No description available.

No description available.

No description available.

Development of the visual system involves the precise orchestration of neural connections between the retina of the eye, the thalamus (dorsal lateral geniculate nucleus; dLGN) and the superior colliculus (SC). During early development, receptor molecules on the growth cones of retinal ganglion cell (RGC) axons sense molecular guidance cues in the extra cellular matrix (ECM) that define their route and branching behaviour within the visual system. Heparan sulphate proteoglycans (HSPGs) are ECM molecules composed of a core protein and a variable number of disaccharide residues that have been implicated in mediating axon guidance. HSPGs are modified by a number of enzymes that contribute to their structural diversity. Based on this structural diversity; the “heparan sulphate code” hypothesis of Bulow and Hobert (2004) postulated that different HSPG modifications confer different axon navigation responses as the growth cones traverse the local environment. To investigate the roles played by specific modifications of HSPG molecules in the guidance of axons, we examined two lines of mutant mice harbouring mutations in the genes encoding HSPG modifying enzymes, Heparan sulphate-6-O-sulphotransferase-1 (Hs6st1) and Heparan sulphate-2-O-sulphotransferase (Hs2st). These two mutant lines were generated through the use of gene trapping. Previous observations of RGC axon development in the two mutant lines revealed distinct axon guidance errors at the optic chiasm. Loss of Hs6st1 sulphation resulted in RGC axons navigating ectopically into the contralateral eye. Loss of Hs2st sulphation resulted in RGC axons navigating outside the normal boundary of the optic chiasm. Early observations suggested that both Hs2st sulphation and Hs6st1 sulphation have distinct, non-overlapping actions and thus, influence different axon guidance signalling pathways at the optic chiasm. Based on our findings and previous work describing the expression patterns and functions of the chemo-repellent axon guidance molecules, Slit1 and Slit2 at the optic chiasm and their Robo2 in the retina, we formulated the hypothesis of an HSPG sulphation code where Hs2st sulphation is specifically required for Slit1-Robo2 signalling and Hs6st1 sulphation is specifically required for Slit2-Robo2 signalling at the optic chiasm. To further our understanding of the roles Hs2st sulphation and Hs6st1 sulphation have on axon guidance, we looked at a number of key choice points that navigating axons encounter and are known to be influenced by Slit signalling. Further observations of RGC axons at the optic chiasm of Hs2st-/- mutants and Hs6st1-/- mutants showed distinct axon guidance phenotypes, both resulting in statistically significant increases in the width of the optic chiasm at the midline. While Hs6st1 sulphation had no effect on RGC axon navigation within the eye (possibly due to 6-O-sulphation compensation by Hs6st3); the loss of Hs6st1 sulphation at the dLGN resulted in a significant increase in the defasciculation of the optic tract. Observations of other axonal tracts influenced by Slit signalling revealed the importance of Hs2st and Hs6st1 sulphation in aiding callosal axons to successfully traverse the midline in corpus callosum development. Observations of the thalamocortical (TCA)/corticothalamic (CTA) tracts revealed that neither Hs2st sulphation nor Hs6st1 sulphation was required for the development of the mouse TCA tract (the latter may be explained by 6-O-sulphation compensation by Hs6st2). To test whether Hs2st and Hs6st1 enzymes have redundant functions in optic chiasm development, we attempted to create Hs2st-/-/Hs6st1-/- double mutants. A PCR genotyping strategy was developed for the identification of Hs6st1 animals and showed that Hs6st1-/- mutants had high postnatal lethality with only 3% of the offspring surviving to weaning while Hs2st-/-/Hs6st1-/- double mutants all died very early during embryonic development. Observations of Hs2st-/-/Hs6st1+/- mutants and Hs2st+/-/Hs6st1-/- mutants that lacked three of the four Hst alleles showed no differences when compared to single Hst knockouts. Finally, we showed that altered Slit expression at the optic chiasm and Robo expression in the retina could not explain the mutant phenotypes observed in Hs2st-/- mutants and Hs6st1-/- mutants, and therefore we hypothesized that Hs2st sulphation and Hs6st1 sulphation regulate distinct aspects of Slit-Robo signalling at the surface of the navigating axon growth cone.

Heparan sulphate proteoglycans (HSPGs) are a family of molecules that are found on the surface of cells or in the extracellular matrix, where they are involved in regulating key signalling events required for normal mammalian brain development. It is thought that specificity of HSPGs for particular signalling processes is encoded by their heparan sulphate (HS) sugar side chains, which can be modified post-translationally to yield huge variation in HS structure. Different sulphation patterns are generated by the action of the heparan sulphate sulfotransferases (HSTs) and sulfatase enzymes, which add or remove sulphate groups to specific positions on residues of the HS side chains. Depending on the expression of these enzymes and the resulting heparan sulphate ‘code’, it is proposed that cells are then able to regulate signals they receive and send in the ligand rich extracellular environment of the developing forebrain. Hs6st1 and Hs2st catalyse 6-O and 2-O HS sulphation, respectively. Following loss of either of these two HSTs, commissural tracts including the corpus callosum fail to develop normally during late mouse embryogenesis. The telencephalic midline environment is perturbed, with a striking mis-positioning of glial cell populations that normally act to guide axons towards the contralateral hemisphere. Too many radial glial cells at the glial wedge (GW) migrate towards the indusium griseum (IG) in mutant embryos. The running hypothesis to explain this phenotype is a change in critical signalling pathways required to set up the correct midline glia environment, such as Fgf8/ERK signalling which has already been identified as up-regulated at the Hs6st1-/- corticoseptal boundary (CSB). In order to further study what changes are occurring at the developing midline of HST-/- embryos compared to WT, we took a hypothesis free approach using RNA-sequencing analysis. RNA extracted from dissected midline regions of WT, Hs2st-/- and Hs6st1-/- mouse embryos at E16.5 was sent for sequencing, and a list of differentially expressed genes obtained. Overall we find few differentially expressed genes at the Hs6st1-/- midline compared to WT. At the Hs2st- /- midline there are a larger number of differentially expressed genes. Following validation studies, we find a significant and specific increase in Fgf17 protein distribution at the CSB of Hs2st-/- embryos compared to WT at E14.5. The results suggest the hypothesis that Hs2st’s normal role is to regulate Fgf17 protein distribution to limit exposure of GW radial glia cells to this translocation signal. When 2-O HS sulphation is lost then in Hs2st-/- embryos, ectopic Fgf17 signalling induces aberrant glia migration which ultimately prevents callosal axons from crossing the telencephalic midline to form the corpus callosum. To test this hypothesis, we used ex vivo slice culture experiments and showed ectopic Fgf17 protein expression is sufficient to trigger precocious translocation of midline glia in WT CSB, phenocopying the glia behaviour of Hs2st-/- embryos. Also consistent with the hypothesis, the Hs2st-/- glia phenotype can be rescued by addition of an FgfR1 inhibitor which reduces number of translocated glia cells. From these results we find for the first time that 2-O sulphated HS plays a remarkably specific role in regulating Fgf17-mediated translocation of midline glia cells at the developing mammalian telencephalic midline.

<p>This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions.</p><p>The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals.</p><p>The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project.</p><p>The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.</p>

This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions. The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals. The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project. The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.

No description available.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.

Heparan sulphate (HS) is critical for embryonic development with involvement in a myriad of biological processes, centrally mediating morphogenic movements and facilitating the specification and differentiation of tissues. Complicated by its structural micro-heterogeneity along with expression on numerous different proteoglycan cores, the plethora of roles for HS in biology and their underlying mechanisms have not yet been fully defined. The discovery and characterisation of new reagents and methods for modification of HS expression and/or structure will aid efforts in elucidating the structure and activity of this glycosaminoglycan. Until now, azido sugars have been utilised as labelling reagents for various types of glycosylation, including N-glycans, O-linked mucin-type glycosylation and O-GlcNAcetylation of proteins. Incorporation of the unnatural azido sugar into the glycan of interest inserts a chemically reactive abiotic azide for subsequent detection via Staudinger ligation or click chemistries. However, to our knowledge, application of these azido sugars has not been explored for glycosaminoglycans. A metabolic labelling approach using Ac4GalNAz yields UDP-GalNAz and UDP-GlcNAz (Boyce et al., 2011), ready to target CS/DS and HS, respectively. We hypothesised that HS synthesis might be altered in the presence of UDP-GlcNAz due to the location of the azide on the acetyl group and the potential for interference with endogenous N-deacetylase-N-sulphotransferase biosynthetic enzyme activity. In mammalian cell culture (Chinese hamster ovary cells), treatment with Ac4GalNAz led to a decrease in total HS abundance accompanied by significant increases in 6-O-sulphation within the chains. Incorporation of a radiolabelled metabolic precursor revealed that average HS chain length was decreased in azido sugar-treated CHO cells. The modifications to HS were dose-dependent and HS inhibition was transient. Following removal of Ac4GalNAz from cell culture, HS expression returned to baseline levels within 24 hours. Previous work from the Bertozzi group has demonstrated the utility of Ac4GalNAz for visualising GalNAc- and O-GlcNAc-modified proteins in vivo. Using Xenopus, we were able to show that treatment of fertilised eggs with Ac4GalNAz decreased the abundance of HS in a similar way to that seen in vitro, with an associated impact on embryonic development. Embryonic axial elongation was impaired, with defective myotomal development and aberrant axonal patterning along the trunk and tail. Posterior somite cell nuclei were disorganised, with loss of distinct chevron patterning and skeletal muscle development was impaired with muscle fibres spanning some of the somite boundaries. Removal of the inhibitor partially rescued tail extension defects, as well as muscle development, but not axonal patterning. Therefore, these experiments illustrate a novel application for Ac4GalNAz as a soluble and reversible inhibitor of HS synthesis for in vitro and in vivo studies. The observed potential for control of inhibition via time- and dose-dependent effects enables targeted and selective inhibition of HS and potentially provides a powerful new inhibitor for the study of HS-mediated events.