ࡱ> 463'`  bjbj ?"     $Y h$ 9   $ ]       K6 s 0 i i i < F0$ $      D Jinzhi Zhang Biomedical Student Joint winner of the 2007 Peter Carlton Jones Award Jinzhi is a 3rd year biochemistry undergraduate student at the University of Bath who submitted an abstract for the PCJ Memorial Award. The abstract is based upon work carried out last year whilst on a six month placement at Dr. Fran Platt's laboratory at the University of Oxford. Jinzhi will be returning to Dr Platts lab for a further 6 month placement this summer to complete the project and will then continue her degree studies. Absence of cholesterol does not prevent NPC phenotype Lay summary: We have discovered that during the early steps that go wrong in NPC1 cells cholesterol does not appear to be needed. Using normal cells that have no cholesterol we were able to change them into cells that mimic NPC1 patient cells. This suggests that cholesterol is not as important in starting the processes that leads to the dysfunction of NPC1 cells, but may still be important during later stages. For this reason we have developed a new method to monitor NPC1 cells treated with a panel of natural substances for any possible improvement. The method relies on monitoring the level of total storage in NPC1 cells as opposed to other methods that follow cholesterol storage alone. We hope that this method will lead to the discovery of novel therapies for NPC1 disease. Absence of cholesterol biosynthesis and exogenous uptake does not prevent induction of a Niemann-Pick C1 phenotype in normal cells Jinzhi Zhang*, Fran Platt & Emyr Lloyd-Evans Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT *Current address: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 2LQ Smith-Lemli-Opitz syndrome (SLOS) is an inherited metabolic disease in which the enzyme involved in the final step of cholesterol biosynthesis (3-hydroxysterol 7-reductase) is defective, causing intracellular accumulation of the cholesterol precursor, 7-dehydrocholesterol (7-DHC). We have found that SLOS cells grown in the absence of extracellular cholesterol have almost no detectable levels of cellular cholesterol and accumulate 7-DHC. In addition, we have found that the accumulation of 7-DHC leads to the elevation of glycosphingolipids, low calcium levels in acidic endosomes, abnormal sphingolipid trafficking and defective early to late endosome transport. These features are all hallmarks of Niemann-Pick C1 (NPC1) disease. It is unlikely that elevated levels of 7-DHC directly cause these cellular phenotypes as treatment of either SLOS or NPC1 cells with the glycosphingolipid biosynthesis inhibitor NB-DGJ corrects the transport defects and partially corrects the calcium defect. Furthermore, initial early elevations in 7-DHC do not give rise to these phenotypes, suggesting a cascade of events following 7-DHC storage. We are currently studying the possibility that 7-DHC behaves as a class II amphiphile and interferes with the function of the NPC1 protein. This work suggests that in the early events leading to cellular pathology of NPC1, cholesterol is expendable, as it does not play a role in generating the cellular phenotypes commonly associated with NPC1. Based on this work we have developed a simple screening test that may be useful for detecting novel NPC1 therapeutics independently of particular storage materials. This microtitre based assay utilises Lysotracker green as a measure of general lysosomal storage rather than storage of a specific substrate and can be adapted for use on any fluorescence plate reader. We have validated the efficacy of this assay using both a U18666A induced macrop h9Uh}W"UVWX C D Q U V YWA gd}W hage model system and human NPC1 fibroblasts and are currently screening novel natural compounds for possible therapeutic benefit. = 0P&P 1F:p+. 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