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Understanding the effect of toxic lyso-lipids on renal and cardiovascular function

Supervisors: Dr Wendy Heywood, Professor Kevin Mills

Background:

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which leads to a deficiency of α-galactosidase A (α-GAL). GLA mutations result in the progressive intracellular accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (Gb3, CTH or GL-3), and contributes to a wide variety of clinical symptoms of FD. In males with the classic phenotype, who have an absent or very low α-GAL activity, gradually develop renal, cerebral and cardiac complications. 
Breakdown of accumulated Gb3 to a lipid called lyso-Gb3 occurs in FD. lyso-Gb3 is not a naturally occurring lipid and has greater association with disease progression than Gb3 [1]. Its is hypothesised that lyso-Gb3 is toxic and may cause many of the symptoms that occur in FD. Exogenously administered lyso-Gb3 has been described to affect healthy sensory neuronal cells which could explain neuronal pain observed in FD [2]. Lyso-gb3 is thought to be generated by the promiscuous action of lysosomal deacylases on accumulated Gb3 and occurs in the lysosome and is released by an unknown mechanism into the circulation. Tissues are then affected by endogenous and exogenous lyso-gb3. The toxicological mechanism of lyso-gb3 is unknown. Lysosomal perturbation occurs not just in LSDs but can occur in normal cells due to stress or exposure to toxic compounds or lysosomotropic drugs [3]. This project would investigate if these environmental exposures could cause the production of low level lyso-gb3 and potentially other lyso-lipids for which over time could contribute to cardiovascular disease. 

Aims/Objectives:
The aim of this project is to confirm and understand the toxicological mechanism of lyso-gb3 on different organ systems in Fabry disease. 

Methods:
Figure 1 outlines plan of the study cell lines that represent the key tissue types affected in Fabry disease will be used as models. The effect of exogenous administered lyso-Gb3 and endogenous created lyso-Gb3 will be studied using an inhibitor for α-GAL to create a Fabry model and an inhibitor of AC to confirm what changes come from accumulated gb3 or lyso-gb3 in FD. Lyso-gb1 which is the lyso-lipid that accumulates in Gaucher disease causing a very different disease phenotype (no cardiac or renal features) will be used as a disease control. The student will use mass spectrometry to perform proteomics and lipid analysis. The data will be analysed using bioinformatics methods that the student will learn from attending UCL bioinformatics courses and from in house expertise in multivariate and gene ontology analysis as previously published by our group [4]. n. Affected pathways will be investigated to confirm the findings. The student will then use lipid analyses and cell culture to investigate lyso-gb3 production in stressful environmental conditions. 

Timeline:
Months 0-6 – cell culture and mass spec training. 
3-8 First cell line experiment and mass spec analysis
6-12 Second cell line experiment and mass spec analysis
10-14 Third cell line experiment and mass spec analysis
14-24 Functional assay development and confirmation pathway
24-33 Environmental exposure experiments and manuscript writing
30-36 Thesis writing  

References:
1.    Nowak A, et al. Mol Genet Metab. 2018 Feb;123(2):148-153. 
2.    Choi L, Vernon J, Kopach O, et al. Neurosci Lett. 2015;594:163-168. 
3.    Lu S, Sung T, Lin N, Abraham RT, Jessen BA (2017) PLoS ONE 12(3): e0173771. 
4. Captur G, Heywood WE, et al .Mol Cell Proteomics. 2020 Jan;19(1):114-127.