Lipid-nanoparticle-induced vacuolization in microglia

Abstract Lipid-containing vacuoles in microglia were discovered more than one hundred years ago in the brain of patients showing neurodegenerative processes. Recently, molecular-biological studies demonstrated specific changes in lipid-metabolism related to neurodegeneration. Despite that already Alzheimer described a distinct glia phenotype having large, lipid-containing vacuoles (Gitterzellen), little is known about how microglia convert lipid metabolites into a vacuolated phenotype. We studied the impact of liver-derived, insoluble, lipid-enriched nanoparticles (Lef-NP) ( ~ 20 nm) and of ceramide-coated Percoll-nanoparticles (Cer-NP) ( ~ 20 nm) on vacuolization in microglia. Lipidomic analysis of Lef-NP revealed numerous distinct lipids, including pro-inflammatory ceramides, which are enriched in the brain of Alzheimer patients. Video microscopy revealed that hepatocyte-derived Lef-NP and Cer-NP enhanced macropinocytosis, followed by macropinosome swelling and formation of the Gitterzellen phenotype. Neither ceramide nor Percoll-nanoparticles induced Gitterzellen-formation. Electron-tomography visualized membrane contact-sites between nanoparticle-loaded endosomes, endoplasmic reticulum cisternae and mitochondria. Suppression of lipid-nanoparticle-induced Gitterzellen-formation by amiloride, which supresses macropinocytosis, and bafilomycin A, an endosomal acidification inhibitor, further confirmed a pinocytotic pathway in Gitterzellen-formation. Bafilomycin A also reversed Gitterzellen to a ramified microglia phenotype. Our experimental findings suggest that lipid-nanoparticles but not emulsified lipids provoke vacuolization in microglia, and provide a simple in-vitro model for a pathogenic process taking years in the human brain.