Identifying and characterizing lysosomal storagedisease phenotypes for utilization in novelscreening and monitoring assays

In the lysosomal storage disease (LSD) field there are very few studies examining large cohorts of LSD samples in order to identify suitable new pan-LSD biomarkers and identify pan-LSD disease mechanisms. This thesis investigated the possibility of using a simple fluorimetric test for lysosomal swelling, simple enzyme assays and the associated accumulation of storage material alongside the presence of unique heavy metal accumulation to identify the majority of LSDs. The results showed that lysosomal swelling is a highly sensitive phenotype and that high-throughput analysis can be achieved using the fluorescent marker lysotracker. This probe can be used to screen LSD cells as both a suitable biomarker and potentially for drug screening to develop new treatments for LSDs. This thesis was also identified that secondary alteration of lysosomal enzymes is a common feature of LSDs. Such secondary lysosomal enzyme alteration could be useful for treatment monitoring and some novel biomarkers for some and potentially all of the LSDs have emerged. I have also conducted the first electron microscopy (EM) study that compares all classes of LSDs. This technique was proven to be useful for characterisation of the lipids and other macromolecules stored both primarily and secondarily in the majority of LSDs. EM also confirmed that alteration of secondary lysosomal enzymes could be the reason behind the accumulation of materials in some LSDs. Divalent cation signalling defects have been reported in several LSDs, I therefore studied Ca2+ and trace element (TEs) ion changes across all the LSDs and discovered that lysosomal Ca2+ defects are common and that changes in Zn2+ and a few other TEs were identified in almost all or specifically altered in some of the LSDs respectively. Our results highlight the possibility of using inductively coupled plasma mass spectrometry (ICP-MS) for monitoring changes in blood TE levels during the course of clinical treatment of CLN5 patients. Finally, evidence points to the NPC1 protein function, in terms of Zn2+ efflux from lysosomes, was inhibited by common storage of sphingoid bases and is a common phenotype across the majority of LSDs that explains the occurrence of secondary lipid accumulation across most of the LSDs. Our findings provide new potential biomarkers, new mechanisms of pathogenesis and new therapeutic targets that are common to all of the LSDs validating the power of studying multiple LSDs together.