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Lipid traffic jam in Niemann Pick type C disease causes altered endocytosis of APP

Background: Cholesterol accumulation upon NPC1 dysfunction in lipid storage disorder Niemann Pick Type C (NPC) causes increased levels of intracellular amyloid-β peptide (Aβ). We have recently shown that Aβ increase in NPC1-/- cells is due to cholesterol-dependent increased β-secretase processing of APP. We have also shown that cholesterol accumulation in NPC1-/- cells causes decreased expression of APP at the cell surface and its accumulation within punctate vesicular structures (Malnar et al. BBA 2010). Since α-secretase shedding primarily occurs at the cell surface, while β-secretase processing occurs mainly within endosomes, we hypothesized that increased Aβ generation upon NPC1 dysfunction is caused by cholesterol-mediated defect in endocytic trafficking of APP. Methods: To test whether increased levels of Aβ in CHO NPC1-/- vs. CHOwt cells are dependent on endocytosis of APP we used APP internalization mutant that lacks entire C-terminal intracellular domain (APPΔC). Localization and trafficking of APP protein within endocytic pathway was analysed in HA-SEAP-APP stably transfected CHO NPC1-/- vs. CHOwt cells by immunocitochemistry and confocal microscopy. To visualize endosomal/lysosomal compartments we used EEA1, TfR, LBPA and LAMP1 antibodies. Results: We show that APPΔC-transfected CHOwt and NPC1-/- cells exhert decreased levels of Aβ compared to APPwt-transfected cells, indicating that APP internalization is required for increased formation of intracellular Aβ in NPC1-/- cells. Immunocitochemistry experiments showed APP accumulation within early and recycling endosomes in NPC1-/- cells, but not in cholesterol rich late endosomes and lysosomes. Furthermore, we show that this accumulation is dependent on high cholesterol levels, since cholesterol depletion in NPC1-/- cells reversed altered APP trafficking to that as in wt cells. We also observed increased internalization and decreased recycling of APP in NPC1-/- cells vs. CHOwt, supporting its accumulation within early/recycling endosomes upon NPC1 dysfunction. Furthermore, blockage of autophagy with bafilomycin A treatment did not lower Aβ levels in NPC1-/- cells, suggesting that increased autophagy in NPC disease is not involved in increased formation of Aβ. Conclusion: In summary, our results support that lipid traffic jam in NPC disease causes accumulation of APP within early/recycling endosome compartments leading to increased formation of Aβ.

amyloid precursor protein (APP); cholesterol; endocytosis

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