Presenilin deficiency leads to altered protein N-glycosylation and endo-lysosomal cholesterol accumulation

Abstract

Major genetic causes of familial Alzheimer disease are mutations in genes encoding presenilin (PS) 1 and 2. PS1 and PS2 are known to constitute the catalytic subunits of the &gamma;-secretase complex, but also for being involved in cellular cholesterol metabolism which is connected to AD development via the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PS affect cellular cholesterol metabolism are not understood. We observed increased cholesterol content in neurons from PS knockout (KO) mice in cortex and CA1 regions and characterize the cholesterol metabolism in PS1- or PS2 KO mouse embryonic fibroblasts (MEFs). <br /> Cholesterol and sterols were measured in MEFs by mass spectrometry (MS) while filipin, a fluorescent cholesterol binding compound was used to visualize free cholesterol. Non-toxic concentrations of inhibitors or chaperone inducer were selected to treat the cells. Protein expression was analyzed by Western blotting (WB) while mRNA levels were measured by 3´mRNa sequencing. <br /> Cellular levels of cholesterol as well as its precursors were high in PS1- and PS2KO MEFs, while filipin revealed intracellular cholesterol accumulation in endo-lysosomal compartments. The expression patterns of proteins involved in cholesterol metabolism were different in PS1- and PS2KO MEFs. Nevertheless, we detected a significant increase in SREBP-2 and CYP51 and a decrease in NPC1 expression level in PS KO MEFs. Deficiency of the cholesterol transporter NPC1 leads to lysosomal cholesterol storage and neurodegeneration in Niemann-Pick´s disease type C. Glycosylation of NPC1 and other membrane proteins, including LRP1, LAMP2 and N-cadherin was impaired in PS KO MEFs. To test whether inhibition of protein glycosylation affects cholesterol metabolism, WT MEFs were treated with inhibitors targeting different enzymes of the process. Inhibition of the early glycosylation step at ER alone was able to decrease NPC1 expression level, induce CYP51 expression and, at the same time, phenocopy the lysosomal cholesterol accumulation observed in PS KO MEFs. <br /> Treatment with a chaperone inducer increasing NPC1 levels significantly attenuated lysosomal cholesterol accumulation and decrease CYP51 level in PS KO MEFs. Overexpression of NPC1 in PS KO MEFs also showed a reduction in lysosomal cholesterol content. <br /> Our results suggest that lack of PS1 or PS2 affects cellular cholesterol metabolism toward upregulation of <em>de novo</em> cholesterol synthesis and lysosomal cholesterol accumulation via impaired protein glycosylation and reduced NPC1 level.