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 amyloidbeta (A␤) peptide. In vitro, A␤1-42, the major alloform of A␤ 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 A␤ concentrations are in the nanomolar range. Here, we show that extracellular soluble A␤ (sA␤) 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, A␤ accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 M) 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 M A␤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 A␤1-40. Homogenates from these A␤1-42-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that A␤ can be taken up by certain cells at low physiologically relevant concentrations of extracellular A␤, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular A␤ 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.amyloid fibrils ͉ late endosomes ͉ lysosomes ͉ plaques A lzheimer's disease (AD), the most common form of dementia in Western countries, involves progressive accumulation of amyloid deposits, neuronal loss, cognitive decline, and eventual death. Senile plaques, a key pathological feature of this disease, are composed primarily of the amyloid-beta (A␤) peptide, and are found throughout the brain (1). A␤ (ranging in length from 39-42 amino acids) is derived from the proteolytic cleavage of an endogenous transmembrane protein known as the amyloid precursor protein (APP). The most common A␤ peptide found in senile plaques is the 42-residue peptide (A␤ 1-42 ) (2), which also shows the strongest propensity for spontaneous aggregation in solution (3). It is widely believed that the aggregation and accumulation of this peptide is involved in disease pathogenesis.A␤ is produced primarily by neurons and secreted into the brain extracellular space where it is normally found in a soluble state (4). A variety of physiological processes, including those associated with neuronal activity, are related to A␤ synthesis and release into the extracellular space (5-7). Under normal physiological conditions and in AD patients, the concentration of A␤ in brain extracellular fluid (interstitial fluid, ISF and cerebrospinal fluid, CSF) is low (10 Ϫ10 M-10 Ϫ9 M) (8...

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

“…This is supported by some recent reports demonstrating that A␤ in culture medium is internalized and aggregated intracellularly into fibrils, eventually causing membrane disruption (47,48). Further studies are required to dissect the structural and molecular mechanisms underlying the critical role of protofibril-monomer interactions in A␤ toxicity.…”

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

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Sequence-dependent Internalization of Aggregating Peptides

“…The internalization of soluble amyloidogenic species has been described previously in cell culture for ␣-synuclein (15,28) and both in cell culture and in vivo for A␤ (14,24) either by specific uptake through receptor-mediated endocytosis or passive diffusion across the cell membrane. In the case of A␤, the internalization of soluble species has been demonstrated to promote maturation into larger aggregates due to the acidic pH and increased concentration generated in the lysosomes.…”