The accumulation of amyloid ␤ peptide(1-42) (A␤(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of A␤(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. A␤ 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 A␤ endocytosis. We visualized aggregate formation of fluorescently labeled A␤(1-42) and tracked its internalization by human neuroblastoma cells and neurons. ␤-Sheet-rich A␤(1-42) aggregates entered the cells at low nanomolar concentration of A␤(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed A␤(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that A␤(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 ␤-sheet-rich aggregates is a prerequisite for A␤(1-42) uptake and cytotoxicity.One of the pathological hallmarks of Alzheimer disease (AD) 2 is the presence of extracellular plaques composed mainly of 42-amino acid amyloid ␤ peptide (A␤(1-42)) (1). The small hydrophobic A␤(1-42) peptide, which is generated by proteolytic cleavage of the amyloid precursor protein, is released as a monomer from the plasma membrane into extracellular space, and tends to aggregate spontaneously into oligomeric, protofibrillar, and fibrillar assemblies (2-4). Oligomeric species of A␤(1-42) are tightly linked to AD pathogenesis and are presumed to be the cause of neuronal damage (5). Several studies have suggested that the reuptake of extracellular A␤(1-42) into neurons may lead to the formation of intracellular aggregates, resulting in neuronal damage and neurotoxicity (6 -8). Endocytosis of misfolded proteins has also been observed in cell models of the tau protein, ␣-synuclein and huntingtin (9, 10), and recent evidence suggests that it may be the initial step in the replication of the misfolded protein structures by prion mechanisms (10 -14). Several possible endocytic pathways, such as macropinocytosis and receptor-mediated endocytosis, have been discussed for A␤ and other misfolded protein aggregates (15-19). However, our understanding of the connection between aggregation and cytotoxicity is still limited. It has not been conclusively determined how and when the A␤(1-42) peptide becomes toxic, whether A␤ aggregates prior to internalization or during the internalization process and, if so, in which intracellular compartments the aggregates form. Elucidating the connection between aggregation and i...

Aggregates of the RNA-binding protein TDP-43 (TAR DNAbinding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergentresistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

Edited by Paul E. FraserIntervention 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, ϳ50% 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-␤ and ␣-synuclein. Systemic light chain amyloidosis (AL)2 is the most common form of systemic amyloidosis (1), but it is still a rare disease with an incidence of 6 -7 in 1,000,000 people (2). Underlying AL pathology is a clonal plasma cell disorder, which produces an immunoglobulin light chain in the bone marrow with a unique sequence. These light chains are released into the blood (monoclonal gammopathy). When elevated LC levels in serum overwhelm renal absorption, LC is also found in the urine. Fulllength LC can be isolated from the urine of these patients (3-5).Clonal plasma cell disorders can present different types of pathologies; light chains form amyloid deposits in AL patients. Here, LC fibrils adopt a conformation characterized by highly stable, intramolecular cross-␤-sheets that stain with Congo red (6) and thioflavin T (ThT) (7-9).In contrast, amyloid deposits are not observed in patients suffering from multiple myeloma (MM), a malignant disease characterized by bone marrow failure and bone destruction. The two contrasting pathologies of LC gammopathies raise the question of which factors determine amyloid formation. Amyloid formation could be initiated (a) by the sequences of the individual light chains, (b) by high levels of light chain expression, (c) by p...

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-β aggregation, as well as the structural remodeling of amyloid-β fibrils by the compound epi-gallocatechin gallate.