Role of Membrane Tension Sensitive Endocytosis and Rho GTPases in the Uptake of the Alzheimer's Disease Peptide Aβ(1-42).

Emelie Wesén,Richard Lundmark,Elin K Esbjörner

doi:10.1021/acschemneuro.0c00053

Emelie Wesén, Richard Lundmark + Show 1 more

Open Access

https://doi.org/10.1021/acschemneuro.0c00053

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Abstract

Intraneuronal accumulation of amyloid-β (Aβ) is an early pathological signum of Alzheimer’s disease, and compartments of the endolysosomal system have been implicated in both seeding and cell–cell propagation of Aβ aggregation. We have studied how clathrin-independent mechanisms contribute to Aβ endocytosis, exploring pathways that are sensitive to changes in membrane tension and the regulation of Rho GTPases. Using live cell confocal microscopy and flow cytometry, we show the uptake of monomeric Aβ(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aβ(1-42) uptake via clathrin independent carriers (CLICs), although overexpression of the bar-domain protein GRAF1, a key regulator of CLICs, had no apparent effect. We furthermore report reduced Aβ(1-42) uptake following overexpression of constitutively active forms of the Rho GTPases Cdc42 and RhoA, whereas modulation of Rac1, which is linked to macropinosome formation, had no effect. Our results confirm that uptake of Aβ(1-42) is clathrin- and dynamin-independent and point to the involvement of a new and distinct clathrin-independent endocytic mechanism which is similar to uptake via CLICs or macropinocytosis but that also appear to involve yet uncharacterized molecular players.

Highlights

Aβ has been suggested as a causative agent of Alzheimer’s disease (AD) pathology;4 many familial forms of AD are associated with mutations that enhance the aggregation propensity of Aβ or alter, disfavorably, its production, processing, and clearance.5−9 Aβ is formed by proteolytic cleavage of the amyloid precursor protein (APP)
Previous work has shown that uptake of monomeric Aβ[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42] is clathrin-independent,13 and in order to further map out the involved endocytic paths and regulators, we here focus on clathrin-independent endocytosis (CIE)
We have used live cell confocal fluorescence microscopy and flow cytometry to study how perturbation of endocytic mechanisms that are sensitive to changes in membrane tension and the regulatory control of Rho GTPases influence the cellular uptake of monomeric Aβ[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42]

Summary

Introduction

Alzheimer’s disease (AD) is characterized by the aggregation of amyloid-β (Aβ) peptides resulting in the formation of extracellular plaque deposits in the brain alongside the formation of intracellular neurofibrillary tau tangles. Aβ has been suggested as a causative agent of AD pathology; many familial forms of AD are associated with mutations that enhance the aggregation propensity of Aβ or alter, disfavorably, its production, processing, and clearance.− Aβ is formed by proteolytic cleavage of the amyloid precursor protein (APP).10This predominantly occurs in endolysosomal organelles whereupon Aβ can be secreted or retained. Extracellular Aβ can efficiently enter cultured cells via endocytosis.− It reportedly enters neurons following tail vein injections into mice with a compromised blood brain barrier, suggesting that the intra- and extracellular Aβ pools are dynamically related. The confinement of Aβ in endolysosomal vesicles subjects the peptide to aggregation promoting conditions, including low pH18 and the presence of lipid membranes. we and others have shown that endocytosed Aβ is aggregating inside living cells, and it has been suggested that endolysosomal compartments could serve as initial sites of Aβ seed formation. Interestingly, in this regard, intraneuronal buildup of Aβ appear as one of the earliest signs of AD, typically manifesting before the formation of extracellular plaques.− For these reasons, as well as the putative importance of endocytosis and the endolysosomal system in the prion-like cell-cell propagation of Aβ aggregation, it is important to better understand how Aβ peptides are endocytosed and accumulated. Aβ has been suggested as a causative agent of AD pathology; many familial forms of AD are associated with mutations that enhance the aggregation propensity of Aβ or alter, disfavorably, its production, processing, and clearance.− Aβ is formed by proteolytic cleavage of the amyloid precursor protein (APP).. Aβ has been suggested as a causative agent of AD pathology; many familial forms of AD are associated with mutations that enhance the aggregation propensity of Aβ or alter, disfavorably, its production, processing, and clearance.− Aβ is formed by proteolytic cleavage of the amyloid precursor protein (APP).10 This predominantly occurs in endolysosomal organelles whereupon Aβ can be secreted or retained..

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