The Neurogenic potential of stem cells is altered in Mucopolysaccharidosis type IIIA

Abstract

Mucopolysaccharidosis type IIIA (MPS IIIA) is one of a series of 11 genetically inherited metabolic disorders and results from a deficiency in the lysosomal enzyme sulphamidase, leading to intracellular and extracellular accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). MPS IIIA is characterised by a profound neurological phenotype and mild skeletal pathology. Currently, the mechanisms leading to disease pathology are poorly understood in MPS IIIA. It has been suggested that the excess amount and aberrant structure of MPS IIIA HS compared to normal HS contributes to disease pathology, due to the vital role of HS in many developmental signalling pathways. GAG accumulation commences prenatally in MPS IIIA, with GAG storage present in the developing CNS in utero, indicating that alterations in CNS development processes may contribute to the neurological pathology of MPS IIIA patients. This thesis aimed to determine the effects of aberrant MPS IIIA HS on one of the earliest processes in CNS development, neurogenesis, through the development of a range of in vitro models of MPS IIIA. Extrinsic, extracellular MPS IIIA HS was found to impair neurogenesis, providing a mechanism of pathology for the severe central nervous system (CNS) pathology prominent in patients. GAGs from a range of other MPS subtypes were found to have diverse effects on neurogenesis, indicating that MPS IIIA HS was distinctly pathogenic. Osteogenesis was similarly impaired by MPS IIIA HS, providing a molecular basis for the mild skeletal pathology observed in patients. To further investigate the effects of MPS IIIA on neurogenesis, two neurogenic MPS IIIA stem cell lines were developed. Mesenchymal stem cells (MSCs) isolated from a mouse model of MPS IIIA and induced pluripotent stem cells (iPSCs) derived from MPS IIIA patient fibroblasts were used to model MPS IIIA neurogenesis in vitro. MPS IIIA murine MSCs and neural progenitor cells derived from MPS IIIA human iPSCs (iPSC-NPCs) displayed decreased proliferative capacity compared to normal cells, indicating a dysfunction in the processes required for stem cell proliferation, many of which are also involved in stem cell differentiation. Both cell lines were then induced along the neural lineage. A significant reduction in the expression of neural marker genes was seen in MPS IIIA murine MSCs when compared to normal murine MSCs, indicating a dysfunction in neurogenesis in MPS IIIA. Similarly, iPSC-derived NSCs induced to form neurons displayed less overt neuronal morphology and a significant decrease in the expression of neuron marker genes. It was hypothesised that alterations in stem cell proliferation and differentiation was a result of MPS IIIA HS disrupting the many HS-dependent signalling pathways involved in stem cell proliferation and differentiation. Overall, through the development of stem cell models with neurogenic properties, this thesis has demonstrated that the MPS IIIA HS impairs neural progenitor proliferation, survival and maturation, likely via alterations in HS-dependent signalling pathways integral to stem cell proliferation and differentiation. Disrupted stem cell proliferation and neurogenesis was identified as a potential mechanism of pathology for the severe neurological pathology prominent in MPS IIIA patients.