Title: Lysosomal phospholipase A2 contributes to the molecular phenotype of NPC1, and modulates cholesterol trafficking as well as mTOR- and COX-2 signaling
Funder: The Academy of Medical Sciences

Abstract: Research background /Visit purpose: Despite being categorized as a “rare lysosomal storage disease”, the genetically caused neurodegenerative Niemann Pick type C 1 disease (NPC1D) has been attracting scientific and clinical attention for decades. This might be explained by both the multi-factorial complexity of the pathology as well as the mechanistic and therapeutic interfaces with major chronic disorders such as atherosclerosis, cancer, and even Ebola- and Marburg virus infection. The molecular phenotype of NPC1D includes a deficiency of the cholesterol transporter protein NPC1, lysosomal accumulation of cholesterol and (sphino)lipids, as well as impaired cholesterol sorting from the endo/lysosomal compartment of the cell to the endoplasmic reticulum. Remarkably long-chained sphingomyelin has long been known to impede lysosomal cholesterol egress from membranes, whereas its degradation products (lyso-sphingomyelin, ceramide and fatty acids) facilitate it. Despite being major constituents of cell membranes, the role of glycerophospholipids in NPC1D has been almost completely ignored. Yet, recent data emerging from lipidomic profiling of lysosomes isolated from NPC1 knock-out cells has proven a multifold accumulation of lysoglycerophospholipids, which are degradation products of lysosomal phospholipases including phospholipase A2. Taking into account the membrane-destabilizing effect of lysolipids, increased activity of phospholipase A2 in NPC1 deficient cells points toward a cellular process aiming at reducing cholesterol buildup and enhancing cholesterol trafficking. Notably, changes in cholesterol homeostasis have recently been shown to be sensed by mTOR, which is a lysosomal signaling machinery contributing to the regulation of lipid and cholesterol metabolism. In addition, emerging data suggest a link between mTOR-, and COX-2 signaling that is responsible for the formation of pro-inflammatory prostaglandins. Thus, lysosomal phospholipase A2 (LPLA2) might modulate mTOR-, and COX-2 signaling. Briefly, my visit aims at shedding some light on the potential role of LPLA2 in the mechanisms underlying NPC1 pathology. Hypothesis: Cells deficient on NPC1 exhibit an enhanced activity of LPLA2 resulting in a significant accumulation of lyso-glycero-phospholipids and free fatty acids in the lysosomes. This high activity is accompanied by modulations of cholesterol trafficking and signaling pathways related to mTOR and COX2. Research plan and Time schedule: In order to achieve the objectives of my visit, I plan to proceed as follows: Month1: 1. Comparing the activity of LPLA2 and a modulatory enzyme called aSMase in NPC1 knock-out fibroblasts with that in wild type ones 2. Downregulation of LPLA2- by RNAi in NPC1 knock-out and wild type fibroblasts 3. Ensuring the success of the down-regulation by means the techniques available in the laboratory (e.g. Western blotting, Activity assays) Month2: •Investigating the effect of the down-regulation on cholesterol trafficking in the RNAi-treated cells as well as in their counterparts by: a. fluorescence microscopy after fluorescent staining of cholesterol b.RT-PCR of key regulatory proteins in cholesterol metabolism (e.g. LPLA2, aSMase, NPC1, NPC2, SREBPs, HMGCoA-reductase, LDLR) c. activity assays of LPLA2 and aSMase Month3: •Investigating the effect of the down-regulation in the RNAi-treated cells and their counterparts on mTOR and COX2 by : a.detection of the mTORC1 kinase activity via Western blotting b.RT-PCR of COX2 and measuring its enzymatic activity


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE