Final Report Summary - STEROLOSOME (Targeting common mechanisms of pathogenesis in diseases of sterol homeostasis associated with lysosome dysfunction; development of novel and rapidly translatable clinical therapies) Project Overview and Objectives: Approximately 6,500 rare diseases collectively affect some 26-30 million European citizens. Rare diseases therefore are an important research area, as very few are understood well enough to be treated effectively in the clinic. Situated within a leading Russell group university, the host laboratory has significant expertise in rare disease research, with an extensive collection of patient-derived cell lines and tissues, in addition to extensive experience in the specialist assays required for this research project. This fellowship focused on elucidating the underlying cell biological mechanisms governing pathogenesis of the rare childhood disease Smith-Lemli-Opitz Syndrome (SLOS), for which there is currently no effective treatment. SLOS is a multiple malformation syndrome attributed to an inborn error of cholesterol metabolism, in which patients present with a spectrum of phenotypes including multiple birth defects, neurological decline and autism spectrum disorder. SLOS is an autosomal recessive disorder caused by mutations in the 7-dehydrocholesterol reductase (DHCR7) gene, leading to defective endoplasmic reticulum (ER) cholesterol biosynthesis and accumulation of the cholesterol precursor, 7-dehydrocholesterol (7DHC), in all tissues. SLOS carrier frequency in Europe is 1/20 with a predicted incidence in central Europe of 1/1,590 live births. However, the actual incidence is 1/60,000 owing to a high degree of maternal loss during the first trimester. Therapeutic options are severely limited, and increased dietary cholesterol is of limited benefit due to defective endocytic transport of dietary (LDL)-derived free cholesterol from lysosomes to the ER in SLOS cells. We have recently discovered a link at the cellular level between SLOS and another rare pediatric disease, Niemann-Pick C (NPC). NPC disease is caused by mutations in either the NPC1 (95% of cases) or NPC2 (5% of cases) genes, encoding the NPC1 and NPC2 proteins, respectively. NPC1 is a 13 transmembrane domain protein of the late endosome and lysosome, while NPC2 is a soluble intra-lysosomal cholesterol transporter. Loss of function of either protein leads to accumulation of cholesterol and other lipids within the lysosomes of cells. Our data suggests that accumulation of a sterol precursor in patient-derived SLOS cells functionally inhibits the NPC1 protein, leading to an NPC disease-like phenotype that negates the therapeutic effect of elevated dietary cholesterol in SLOS. To this end, this fellowship employed a multidisciplinary and cross-collaborative research approach to address the following project objectives: (1) Comprehensively characterise the similarity between NPC and SLOS at the cellular and whole organism level. (2) Determine the mechanism by which sterol accumulation inhibits NPC1 function and elucidate their role in regulating NPC1 activity. (3) Develop rapidly translatable therapies for SLOS based on currently available NPC therapies. (4) Identify suitable clinical markers to measure the efficacy of novel therapeutic interventions in SLOS. Work Performed and Main Results: Clinically, the majority of patients diagnosed with SLOS are classified as either severe or classical/moderate. While mildly affected patients do not present with the birth defects, they do present with the characteristic autistic spectrum disorders observed in all SLOS patients. Using biochemical (including enzymatic assays, thin layer chromatography, high-performance liquid chromatography, and magnetite-organelle purification) and cell biological (including live cell Ca2+ imaging, immunocytochemistry, and intracellular lipid trafficking assays) techniques we determined the extent of the cellular phenotypes across the SLOS severity range, as this will impact upon the potential efficacy of future therapies. Intracellular trafficking was impaired in both severe and classical SLOS patient fibroblasts, but was unaffected in fibroblasts from mild SLOS patients. Analysis of glycosphingolipids (GSLs) by HPLC and TLC showed that levels were significantly elevated across the clinical spectrum, and correlated with decreasing 7-dehydrocholesterol reductase (Dhcr7) enzymatic activity. Importantly, we demonstrated that the NPC disease-like phenotypes observed in SLOS cells are not observed in other sterol biosynthetic diseases, and that these specific defects are due to accumulation of 7-dehydrocholesterol (7DHC) and not 8-dehydrocholesterol (8DHC), which has also been reported to accumulate in SLOS cells. Furthermore, lipid storage profiling of tissues isolated from a validated disease model (Dhcr7-/-) of SLOS, revealed that total GSLs are significantly increased in the central nervous system of Dhcr7-/- disease models compared to their wild type counterparts, correlating with previous data from Lloyd-Evans et al. (2008) showing lipids are elevated in NPC1 disease models. Analysis of lipids, which were solvent extracted from cerebrospinal fluid (CSF) and serum from SLOS patients spanning the clinical severity range showed a strong positive correlation between residual Dhcr7 enzymatic activity and both sphingosine and cholesterol in the CSF of SLOS patients, as well as a negative correlation in 7DHC levels. In order to identify the specific sterol(s) that may be inhibiting NPC1 protein function, we optimised a novel lysosomal purification method developed in the Lloyd-Evans laboratory to isolate purified lysosomes from control and Dhcr7-/- mouse embryonic fibroblasts (MEFs). Analysis of these samples will allow the determination of the particular sterol(s) that accumulates in the lysosomes of SLOS cells, thus facilitating future structure-function studies to determine its effect on the lysosomal NPC1 protein. Furthermore, it may provide better genotype-phenotype analysis for patient samples, and ultimately may identify a novel target for drug intervention. Currently, SLOS is treated by increasing dietary cholesterol. However, as cholesterol cannot cross the blood-brain barrier (BBB) and cannot be utilised in other tissues due to trafficking defects, this therapeutic approach is inefficacious. As such, this fellowship sought to identify improved treatments for SLOS, that are rapidly translatable, based on our current knowledge of NPC. Miglustat is currently the only European Medicines Agency (EMEA) approved therapy for NPC. It has been shown to reduce sphingolipid accumulation and delay neurodegeneration. Our preliminary data are promising, and indicate potential corrective effects of miglustat in an in vitro model of SLOS. Further experiments are underway to validate these findings in parallel with in vivo studies in relevant disease models. Additionally, our laboratory is currently in the process of developing a zebrafish (Danio rerio) model of SLOS that will greatly facilitate the rapid screening of novel compounds in vivo, and allow us to perform dose range finding experiments with greater ease. In vitro studies, investigating the therapeutic potential of two other experimental compounds (curcumin and cyclodextrin) that have shown in vitro and in vivo efficacy in the treatment of NPC, demonstrated that the Ca2+ modulator curcumin significantly improved the intracellular trafficking defects observed in SLOS cells, while cyclodextrin has no significant effect on lipid storage compared to control cells. We hypothesise that the lack of efficacy of cyclodextrin is due to the abnormal membrane fluidity that has been reported in SLOS cells. Future work is focused on combination therapies; once generated, the zebrafish model will allow rapid screening of early embryonic phenotypes and complement further in vivo studies by our collaborators. Based on previous work from this laboratory, we sought to determine whether lysotracker staining could also be used as an effective clinical biomarker for SLOS, given the phenotypic similarity (lipid storage) between NPC and SLOS at the cellular level. A fluorescence assay based on quantifying lysosomal volume using lysotracker staining, optimised for 96-well plate format, was established in the laboratory. We observed a significant 3-fold increase in fluorescence in Dhcr7-/- MEFs/patient cells compared to control cells. As expected, lysotracker staining was also significantly increased when these cells were observed under the fluorescence microscope. This assay will greatly facilitate the rapid screening of potential new SLOS therapies/combination therapies. It could also be used as a potential clinical biomarker in addition to the sphingolipids, and has been validated in our laboratory for a panel of other lysosomal storage disorders. Potential Impact: SLOS is most prevalent in central Europeans. Treatment costs are high; severely affected patients require sophisticated feeding care and corrective surgery for complex birth defects. Additionally, debilitating birth defects, ill health and neurological symptoms associated with autism spectrum disorders pose a sub-optimal quality of life for patients. Apart from increasing dietary cholesterol to correct for the endogenous biosynthetic defect (which is of no clinical benefit), there is no therapy for any form of this disease. This fellowship has significantly contributed to our understanding of SLOS pathophysiology, and has made a substantial impact in this area of unmet clinical need. We provided the first complete phenotypic characterisation of SLOS at the cellular and whole organism level, yielding significant insight into the mechanism that contributes to disease pathogenesis and potential identification of novel therapeutic targets for clinical intervention. Findings from this fellowship have major basic and translational implications for SLOS research and rare disease research worldwide, meeting an important and current FP7 Health thematic priority namely “Translating research for human health”.