Investigating Endothelial Dysfunction in a CRISPR/Cas9 Model of Fabry Disease

Abstract

Fabry disease is a rare, X-linked lysosomal storage disease arising from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA). Reduced GLA activity disrupts glycosphingolipid (GSL) catabolism, leading to widespread accumulation of neutral GSLs in cells resulting in clinical manifestations such as stroke, vasculopathy, cardiomyopathy, and renal failure. The link between GLA deficiency and accumulation of GSLs with vascular dysfunction is unclear. Past work in the Shayman lab has led to the formulation of an overarching hypothesis for the mechanism of the vasculopathy in Fabry disease, namely that GLA deficiency leads to uncoupling of endothelial nitric oxide synthase (eNOS) and formation of reactive oxidants resulting in protein nitrosylation. The primary cause of eNOS uncoupling in the setting of GLA deficiency is unknown. In this thesis, I used the CRISPR/Cas9 method for gene editing to mutate Gla in the human endothelial cell line, EA.hy926, to obtain GLA-deficient cells. Neutral GSLs were extracted and analyzed with thin layer chromatography (TLC). NOS activity was measured by monitoring the conversion of [3H]L-arginine to [3H]L-citrulline. Contrary to what is observed with siRNA knockdown of GLA expression in EA.hy926 cells, GLA-deficient cells did not accumulate the primary substrate for GLA, globotriaosylceramide (Gb3). On the other hand, further analysis showed that these GLA-deficient cells accumulate the downstream GSL, globotetraosylceramide (Gb4). However, both the siRNA and CRISPR/Cas9 models have elevated globotriaosylsphingosine (lyso-Gb3). As observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had less NOS activity. Two different methods of reducing globo series GSLs had opposite effects on NOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved NOS activity. In contrast, treating cells with a glucosylceramide synthase inhibitor (inhibits the synthesis of downstream GSLs) decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated GSLs. Other studies showed that NOS activity is improved in GLA-deficient cells by increasing the availability of the eNOS cofactor, tetrahydrobiopterin, suggesting that GLA-deficient cells have a limited supply of endogenous BH4. Finally, it was observed that lyso-Gb3 inhibits NOS activity.