Brain sweet brain: importance of sugars for the cerebral microenvironment and tumor development

The extracellular matrix (ECM) in the brain tissue is a complex network of glycoproteins and proteoglycans that fills the intercellular space serving as scaffolding to provide structural framework for the tissue and regulate the behavior of cells via specific receptors - integrins. There is enormous structural diversity among proteoglycans due to variation in the core protein, the number of glycosaminoglycans chains, the extent and position of sulfation. The lectican family of proteoglycans interacts with growth factors, hyaluronan and tenascin forming a complex structure that regulates neuronal plasticity and ion homeostasis around highly active neurons. In this review, we will discuss the latest insights into the roles of brain glycoproteins as modulators of cell adhesion, migration, neurite outgrowth and glial tumor invasion.     

Characterization of glycosaminoglycans produced by primary astrocytes in vitro.

Quantitative biosynthetic studies with cultures highly enriched for glial fibrillary acidic protein (GFAP+) cells of neonatal mammalian brain demonstrated production of four proteoglycans: hyaluronate (HA), heparan sulphate (HS), chondroitin sulphate (CS), and dermatan sulphate (DS). The glycosaminoglycans were present in cell conditioned medium and in the cellular compartment. There were qualitative differences in the subcellular disposition of the various proteoglycans. The ratio of HS to CS/DS in cell extracts was 1:1, while in medium this ratio was 1:6. All of the glycosaminoglycans were associated with core proteins that were integral to the cell membrane and associated with the cell surface by non-covalent interactions involving glycosaminoglycans. Less than 20% of the HS was non-covalently associated with the astrocyte cell surface reflecting in part the proportionately smaller amounts of this proteoglycan released to astrocyte conditioned medium. HS released to medium was undersulphated relative to that associated with cells. The astrocyte can contribute proteoglycans to the extracellular milieu and displays cell surface proteoglycans that have the potential to provide appropriate substrates for neuron adhesion, process extension, and other cell-cell interactions.     

A 'GAG' reflex prevents repair of the damaged CNS

The extracellular matrix of the central nervous system (CNS) serves as both a supporting structure for cells and a rich source of signaling molecules that can influence cell proliferation, survival, migration and differentiation. A large proportion of this matrix is composed of proteoglycans--proteins with long chains of polysaccharides, called glycosaminoglycans (GAGs), covalently attached. Although many of the activities of proteoglycans depend on their core proteins, GAGs themselves can influence cell signaling. Here we review accumulating evidence that two GAGs, chondroitin sulfate and hyaluronan, play essential roles during nervous system development but also accumulate in chronic CNS lesions and inhibit axonal regeneration and remyelination, making them significant hindrances to CNS repair. We propose that the balance between the synthesis and degradation of these molecules dictates, in part, how regeneration and recovery from CNS damage occurs.     

Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders

Proteoglycans are associated with all kinds of amyloid deposits in the human body. These complex macromolecules, in particular heparan sulphate proteoglycans, have also been implicated in several features of the pathogenesis of Alzheimer's disease (AD), including the genesis of senile plaques, cerebrovascular amyloid, and neurofibrillary tangles. In this review we focus on the role of proteoglycans and glycosaminoglycans in amyloidogenesis in general and in AD in particular. Heparan sulphate proteoglycans may promote amyloid-beta peptide (Abeta) or tau fibrillisation on the one hand, and provide resistance against proteolytic breakdown on the other. Knowledge about the role of proteoglycans in AD pathology may eventually be of therapeutic use, because small polysulphated compounds, which can interfere with the interaction between proteoglycan and Abeta, have been shown to stop or even prevent amyloidogenesis.     

Interleukin-1 and nerve growth factor induce hypersecretion and hypersulfation of neuroblastoma proteoglycans which bind β-amyloid

Inflammation and the response to injury may play an important role in the process of amyloidosis in Alzheimer's disease. We investigated the effect of interleukin-1 (IL-1) and nerve growth factor (NGF) on the metabolism of neuroblastoma proteoglycans. IL-1 and NGF increased the net charge and the net secretion of neuroblastoma proteoglycans. NGF also specifically increased the relative amount of cell-associated and secreted heparan sulfate proteoglycans in these cells. We previously demonstrated that neuroblastoma heparan sulfate proteoglycan binds specifically to the amyloid β-amyloid peptide involved in Alzheimer's disease. Heparan sulfate glycosaminoglycans synthesized by IL-1-stimulated cells demonstrated an increased relative binding affinity for the β-amyloid peptide. Thus, IL-1 and NGF induce the hypersecretion and hypersulfation of neuroblastoma heparan sulfate proteoglycans which bind β-amyloid. These studies link the process of inflammation and repair with alterations in the metabolism of heparan sulfate proteoglycans and amyloid formation in Alzheimer's disease and other disorders.     

Glycosaminoglycan antigens in peripheral nerve: Studies with antibodies from a patient with neuropathy and monoclonal gammopathy

An immunoblot staining procedure was developed for the detection of antibody binding to glycosaminoglycans (GAGs). The method was used to study the binding of a human monoclonal antibody (M-protein) from a patient with peripheral neuropathy, previously found to react with proteoglycans (PGs). GAGs prepared from human peripheral nerve were separated by electrophoresis on cellulose acetate membranes, transferred onto nitrocellulose sheets, and immunostained with the M-protein. The M-protein bound to a single GAG band with intermediate mobility which eluted with 1.25 N NaCl on ion-exchange chromatography and was chondroitinase sensitive. The M-protein appeared to bind to chondroitin sulfate containing proteoglycans in peripheral nerve.     

Expression of chondroitin sulfate and keratan sulfate proteoglycans in the path of growing retinal axons in the developing chick

Previous investigations have identified proteoglycans in the central nervous system during development and have implicated some proteoglycans as axon guidance molecules that act by inhibiting axon extension. The present study investigated the pattern of immunoreactivity for several glycosaminoglycans common to certain proteoglycans relative to growing retinal axons in the developing chick visual system and in retinal explant cultures. Immunostaining for chondroitin-6-sulfate, chondroitin-4-sulfate, and keratan sulfate was observed to colocalize with retinal axons throughout the retinofugal pathway during the entire period of retinal axon growth. The proteoglycan form of collagen IX, however, was only observed in the retina, primarily peripheral to the areas with actively growing axons, The pattern of immunostaining for chondroitin sulfate in tissue sections suggested that the retinal axons might be a source for some of the chondroitin sulfate immunostaining in the developing visual pathway. This was confirmed in that chondroitin sulfate immunostaining was also observed on neurites emanating from cultured retinal explants. These findings indicate that retinal axons grow in the presence of chondroitin sulfate and keratan sulfate proteoglycans and that these proteoglycans in the developing chick visual pathway have functions other than to inhibit axon growth. © 1995 Wiley-Liss, Inc.     

Glycosaminoglycan levels and proteoglycan expression are altered in the hippocampus of patients with mesial temporal lobe epilepsy

Extracellular matrix proteoglycans (PGs) and glycosaminoglycans (GAGs) play a crucial role in cell differentiation and synaptogenesis by modulating neurite outgrowth. The chondroitin sulfate (CS)-rich PG, the receptor protein tyrosine phosphatase zeta/beta (RPTP zeta/beta), has been related to neural morphogenesis and axon guidance. Hippocampal sclerosis is the most frequent pathologic finding in patients with intractable mesial temporal lobe epilepsy (MTLE), which is associated with neuron loss, reactive gliosis, and mossy fiber sprouting. In the present study, we investigated the concentration of CS, heparan sulfate (HS) and hyaluronic acid (HA) in the hippocampus and temporal neocortex as well as RPTP zeta/beta expression in the hippocampus of patients with MTLE. Compared to autopsy control tissue, epileptic hippocampi showed a significantly increased concentration of CS (224%; p=0.0109) and HA (146%; p=0.039). HS was instead similar to control values. No differences were found in the concentration of CS, HS, or HA in the temporal neocortex of epileptic patients when compared to control values. In contrast, RPTP zeta/beta immunoreactivity was induced in astrocytes of the inner molecular layer of the dentate gyrus of the sclerotic hippocampus. Because matrix compounds have been associated with tissue injury and repair, the present findings suggest that changes in PGs and GAGs might be related to damage-induced gliosis and neuronal reorganization in the hippocampus of MTLE patients.     

Semaphorin 3A displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix

Secreted semaphorins are essential for neural development and continue to be expressed in subpopulations of adult neurons, where they subserve as yet unknown functions. We employed functional myc- and GFP-tagged Sema3A proteins to obtain insight in the localization of Sema3A in neuronal cells. Sema3A localized to both axons and dendrites of cortical neurons. GFP-Sema3A exhibited a characteristic punctate distribution on the surface of Neuro-2a cells, localized to migratory pathways of cultured cells, and co-localized with and induced clustering of its receptor component neuropilin-1. Treatment with excess glycosaminoglycans and chondroitinase ABC resulted in the removal of cell surface Sema3A. Heparin enhanced Sema3A's binding to neuropilin-1-expressing cells and potentiated its growth cone collapsing activity. Together, these results indicate that association with proteoglycans in the extracellular matrix of neuronal cells plays an important role in the localization of the chemorepulsive guidance cue Sema3A, and that this interaction may enhance its biological activity.     

Neuronal growth cones and regeneration: gridlock within th extracellular matrix

The extracellular matrix is a diverse composition of glycoproteins and proteoglycans found in all cellular systems.The extracellular matrix,abundant in the mammalian central nervous system,is temporally and spatially regulated and is a dynamic"living"entity that is reshaped and redesigned on a continuous basis in response to changing needs.Some modifications are adaptive and some are maladaptive.It is the maladaptive responses that pose a significant threat to successful axonal regeneration and/or sprouting following traumatic and spinal cord injuries,and has been the focus of a myriad of research laboratories for many years.This review focuses largely on the extracellular matrix component,chondroitin sulfate proteoglycans,with certain comparisons to heparan sulfate proteoglycans,which tend to serve opposite functions in the central nervous system.Although about equally as well characterized as some of the other proteoglycans such as hyaluronan and dermatan sulfate proteoglycan,chondroitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the field of axonal injury and regeneration.Four laboratories discuss various aspects of chondroitin sulfate proteoglycans and proteoglycans in general with respect to their structure and function(Beller and Snow),the recent discovery of specific chondroitin sulfate proteoglycan receptors and what this may mean for increased advancements in the field(Shen),extracellular matrix degradation by matrix metalloproteinases,which sculpt and resculpt to provide support for outgrowth,synapse formation,and synapse stability(Phillips et al.),and the perilesion microenvironment with respect to immune system function in response to proteoglycans and central nervous system injuries(Jakeman et al.).     

Motor nerve regulates muscle extracellular matrix proteoglycan expression

Denervation of rat leg muscles caused a 2-3-fold increase in 35S-sulfate and 3H-glucosamine incorporation into proteoglycans of the muscle extracellular matrix. The size of the proteoglycans and the glycosaminoglycan chain length and degree of sulfation were unchanged. Because the rate of degradation of proteoglycans was also unchanged by denervation, we infer that denervation increases proteoglycan synthesis. Muscle reinnervation restored the original rate of synthesis of proteoglycans. Paralysis of innervated muscle caused increased incorporation of sulfate comparable to that seen in denervation. Thus motor nerve activity appears to regulate the level of proteoglycans in the muscle extracellular matrix.     

Activated Macrophages and the Blood–Brain Barrier: Inflammation after CNS Injury Leads to Increases in Putative Inhibitory Molecules

The cellular responses to spinal cord or brain injury include the production of molecules that modulate wound healing. This study examined the upregulation of chondroitin sulfate proteoglycans, a family of molecules present in the wound healing matrix that may inhibit axon regeneration in the central nervous system (CNS) after trauma. We have demonstrated increases in these putative inhibitory molecules in brain and spinal cord injury models, and we observed a close correlation between the tissue distribution of their upregulation and the presence of inflammation and a compromised blood–brain barrier. We determined that the presence of degenerating and dying axons injured by direct trauma does not provide a sufficient signal to induce the increases in proteoglycans observed after injury. Activated macrophages, their products, or other serum components that cross a compromised blood–brain barrier may provide a stimulus for changes in extracellular matrix molecules after CNS injury. While gliosis is associated with increased levels of proteoglycans, not all reactive astrocytes are associated with augmented amounts of these extracellular matrix molecules, which suggests a heterogeneity among glial cells that exhibit a reactive phenotype. Chondroitin sulfate also demarcates developing cavities of secondary necrosis, implicating these types of boundary molecules in the protective response of the CNS to trauma.     

Functional interactions of neuronal heparan sulphate proteoglycans with laminin

Quantitative biosynthetic studies using cellular extracts and neuron conditioned medium demonstrated that heparan sulphate proteoglycans (HSPGs) comprised 20-25% of the sulphated proteoglycans produced by neurons while the remainder consisted of chondroitin sulphate proteoglycans (CSPGs). When chromatographic fractions containing guanidine extracted and partially purified proteoglycans from culture medium conditioned by neurons (NCM) were used to pretreat a laminin substrate, neurite formation by sensory neurons was enhanced. Enhanced neurite promoting activity was not apparent if, during the pretreatment of the laminin substrate with NCM, heparan sulphate glycosaminoglycans (HS) were present. To determine the molecular basis of cell surface HSPG interactions with immobilized laminin, adhesion and neurite growth by dissociated sensory neurons were quantified at 4 h in vitro--a time at which there was no apparent contribution of released proteoglycans to neurite growth. Whereas adhesion was not influenced, neurite growth was partially inhibited in a dose-dependent manner if the sensory neurons were coincubated with HS, and if the cells were pretreated, prior to seeding, with heparitinase. The inhibitory effect produced by coincubation with saturating concentrations of HS was no longer apparent if the cells had been pretreated with heparitinase. These findings distinguish quantitatively between neurite growth on laminin and on laminin-HSPG complexes, and suggest that some neuronal cell surface and released HSPGs are involved in neurite growth by virtue of non-covalent interactions with glycosaminoglycan binding domains of laminin.     

Ultrastructural visualization of primate cone photoreceptor matrix sheaths

Glycoconjugates, including glycolipids, glycoproteins, and proteoglycans, are present in the plasma membrane of photoreceptor cells and in the interphotoreceptor matrix surrounding photoreceptor cell ellipsoids and outer segments. Although the precise function of these molecules is unknown, they may be important in mediating photoreceptor-pigment epithelial cell interactions, outer segment membrane assembly, and/or disc shedding. Lectins, affinity ligands for defined carbohydrate sequences, have proven particularly useful in studying the glycoconjugate composition of the interphotoreceptor matrix. The peanut lectin selectively binds to domains of the interphotoreceptor matrix surrounding cone ("cone matrix sheaths"), but not rod inner and outer segments. This is evidence for the existence of chemical and structural heterogeneity within the interphotoreceptor matrix. The studies described herein utilized ultrastructural pre-embedding histochemical labeling to assess whether, in addition to the surrounding interphotoreceptor matrix, peanut lectin binding is associated directly with that plasma membrane of cone inner and outer segments. This study confirms that ferritin-conjugated peanut agglutinin binds to cone matrix sheaths, and, in addition, provides ultrastructural evidence for the presence of binding to the plasma membrane surrounding cone inner and outer segments. The data suggest that cone membrane-associated peanut agglutinin-binding molecules may differ from those located within cone matrix sheaths.     

Release of heparan sulfate proteoglycans from cultured aortic endothelial cells by thrombin

Anticoagulant heparan sulfate proteoglycans have been shown to be released from cultured endothelial cells. The effect of thrombin on their release was investigated. Thrombin t more than one unit/ml accelerated the release of [as]sulfate-labeled glycosaminoglycans from cultured porcine aortic endothelial cells. The effect of thrombin reached a maximum after one hour of incubation, and was dependent on the enzyme concentration. 10 unit/ml of thrombin released approximately twice as much amount of J'S-glycosaminoglycans as did Hanks' balanced salt solution alone. When the active site of thrombin was blocked by either diisopropylfluorophosphate or hirudin, the enzyme effect was completely abolished. Released glycosaminoglycans were resistant to chondroitin ABC lyase digestions, but degraded by either heparitinase or nitrous acid treatments. Released ³⁵S-materials were precipitated with trichloroacetic acid and shown to be degraded into smaller molecules after alkali treatment on Sepharose CL-6B gel filtration chromatography. On the other hand, thrombin treatment of Cr-labeled cells did not cause the release of radioactivity. These results indicate that thrombin potentiates the release of heparan sulfate proteoglycans from cultured aortic endothelial cells without causing an appreciable damage to the cells. The effect of thrombin is active site-dependent and requires a relatively high enzyme concentration.     

Neuronal growth cones and regeneration:gridlock within the extracellular matrix

The extracellular matrix is a diverse composition of glycoproteins and proteoglycans found in all cellular systems. The extracellular matrix, abundant in the mammalian central nervous system, is temporally and spatially regulated and is a dynamic ＂living＂ entity that is reshaped and redesigned on a continuous basis in response to changing needs. Some modifications are adaptive and some are maladaptive. It is the maladaptive responses that pose a significant threat to successful axonal regeneration and/or sprouting following traumatic and spinal cord injuries, and has been the focus of a myriad of research laboratories for many years. This review focuses largely on the extracellular matrix component, chondroitin sulfate proteoglycans, with certain comparisons to heparan sulfate proteoglycans, which tend to serve opposite functions in the central nervous system. Although about equally as well characterized as some of the other proteoglycans such as hyaluronan and dermatan sulfate proteoglycan, chondroitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the field of axonal injury and regeneration. Four laboratories discuss various aspects of chondroitin sulfate proteoglycans and proteoglycans in general with respect to their structure and function （Beller and Snow）, the recent discovery of specific chondroitin sulfate proteoglycan receptors and what this may mean the field （Shen）, extracellular for increased advancements in matrix degradation by matrix metalloproteinases, which sculpt and resculpt to provide support for outgrowth, synapse formation, and synapse stability （Phillips et al.）, and the perilesion microenvironment with respect to immune system function in response to proteoglycans and central nervous system injuries （Jakeman et al.）.     

Dermatan sulfate and de-sulfated heparin solubilized collagen-tailed acetylcholinesterase from the rat neuromuscular junction

We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (AChE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed AChE. Taking into account that the solubilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina.     

Glioma infiltration and extracellular matrix: key players and modulators

An outstanding characteristic of gliomas is their infiltration into brain parenchyma, a property that impairs complete surgical resection; consequently, these tumors might recur, resulting in a high mortality rate. Gliomas invade along preferential routes, such as those along white matter tracts and in the perineuronal and perivascular spaces. Brain extracellular components and their partners and modulators play a crucial role in glioma cell invasion. This review presents an extensive survey of the literature, showing how the brain extracellular matrix (ECM) is modulated during the glioma infiltration process. We explore aspects of ECM interaction with glioma cells, reviewing the main glycosaminoglycans, glycoproteins and proteoglycans. We discuss the roles of ECM‐binding proteins, including CD44, RHAMM, integrins and axonal guidance molecules, and highlight the role of proteases and glycosidases in glioma infiltration; in binding and release chemokines, cytokines and growth factors ; and in generating new bioactive ECM fragments. We also consider the roles of cytoskeletal signaling, angiogenesis, miRNAs and the glial‐to‐mesenchymal transition linked to glioma invasion. We closely discuss therapeutic approaches based on the modulation of the extracellular matrix, targeting the control of glioma infiltration, its relative failure in clinical trials, and potential means to overcome this difficulty.     

Glycosaminoglycans co-administration enhance insulin-like growth factor-I neuroprotective and neuroregenerative activity in traumatic and genetic models of motor neuron disease: a review.

In this report it is shown how glycosaminoglycans and insulin-like growth factor-I (IGF-I) promote muscle reinnervation and prevent motor neuron death in experimental models of motor neuron disease. Such effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation by means of subcutaneous injections of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve, glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy, whereas, glycosaminoglycan treatment of lesioned rats increased IGF-I mRNA and protein in the reinnervated muscle, and IGF-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of lesioned rats with IGF-I promoted muscle reinnervation, and prevented muscle fibre atrophy, higher levels of IGF-I in the reinnervated muscle, of IGF-I, and insulin-like growth factor binding proteins in plasma. In the wobbler mouse IGF-I and glycosaminoglycans alone promote only a partial motor neuron survival and the preservation of forelimb function decays after 3 weeks of treatment. However when glycosaminoglycans and insulin-like growth factor are administered together the motor neuron disease in the wobbler mouse is halted and there is no more loss of motor neurons.     

Extracellular matrix of peripheral nerves in diabetes

Peripheral nerves are susceptible to develop multiple changes in their morphology and biochemical composition as consequences of diabetes mellitus. This review focuses on diabetes-induced alterations of the extracellular matrix of the peripheral nerves, and on the potential molecular mechanisms causing these changes. The interest towards the extracellular matrix of peripheral nerves of diabetic patients is highlighted by the fact that the extracellular matrix does not only mechanically support the cells which it surrounds, but it also regulates their behavior through specific interactions mediated via molecules on the cell surface, such as integrin receptors and cell surface proteoglycans. Thus, changes in the structure and composition of the extracellular matrix may alter cellular functions in multiple ways. At the ultrastructural level, these changes include e.g. thickening of vascular, perineurial and Schwann cell associated basement membranes; accumulation of microfibrillar material in the vicinity of perineurial cells; and increased diameter of endoneurial collagen fibrils. At the molecular level, the changes may be associated with altered metabolism of various collagen types, such as type I, III, IV and VI collagens.