Assembly Of Amyloid Beta-protein Research Articles

α-Helical Motif as Inhibitors of Toxic Amyloid-β Oligomer Generation via Highly Specific Recognition of Amyloid Surface.

SummaryAmyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

Do We Need Anti-Prion Compounds to Treat Alzheimer's Disease?

Background: While phase III clinical trials for the treatment of Alzheimer’s disease (AD) keep failing regardless of the target, more and more data suggest that the toxic protein assemblies of amyloid-beta protein (Aβ) and tubulin binding protein (TAU) behave like prions. Irrespective of the question of whether AD is theoretically or practically contagious, the presence of a self-replicating toxic etiologic agent in the brains of AD patients must have decisive consequences for drug development programs and clinical trial designs. Objectives: We intend to challenge the hypothesis that the underlying etiologic agent of AD is behaving prion-like. We want to discuss whether the outcome of clinical trials could have been predicted based on this hypothesis, and whether compounds that directly disassemble the toxic prion could be more beneficial for AD treatment. Method: We collected publicly accessible pre-clinical efficacy data of Aβ targeting compounds that failed or still are in phase III clinical trials. We describe the desired properties of an anti-prionic compound and compare it the properties of past and current phase III drug candidates. Results: We could not find convincing and reproducible pre-clinical efficacy data of past and current phase III drug candidates on cognition other than in preventive treatment settings. The desired properties of an anti-Aβ-prionic compound are fulfilled by the drug candidate RD2, which has been developed to directly disassemble toxic Aβ oligomers. Conclusion: RD2 is the first anti-prionic drug candidate. It is able to enhance cognition and impede neurodegeneration in three different transgenic AD mouse models, even under truly non-preventive conditions and even when applied orally. In addition, it is safe in humans.

Amyloid-β fibrils assembled on ganglioside-enriched membranes contain both parallel β-sheets and turns

Some protein and peptide aggregates, such as those of amyloid-β protein (Aβ), are neurotoxic and have been implicated in several neurodegenerative diseases. Aβ accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer's disease. Although the presence of Aβ deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between Aβ secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that Aβ40 fibrils can be obtained by incubating seed-free Aβ40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection-absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel β-sheets, whereas self-assembled Aβ40 molecules formed antiparallel β-sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn Aβ40 Our findings indicate that Aβ generally self-assembles into antiparallel β-structures but can also form protofibrils with parallel β-sheets by interacting with ganglioside-bound Aβ. We concluded that by promoting the formation of parallel β-sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of Aβ fibrils. These results advance our understanding of ganglioside-induced Aβ fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer's disease.

Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration

The accumulation of amyloid-β (Aβ) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer's disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aβ oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aβ oligomers in vivo.

The Role of Di-Tyrosine Bonding in Amyloid Beta-Protein Assembly

Using a coarse-grained protein model combined with discrete molecular dynamics, the proteins involved in Alzheimer's disease (AD) may be studied in a variety of ways at atomic resolution. AD is caused by the misfolding of amyloid beta-protein (AB). We studied the effect of di-tyrosine bonding on folding and oligomer formation of two predominant AB alloforms, AB40 and AB42, which contain tyrosine (TYR) at position 10. To explore the possible effects of a TYR-TYR bond, Virtual Molecular Dynamics and TCL scripting were used to study the oligomer distribution, secondary and tertiary structure and the rate at which tyrosine bonds form as a function of time. It was found that TYR-TYR bonds increase the propensity to form pentamers and hexamers in AB40, which are possible starting points for the protofibrils that form the basis of amyloid plaque. This piece of information suggests that di-tyrosine bonding may play a role in AD, but does not significantly affect the structure of resulting AB oligomers.

Insulin inhibits Aβ fibrillogenesis through a decrease of the GM1 ganglioside‐rich microdomain in neuronal membranes

Type 2 diabetes is a risk factor for late-onset Alzheimer's disease. However, the underlying mechanisms remain unknown. To investigate whether insulin is associated with the assembly of amyloid beta-protein from the cell surface, we treated nerve growth factor (NGF)-treated rat pheochromocytoma 12 (PC12) cells with insulin, which is related to the development of diabetes. Insulin treatment induced a decrease in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of NGF-treated PC12 cells. The insulin-induced effects on GM1 levels were regulated by a phosphatidylinositol 3-kinase inhibitor, but not by an extracellular signal-regulated kinase inhibitor. Pre-treatment with a protein synthesis inhibitor did not inhibit the decrease in GM1 levels induced by insulin. In addition, insulin failed to induce formation of fibrils from soluble amyloid beta-protein or to accelerate GM1-induced fibril formation. Furthermore, assembly of amyloid beta-protein in cultures of NGF-treated PC12 cells was significantly decreased by insulin. These results suggest that insulin inhibits amyloid beta-protein assembly by decreasing GM1 expression in detergent-resistant membrane microdomains of neuronal membranes.

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer’s disease

The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.

1SA2-05 強い神経細胞死活性を持つアルツハイマー病患者に由来するアミロイドβ凝集体(1SA2 プリオンとウィルスの感染(BSJ&ABA連携国際シンポジウム),第47回日本生物物理学会年会)

1SA2-05 強い神経細胞死活性を持つアルツハイマー病患者に由来するアミロイドβ凝集体(1SA2 プリオンとウィルスの感染(BSJ&ABA連携国際シンポジウム),第47回日本生物物理学会年会)

HDL: Close to Our Memories?

The last decade has witnessed an explosion in studies of the role of lipoproteins in brain function. Neurons require a continuous supply of lipids for membrane synthesis and acetylcholine production. Indeed, the brain is a site of intense lipid turnover—even though the central nervous system (CNS) accounts for only 2.1% of body weight, it contains 23% of total body cholesterol.1 Lipid metabolism in the brain is tightly controlled locally, as plasma lipoproteins are shielded from the brain by the blood-brain barrier. Although neuronal cells are capable of de novo synthesis of a wide spectrum of molecular species of lipids, they rely heavily on exogenous sources and readily bind and internalize lipoproteins of the extracellular fluid.2 Equally, neurons need to dispose of excess lipids; lipoprotein-mediated lipid transport is therefore bidirectional and includes cellular efflux of cholesterol.3 See accompanying article on page 1556 Human cerebrospinal fluid (CSF) primarily contains spherical lipoproteins of approximately 10 to 12 nm in diameter with hydrated density in the range 1.063 to 1.25 g/mL, thereby resembling HDL in human plasma.3,4⇓ Lipid concentrations in CSF are however much lower (eg, 300- to 400-fold for total cholesterol and phospholipids) as compared to the plasma compartment.4 Apolipoproteins (apo) E and A-I are the major apolipoproteins in human CSF (typical concentration range: 0.1 to 0.4 mg/dL3,4⇓), with apoA-II, A-IV, J, D, C-II, C-III, C-IV, and H equally present.3,4⇓ Importantly, CSF lipoproteins carry amyloid-β (Aβ), a 39- to 43-aa peptide produced in neuronal cells, which is the major component of senile amyloid plaques.5 The metabolism of CSF lipoproteins remains poorly characterized but seems to be distinct from that of plasma lipoproteins. ApoE-rich HDL are synthesized locally in CNS and secreted by astrocytes as discoidal complexes enriched in free cholesterol.6 High …

Transglutaminase Induces Protofibril-like Amyloid β-Protein Assemblies That Are Protease-resistant and Inhibit Long-term Potentiation

An increasing body of evidence suggests that soluble assemblies of amyloid beta-protein (Abeta) play an important role in the initiation of Alzheimer disease (AD). In vitro studies have found that synthetic Abeta can form soluble aggregates through self-assembly, but this process requires Abeta concentrations 100- to 1000-fold greater than physiological levels. Tissue transglutaminase (TGase) has been implicated in neurodegeneration and can cross-link Abeta. Here we show that TGase induces rapid aggregation of Abeta within 0.5-30 min, which was not observed with chemical cross-linkers. Both Abeta40 and Abeta42 are good substrates for TGase but show different aggregation patterns. Guinea pig and human TGase induced similar Abeta aggregation patterns, and oligomerization was observed with Abeta40 concentrations as low as 50 nm. The formed Abeta40 species range from 5 to 6 nm spheres to curvilinear structures of the same width, but up to 100 nm in length, that resemble the previously described self-assembled Abeta protofibrils. TGase-induced Abeta40 assemblies are resistant to a 1-h incubation with either neprilysin or insulin degrading enzyme, whereas the monomer is rapidly degraded by both proteases. In support of these species being pathological, TGase-induced Abeta40 assemblies (100 nm) inhibited long term potentiation recorded in the CA1 region of mouse hippocampus slices. Our data suggest that TGase can contribute to AD by initiating Abeta oligomerization and aggregation at physiological levels, by reducing the clearance of Abeta due to the generation of protease-resistant Abeta species, and by forming Abeta assemblies that inhibit processes involved in memory and learning. Our data suggest that TGase might constitute a specific therapeutic target for slowing or blocking the progression of AD.

Amyloid Activates GSK-3β to Aggravate Neuronal Tauopathy in Bigenic Mice

The hypothesis that amyloid pathology precedes and induces the tau pathology of Alzheimer's disease is experimentally supported here through the identification of GSK-3 isozymes as a major link in the signaling pathway from amyloid to tau pathology. This study compares two novel bigenic mouse models: APP-V717I x Tau-P301L mice with combined amyloid and tau pathology and GSK-3beta x Tau-P301L mice with tauopathy only. Extensive and remarkable parallels were observed between these strains including 1) aggravation of tauopathy with highly fibrillar tangles in the hippocampus and cortex; 2) prolonged survival correlated to alleviated brainstem tauopathy; 3) development of severe cognitive and behavioral defects in young adults before the onset of amyloid deposition or tauopathy; and 4) presence of pathological phospho-epitopes of tau, including the characteristic GSK-3beta motif at S396/S404. Both GSK-3 isozymes were activated in the brain of parental APP-V717I amyloid mice, even at a young age when cognitive and behavioral defects are evident but before amyloid deposition. The data indicate that amyloid induces tauopathy through activation of GSK-3 and suggest a role for the kinase in maintaining the functional integrity of adult neurons.

Cross‐seeding of wild‐type and hereditary variant‐type amyloid β‐proteins in the presence of gangliosides

We investigated the molecular mechanism underlying the ganglioside-induced initiation of the assembly of wild and hereditary variant-type amyloid beta-proteins, including Arctic-, Dutch-, and Flemish-type amyloid beta-proteins. We monitored the assembly of amyloid beta-protein by thioflavin-T assay, western blotting and electron microscopy. We also examined how externally added amyloid beta-protein assembles in a cell culture. The assembly of wild-, Arctic-, Dutch-, and Flemish-type amyloid beta-proteins were accelerated in the presence of GM1, GM1, GM3 and GD3 gangliosides. Notably, all of these amyloid beta-proteins accelerated the assembly of different type of amyloid beta-protein, following prior binding to a specific ganglioside. A specific-ganglioside-bound form of variant-type amyloid beta-protein was recognized by the antibody (4396C) specific to the GM1-ganglioside-induced altered conformation of wild-type amyloid beta-protein. Moreover, the assembly of these amyloid beta-proteins in the presence of a specific ganglioside was markedly suppressed by coincubation with 4396C. This study suggests that cross-seeding can occur between wild and hereditary variant-type amyloid beta-proteins despite differences in their amino acid sequences.