KSGal6ST Is Essential for the 6-Sulfation of Galactose within Keratan Sulfate in Early Postnatal Brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Keratan sulfate (KS) proteoglycan side chains are abundant in the human cartilage matrix, but these chains have been said to be absent in murine skeletal tissues. We previously showed that KS suppresses cartilage damage and ameliorates inflammation in mice arthritis model. Because mice deficient of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) (KS biosynthesis enzyme) are now available, we decided to do further examinations.We examined, in culture, the difference between GlcNAc6ST-1^−/− and wild-type (WT) mice for interleukin (IL)-1α-induced glycosaminoglycan (GAG) release from the articular cartilage. Arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail and subsequent intraperitoneal injection of lipopolysaccharide. We examined the differences in arthritis severities in the two genotypes. After intraperitoneal KS administration in phosphate-buffered saline (PBS) or PBS alone, we evaluated the potential of KS in ameliorating arthritis and protecting against cartilage damage in deficient mice.GAG release induced by IL-1α in the explants, and severity of arthritis were greater in GlcNAc6ST-1^−/− mice than their WT littermates. Intraperitoneal KS administration effectively suppressed arthritis induction in GlcNAc6ST-1^−/− mice. Thus, GlcNAc6ST-1^−/− mice cartilage is more fragile than WT mice cartilage, and exogenous KS can suppress arthritis induction in GlcNAc6ST-1^−/− mice. Vestigial KS chain or altered glycosylation in articular cartilage in GlcNAc6ST-1^−/− mice may be protective against arthritis and associated cartilage damage as well as cartilage damage in culture. KS may offer therapeutic opportunities for chondroprotection and suppression of joint damage in inflammatory arthritis and may become a therapeutic agent for treating rheumatoid arthritis.     

6-Sulphated chondroitins have a positive influence on axonal regeneration

Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.     

Sulfation of Colonic Mucins by N-Acetylglucosamine 6-O-Sulfotransferase-2 and Its Protective Function in Experimental Colitis in Mice

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST-2) catalyzes the sulfation of mucin-like glycoproteins, which function as ligands for a lymphocyte homing receptor, L-selectin, in the lymph node high endothelial venules (HEVs). We previously showed that GlcNAc6ST-2 is expressed not only in lymph node HEVs but also in the colonic epithelial cells in mice. Here we investigated the regulatory mechanism and physiological significance of colonic expression of GlcNAc6ST-2 in mice. Treatment of a mouse colonic epithelial cell line with butyrate, a short-chain fatty acid produced by anaerobic bacteria, induced GlcNAc6ST-2 expression in the presence of epidermal growth factor. Administration of butyrate in the drinking water stimulated GlcNAc6ST-2 expression in the mouse intestine, indicating that butyrate could serve as a regulatory molecule for the GlcNAc6ST-2 expression in vivo. Immunohistochemical analysis indicated that the sulfation of colonic mucins was greatly diminished in GlcNAc6ST-2-deficient mice. Liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type and GlcNAc6ST-2-deficient mice showed that GlcNAc-6-O-sulfation was the predominant sulfate modification of these mucins, and it was exclusively mediated by GlcNAc6ST-2. After colitis induction by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of wild-type mice, indicating that the sulfation of colonic mucins by GlcNAc6ST-2 has a protective function in experimental colitis. These findings indicate that GlcNAc6ST-2, whose expression is regulated by butyrate, is a major sulfotransferase in the biosynthesis of sulfomucins in the mouse colon, where they serve as a mucosal barrier against colonic inflammation.     

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.     

Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.     

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.     

Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.     

N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing

Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable.     

Ablation of Keratan Sulfate Accelerates Early Phase Pathogenesis of ALS

Biopolymers consist of three major classes, i.e., polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (sugar chains). It is widely accepted that polynucleotides and polypeptides play fundamental roles in the pathogenesis of neurodegenerative diseases. But, sugar chains have been poorly studied in this process, and their biological/clinical significance remains largely unexplored. Amyotrophic lateral sclerosis (ALS) is a motoneuron-degenerative disease, the pathogenesis of which requires both cell autonomous and non-cell autonomous processes. Here, we investigated the role of keratan sulfate (KS), a sulfated long sugar chain of proteoglycan, in ALS pathogenesis. We employed ALS model SOD1^G93A mice and GlcNAc6ST-1^−/− mice, which are KS-deficient in the central nervous system. Unexpectedly, SOD1^G93AGlcNAc6ST-1^−/− mice exhibited a significantly shorter lifespan than SOD1^G93A mice and an accelerated appearance of clinical symptoms (body weight loss and decreased rotarod performance). KS expression was induced exclusively in a subpopulation of microglia in SOD1^G93A mice, and became detectable around motoneurons in the ventral horn during the early disease phase before body weight loss. During this phase, the expression of M2 microglia markers was transiently enhanced in SOD1^G93A mice, while this enhancement was attenuated in SOD1^G93AGlcNAc6ST-1^−/− mice. Consistent with this, M2 microglia were markedly less during the early disease phase in SOD1^G93AGlcNAc6ST-1^−/− mice. Moreover, KS expression in microglia was also detected in some human ALS cases. This study suggests that KS plays an indispensable, suppressive role in the early phase pathogenesis of ALS and may represent a new target for therapeutic intervention.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K+ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K⁺ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).     

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4^?/?) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4^?/? mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4^?/? mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.     

Promoting Optic Nerve Regeneration in Adult Mice with Pharmaceutical Approach

Our previous research has suggested that lack of Bcl-2-supported axonal growth mechanisms and the presence of glial scarring following injury are major impediments of optic nerve regeneration in postnatal mice. Mice overexpressing Bcl-2 and simultaneously carrying impairment in glial scar formation supported robust optic nerve regeneration in the postnatal stage. To develop a therapeutic strategy for optic nerve damage, the combined effects of chemicals that induce Bcl-2 expression and selectively eliminate mature astrocytes—scar forming cells—were examined in mice. Mood-stabilizer, lithium, has been shown to induce Bcl-2 expression and stimulate axonal outgrowth in retinal ganglion cells in culture and in vivo. Moreover, astrotoxin (alpha-aminoadipate), a glutamate analogue, selectively kills astrocytes while has minimal effects on surrounding neurons. In the present study, we sought to determine whether concurrent applications of lithium and astrotoxin were sufficient to induce optic nerve regeneration in mice. Induction of Bcl-2 expression was detected in the ganglion cell layer (GCL) of mice that received a lithium diet in compared with control-treated group. Moreover, efficient elimination of astrocytes and glial scarring was observed in the optic nerve of mice treated with astrotoxin. Simultaneous application of lithium and astrotoxin, but not any of the drugs alone, induced robust optic nerve regeneration in adult mice. These findings further support that a combinatorial approach of concurrent activation of Bcl-2-supported growth mechanism and suppression of glial scarring is required for successful regeneration of the severed optic nerve in adult mice. They suggest a potential therapeutic strategy for treating optic nerve and CNS damage. Special issue article in honor of Dr. Ji-Sheng Han.     

Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.     

Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate

Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-5/GST4beta) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4alpha) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/GST4) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce keratan sulfate (Akama, T. O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 16271-16278). In this study, we analyzed the substrate specificities of these sulfotransferases in vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of keratan sulfate. To clarify further the involvement of hCGn6ST in biosynthesis of keratan sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, beta1,4-galactosyltransferase-I, beta1,3-N-acetylglucosaminyltransferase-2, hCGn6ST, and keratan sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of keratan sulfate. Based on these results, we propose a biosynthetic pathway for N-linked keratan sulfate on corneal proteoglycans.     

Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re‐growth across the gradient. The use of selective enzyme inhibitors revealed that MMP‐2 enables immature astrocytes to cross the proteoglycan rim. The bridge‐building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC‐induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced. © 2010 Wiley Periodicals, Inc.Develop Neurobiol 70: 826–841, 2010     

Attenuation of scratch-induced reactive astrogliosis by novel EphA4 kinase inhibitors

The EphA4 receptor and its ephrin ligands are involved in astrocytic gliosis following CNS injury. Therefore, a strategy aimed at the blockade of EphA4 signaling could have broad therapeutic interest in brain disorders. We have identified novel small molecule inhibitors of EphA4 kinase in specific enzymatic and cell-based assays. In addition, we have demonstrated in two in vitro models of scratch injury that EphA4 receptor kinase is activated through phosphorylation and is involved in the repopulation of the wound after the scratch. A potent EphA4 kinase inhibitor significantly inhibited wound closure and reduced the accumulation of the reactive astrocytes inside the scratch. We have also shown that after the transient focal cerebral ischemia in rats, a large glial scar is formed by the accumulation of astrocytes and chondroitin sulfate proteoglycan surrounding the infarcted tissue at 7 days and 14 days of reperfusion. EphA4 protein expression is highly up-regulated in the same areas at these time points, supporting its potential role in the glial scar formation and maintenance. Taken together, these results suggest that EphA4 kinase inhibitors might interfere with the astrogliosis reaction and thereby lead to improved neurological outcome after ischemic injury.