Ciampa, Mariangela (2025) Iminosugars as pharmacological chaperone therapy for MPS IIIB. [Tesi di dottorato]

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Abstract

Mucopolysaccharidosis type IIIB (MPS IIIB), also known as Sanfilippo B syndrome, is an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU), which is involved in heparan sulfate (HS) catabolism. Accumulation of HS in the lysosomes and other cellular compartments leads to tissue and organ dysfunctions, resulting in a wide range of clinical symptoms, including severe neurodegeneration. Current therapies for MPS IIIB aim to alleviate symptoms; however, they do not address the neurological phenotype of the disease. Here, we describe the ability of seven newly synthesized N-substituted L-iminosugars to reduce substrate storage and lysosomal dysfunctions in MPS fibroblasts and in a neuronal cellular model of the MPS IIIB. HS levels and lysosomal defects were assessed by immunofluorescence, while the mechanism of action of the selected L-iminosugars was investigated through western blotting analysis and fluorometric enzymatic assays. We found that four of these N-substituted L-iminosugars increase NAGLU protein levels and enzymatic activity and promote endogenous mutated NAGLU sorting toward the lysosomal compartment. Furthermore, we demonstrated that the active L-iminosugars decrease HS accumulation by downregulating exostosin glycosyltransferase protein levels. In particular, to investigate the mechanism of action and the therapeutic potential of the iminosugar N-butyl L-DNJ (L-NBDNJ), we demonstrated that this compound stabilizes both mutated and recombinant NAGLU (rhNAGLU) enzymes without inhibiting their activity, thus functioning as an allosteric chaperone. Finally, we evaluated the therapeutic effects of L-NBDNJ in Naglu-/- mice and found that L-NBDNJ treatment improves cognitive and motor functions, along with a reduction in HS and lysosome accumulation, a rescue of autophagy impairment, and decreased neuroinflammation in the cortex and hippocampus of L-NBDNJ–treated Naglu-/- mice. The findings of this work pave the way for the development of novel therapeutic approaches for the treatment of MPS IIIB based on the use of L-iminosugars acting as pharmacological chaperones for the NAGLU enzyme.

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