Single-Molecule Imaging Reveals Aβ42:Aβ40 Ratio-Dependent Oligomer Growth on Neuronal Processes

Johnson, Robin; Schauerte, Joseph A.; Chang, Chun Chieh; Wisser, Kathleen; Althaus, J. Christian; Carruthers, Christopher; Sutton, Michael A.; Steel, Duncan G.; Gafni, Ari

doi:10.1016/j.bpj.2012.12.051

Cited by 27 publications

(42 citation statements)

References 45 publications

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“…It is tempting to speculate that small oligomers may have a higher affinity to the plasma membrane than the monomeric peptide, facilitating the conversion to ␤-sheet-rich structures and subsequent internalization. Our results demonstrate that, unlike monomers, preexisting A␤ aggregates are internalized at low nanomolar concentrations, which corresponds to previously observed binding of A␤ oligomers to neuronal plasma membranes at nanomolar concentration (31,32).…”

Section: Discussionsupporting

confidence: 62%

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

Jin

Kedia

Illes‐Toth

et al. 2016

Journal of Biological Chemistry

116

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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...

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Section: Discussionsupporting

confidence: 62%

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

Jin

Kedia

Illes‐Toth

et al. 2016

Journal of Biological Chemistry

116

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“…FRET experiments with HL555 Aβ40 and HL647 Aβ42 confirmed that mixed oligomers do in fact form . Following this observation, we continued to utilize these two peptides to further examine the mechanism of oligomer growth on neurites exposed to mixed Aβ40 and Aβ42.…”

Section: Binding and Oligomer Formation: Live Cell Membranesmentioning

confidence: 81%

“…Further experiments using neurites of primary rat hippocampal cells gave initially similar results at even lower Aβ concentrations . Plated cells were incubated with 1 n M HL647 Aβ40 or Aβ42 for 10 minutes, gently washed three times so as to preserve the cells' adherence to the slides, and imaged in confocal mode.…”

Section: Binding and Oligomer Formation: Live Cell Membranesmentioning

confidence: 84%

“…D: Diagram showing a region of interest around a single oligomer (shown zoomed in at right as a smoothed three dimensional surface plot of an actual cellbound oligomer from Ref. ). Confocal mode peak fluorescence intensity or integrated fluorescence intensity of the oligomer's intensity profile can be used to measure oligomer size.…”

Section: Methodsmentioning

confidence: 99%

“…A number of recent studies have raised interest in the role of Aβ42:Aβ40 ratio in peptide aggregation and interaction with cells . We explored this issue by comparing on‐membrane oligomer growth of a 1:1 mixture of the two peptides to that observed for each peptide alone . Relative to homogeneous Aβ40 or Aβ42, the size distribution for cell‐bound mixed 1 n M peptide after 10 minutes was significantly shifted toward monomers and dimers.…”

Section: Binding and Oligomer Formation: Live Cell Membranesmentioning

confidence: 99%

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Structural evolution and membrane interactions of Alzheimer's amyloid‐beta peptide oligomers: New knowledge from single‐molecule fluorescence studies

2014

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Amyloid-b peptide (Ab) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single-molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid-b, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar-to-picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Ab40 to Ab42. Here, we discuss several types of single-molecule imaging that have been applied to the study of Ab oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief Abbreviations: AD, Alzheimer's Disease; ADDL, amyloid-derived diffusible ligand; Ab, amyloid-b peptide; Ab40, 40-residue version of Ab; Ab42, 42-residue version of Ab; Ab*56, dodecameric Ab oligomer; APP, amyloid precursor protein; cTCCD, confocal two-color coincidence detection; D, diffusion coefficient; FAM, carboxyfluorescein; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GUV, giant unilamellar vesicle; hAPP, human Amyloid Precursor Protein; HFIP, hexafluoroisopropanol; HL488, HiLyte Fluor 488; HL555, HiLyte Fluor 555; HL647, HiLyte Fluor 647; MSD, mean square displacement; SMM, single-molecule microscopy; TAMRA, carboxytetramethylrhodamine; TIRF, total internal reflection fluorescence. overview of the current limitations of the technique, including the lack of sensitive assays for Ab-induced toxicity, is included in hopes of inspiring future development in this area of research.

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Not Oligomers but Amyloids are Cytotoxic in the Membrane‐Mediated Amyloidogenesis of Amyloid‐β Peptides

et al. 2018

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The formation of neurotoxic aggregates by amyloid-β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aβ. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Here, aggregation processes of Aβ on living cells and cytotoxic events were monitored by fluorescence techniques. Aβ formed amyloids after forming oligomers composed of ≈10 Aβ molecules. The formation of amyloids was necessary to activate apoptotic caspase-3 and reduce the ability of the cell to proliferate; this indicated that amyloid formation is a key event in Aβ-induced cytotoxicity.

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Single-Molecule Imaging Reveals Aβ42:Aβ40 Ratio-Dependent Oligomer Growth on Neuronal Processes

Cited by 27 publications

References 45 publications

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

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

Structural evolution and membrane interactions of Alzheimer's amyloid‐beta peptide oligomers: New knowledge from single‐molecule fluorescence studies

Not Oligomers but Amyloids are Cytotoxic in the Membrane‐Mediated Amyloidogenesis of Amyloid‐β Peptides

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