Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Dans cette thèse, nous avons étudié l'influence de plusieurs variants génétiques situés dans les régions chromosomiques 5q31-q33, 6p21, et 17p12, pour lesquelles une liaison génétique avec des phénotypes de paludisme a été montrée.Les gènes NCR3 et TNF, qui sont situés dans la région chromosomique 6p21, ont été associés au paludisme dans une population vivant au Burkina Faso. Nous avons répliqué ces études dans une population congolaise afin deconfirmer les associations des polymorphismes avec les accès palustres simples et la parasitémie symptomatique. Nos résultats montrent que le polymorphismeNCR3-412 est associé avec les accès palustres simples au Congo, et que les polymorphismes TNF-308, TNF-244, et TNF-238 sont associés avec les accès palustres simples ou la parasitémie symptomatique. En outre, nos analyses bioinformatiques suggèrent que les polymorphismes TNF-244 et TNF-238 agissent en synergie pour modifier le site de fixation pour au moins un facteur de transcription.Les deux gènes HS3ST3A1 et HS3ST3B1, qui sont situés dans la région chromosomique 17p12, sont impliqués dans la biosynthèse des heparanes sulfates. Dans cette étude, nous avons étudié l'association d’un polymorphisme situé dans le promoteur de HS3ST3A1 avec les accès palustres simples et la parasitémie symptomatique, et n’avons détecté aucune association. Nous avons étudié en outre le gène NDST1, situé dans la région chromosomique 5q31-q33, et qui code également pour une enzyme impliquée dans la voie héparane sulfate. Des résultats préliminaires encourageants soutiennent l'hypothèse que la variation génétique de NDST1 influence la parasitémie asymptomatique. / In this thesis, we investigated the influence of some genetic variants located within chromosomes 5q31-q33, 6p21, and 17p12, which have been shown to be linked to malaria phenotypes. The genes NCR3 and TNF, which are located in the chromosomal region 6p21, have been reported to be associated with malaria in Burkina Faso population. We have replicated those studies in Congolese population to evaluate the associations of the SNPs in those genes with mild malaria attack and Plasmodium parasitemia. The results showed that the variant NCR3-412 is associated with mild malaria in Congo, and TNF-308, TNF-244, and TNF-238 are associated with mild malaria attack, maximum parasitemia, or both. In addition, bioinformatic studies suggest that TNF-244 and TNF-238 synergise to alter the binding of transcription factors.The two genes HS3ST3A1 and HS3ST3B1, which are located in chromosomal regions 17p12, are involved in the heparan sulfate proteoglycan biosynthesis. In this study, we further investigated the association of the polymorphisms in these genes with mild malaria attack and maximum parasitemia. However no association was found. We further studied the NDST1 gene, which is located within chromosome 5q31-q33, and which encodes the bifunctional enzyme N-deacetylase/ N-sulfotransferase 1, and also participates in the heparan sulfate synthesis . Encouraging results support the hypothesis that NDST1 variation influence controlling parasitemia. Further association and functional studies are needed to validate the role of NDST1 in malaria infection. More generally, the enzymes involved in the heparan sulfate pathway might play a key role in controlling malaria infection.

In the brain, axons are wrapped by myelin sheaths which ensure fast saltatory conduction of impulses and provide metabolic support. In multiple sclerosis (MS), the myelin sheaths are lost which leaves the axon denuded. This not only results in slower conduction of action potentials, but if prolonged, can also lead to axon death due to the loss of metabolic support. This neurodegeneration is the main cause of permanent disability in multiple sclerosis patients. The axon death and disability which stem from it could be prevented by restoring the myelin wrap before axon damage has occurred. This remyelination process is carried out by oligodendrocyte precursor cells which are present throughout life. To remyelinate, OPCs migrate to the area of damage and differentiate into myelinating oligodendrocytes which ensheathe axons with new myelin. In multiple sclerosis, this process occurs but is insufficient to overcome the damage. Therefore, central to the therapeutic efforts in multiple sclerosis is the aim to improve endogenous remyelination. Enhancing recruitment of oligodendrocyte precursor cells (OPCs) to the areas of damage is a clinically unexplored target. To investigate the therapeutic potential of OPC recruitment modulators, I have looked at 2 different targets involved in migration NDST1/HS and Sema3A/NP1. The first target, heparan sulfate (HS) is a proteoglycan which is important to OPC migration, investigated by Pascale Durbec's group in France. In a demyelinating mouse model, its key synthesising enzyme, NDST1, is upregulated by oligodendroglia in a belt around the lesion to aid OPC recruitment. Loss of NDST1 in oligodendrocytes was found to impair remyelination and reduce OPC migration in mice. In collaboration with them, I investigated the relevance of this molecule in post-mortem MS human tissue. I found that in human as well as mouse, NDST1 was primarily expressed by oligodendroglia. The protein level and the proportion of oligodendroglia expressing NDST1 was increased in MS compared to control indicating NDST1 upregulation as a disease response in human. We also found that low numbers of NDST1+ oligodendroglia correlate with bigger sizes of lesions and chronic lesion types that fail to repair, highlighting its importance in repair. Moreover, high numbers of NDST1+ cells in a patient correlated with increased remyelination potential. This indicates that in human, intra-patient variation in NDST1 level may explain differences in potential for endogenous repair. Secondly, I looked at Sema3A, a chemorepulsive molecule which is upregulated in demyelinated injury rodent models aswell as multiple sclerosis lesions, particularly in OPC-depopulated chronic active lesions. Research has consistently found that the level of Sema3A negatively correlates to remyelination because Sema3A hinders OPC migration. This has highlighted Sema3A as a potential target to improve OPC recruitment in MS however the size and shape of the molecule make it hard to design therapeutics against it. Therefore, I looked at its druggable receptor, Neuropilin 1 (NP1), to see whether inhibition of NP1 had the same positive effect on OPC recruitment and remyelination as lowering the level of Sema3A. NP1 is a tyrosine kinase receptor for both Sema3A and vascular endothelial growth factor (VEGF) and is found in many cell types. To check if NP1 inhibition is beneficial, I assessed remyelination in a mouse where the Sema3A binding site of NP1 has been mutated to prevent Sema3A binding and exerting its effect. This is a proxy for a (currently unavailable) ideal NP1 inhibitor of the Sema3A site only. Contrary to my expectations, OPC recruitment and remyelination in the mutant mice were not improved. However, the NP1 mutation resulted in an altered immune response. To exclude the possibility that no improvement in the OPC recruitment and remyelination of those mice was seen because it was negated by the altered immune response, I explored a cell specific mutant mouse in which NP1 was deleted in oligodendroglia only. In this mutant as well, I did not see improvement of OPC recruitment and remyelination. I therefore propose that Neuropilin 1 is not imperative for Sema3As action in remyelination and is not suitable as a therapeutic target in multiple sclerosis. Loss of the whole NP1, but not loss of the Sema3A site also resulted in biggermyelinated and unmyelinated axons as well as a different myelin thickness post remyelination. This showed that VEGF and the VEGF site on NP1 in oligodendroglia have a previously unknown but important role in determining axon size and myelin thickness which should be further investigated. To further elucidate those results in a simple system, I looked at how Sema3A, NP1-Sema3A inhibitors, VEGF and NP1-VEGF inhibitor affect OPC behaviour. I confirmed Sema3As chemorepulsive effect but also showed that at different concentrations it can improve proliferation and survival of OPCs. Inhibiting the Sema3A site and the VEGF site of NP1 by specific blocking antibodies also affects OPC proliferation and maturation. This suggested that NP1s ligands are involved in more than just OPC migration. In summary, this work supports the relevance of the mouse findings that NDST1 is upregulated in demyelination and important for repair for human illustrating that it might be a suitable therapeutic target to investigate. However, despite the importance of Sema3A in MS models, its only reported receptor, NP1, is not essential for Sema3As action. Therefore, it is an unsuitable therapeutic target. The fact that NP1 is an inappropriate drug target for MS is further demonstrated by the involvement of its ligands in multiple OPC behaviours both in positive and negative aspects.

Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces. In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do. Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized. A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.