Glucose directs amyloid-beta into membrane-active oligomers

Oligomeric amyloid-β 1-42 (Aβ-42) peptides are considered to be the most toxic species connected to the occurrence of Alzheimer's disease. However, not all aggregation conditions promote oligomer formation in vitro, raising the question whether oligomer formation in vivo also requires a specific suitable cellular environment. We recently found that interaction with neuronal membranes initiates aggregation of Aβ-42 and neuronal uptake. Our data suggest that small molecules in the extracellular space can facilitate the formation of membrane-active Aβ-42 oligomers. We analyzed the early stage of Aβ-42 aggregation in the presence of glucose and sucrose and found that these sugars strongly favor Aβ-42 oligomer formation. We characterized oligomers by dynamic light scattering, atomic force microscopy, immuno-transmission electron microscopy and fluorescence cross correlation spectroscopy. We found that Aβ-42 spontaneously and rapidly forms low molecular weight oligomers in the presence of sugars. Slightly acidic pH (6.7-7) greatly favors oligomer formation when compared to the extracellular physiological pH (7.4). Circular dichroism demonstrated that these Aβ-42 oligomers did not adopt a β-sheet structure. Unstructured oligomeric Aβ-42 interacted with membrane bilayers of giant unilamellar vesicles (GUV) and neuronal model cells, facilitated cellular uptake of Aβ-42, and inhibition of mitochondrial activity. Our data therefore suggest that elevated concentrations of glucose within the range observed in diabetic individuals (10 mM) facilitate the formation of membrane-active Aβ-42 oligomers

Aggregation of full length immunoglobulin light chains from AL amyloidosis patients is remodeled by epigallocatechin-3-gallate

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol Epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and Multiple Myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from urine of AL and MM patients. We quantified their thermodynamic stabilities, and monitored their aggregation under physiological conditions by ThT fluorescence, light scattering, SDS-stability and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ~50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-{beta} and {alpha}-synuclein

670 nm laser light and EGCG complementarily reduce amyloid-β aggregates in human neuroblastoma cells: basis for treatment of Alzheimer's disease?

Objective: The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimer's disease (AD). Background Data: Despite intensive research efforts, there is no treatment for AD. One root cause of AD is the extra- and intracellular deposition of amyloid-beta (A{beta}) fibrils in the brain. Recently, it was shown that extracellular A{beta} can enter brain cells, resulting in neurotoxicity. Methods: After internalization of A{beta}(42) into human neuroblastoma (SH-EP) cells, they were irradiated with moderately intense 670-nm laser light (1000 Wm(-2)) and/or treated with epigallocatechin gallate (EGCG). Results: In irradiated cells, A{beta}(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, A{beta}(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with A{beta}(42) and EGCG, there was a significant increase in cell numbers in response to laser irradiation. EGCG alone had no effect on cell proliferation. Laser irradiation significantly increased ATP levels in A{beta}(42)-free cells, when compared to nonirradiated cells. Laser-induced clearance of Aβ(42) aggregates occurred at the expense of cellular ATP. Conclusions: Irradiation with moderate levels of 670-nm light and EGCG supplementation complementarily reduces A{beta} aggregates in SH-EP cells. Transcranial penetration of moderate levels of red to near-infrared (NIR) light has already been amply exploited in the treatment of patients with acute stroke; the blood-brain barrier (BBB) penetration of EGCG has been demonstrated in animals. We hope that our approach will inspire a practical therapy for AD

Amyloid-β(1-42) aggregation initiates its cellular uptake and cytotoxicity

The accumulation of amyloid beta peptide(1-42) (Abeta(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Abeta(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Abeta may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Abeta endocytosis. We visualized aggregate formation of fluorescently labeled Abeta(1-42) and tracked its internalization by human neuroblastoma cells and neurons. beta-Sheet-rich Abeta(1-42) aggregates entered the cells at low nanomolar concentration of Abeta(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Abeta(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Abeta(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of beta-sheet-rich aggregates is a prerequisite for Abeta(1-42) uptake and cytotoxicity

The green tea polyphenol (-)-epigallocatechin gallate prevents the aggregation of tau protein into toxic oligomers at substoichiometric ratios

The accumulation of amyloid-beta (Abeta) and tau aggregates is a pathological hallmark of Alzheimer's disease. Both polypeptides form fibrillar deposits, but several lines of evidence indicate that Abeta and tau form toxic oligomeric aggregation intermediates. Depleting such structures could thus be a powerful therapeutic strategy. We generated a fragment of tau (His-K18DeltaK280) that forms stable, toxic, oligomeric tau aggregates in vitro. We show that (-)-epigallocatechin gallate (EGCG), a green tea polyphenol that was previously found to reduce Abeta aggregation, inhibits the aggregation of tau K18DeltaK280 into toxic oligomers at ten- to hundred-fold substoichiometric concentrations, thereby rescuing toxicity in neuronal model cells

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease

Amyloid-β(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity

The accumulation of amyloid β peptide(1-42) (Aβ(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1-42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1-42) aggregates entered the cells at low nanomolar concentration of Aβ(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1-42) uptake and cytotoxicity

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-β and α-synuclein

Amyloid-β(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity

The accumulation of amyloid β peptide(1-42) (Aβ(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1-42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1-42) aggregates entered the cells at low nanomolar concentration of Aβ(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1-42) uptake and cytotoxicity

Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-{beta} (A{beta}) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of A{beta} fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in A{beta} peptides and stabilizes the self-assembly of seeding-competent, {beta}-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic A{beta} oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by A{beta} oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells