Abstract
Intracellular amyloid β peptide (Aβ) accumulation has drawn attention in relation to the pathophysiology of Alzheimer’s disease in addition to its extracellular deposition as senile plaque. Cellular uptake of extracellular Aβ is one of the possible mechanisms by which intracellular Aβ deposits form. Given the relevance of Aβ inside cells, it is important to understand the mechanism by which it is taken up by them. In this study, we elucidated that Neuro2A and SH-SY5Y cells internalize specifically oligomerized Aβ in a time- and dose-dependent manner. The depletion of plasma membrane cholesterol with methyl-β-cyclodextrin or treatment with trypsin diminished the internalization of oAβ, suggesting that the oAβ uptake might be both a lipid raft-dependent and heparan sulfate proteoglycan-mediated process. Treatment with a macropinocytosis inhibitor (ethylisopropyl amiloride and wortmannin) also drastically reduced the uptake of oligomer-Aβ (oAβ). oAβ-treated cells exhibited an increase in Rac1 activity, indicating that macropinocytosis induced by oAβ is regulated by these small GTPases. These findings suggest that macropinocytosis is a major endocytic route through which oAβ42 enters cells.
Highlights
Alzheimer’s disease (AD) is the most common form of dementia, which is histopathologically characterized by the presence of senile plaques and neurofibrillary tangles (NFTs; Belyaev et al, 2010; Viola and Klein, 2015; Frost and Li, 2017)
amyloid β peptide (Aβ) is a 40- or 42-amino-acid peptide produced through the cleavage of amyloid precursor protein by β- and γ-secretase (Belyaev et al, 2010; Serrano-Pozo et al, 2011; Abbreviations: AD, Alzheimer’s disease; Aβ, amyloid beta; ANOVA, analysis of variance; Cholera Toxin B subunit (CTB), cholera toxin B subunit; DAPI, 4’,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle Medium; ethylisopropyl amiloride (EIPA), 5-(N-ethyl-N-isopropyl)amiloride; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HSPG, heparan sulfate proteoglycan; MβCD, methyl-β-cyclodextrin; NFT, neurofibrillary tangle; oAβ, oligomeric amyloid beta
MO, USA); ethylisopropyl amiloride (EIPA) a blockef of macropinocytosis, SecinH3, an indirect inhibitor of ADP ribosylation factor protein 6 (Arf6), Grassofermata (NAV 2729) a direct inhibitor of Arf6, and EHT1864, a Rac1 inhibitor of were purchased from Cayman chemical (Ann Arbor, MI, USA); dynasore, a dynamin inhibitor was from Abcam (Cambridge, MA, USA), Alexa Fluor 488 transferrin and phalloidin were obtained from Invitrogen (Carlsbad, CA, USA); wortmannin, a phosphoinositide 3-kinase inhibitor, Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium, phenol red-free Ham’s F12 Medium, 0.25% trypsin-EDTA, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan); fetal bovine serum was from COSMO BIO (Tokyo, Japan); and Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI), were from Dojindo (Kumamoto, Japan)
Summary
Alzheimer’s disease (AD) is the most common form of dementia, which is histopathologically characterized by the presence of senile plaques and neurofibrillary tangles (NFTs; Belyaev et al, 2010; Viola and Klein, 2015; Frost and Li, 2017). Regarding the mechanism behind the appearance of intracellular Aβ42, several studies have reported that the generation of Aβ42 might take place inside intracellular vesicles such as endosomes (Zhang and Song, 2013; Schützmann et al, 2021). Another plausible manner by which intracellular Aβ42 will appear is that cells actively take up the secreted Aβ42 and traffic it to multivesicular bodies (Friedrich et al, 2010). Aβ42 might appear inside cells not in an exclusive manner, information on the mechanism by which Aβ42 can be internalized should be valuable in the search for future therapeutics for AD
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