repeat lengths. Fibril stability also increases with repeat length in a nonlinear fashion.

Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

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.

Polyglutamine amyloid core boundaries and flanking domain dynamics in huntingtin fragment fibrils determined by solid-state nuclear magnetic resonance.

In Huntington's disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A "protective" C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (htt(NT)) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the ß-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt ß-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the htt(NT) α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

Improved chemical synthesis of hydrophobic Aß peptides using addition of C-terminal lysines later removed by carboxypeptidase B.

Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid-phase synthesis of the Alzheimer's plaque peptide Aß40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aß42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid-phase synthesis of Aß peptides. In our method, Lys residues are linked to the desired peptide's C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B (CPB). With this method we obtained both Aß42 and Aß46 of superior quality that, for Aß42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with CPB removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aß46, which is extended from the sequence Aß42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aß42 or Aß40. The enhanced amyloidogenicity of Aß46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aß amyloid formation.

A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers.

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-ß core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington's disease (HD), the httNT segment on the polyQ's N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer - either the tetramer or a larger multimer - plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein-protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

A serendipitous survey of prediction algorithms for amyloidogenicity.

The 17- amino acid N-terminal segment of the Huntingtin protein, htt(NT), grows into stable α-helix rich oligomeric aggregates when incubated under physiological conditions. We examined 15 scrambled sequence versions of an htt(NT) peptide for their stabilities against aggregation in aqueous solution at low micromolar concentration and physiological conditions. Surprisingly, given their derivation from a sequence that readily assembles into highly stable α-helical aggregates that fail to convert into ß-structure, we found that three of these scrambled peptides rapidly grow into amyloid-like fibrils, while two others also develop amyloid somewhat more slowly. The other 10 scrambled peptides do not detectibly form any aggregates after 100 h incubation under these conditions. We then analyzed these sequences using four previously described algorithms for predicting the tendencies of peptides to grow into amyloid or other ß-aggregates. We found that these algorithms-Zyggregator, Tango, Waltz, and Zipper-varied greatly in the number of sequences predicted to be amyloidogenic and in their abilities to correctly identify the amyloid forming members of this scrambled peptide collection. The results are discussed in the context of a review of the sequence and structural factors currently thought to be important in determining amyloid formation kinetics and thermodynamics.

Kinetically competing huntingtin aggregation pathways control amyloid polymorphism and properties.

In polyglutamine (polyQ) containing fragments of the Huntington's disease protein huntingtin (htt), the N-terminal 17 amino acid htt(NT) segment serves as the core of α-helical oligomers whose reversible assembly locally concentrates the polyQ segments, thereby facilitating polyQ amyloid nucleation. A variety of aggregation inhibitors have been described that achieve their effects by neutralizing this concentrating function of the htt(NT) segment. In this paper we characterize the nature and limits of this inhibition for three means of suppressing htt(NT)-mediated aggregation. We show that the previously described action of htt(NT) peptide-based inhibitors is solely due to their ability to suppress the htt(NT)-mediated aggregation pathway. That is, under htt(NT) inhibition, nucleation of polyQ amyloid formation by a previously described alternative nucleation mechanism proceeds unabated and transiently dominates the aggregation process. Removal of the bulk of the htt(NT) segment by proteolysis or mutagenesis also blocks the htt(NT)-mediated pathway, allowing the alternative nucleation pathway to dominate. In contrast, the previously described immunoglobulin-based inhibitor, the antihtt(NT) V(L) 12.3 protein, effectively blocks both amyloid pathways, leading to stable accumulation of nonamyloid oligomers. These data show that the htt(NT)-dependent and -independent pathways of amyloid nucleation in polyQ-containing htt fragments are in direct kinetic competition. The results illustrate how amyloid polymorphism depends on assembly mechanism and kinetics and have implications for how the intracellular environment can influence aggregation pathways.

Assays for studying nucleated aggregation of polyglutamine proteins.

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.

Apolipoprotein A-I deficiency increases cerebral amyloid angiopathy and cognitive deficits in APP/PS1DeltaE9 mice.

A hallmark of Alzheimer disease (AD) is the deposition of amyloid ß (Aß) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aß(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 Aß(42) and decreases Aß(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 Aß oligomer levels, Aß plaque load, or levels of insoluble Aß in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aß isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I(KO) mice, insoluble Aß(40) is increased more than 10-fold, and Aß(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 Aß 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 aggregation-enhancing huntingtin N-terminus is helical in amyloid fibrils.

The 17-residue N-terminus (htt(NT)) directly flanking the polyQ sequence in huntingtin (htt) N-terminal fragments plays a crucial role in initiating and accelerating the aggregation process that is associated with Huntington's disease pathogenesis. Here we report on magic-angle-spinning solid-state NMR studies of the amyloid-like aggregates of an htt N-terminal fragment. We find that the polyQ portion of this peptide exists in a rigid, dehydrated amyloid core that is structurally similar to simpler polyQ fibrils and may contain antiparallel ß-sheets. In contrast, the htt(NT) sequence in the aggregates is composed in part of a well-defined helix, which likely also exists in early oligomeric aggregates. Further NMR experiments demonstrate that the N-terminal helical segment displays increased dynamics and water exposure. Given its specific contribution to the initiation, rate, and mechanism of fibril formation, the helical nature of htt(NT) and its apparent lack of effect on the polyQ fibril core structure seem surprising. The results provide new details about these disease-associated aggregates and also provide a clear example of an amino acid sequence that greatly enhances the rate of amyloid formation while itself not taking part in the amyloid structure. There is an interesting mechanistic analogy to recent reports pointing out the early-stage contributions of transient intermolecular helix-helix interactions in the aggregation behavior of various other amyloid fibrils.

Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease.

Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Structural variations in the cross-beta core of amyloid beta fibrils revealed by deep UV resonance Raman spectroscopy.

Understanding fibrillogenesis at a molecular level requires detailed structural characterization of amyloid fibrils. The combination of deep UV resonance Raman (DUVRR) spectroscopy and post mortem hydrogen-deuterium exchange (HX) was utilized for probing parallel vs antiparallel beta-sheets in fibrils prepared from full-length Abeta(1-40) and Abeta(34-42) peptides, respectively. Using previously published structural data based on solid-state NMR analysis, we verified the applicability of Asher's approach for the quantitative characterization of peptide conformation in the Abeta(1-40) fibril core. We found that the conformation of the parallel beta-sheet in the Abeta(1-40) fibril core is atypical for globular proteins, while in contrast, the antiparallel beta-sheet in Abeta(32-42) fibrils is a common structure in globular proteins. In contrast to the case for globular proteins, the conformations of parallel and antiparallel beta-sheets in Abeta fibril cores are substantially different, and their differences can be distinguished by DUVRR spectroscopy.

Therapeutic versus neuroinflammatory effects of passive immunization is dependent on Aß/amyloid burden in a transgenic mouse model of Alzheimer's disease.

BACKGROUND: Passive immunization with antibodies directed to Aß decreases brain Aß/amyloid burden and preserves memory in transgenic mouse models of Alzheimer's disease (AD). This therapeutic strategy is under intense scrutiny in clinical studies, but its application is limited by neuroinflammatory side effects (autoimmune encephalitis and vasogenic edema). METHODS: We intravenously administered the monoclonal Aß protofibril antibody PFA1 to aged (22 month) male and female 3 × tg AD mice with intermediate or advanced AD-like neuropathologies, respectively, and measured brain and serum Aß and CNS cytokine levels. We also examined 17 month old 3 × tg AD female mice with intermediate pathology to determine the effect of amyloid burden on responses to passive immunization. RESULTS: The 22 month old male mice immunized with PFA1 had decreased brain Aß, increased serum Aß, and no change in CNS cytokine levels. In contrast, 22 month old immunized female mice revealed no change in brain Aß, decreased serum Aß, and increased CNS cytokine levels. Identical experiments in younger (17 month old) female 3 × tg AD mice with intermediate AD-like neuropathologies revealed a trend towards decreased brain Aß and increased serum Aß accompanied by a decrease in CNS MCP-1. CONCLUSIONS: These data suggest that passive immunization with PFA1 in 3 × tg AD mice with intermediate disease burden, regardless of sex, is effective in mediating potentially therapeutic effects such as lowering brain Aß. In contrast, passive immunization of mice with a more advanced amyloid burden may result in potentially adverse effects (encephalitis and vasogenic edema) mediated by certain proinflammatory cytokines.

Polymorphism in the intermediates and products of amyloid assembly.

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.

Structures of Abeta-related peptide--monoclonal antibody complexes.

Passive immunotherapy (PI) is being explored as a potential therapeutic against Alzheimer's disease. The most promising antibodies (Abs) used in PI target the EFRH motif of the Abeta N-terminus. The monoclonal anti-Abeta Ab PFA1 recognizes the EFRH epitope of Abeta. PFA1 has a high affinity for Abeta fibrils and protofibrils (0.1 nM), as well as good affinity for Abeta monomers (20 nM). However, PFA1 binds the toxic N-terminally modified pyroglutamate peptide pyro-Glu3-Abeta with a 77-fold loss in affinity compared to the WT Abeta(1-8). Furthermore, our earlier work illustrated PFA1's potential for cross-reactivity. The receptor tyrosine kinase Ror2, which plays a role in skeletal and bone formation, possesses the EFRH sequence. PFA1 Fab binds the Ror2(518-525) peptide sequence REEFRHEA with a 3-fold enhancement over WT Abeta(1-8). In this work, the crystal structures of the hybridoma-derived PFA1 Fab in complex with pyro-Glu3-Abeta peptide and with a cross-reacting peptide from Ror2 have been determined at resolutions of 1.95 and 2.7 A, respectively. As with wild-type Abeta, these peptides bind to the Fab via a combination of charge- and shape-complementarity, hydrogen-bonding, and hydrophobic interactions. Comparison of the structures of the four peptides Abeta(1-8), Grip1, pyro-Glu3-Abeta(3-8), and Ror2 in complex with PFA1 shows that the greatest conformational flexibility occurs at residues 2 to 3 and 8 of the peptide. These structures provide a molecular basis of the specificity tolerance of PFA1 and its ability to recognize Abeta N-terminal heterogeneity. The structures provide clues to improving mAb specificity and affinity for pyroglutamate Abeta.