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Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide.

ABSTRACT: One of the neuropathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque, primarily composed of aggregated amyloid-beta (Abeta) peptide. In vitro, Abeta(1-42), the major alloform of Abeta found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and Abeta concentrations are in the nanomolar range. Here, we show that extracellular soluble Abeta (sAbeta) at concentrations as low as 1 nM was taken up by murine cortical neurons and neuroblastoma (SHSY5Y) cells but not by human embryonic kidney (HEK293) cells. Following uptake, Abeta accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 microM) were greater than two orders of magnitude higher than that in the extracellular fluid (25 nM), as quantified by fluorescence intensity using laser scanning confocal microscopy. Furthermore, SHSY5Y cells incubated with 1 muM Abeta(1-42) for several days demonstrated a time-dependent increase in intracellular high molecular weight (HMW) (>200 kDa) aggregates, which were absent in cells grown in the presence of Abeta(1-40). Homogenates from these Abeta(1-42)-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that Abeta can be taken up by certain cells at low physiologically relevant concentrations of extracellular Abeta, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular Abeta aggregates to form HMW species which are capable of seeding amyloid fibril growth. We speculate that extrusion of these aggregates may seed extracellular amyloid plaque formation during AD pathogenesis.

SUBMITTER: Hu X

PROVIDER: S-EPMC2787156 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide.

Hu Xiaoyan X Crick Scott L SL Bu Guojun G Frieden Carl C Pappu Rohit V RV Lee Jin-Moo JM

Proceedings of the National Academy of Sciences of the United States of America 20091112 48

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque, primarily composed of aggregated amyloid-beta (Abeta) peptide. In vitro, Abeta(1-42), the major alloform of Abeta found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and Abeta concentrations are in the nanomolar range. Here, we show that extracellular soluble Abeta (sAbeta) at c ...[more]

PMID: 19910533

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Project description:Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in β-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid β peptide (Aβ1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic β-hairpin like structures built on a piperidine-pyrrolidine β-turn inducer are described. By linking to this β-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

Dataset Information

Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide.

Publications

Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide.

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