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Concept: Heparan sulfate

178

The E4 isoform of apolipoprotein (apoE4) is known to be a major risk factor for Alzheimer’s Disease (AD). Previous in vitro studies have shown apoE4 to have a negative effect on neuronal outgrowth when incubated with lipids. The effect of apoE4 itself on the development of neurons from the central nervous system (CNS), however, has not been well characterized. Consequently, apoE4 alone has not been pursued as a substrate for neuronal cultures. In this study, the effect of surface-bound apoE4 on developmental features of rat hippocampal neurons was examined. We show that apoE4 substrates elicit significantly enhanced values in all developmental features at day 2 of culture when compared to laminin (LN) substrates, which is the current substrate-of-choice for neuronal cultures. Interestingly, the adhesion of hippocampal neurons was found to be significantly lower on LN substrates than on glass substrates, but the axon lengths on both substrates were similar. In addition, this study demonstrates that the adhesion- and growth-enhancing effects of apoE4 substrates are not mediated by heparan sulfate proteoglycans (HSPGs), proteins that have been indicated to function as receptors or co-receptors for apoE4. In the absence of lipids, apoE4 appears to use an unknown pathway for up-regulating neuronal adhesion and neurite outgrowth. Our results indicate that apoE4 is better than LN as a substrate for primary cultures of CNS neurons and should be considered in the design of tissue engineered CNS.

Concepts: Alzheimer's disease, Central nervous system, Nervous system, Neuron, Brain, Axon, Synapse, Heparan sulfate

28

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Concepts: Immune system, Chondroitin sulfate, Extracellular matrix, Cell membrane, Rheumatoid arthritis, Proteoglycan, Cartilage, Heparan sulfate

28

Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels.

Concepts: Chondroitin sulfate, Heparan sulfate, Heparin, Glycosaminoglycan, Dermatan sulfate, Glycosaminoglycans, Mucopolysaccharidosis, Iduronic acid

28

Heparan sulfate proteoglycans (HSPGs) play pivotal roles in the regulation of Wnt signaling activity in several tissues. At the Drosophila melanogaster neuromuscular junction (NMJ), Wnt/Wingless (Wg) regulates the formation of both pre- and postsynaptic structures; however, the mechanism balancing such bidirectional signaling remains elusive. In this paper, we demonstrate that mutations in the gene of a secreted HSPG, perlecan/trol, resulted in diverse postsynaptic defects and overproduction of synaptic boutons at NMJ. The postsynaptic defects, such as reduction in subsynaptic reticulum (SSR), were rescued by the postsynaptic activation of the Frizzled nuclear import Wg pathway. In contrast, overproduction of synaptic boutons was suppressed by the presynaptic down-regulation of the canonical Wg pathway. We also show that Trol was localized in the SSR and promoted postsynaptic accumulation of extracellular Wg proteins. These results suggest that Trol bidirectionally regulates both pre- and postsynaptic activities of Wg by precisely distributing Wg at the NMJ.

Concepts: Action potential, Heparan sulfate, Chemical synapse, Neuromuscular junction, Perlecan, Agrin

28

Mucopolysaccharidosis II (Hunter syndrome) is a rare x-linked disorder caused by a deficiency in the lysosomal enzyme iduronate-2-sulphatase, leading to an accumulation of the glycosaminoglycans (GAGs) dermatansulphate and heparan sulphate. The consequence of GAGs accumulation is progressive, multiorgan disease. Enzyme-replacement therapy is hypothesised to result in disease stabilisation and improved prognosis. We present a severe case of Hunter syndrome diagnosed at age 2 years and 4 months, who started enzyme-replacement therapy at the age of 3 years and 3 months. We report his evolution after 1 year of treatment. The treatment response was good and there was significant improvement in the quality of life. Owing to the rarity of Hunter syndrome, the multisystem nature and the heterogeneity of disease progression, patient care implies interdisciplinary consultations with a wide range of specialists. The best management can be provided in reference centres for metabolic diseases.

Concepts: Medicine, Chondroitin sulfate, Lysosomal storage disease, Heparan sulfate, Heparin, Glycosaminoglycan, Mucopolysaccharidosis, Hurler syndrome

27

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype, and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2 which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine if the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from the patients and healthy individuals, separated it into plasma and cellular fractions, and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method by a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution, and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µl). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of heparan sulfate in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of heparan sulfate to chondroitin sulfate (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amount and the HS/CS ratios, which could be used as a diagnostic biomarker for HME.

Concepts: Chondroitin sulfate, Proteoglycan, Chromatography, High performance liquid chromatography, Heparan sulfate, Heparin, Glycosaminoglycan, Glycosaminoglycans

27

Heparan sulfate proteoglycans (HSPGs) have essential functions during embryonic development and throughout postnatal life. To exert these functions, they undergo a series of processing reactions by heparan sulfate modifying enzymes (HSMEs), which endows them with highly modified HS domains that provide specific docking sites for a large number of bio-active molecules. The development and antigen-dependent differentiation of normal B lymphocytes, as well as the growth and progression of B-lineage malignancies, are orchestrated by an array of growth factors, cytokines, and chemokines many of which display HS binding. As discussed in this review, tightly regulated HSPG expression is a requirement for normal B cell maturation, differentiation and function. In addition, the HSPG syndecan-1 functions as a versatile co-receptor for signals from the bone marrow microenvironment, essential for the survival of long-lived plasma cells and multiple myeloma (MM) plasma cells. Targeting of HSMEs or HS chains on MM cells increases their sensitivity to drugs currently used in MM treatment, including bortezomib, lenalidomide or dexamethasone. Taken together, these findings render the HS-biosynthetic machinery a promising target for MM treatment. © 2013 The Authors Journal compilation © 2013 FEBS.

Concepts: Immune system, Protein, Multiple myeloma, Bone marrow, Thalidomide, Lenalidomide, B cell, Heparan sulfate

25

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

Concepts: Cholesterol, Atherosclerosis, Coronary artery disease, Familial hypercholesterolemia, Atheroma, Low density lipoprotein receptor gene family, Liver, Heparan sulfate

25

Bi-allelic variants in CHST14, encoding dermatan-4-O-sulfotransferase-1 (D4ST1), cause musculocontractural EDS (MC-EDS), a recessive disorder characterized by connective tissue fragility, craniofacial abnormalities, congenital contractures and developmental anomalies. Recently, the identification of bi-allelic variants in DSE, encoding dermatan sulfate epimerase-1 (DS-epi1), in a child with MC-EDS features, suggested locus heterogeneity for this condition. DS-epi1 and D4ST1 are crucial for biosynthesis of dermatan sulfate (DS) moieties in the hybrid chondroitin sulfate (CS)/DS glycosaminoglycans (GAGs). Here we report four novel families with severe MC-EDS caused by unique homozygous CHST14 variants and the second family with a homozygous DSE missense variant, presenting a somewhat milder MC-EDS phenotype. The glycanation of the dermal DS proteoglycan decorin is impaired in fibroblasts from D4ST1- as well as DS-epi1-deficient patients. However, in D4ST1-deficieny the decorin GAG is completely replaced by CS, whereas in DS-epi1-deficiency still some DS moieties are present. The multisystemic abnormalities observed in our patients support a tight spatiotemporal control of the balance between CS and DS, which is crucial for multiple processes including cell differentiation, organ development, cell migration, coagulation and connective tissue integrity. This article is protected by copyright. All rights reserved.

Concepts: Chondroitin sulfate, Collagen, Proteoglycan, Cartilage, Heparan sulfate, Glycosaminoglycan, Dermatan sulfate, Ehlers-Danlos syndrome

25

Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency ~3-fold compared with wild-type (WT) AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to ~5-fold, by the mutagenesis of surface exposed tyrosine and threonine residues involved in intracellular trafficking of AAV vectors. Thus, AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD-peptide and tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency to ~8-fold in vitro. Additionally, we mutated lysine (K531E) to impair affinity of AAV6 vectors to heparan sulfate proteoglycan (HSPG). Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V-K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells.Gene Therapy accepted article preview online, 13 August 2015. doi:10.1038/gt.2015.89.

Concepts: Gene, Cancer, Mutation, Oncology, Microbiology, Prostate cancer, Heparan sulfate, Virology