Dataset Information

Endocytic uptake of monomeric amyloid-? peptides is clathrin- and dynamin-independent and results in selective accumulation of A?(1-42) compared to A?(1-40).

ABSTRACT: Intraneuronal accumulation of amyloid-? (A?) peptides represent an early pathological feature in Alzheimer's disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of A?(1-40) and A?(1-42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of A?(1-42) compared to A?(1-40); an imaging-based quantification showed that cells treated with 1?µM peptide for 8?h contained 800,000 peptides of A?(1-42) and 400,000 of A?(1-40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of A?(1-42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the A? variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced A?(1-40) and A?(1-42) uptake considerably. This study clarifies mechanisms of A?(1-40) and A?(1-42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal A? in AD.

SUBMITTER: Wesen E

PROVIDER: S-EPMC5435687 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1-42) compared to Aβ(1-40).

Wesén Emelie E Jeffries Gavin D M GDM Matson Dzebo Maria M Esbjörner Elin K EK

Scientific reports 20170517 1

Intraneuronal accumulation of amyloid-β (Aβ) peptides represent an early pathological feature in Alzheimer's disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of Aβ(1-40) and Aβ(1-42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreove ...[more]

PMID: 28515429

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Project description:UnlabelledBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.ImportanceBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

Dataset Information

Endocytic uptake of monomeric amyloid-? peptides is clathrin- and dynamin-independent and results in selective accumulation of A?(1-42) compared to A?(1-40).

Publications

Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1-42) compared to Aβ(1-40).

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