Evaluation of the effects of two compounds on the endo-lysosomal system in Mucopolysaccharidosis type II disease models

Lysosomal storage disorders (LSDs) are a group of diseases characterized by the accumulation of substrates, due to the defective function of lysosomes. Among LSDs, Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare disorder caused by a deficit of the lysosomal enzyme iduronate-2-sulfatase (IDS). As a result, heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycans (GAGs) pathologically accumulate in different organs. Recent evidence, however, points out that lysosomal dysfunction and GAGs accumulation may be only part of the problem and the alteration of several cellular pathways might participate in the disease pathogenesis. To date, enzyme replacement therapy (ERT) is available for these patients, but the recombinant enzyme is not able to cross the blood-brain barrier (BBB) and thus this therapy is not effective in treating the neurological symptoms, which characterize the most severe forms of the disease. Based on this unmet therapeutic need, my thesis project was about testing the potential beneficial effects of two candidate compounds, namely delta-tocopherol (DT) and NK1. DT is a natural isoform of vitamin E, well-known for its anti-oxidant properties. In addition to its conventional anti-oxidant role, however, recent studies on LSDs showed a beneficial effect of this compound on GAGs accumulation, probably by enhancing lysosomal exocytosis. NK1, instead, is a recombinant form of the natural splice variant of the hepatocyte growth factor (HGF), which was demonstrated to bind extracellular HS and DS with high affinity and specificity, thereby allowing to restore the proper ligand-receptor balance and downstream signalling pathways. I assessed the potential biological effects of DT and NK1 in MPS II both in vitro, using an IDS mutant neuronal cell line, and in vivo, using an established ids mutant zebrafish line. I first performed viability assays in both experimental models to evaluate any potential cytotoxicity associated with the two compounds and determined the optimal range of concentrations and a temporal window for the treatments. I next carried out Western Blot analyses to examine the effects of DT and NK1 on the endo-lysosomal system, focusing my attention on well-known markers such as Lamp1 and Rab7. In addition to this, I preliminarily tested the effects of DT and NK1 on autophagy. Regarding the in vitro model while none of the two selected compounds exhibited significant effects on the rescue of neuronal differentiation defects, DT showed a slight recover of the decreased RAB7 and LAMP1 protein levels in mutant cells. Moreover, DT demonstrated to further increase autophagosomal maturation in MPS II neurons. On the other hand, when examining the in vivo model, while no evident effects could be detected on Rab7 protein levels by both compounds, I could observe a slight, although not significant, recovery of autophagosomal maturation in DT-treated larvae. Although still preliminary, my results point to potential beneficial effects of DT on target cellular abnormalities triggered by IDS loss of function.