Traffic lights for axon growth: proteoglycans and their neuronal receptors

Axon growth is a central event in the development and post-injury plasticity of the nervous system. Growing axons encounter a wide variety of environmental instructions. Much like traffic lights in controlling the migrating axons, chondroitin sulfate proteoglycans （CSPGs） and heparan sulfate proteoglycans （HSPGs） often lead to ＂stop＂ and ＂go＂ growth responses in the axons, respectively. Recently, the LAR family and NgR family molecules were identified as neuronal receptors for CSPGs and HSPGs. These discoveries provided molecular tools for further study of mechanisms underlying axon growth regulation. More importantly, the identification of these proteoglycan receptors offered potential therapeutic targets for promoting post-injury axon regeneration.     

Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans

The current dogma in neural regeneration research implies that chondroitin sulfate proteoglycans(CSPGs) inhibit plasticity and regeneration in the adult central nervous system(CNS). We argue that the role of the CSPGs can be reversed from inhibition to activation by developmentally expressed CSPG-binding factors. Heparin-binding growth-associated molecule(HB-GAM; also designated as pleiotrophin) has been studied as a candidate molecule that might modulate the role of CSPG matrices in plasticity and regeneration. Studies in vitro show that in the presence of soluble HB-GAM chondroitin sulfate(CS) chains of CSPGs display an enhancing effect on neurite outgrowth. Based on the in vitro studies, we suggest a model according to which the HB-GAM/CS complex binds to the neuron surface receptor glypican-2, which induces neurite growth. Furthermore, HB-GAM masks the CS binding sites of the neurite outgrowth inhibiting receptor protein tyrosine phosphatase sigma(PTPσ), which may contribute to the HB-GAM-induced regenerative effect. In vivo studies using two-photon imaging after local HB-GAM injection into prick-injury of the cerebral cortex reveal regeneration of dendrites that has not been previously demonstrated after injuries of the mammalian nervous system. In the spinal cord, two-photon imaging displays HB-GAM-induced axonal regeneration. Studies on the HB-GAM/CS mechanism in vitro and in vivo are expected to pave the way for drug development for injuries of brain and spinal cord.     

Proteoglycans: Road Signs for Neurite Outgrowth

Proteoglycans in the central nervous system play integral roles as ＂traffic signals＂ for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-neurite interaction is necessary for successfully promoting regeneration in the iniured central nervous system.     

Extracellular matrices,artificial neural scaffolds and the promise of neural regeneration

Over last 20 years, extracellular matrices have been shown to be useful in promoting tissue regeneration. Recently, they have been used and have had success in achieving neurogenesis. Recent developments in extracellular matrix design have allowed their successful in vivo incorporation to engender an environment favorable for neural regeneration in animal models. Promising treatments under investigation include manipulation of the intrinsic extracellular matrix and incorporation of engineered naometer-sized scaffolds through which inhibition of molecules serving as barriers to neuroregeneration and delivery of neurotrophic factors and/or cells for successful tissue regeneration can be achieved. Further understanding of the changes incurred within the extracellular matrix following central nervous system injury will undoubtedly help design a clinically efficacious extracellular matrix scaffold that can mitigate or reverse neural degeneration in the clinical setting.     

Analyzing the role of extracellular matrix during nervous system development to advance new regenerative strategies

<正>Regeneration in the central nervous system(CNS)is limited,and CNS damage often leads to cognitive impairment or permanent functional motor and sensory loss.Impaired regenerative capacity is multifactorial and includes inflammation,loss of the bloodbrain barrier,and alteration in the extracellular matrix(ECM).One of the main problems is the formation of a glial scar and the production of inhibitory ECM,such as proteoglycans,that     

Co-culture of oligodendrocytes and neurons can be used to assess drugs for axon regeneration in the central nervous system

We present a novel in vitro model in which to investigate the efficacy of experimental drugs for the promotion of axon regeneration in the central nervous system. We co-cultured rat hippocampal neurons and cerebral cortical oligodendrocytes, and tested the co-culture system using a Nogo-66 receptor antagonist peptide(NEP1–40), which promotes axonal growth. Primary cultured oligodendrocytes suppressed axonal growth in the rat hippocampus, but NEP1–40 stimulated axonal growth in the co-culture system. Our results confirm the validity of the neuron-oligodendrocyte co-culture system as an assay for the evaluation of drugs for axon regeneration in the central nervous system.     

Promoting axon regeneration in the central nervous system by increasing PI3-kinase signaling

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP₃ from PIP₂(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP₃ as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.     

The link between olfactory ensheathing cell survival and spinal cord injury repair: a commentary on common limitations of contemporary research

Olfactory ensheathing cells(OECs)are crucial players in the continuous regeneration of the olfactory nervous system that occurs through out life and are thought to have unique growth-promoting properties.For this reason,OEC transplantation has been thoroughly explored for the potential to promote neural repair after both central and peripheral nervous system injuries.Numerous studies have shown that OEC transplantation is safe and can promote recovery after spinal cord injury(SCI),both in animal models and in human clinical trials.To date,a variety of injury types and time-points after injury,as well as different delivery methods,have been tested.Outcomes have been encouraging(in rodent models including,for example,restoration of locomotion,breathing and climbing ability along with induction of axonal sprouting and some axonal regeneration)but highly variable(Barnett and Riddell,2007;Gomez et al.,2018).     

Decorin treatment of spinal cord injury

The scarring response after a penetrant central nervous system injury results from the interaction between invading leptominingeal/pericyte-derived fibroblasts and endogenous reactive astrocytes about the wound margin. Extracellular matrix and scar-derived axon growth inhibitory mole- cules fill the lesion site providing both a physical and chemical barrier to regenerating axons. Dec orin, a small leucine-rich chondroitin-dermatan sulphate proteoglycan expressed by neurons and astrocytes in the central nervous system, is both anti-fibrotic and anti-inflammatory and attenu- ates the formation and partial dissolution of established and chronic scars. Here, we discuss the potential of using Decorin to antagonise scarring in the central nervous system.     

Restoring axonal localization and transport of transmembrane receptors to promote repair within the injured CNS:a critical step in CNS regeneration

Each neuronal subtype is distinct in how it develops, responds to environmental cues, and whether it is capable of mounting a regenerative response following injury. Although the adult central nervous system (CNS) does not regenerate, several experimental interventions have been trialled with successful albeit lim-ited instances of axonal repair. We highlight here some of these approaches including extracellular matrix (ECM) modiifcation, cellular gratfing, gene therapy-induced replacement of proteins, as well as application of biomaterials. We also review the recent report demonstrating the failure of axonal localization and trans-port of growth-promoting receptors within certain classes of mature neurons. More speciifcally, we discuss an inability of integrin receptors to localize within the axonal compartment of mature motor neurons such as in the corticospinal and rubrospinal tracts, whereas in immature neurons of those pathways and in mature sensory tracts such as in the optic nerve and dorsal column pathways these receptors readily local-ize within axons. Furthermore we assert that this failure of axonal localization contributes to the intrinsic inability of axonal regeneration. We conclude by highlighting the necessity for both combined therapies as well as a targeted approach speciifc to both age and neuronal subtype will be required to induce substantial CNS repair.     

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.).     

Matrix metalloproteinases and proteoglycans in axonal regeneration

After an injury to the adult mammalian central nervous system (CNS), a variety of growth-inhibitory molecules are upregulated. A glial scar forms at the site of injury and is composed of numerous molecular substances, including chondroitin sulfate proteoglycans (CSPGs). These proteoglycans inhibit axonal growth in vitro and in vivo. Matrix metalloproteinases (MMPs) can degrade the core protein of some CSPGs as well as other growth-inhibitory molecules such as Nogo and tenascin-C. MMPs have been shown to facilitate axonal regeneration in the adult mammalian peripheral nervous system (PNS). This review will focus on the various roles of proteoglycans and MMPs within the injured nervous system. First, we will present a general background on the injured central nervous system and explore the roles that proteoglycans play in the injured PNS and CNS. Second, we will discuss the various functions of MMPs within the injured PNS and CNS. Special attention will be paid to the possibility of how MMPs might modify the growth-inhibitory extracellular environment of the injured adult mammalian spinal cord and facilitate axonal regeneration in the CNS.     

Differential outgrowth of retinal neurites on purified extracellular matrix molecules

Organotypic cultures of the embryonic retina were used to study the influence of extracellular matrix molecules on neurite elongation during development of the central nervous system. Microexplants from the chick retina (embryonic day 6) were grown in medium containing appropriate trophic support on purified matrix molecules adsorbed to plastic at various concentrations. The maximum neurite length obtained on each type of substratum was measured on day 4 of culture. No fiber outgrowth occurred on substrata of vitronectin or a hyaluronate-binding chondroitin sulfate proteoglycan. In contrast, neurite elongation was strongly promoted on laminin in a dose-dependent manner. Fibronectin elicited a neurite outgrowth corresponding to about one-third the length of the outgrowth on laminin. A 31,000-dalton fibronectin fragment representing the heparin-binding domain elicited neurite elongation comparable to that promoted by the intact fibronectin molecule. Other isolated domains of fibronectin, including the 105,000-dalton "cell-binding" domain, did not allow neurite outgrowth. Furthermore, preincubation of fibronectin substratum with antibodies to the heparin-binding fibronectin fragment entirely prevented outgrowth. Fiber outgrowth was also evoked on substrata of platelet factor 4, a protein binding heparan sulfate. Adding increasing concentrations of heparin progressively inhibited the neurite extension on laminin, whereas similar addition of soluble chondroitin sulfate proteoglycan had no effect. The results indicate that growing retinal neurites show strong preference for laminin versus fibronectin. Moreover, the outgrowth-promoting activity of both cell adhesion proteins seems to be localized to their heparin-binding regions. It is suggested that during development of the visual system, elongating retinal neurites can actively discriminate between different extracellular molecules by a mechanism that may involve participation of cell surface heparan sulfate proteoglycans.     

Chondroitin Sulfate Proteoglycans Are Associated with the Lesions of Alzheimer's Disease

Chondroitin sulfate proteoglycans (CSPG) are extracellular matrix proteins inhibitory to neurite outgrowth in vitro and correlated with decreased neurite outgrowth after CNS injury. Previously, heparan sulfate proteoglycan and dermatan sulfate proteoglycan have been shown to be associated with senile plaques (SPs) and neurofibrillary tangles (NFTs) but CSPG was not. In an immunocytochemical study, three monoclonal antibodies to different sulfation states of the chondroitin glycosaminoglycan were used to localize CSPG in cases of Alzheimer's disease. Chondroitin 4-sulfate was found in both SPs and NFTs. An antibody to unsulfated chondroitin strongly immunostained intracellular NFTs and the dystrophic neurites of SPs. Chondroitin 6-sulfate was found in NFTs and the area around SPs. These results suggest that CSPG, in addition or as an alternative to β-amyloid protein, could be responsible for the regression of neurites around senile plaques in Alzheimer's disease.     

REGULATED EXPRESSION OF CHONDROITIN SULFATES AT SITES OF EPITHELIAL-MESENCHYMAL INTERACTION: SPATIO-TEMPORAL PATTERNING IDENTIFIED WITH ANTI-CHONDROITIN SULFATE MONOCLONAL ANTIBODIES

Chondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non-neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up-regulated or down-regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti-chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non-randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non-reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS-56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial-mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial—mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system. Copyright © 1996. Published by Elsevier Science Ltd.     

A novel diketopiperazine stimulates sprouting of spinally projecting axons

Following spinal cord injury, spared axonal projections undergo spontaneous compensatory sprouting in an attempt to reinnervate synaptic targets that were deinnervated as a result of injury. However, compensatory sprouting is hindered by the expression of a myriad of inhibitory molecules throughout the adult central nervous system, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated inhibitory proteins (MAIPs). In this study, we have identified a diketopiperazine designated DKP101516 that can overcome the inhibitory effects of MAIPs and CSPGs on neurite outgrowth and branching. In vivo analysis demonstrates that DKP101516 enhances the plasticity of various axonal populations following septuple dorsal rhizotomy by overcoming the inhibitory effects of CSPGs and MAIPs. Our results suggest that DKP101516 may encourage spinal cord repair by stimulating compensatory sprouting of intact axonal projections.     

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.     

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.