ࡱ> 574'` bjbj ?"@ @ @ @ L d d d d d d d d $Kh: d d :d d O d d    d X ;6@ : e0 q q q Dd Z @ 42 d d d :: d d d  || The Peter Carlton Jones Memorial Award 2006 Winning Submission by Emyr Lloyd Evans Read Emyr's submission Lay summary: We have discovered that NPC1 lysosomes have less calcium than normal lysosomes. This lysosomal calcium problem occurs as an early step in the disease process leading to the lipid accumulation, including the build up of cholesterol and glycolipids. If we compensate for the calcium problem in the NPC1 mouse by treating it with a drug that alters calcium levels the mice survive longer and the disease process is slowed. This research suggests that treating the calcium problem in NPC1 may offer a new route to the development of an effective therapy. Defective late endosomal/lysosomal calcium homeostasis is an early event and a potential therapeutic intervention point in Niemann-Pick C1 disease pathogenesis. Emyr Lloyd-Evans, Dan Sillence and Fran Platt. Department of Pharmacology, University of Oxford, South Parks Rd., Oxford, OX1 3QT. Niemann-Pick type C1 (NPC1) is an autosomal recessive neurodegenerative lysosomal storage disorder. NPC1 is caused by mutations in a late endosomal/lysosomal protein (NPC1) of unknown function leading to accumulation of glycosphingolipids, sphingosine, sphingomyelin and cholesterol in these compartments. How mutations in the NPC1 protein leads to lipid storage and how lipid storage leads to neuropathology remains unclear. Some of the sphingolipids stored in NPC1 cells and tissues have been reported to alter endoplasmic reticulum (ER) calcium homeostasis in primary sphingolipid storage disorders, contributing to neuropathology (Gaucher, Sandhoff and Niemann-Pick A). In order to see whether NPC1 cells share common calcium induced neuropathological mechanisms with the primary sphingolipidoses we analyzed the calcium content of intracellular NPC1 organelles using the intracellular fluorimetric probe Fura-2AM. We have discovered that NPC1 null cells have a decrease in total cellular calcium. Calcium uptake and release from the ER, the largest calcium store in the cell, is normal. Mitochondrial calcium content is also normal, indicating that NPC1 mitochondria can correctly sequester calcium despite a reported defect in their membrane potential. We discovered that the defect is caused by a large reduction (~70-80%) in the late endosomal/lysosomal acidic calcium pool. This novel finding was confirmed using multiple pharmacological agents to specifically empty those stores. In order to discover the relevance of this finding to the disease process we used secondary amines (U18666A, sphingosine) and siRNA against NPC1, to induce the NPC1 phenotype in control RAW macrophage cells, and recorded the events that occurred over a 48h time course. Perturbation of late endosomal/lysosomal calcium is the first event that we detect following amine treatment. This is followed by abnormal transport of a fluorescent glycosphingolipid, BODIPY-lactosylceramide, to late endosomes, with cholesterol and glycosphingolipid storage occurring last in this sequence of events. Silencing of the NPC1 gene leads to the same chronology, indicating the validity of using secondary amines to mimic early events in NPC1 pathogenesis. To validate the possibility that late endosomal/lysosomal calcium depletion is an early event in NPC1 pathogenesis we treated control RAW macrophages with BAPTA-dextran (a plasma membrane impermeable calcium chelator that can than be internalized by endocytosis and chased into different endocytic compartments) to chelate calcium in the late endosomal/lysosomal system. Chelation of calcium in these compartments resulted in mistrafficking of sphingolipids and storage of free cholesterol, indicative of NPC1. As depletion of calcium could induce an NPC1 phenotype we determined whether increasing cytosolic calcium by treating NPC1 cells with calcium modulating agents (calcitriol, thapsigargin, curcumin) could reverse the cellular abnormalities. All three reagents were able to reverse the endosomal transport defects leading to a reduction in lipid storage back to control levels. We are currently testing in the NPC1 mouse model the efficacy of curcumin, a natural product that is tolerated at high doses. Treatment with curcumin leads to an elevation in cytosolic calcium that corrects defective vesicular fusion and transport at the level of the late endosome (potentially caused by reduced calcium efflux out of the organelle). Early results indicate improved functionality and increased life expectancy in the curcumin treated NPC1 mouse. This study indicates that abnormal late endosomal/lysosomal calcium in NPC1 cells is an initiating factor in pathogenesis and a target for therapeutic intervention with drugs that modulate calcium homeostasis. h9h-TUmnopq~ G H w w dgd= 0P&P 1F:p+. 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