Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Microglial INPP5D limits plaque formation and glial reactivity in the PSAPP mouse model of Alzheimer's disease.

INTRODUCTION: The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimer's disease (LOAD). METHODS: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL /PSEN1Δexon9 (PSAPP) mice, we injected 3-month-old Inpp5dfl/fl /Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl /Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. RESULTS: At age 6 months, we found that the percent area of 6E10+ deposits and plaque-associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown. DISCUSSION: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. HIGHLIGHTS: Inpp5d knockdown increases plaque burden and plaque-associated microglia number. Spatial transcriptomics identifies an expanded plaque-specific gene expression profile. Plaque-induced gene expression is altered by Inpp5d knockdown in microglia. Our plaque-associated gene signature overlaps with human Alzheimer's disease gene networks.

Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease.

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure.

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.

Common fibrillar spines of amyloid-ß and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors.

Amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aß and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aß and hIAPP, termed Aß(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aß and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aß(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aß and hIAPP.

Atomic-resolution structure of a disease-relevant Aß(1-42) amyloid fibril.

Amyloid-ß (Aß) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aß(1-40) and Aß(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aß(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aß oligomers are believed to be the neurotoxic species, Aß amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aß(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-ß-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a ß-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

Familial Alzheimer's Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-ß Peptide.

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-ß (Aß) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aß sequence is due to differences in the conformations adopted by mutant Aß peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aß peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aß, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aß. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aß sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aß to form immunologically and morphologically distinct amyloid structures.

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-ß.

Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.

Structural differences between amyloid beta oligomers.

In Alzheimer's disease, soluble Aß oligomers are believed to play important roles in the disease pathogenesis, and their levels correlate with cognitive impairment. We have previously shown that Aß oligomers can be categorized into multiple structural classes based on their reactivity with conformation-dependent antibodies. In this study, we analyzed the structures of Aß40 oligomers belonging to two of these classes: fibrillar and prefibrillar oligomers. We found that fibrillar oligomers were similar in structure to fibrils but were less stable towards denaturation while prefibrillar oligomers were found to be partially disordered. These results are consistent with previously proposed structures for both oligomer classes while providing additional structural information.

Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes.

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Dietary DHA supplementation in an APP/PS1 transgenic rat model of AD reduces behavioral and Aß pathology and modulates Aß oligomerization.

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on ß-amyloid (Aß) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aß oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aß indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aß plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aß oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aß oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aß oligomers and decreased prefibrillar (i.e. putatively more toxic) Aß oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aß aggregation by stabilizing soluble fibrillar Aß oligomers and thus reduce the formation of both Aß plaques and prefibrillar Aß oligomers. However, since fibrillar Aß oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aß oligomer levels for more effective prevention of AD in clinical settings.

Out-of-register ß-sheets suggest a pathway to toxic amyloid aggregates.

Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing ß-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register ß-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register ß-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register ß-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.

A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric aß, and frank neuronal loss.

Alzheimer's disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The "amyloid cascade hypothesis" posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aß peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.

Intracellular amyloid and the neuronal origin of Alzheimer neuritic plaques.

Genetic analysis of familial forms of Alzheimer's disease (AD) causally links the proteolytic processing of the amyloid precursor protein (APP) and AD. However, the specific type of amyloid and mechanisms of amyloid pathogenesis remain unclear. We conducted a detailed analysis of intracellular amyloid with an aggregation specific conformation dependent monoclonal antibody, M78, raised against fibrillar Aß42. M78 immunoreactivity colocalizes with Aß and the carboxyl terminus of APP (APP-CTF) immunoreactivities in perinuclear compartments at intermediate times in 10month 3XTg-AD mice, indicating that this represents misfolded and aggregated protein rather than normally folded APP. At 12months, M78 immunoreactivity also accumulates in the nucleus. Neuritic plaques at 12months display the same spatial organization of centrally colocalized M78, diffuse chromatin and neuronal nuclear NeuN staining surrounded by peripheral M78 and APP-CTF immunoreactivity as observed in neurons, indicating that neuritic plaques arise from degenerating neurons with intracellular amyloid immunoreactivity. The same staining pattern was observed in neuritic plaques in human AD brains, showing elevated intracellular M78 immunoreactivity at intermediate stages of amyloid pathology (Braak A and B) compared to no amyloid pathology and late stage amyloid pathology (Braak 0 and C, respectively). These results indicate that intraneuronal protein aggregation and amyloid accumulation is an early event in AD and that neuritic plaques are initiated by the degeneration and death of neurons by a mechanism that may be related to the formation of extracellular traps by neutrophils.

Systemic vaccination with anti-oligomeric monoclonal antibodies improves cognitive function by reducing Aß deposition and tau pathology in 3xTg-AD mice.

Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid-beta (Aß) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aß from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aß is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti-Aß immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence-independent non-fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg-AD mice. Anti-oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer-specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg-AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease.

CD45 deficiency drives amyloid-ß peptide oligomers and neuronal loss in Alzheimer's disease mice.

Converging lines of evidence indicate dysregulation of the key immunoregulatory molecule CD45 (also known as leukocyte common antigen) in Alzheimer's disease (AD). We report that transgenic mice overproducing amyloid-ß peptide (Aß) but deficient in CD45 (PSAPP/CD45(-/-) mice) faithfully recapitulate AD neuropathology. Specifically, we find increased abundance of cerebral intracellular and extracellular soluble oligomeric and insoluble Aß, decreased plasma soluble Aß, increased abundance of microglial neurotoxic cytokines tumor necrosis factor-α and interleukin-1ß, and neuronal loss in PSAPP/CD45(-/-) mice compared with CD45-sufficient PSAPP littermates (bearing mutant human amyloid precursor protein and mutant human presenilin-1 transgenes). After CD45 ablation, in vitro and in vivo studies demonstrate an anti-Aß phagocytic but proinflammatory microglial phenotype. This form of microglial activation occurs with elevated Aß oligomers and neural injury and loss as determined by decreased ratio of anti-apoptotic Bcl-xL to proapoptotic Bax, increased activated caspase-3, mitochondrial dysfunction, and loss of cortical neurons in PSAPP/CD45(-/-) mice. These data show that deficiency in CD45 activity leads to brain accumulation of neurotoxic Aß oligomers and validate CD45-mediated microglial clearance of oligomeric Aß as a novel AD therapeutic target.

Amyloid-ß annular protofibrils evade fibrillar fate in Alzheimer disease brain.

Annular protofibrils (APFs) represent a new and distinct class of amyloid structures formed by disease-associated proteins. In vitro, these pore-like structures have been implicated in membrane permeabilization and ion homeostasis via pore formation. Still, evidence for their formation and relevance in vivo is lacking. Herein, we report that APFs are in a distinct pathway from fibril formation in vitro and in vivo. In human Alzheimer disease brain samples, amyloid-ß APFs were associated with diffuse plaques, but not compact plaques; moreover, we show the formation of intracellular APFs. Our results together with previous studies suggest that the prevention of amyloid-ß annular protofibril formation could be a relevant target for the prevention of amyloid-ß toxicity in Alzheimer disease.

Heterodivalent linked macrocyclic ß-sheets with enhanced activity against Aß aggregation: two sites are better than one.

This paper reports a series of heterodivalent linked macrocyclic ß-sheets 6 that are not only far more active against amyloid-ß (Aß) aggregation than their monovalent components 1a and 1b but also are dramatically more active than their homodivalent counterparts 4 and 5. The macrocyclic ß-sheet components 1a and 1b comprise pentapeptides derived from the N- and C-terminal regions of Aß and molecular template and turn units that enforce a ß-sheet structure and block aggregation. Thioflavin T fluorescence assays show that heterodivalent linked macrocyclic ß-sheets 6 delay Aß(1-40) aggregation 6-8-fold at equimolar concentrations and substantially delay aggregation at substoichiometric concentrations, while homodivalent linked macrocyclic ß-sheets 4 and 5 and monovalent macrocyclic ß-sheets 1a and 1b only exhibit more modest effects at equimolar or greater concentrations. A model to explain these observations is proposed, in which the inhibitors bind to and stabilize the early ß-structured Aß oligomers and thus delay aggregation. In this model, heterodivalent linked macrocyclic ß-sheets 6 bind to the ß-structured oligomers more strongly, because N-terminal-derived component 1a can bind to the N-terminal-based core of the ß-structured oligomers, while the C-terminal-derived component 1b can achieve additional interactions with the C-terminal region of Aß. The enhanced activity of the heterodivalent compounds suggests that polyvalent inhibitors that can target multiple regions of amyloidogenic peptides and proteins are better than those that only target a single region.