P1‐455: Subcellular distribution of APP/C99 and amyloid‐beta peptide is altered in CHO NPC1‐null cells compared to CHOwt

Abstract

Background: Alzheimer's disease is associated with extracellular deposits of amyloid-peptides (A) in the brain. Sequential proteolytic processing of the-amyloid precursor protein (APP) by secretases leads to generation of A. APP is first cleaved by either-secretase or-secretase which releases most of ectodomain leaving behind short c-terminal fragment (APP-CTF) tethered within membrane. APP-CTF acts as substrate to-secretase to release A. Several studies have indicated dysregulation of protein transport and lipid metabolism as an important aspect of AD. Previously, we also showed that the inhibition of glycosphingolipid (GSL) biosynthesis reduces secretion of A. Sphingolipids are enriched in the plasma membrane, from here they are transported to late endocytic compartments where they are degraded. Inherited defects in degradation of these lipids cause sphingolipid storage disorders (SLSDs) marked by their accumulation in neurons and neurodegeneration. Both, AD as well as SLSDs are associated with intraneuronal tau tangles and inflammation. There is a common defect in lipid transport along the endocytic pathway in SLSDs and the processing of APP also occurs predominantly in post-Golgi secretory and endocytic compartments, therefore we investigated the effect of accumulation of sphingolipids on transport and processing of APP. Overall objective of the study was to address the common mechanisms involved in SLSDs and AD, if any. Methods: We used cultured cells and increased GSLs levels by incubation with bovine brain gangliosides. The processing of APP and its derivatives was studied by immunoblotting. Pulse chase experiments were performed to analyze the turnover of APP metabolites. Subcellular distribution of APP and its products was analysed by density gradient and immunocytochemistry. Results: The accumulation of sphingolipids markedly increased the secretion of A and lead to strong increase in APP-CTF levels. However-secretase activity was not affected by GSLs in in vitro assays. We demonstrate that the increased levels of cellular GSLs altered the distribution and stability APP-CTFs. Similar results were also obtained in independent genetic models. Conclusions: Alteration in sphingolipid metabolism with associated accumulation of APP-CTFs might underlie the pathophysiologic events in AD as well as sphingolipid storage disorders along with other reported factors. Background: A sphingolipid storage disease (SLSD) Niemann Pick type C (NPC) is caused by dysfunction of NPC1 protein which leads to accumulation of free cholesterol and glycosphingolipids in endosomal/lysoso-mal compartments. It has been recently shown that this defect leads to increased formation of amyloid-beta (Abeta) peptide, and is accompanied by altered localization of presenilin 1 to early/late endosomes. We hypothesized that cholesterol accumulation upon NPC1 loss of function leads to increased APP/C99 localization in endosomes and increased formation of Abeta in these compartments. To test this we monitored subcellular local-ization of APP/C99...