Implication of protein glycosylation impairment in cellular cholesterol accumulation caused by Presenilin deficiency
Authors: Fabiano Marietta , Oikawa Naoto , Lütjohann Dieter , Walter Jochen 
Published: April 2020

BackgroundMajor genetic causes of familial Alzheimer disease (FAD) are mutations in genes encoding presenilin (PS) 1 and 2, respectively. PS1 and PS2 are transmembrane proteins known to constitute the catalytic subunits of the γ‐secretase complex, a membrane protease involved in proteolytic processing of amyloid precursor protein (APP) that gives rise to amyloid β‐protein (Aβ) secretion. In addiction, PSs are known to be involved in cellular cholesterol metabolism, which is fundamental for physiological cellular functions. Alterations in cholesterol metabolism could be implicated in AD development, indicated by the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PSs play a role in cellular cholesterol metabolism is still not fully clarified. In this study, we characterized cholesterol metabolism in the cells deficient in either PS1 or PS2.MethodsWe have studied PS1‐ or PS2‐knock out (KO) MEFs by mass spectrometry (MS) for sterols including cholesterol and by microscopy with a cholesterol binding compound, filipin. Expression level of proteins involved in cellular cholesterol metabolism was analyzed by Western blotting (WB).ResultsCellular level of cholesterol as well as its precursors, desmosterol and lathosterol, were elevated in PS1‐ and PS2‐KO as compared to WT MEFs. Fluorescence microscopy also revealed intracellular cholesterol accumulation in PS1 and PS2 KO cells. Interestingly, the subcellular distribution of cholesterol was different between PS1‐ and PS2‐KO MEFs. We further analyzed expression of proteins involved in cellular cholesterol metabolism, including NPC1, an intracellular cholesterol transporter whose deficiency leads to lysosomal cholesterol storage and neurodegeneration in Niemann‐Pick disease type C. The deficiency of PS1‐ and PS2‐KO MEFs was associated with altered glycosylation of NPC1. The glycosylation and expression of other membrane proteins, such as N‐cadherin, LAMP2, and LRP1 was also dependent on the PS genotype. To investigate the role of protein glycosylation in cellular cholesterol metabolism, we chronically treated wild‐type MEFs with several inhibitors of protein glycosylation targeting different steps of glycosylation processes. The inhibitors differentially affected protein glycosylation pattern and subcellular cholesterol distribution. Importantly, some of the inhibitors partially mimicked the cellular cholesterol accumulation as well as the protein glycosylation pattern observed in PS1‐ and PS2‐KO MEFs.Discussion/ConclusionsThese results suggest that deficiency of PS1 or PS2 differentially affect cellular cholesterol metabolism via impairment of protein glycolsylation. The exact mechanisms of altered expression of NPC1 as well as γ‐secretase activity in the impairment of cholesterol metabolism and protein glycosylation remain to be elucidated. It will be interesting to further dissect the role of protein glycosylation and cholesterol mismetabolism in the pathogenesis of AD.Support or Funding InformationGerman Research Foundation (DFG) (Project number: 348207315, Naoto Oikawa)