LEVERAGING THE ZEBRAFISH MODEL TO BREAK DOWN GLIAL MOLECULAR CHANGES UNDERLYING NEURONOPATHIC MUCOPOLYSACCHARIDOSIS TYPE II

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the enzyme iduronate-2-sulfatase (IDS), leading to the ubiquitous accumulation of heparan sulfate (HS) and dermatan sulfate (DS). The most severe phenotype is characterized by pronounced systemic development, including progressive impairment of central nervous system (CNS) functions. Given the still poorly characterized contribution of glial cells to MPS II neuropathogenesis, this study aimed to further investigate their role by taking advantage of an established ids-mutant zebrafish model. A time-course western blot analysis revealed a stepwise astrocytic response, initially marked by an increase in GFAP (glial fibrillary acidic protein) expression at 15 days post-fertilization (dpf) in midbrains isolated from ids-mutant fish, followed by the activation of distinct astrocyte subpopulations. To refine the astrocyte-specific contribution to MPS II neuropathogenesis at 15 dpf, a fluorescence-activated cell sorting (FACS) protocol was developed using midbrains from a GFAP reporter line. Proteomic analysis of the resulting GFP-positive and GFP-negative populations was performed to identify molecular drivers of the astrogliosis observed in mutant fish. Concomitantly, an increased neuronal cell death was detected in ids-mutant midbrains compared with age-matched controls, as assessed by TUNEL assay and double immunofluorescence labeling, suggesting a link between reduced neuronal viability and enhanced astrocytic activation. Finally, to investigate the contribution of microglia to CNS degeneration, Neutral Red staining and immunofluorescence analyses were performed to evaluate microglial reactive morphology in 15 dpf ids-mutant zebrafish, with the aim of identifying early molecular events triggering neuroinflammation in MPS II.