Investigating neuronal pathogenesis in Mucopolysaccharidosis type II and Mucolipidosis type II disease models

Lysosomal storage disorders (LSDs) are genetic diseases affecting lysosomal functions characterized by accumulation of undegraded substrates. However, lysosomes participate in various cellular processes such as intracellular trafficking and cell signaling. Despite their genetic and clinical variability, these disorders frequently present common neurological alterations. In this context, I focused my attention on the characterization of the neuropathogenesis of two LSDs, Mucopolysaccharidosis type II (MPS II) and Mucolipidosis type II (ML II), both exhibiting accumulation of glycosaminoglycans and severe neurological abnormalities. MPS II patients’ genetic defects affect the lysosomal hydrolase iduronate-2-sulfatase (IDS), involved in glycosaminoglycans catabolism. Considering the role of Wnt signaling in neurodevelopment I investigated its possible dysregulation in the central nervous system of a previously generated MPS II zebrafish model. I found altered protein levels for phospho-β-catenin (S552), the transcriptionally active form of the major Wnt signaling mediator β-catenin. However, no changes of protein levels for phospho-Gsk3β (S9), the inactivated form of Gsk3β, and abnormalities of Wnt/β-catenin-dependent transcriptional activity have been detected. Therefore, I checked for secondary pathways affecting phospho-β-catenin (S552) expression by measuring N-cadherin and Akt protein levels. Finally, I preliminarily studied synaptic dysfunction in ids larvae detecting an upregulation of the post-synaptic marker Psd-95, despite no variations in its intracellular localization. In addition, I generated a novel human neuronal MPS II model by applying the CRISPR/Cas9 protocol on LUHMES cells retrieving two independent clones. I detected early impairment of the endo-lysosomal system in differentiated IDS mutant clones, characterized by decreased RAB7 and LAMP1 levels as well as lysosomal alkalinization. Additionally, checking for autophagic defects, while LC3-II/I were unaltered, p62 was significantly reduced in one of the mutant clones. Furthermore, I detected a secondary accumulation of lipids in mutant cells, consistent with LSDs pathogenic storage of additional substrates. Finally, I observed a reduced differentiation potential of the IDS knock-out clones, supporting the fundamental role of lysosomes during neuronal differentiation. As for ML II, it is caused by mutations in the GNPTAB gene encoding N-acetylglucosamine (GlcNAc)-1-phosphotransferase, the enzyme that tags lysosomal proteins with a GlcNAc-1-phosphate on mannose residues. A subsequent reaction removes the GlcNAc residue exposing the mannose-6-phosphate tag required to target lysosomal proteins to lysosomes. Deficiency of this enzyme leads to hypersecretion of lysosomal hydrolases in the extracellular space and their depletion into lysosomes. To investigate ML II neuropathogenesis, I characterized two independent GNPTAB knock-out clones generated in human medulloblastoma cells. Increased LAMP2 levels and Lysotracker staining showed a higher number of lysosomes in mutant cells. Additionally, I detected cholesterol accumulation into enlarged lysosomes and a significant reduction of acid hydrolases activities in mutant cells. Next, I performed an RNA-seq analysis to provide a dataset of altered pathways in GNPTAB mutants. Differentially expressed genes identified between mutant and control cells were related to extracellular matrix organization, signal transduction and cholesterol biosynthesis. Finally, I performed proteomics of cell surface glycosylated proteins to find target proteins which trafficking and cell membrane localization might be affected by lysosomal dysfunction. Among the top differentially expressed proteins, p62 presented the highest fold change, suggesting an autophagic impairment downstream to GNPTAB loss of function.