engleski

Loss of NPC1 leads to decreased expression of APP at the cell surface and alters APP distribution within endosome compartments

Background: Dysfunction of NPC1 protein causes Niemann Pick type C disease (NPC) and is characterized by accumulation of cholesterol and glycosphingolipids in endosomal/lysosomal compartments. It has been recently demonstrated that NPC exhibits an AD-like pathology showing tau hyperphosphorylation and increased amyloid-beta peptide (Abeta) levels, in addition to a shift in presenilin 1 distribution towards early/late endosomes. In this work we analyzed trafficking of APP/C99 in NPC cells. We hypothesized that cholesterol accumulation upon NPC1 dysfunction leads to increased APP/C99 in endosome compartments and, thus, increased Abeta generation. Methods: The levels of endogenous APP at the cell surface of CHOwt and CHO NPC1-null cells (NPC) were analyzed by biotinylation assay using EZ-Link NHS-SS-Biotin (Pierce). Endosome compartmentalization of APP/C99 in wt and NPC cells was monitored by endosome fractionation and by confocal microscopy. Endosome fractionation was performed in a 25-35% sucrose gradient. Early and late endosome fractions were detected by EEA1 (early) and Rab7 (late) staining. The same antibodies were used to analyze colocalization of endogenous APP/C99 in early or late endosomes by confocal microscopy. Results: Biotinylation assays show a marked decrease of APP at the cell surface in NPC cells compared to wt cells, suggesting that NPC1 loss may cause a shift in APP distribution within subcellular compartments. Indeed, endosome fractionation and confocal microscopy confirmed that in NPC cells there is more APP/C99 within early/late endosomal compartments compared to wt cells. In addition, we observed altered EEA1 and Rab7 staining between NPC and wt cells, indicating that altered endosomal distribution of APP/C99 upon NPC1 loss may be due to dysfunction of endosomal compartments in NPC cells. Conclusion: Our results show that loss of NPC1 function causes altered trafficking of APP/C99 towards endosome compartments. Finding a markedly decreased levels of APP at the cells surface and its increased compartmentalization within early/late endosomes in NPC cells, suggests that increased Abeta in NPC disease may be due to increased coupling of presenilin 1 and its substrate C99 in endosome compartments. This work was funded by the grants: NIH-FIRCA 1R03TW007335-01 (A.G.) and Ministry of Science of the Republic of Croatia 098-0982522-2525 (S.H.).

Alzheimer’s disease; APP; cholesterol; endosome; Niemann Pick type C disease; NPC1

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