Inhibition of central axon regeneration:perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury

Background:Failure of axon regeneration after spinal cord injury(SCI)underlies the paralysis that so profoundly affects patients’quality of life.Many factors are involved in the regeneration failure.Chondroitin sulfate proteoglycans(CSPGs),normal constituents of the perineuronal nets in central nervous system(CNS),are secreted at the injury site and initially were thought to act as a purely physical barrier.In the past decade,the receptor-like protein tyrosine phosphatases,protein tyrosine phosphatase sigma(PTPσ),and leukocyte common antigen-related phosphatase(LAR),have been identified as transmembrane receptors for CSPGs.The two receptors for myelin-associated growth inhibitors,Nogo receptors 1 and 3(NgR1 and NgR3)also have been found to bind with CSPGs(Sharma et al.,2012).These findings suggest that CSPGs inhibit regeneration by interacting with these receptors,initiating downstream inhibitory signaling(Figure 1).     

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.）.     

Elucidating the molecular mechanisms of CSPG-mediated axon growth inhibition through phosphoproteomics analysis

<正>Chondroitin sulfate proteoglycans(CSPGs)are key components of the extracellular matrix in the central nervous system(CNS).High levels of CSPGs are expressed in the embryonic mammalian CNS,where they play important roles in axon guidance and pathfinding.In the adult CNS,CSPGs are found in the perineu-     

Targeting cell surface receptors for axon regeneration in the central nervous system

Axon regeneration in the CNS is largely unsuccessful due to excess inhibitory extrinsic factors within lesion sites together with an intrinsic inability of neurons to regrow following injury. Recent work demonstrates that forced expression of certain neuronal transmembrane receptors can recapitulate neuronal growth resulting in successful growth within and through inhibitory lesion environments. More specifically, neuronal expression of integrin receptors such as alpha9beta1 integrin which binds the extracellular matrix glycoprotein tenascin-C, trk receptors such as trk B which binds the neurotrophic factor BDNF, and receptor PTPσ which binds chondroitin sulphate proteoglycans, have all been show to significantly enhance regeneration of injured axons. We discuss how reintroduction of these receptors in damaged neurons facilitates signalling from the internal environment of the cell with the external environment of the lesion milieu, effectively resulting in growth and repair following injury. In summary, we suggest an appropriate balance of intrinsic and extrinsic factors are required to obtain substantial 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.     

MicroRNA-based therapeutics for optic neuropathy: opportunities and challenges

Optic nerve degeneration is a major cause of irreversible blindness worldwide with glaucoma being the most common optic neuropathy,affecting approximately 76 million people worldwide in 2020.The optic nerve comprises axons of retinal ganglion cells(RGCs),the output neurons of the inner retina.Protecting RGCs and axons from degeneration and regenerating RGC axons to preserve and recover vision in patients with progressive optic neuropathy is an unmet need.Unlike embryonic neurons,mature neurons of the mammalian central nervous system fail to regenerate their axons following injury.The age-related loss of axon regenerative capacity of RGCs over time renders vision loss from optic neuropathy irreversible.The failure of injured RGCs to regenerate axons is largely attributed to inhibitory molecules in the extrinsic environment and a change in the intrinsic molecular makeup of aging cells.Early studies have demonstrated that RGCs require specific molecular signals for the stimulation of axon growth even without inhibitory molecules in the extrinsic environment,leading successive efforts to focus on uncovering the intrinsic signaling pathways that control axon extension during RGC development.     

Beyond taxol： microtubule-based strategies for promoting nerve regeneration after injury

<正>After injury,damaged axons have the capacity to regenerate,but the regenerative capacity of the axon,particularly axons of the central nervous system,is quite limited.This is because the damaged axons tend to retract,because they encounter obstacles such as scar tissue and inhibitory molecules,and because their growth rates simply do not match those of a juvenile axon.In recent years,there has been a focus on microtubules as among the most important factors in encouraging injured adult axons to regenerate.Microtubules are hollow polymeric filaments composed of tubulin subunits that provide structural support for the     

RAFting the rapids of axon regeneration signaling

<正>The functional regeneration of damaged axons and severed connections in the mature central nervous system(CNS)remains a challenging goal of neurological research.Mature CNS neurons are refractory to axon regeneration for two major reasons,one,because the activity of cell-intrinsic mechanisms that drive axon growth     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons

During the initial stages of development, the notochord provides repulsive signals for dorsal root ganglion (DRG) axons via semaphorin 3A/neuropilin-1, axonin-1/SC2, and other unknown repulsive molecules. The notochord is known to produce aggrecan, one of the chondroitin sulfate proteoglycans (CSPGs). We report here that adding aggrecan to the culture medium cannot only induce DRG growth cone collapse, but also inhibit DRG axonal growth. Using cocultures composed of tissues derived from chick embryos or neuropilin-1-deficient mice treated with chondroitinase ABC, we show the direct evidence that CSPGs are involved in notochord-derived repulsion for DRG axons. At later developmental stages, CSPGs are involved in perinotochordal sheath-derived axon repulsion, but not in notochord core-derived repulsion. We further demonstrate that TAG-1/axonin-1/SC2 is not involved in mediating repulsive activities by CSPGs, but is required for notochord core-derived axon repulsion. Thus, notochord-derived multiple axon repulsions act in a spatiotemporal-specific manner to shape the initial trajectories of DRG axons.     

Scar-mediated inhibition and CSPG receptors in the CNS

Severed axons in adult mammals do not regenerate appreciably after central nervous system (CNS) injury due to developmentally determined reductions in neuron-intrinsic growth capacity and extracellular environment for axon elongation. Chondroitin sulfate proteoglycans (CSPGs), which are generated by reactive scar tissues, are particularly potent contributors to the growth-limiting environment in mature CNS. Thus, surmounting the strong inhibition by CSPG-rich scar is an important therapeutic goal for achieving functional recovery after CNS injuries. As of now, the main in vivo approach to overcoming inhibition by CSPGs is enzymatic digestion with locally applied chondroitinase ABC (ChABC), but several disadvantages may prevent using this bacterial enzyme as a therapeutic option for patients. A better understanding of the molecular mechanisms underlying CSPG action is needed in order to develop more effective therapies to overcome CSPG-mediated inhibition of axon regeneration and/or sprouting. Because of their large size and dense negative charges, CSPGs were thought to act by non-specifically hindering the binding of matrix molecules to their cell surface receptors through steric interactions. Although this may be true, recent studies indicate that two members of the leukocyte common antigen related (LAR) phosphatase subfamily, protein tyrosine phosphatase σ (PTPσ) and LAR, are functional receptors that bind CSPGs with high affinity and mediate CSPG inhibitory effects. CSPGs also may act by binding to two receptors for myelin-associated growth inhibitors, Nogo receptors 1 and 3 (NgR1 and NgR3). If confirmed, it would suggest that CSPGs have multiple mechanisms by which they inhibit axon growth, making them especially potent and difficult therapeutic targets. Identification of CSPG receptors is not only important for understanding the scar-mediated growth suppression, but also for developing novel and selective therapies to promote axon sprouting and/or regeneration after CNS injuries, including spinal cord injury (SCI).     

Limited growth of severed CNS axons after treatment of adult rat brain with hyaluronidase

Many chondroitin sulfate proteoglycans (CSPGs) have been shown to influence CNS axon growth in vitro and in vivo. These interactions can be mediated through the core protein or through the chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains. We have shown previously that degrading CS GAG side chains using chondroitinase ABC enhances dopaminergic nigrostriatal axon regeneration in vivo. We test the hypothesis that interfering with complete CSPGs also limit axon growth in vivo. Neurocan, versican, aggrecan, and brevican CSPGs may be anchored within extracellular matrix through binding to hyaluronan glycosaminoglycan. We examine whether degradation of hyaluronan using hyaluronidase might release these inhibitory CSPGs from the extracellular matrix and thereby enhance regeneration of cut nigrostriatal axons. Anesthetized adult rats were given knife cut lesions of the right hemisphere nigrostriatal tract and cannulae were secured transcranially thereby allowing repeated perilesional infusion of saline or saline containing hyaluronidase once daily for 10 days post-axotomy. Eleven days post-transection brains from animals under terminal anesthesia were recovered for histological evaluation. Effective delivery of substance was inferred from the observed reduction in perilesional immunoreactivity for neurocan and versican after treatment with hyaluronidase (relative to saline). Immunolabeling using antibodies against tyrosine hydroxylase was used to examine the response of cut dopaminergic nigral neurons. After transection and treatment with saline, dopaminergic nigral neurons sprouted in a region lacking astrocytes, neurocan and versican. Axons did not regenerate into the lesion surround that contained astrocytes and abundant neurocan and versican. After transection and treatment with hyaluronidase, there was a significant increase in the number of cut dopaminergic nigral axons growing up to 800 microm anterior to the site of transection. However, cut dopaminergic nigral axons still did not regenerate into the lesion surround that contained reduced (albeit residual) neurocan and versican immunoreactivity. Thus, partial degradation of hyaluronan and chondroitin sulfate and depletion of hyaluronan-binding CSPGs enhances local sprouting of cut CNS axons, but long-distance regeneration fails in regions containing residual hyaluronan-binding CSPGs. Hyaluronan, chondroitin sulfate and hyaluronan-binding CSPGs therefore likely contribute toward the failure of spontaneous axon regeneration in the injured adult mammalian brain and spinal cord.     

Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons

Inhibitory chondroitin sulfate proteoglycans (CSPGs) and myelin-associated molecules are major impediments to axon regeneration within the adult central nervous system (CNS). Decorin infusion can however suppress the levels of multiple inhibitory CSPGs and promote axon growth across spinal cord injuries [Davies, J.E., Tang, X., Denning, J.W., Archibald, S.J., and Davies, S.J., 2004. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur. J. Neurosci. 19, 1226-1242]. A question remained as to whether decorin can also increase axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. We have therefore conducted an in vitro analysis of neurite extension by decorin-treated adult dorsal root ganglion (DRG) neurons cultured on substrates of inhibitory CSPGs or myelin membranes mixed with laminin. Decorin treatment promoted 14.5 and 5-fold increases in average neurite length/neuron over untreated controls on CSPGs or myelin membranes respectively. In addition to suppressing inhibitory scar formation, our present data shows that decorin can directly boost the ability of neurons to extend axons within CSPG or myelin rich environments.     

Axon regeneration in peripheral nerves is enhanced by proteoglycan degradation

Regeneration of axons in the peripheral nervous system is enhanced by the removal of glycosaminoglycan side chains (GAGs) of chondroitin sulfate proteoglycans. However, some axons regenerate poorly despite such treatment, suggesting the existence of additional inhibitors. We compared the effects of enzymatic removal of GAGs from chondroitin sulfate proteoglycans versus two other proteoglycan species, heparan sulfate and keratan sulfate proteoglycans, on the regeneration of peripheral axons. Common fibular (CF) nerves of thy-1-YFP-H mice were cut and repaired using short segments of CF nerves harvested from wild-type littermates and pre-treated with a GAG-degrading enzyme for 1 h prior to nerve repair. Axonal regeneration was assayed by measuring the lengths of profiles of YFP+ axons in optical sections of the grafted nerves 1 week later. Except for grafts treated with keratanase, more and longer axon profiles were encountered in enzyme-treated grafts than in control grafts. Heparinase III treatments induced the greatest number of axons to enter into the graft. The proportions of axon profiles longer than 1000 microm were greater in grafts treated with chondroitinase ABC or heparinase I, but not with either keratanase or heparinase III. More regenerative sprouts were observed after treatment with heparinase I than any other enzymes. Treatment with a mixture of all four enzymes resulted in an enhancement of axon regeneration which was greater than that observed after treatment with any of the enzymes individually. The effects of chondroitinase ABC and heparinase III were correlated with specific GAG degradation. We believe that enzymatic removal of GAGs is especially effective in promoting the ability of regenerating axons to select their pathway in the distal stump (or nerve graft) and, in the case of chondroitinase ABC or heparinase I, it may also promote growth within that pathway.     

Degradation of chondroitin sulfate proteoglycans potentiates transplant-mediated axonal remodeling and functional recovery after spinal cord injury in adult rats

Transplantation of growth-permissive cells or tissues was used to bridge a lesion cavity and induce axonal growth in experimental spinal cord injury (SCI). Axonal interactions between host and transplant may be affected by upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) following various transplantation strategies. The extent of axonal growth and functional recovery after transplantation of embryonic spinal cord tissue decreases in adult compared to neonatal host. We hypothesized that CSPGs contribute to the decrease in the extent to which transplant supports axonal remodeling and functional recovery. Expression of CSPGs increased after overhemisection SCI in adult rats but not in neonates. Embryonic spinal cord transplant was surrounded by CSPGs deposited in host cord, and the interface between host and transplant seemed to contain a large amount of CSPGs. Intrathecally delivered chondroitinase ABC (C'ase) improved recovery of distal forelimb usage and skilled motor behavior after C4 overhemisection injury and transplantation in adults. This behavioral recovery was accompanied by an increased amount of raphespinal axons growing into the transplant, and raphespinal innervation to the cervical motor region was promoted by C'ase plus transplant. Moreover, C'ase increased the number of transplanted neurons that grew axons to the host cervical enlargement, suggesting that degradation of CSPGs supports remodeling not only of host axons but also axons from transplanted neurons. Our results suggest that CSPGs constitute an inhibitory barrier to prevent axonal interactions between host and transplant in adults, and degradation of the inhibitory barrier can potentiate transplant-mediated axonal remodeling and functional recovery after SCI.     

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.     

Chondroitin sulfate proteoglycan expression pattern in hippocampal development: potential regulation of axon tract formation

A variety of molecular influences in the extracellular matrix (ECM) interact with developing axons to guide the formation of hippocampal axon pathways. One of these influences may be chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal extension during development and following central nervous system injury. In this study, we examined the role of CSPGs and cell adhesion molecules in the regulation of axon tract formation during hippocampal development. We used indirect immunofluorescence to examine the developmental pattern of CSPG expression relative to axon tracts that express the cell adhesion molecule L1. Additionally, we used dissociated and explant cell cultures to examine the effects of CSPGs on hippocampal axon development in vitro. In vivo, we found that the CSPG neurocan is expressed throughout the alveus, neuropil layers, and parts of the dentate gyrus from E16 to P2. The CSPG phosphacan is expressed primarily in the neuropil layers at postnatal stages. After E18, intense labeling of neurocan was observed in regions of the alveus surrounding L1-expressing axon fascicles. In vitro, axons from brain regions that project through the alveus during development would not grow across CSPG substrata, in a concentration-dependent manner. In addition, hippocampal axons from dissociated neuron cultures only traveled across CSPG substrata as fasciculated axon bundles. These findings implicate CSPG in the regulation of axon trajectory and fasciculation during hippocampal axon tract formation.     

Chronotopic fiber reordering and the distribution of cell adhesion and extracellular matrix molecules in the optic pathway of fetal ferrets

We have examined the age-related reordering of optic axons as they pass through the chiasmatic region in fetal ferrets. Proportions of young and old optic axons were determined from electron micrographs taken sequentially through the prechiasmatic nerve, chiasm, and tract. This “chronotopic” reordering of axons was shown to emerge gradually, beginning rostral to the fusion of the two optic nerves, but continuing to develop caudal to the chiasmatic midline. Segregation of young from old optic axons was most pronounced within the optic tract. We then compared the emergence of this fiber reorganization to the distribution of cell adhesion and extracellular matrix molecules and to the glial architecture within the pathway. Using immunohistochemistry, the distributions of the cell adhesion molecules L1, NCAM, and TAG-1 and the extracellular matrix molecules laminin-1 and chondroitin sulfate proteoglycans (CSPGs) were determined. Among these, only the distribution of CSPGs was observed to change in a manner that complemented the segregation of young from old optic axons. CSPGs were densest in the deeper parts of the optic tract, coincident with radial glial fibers that turn to course within the region of the oldest optic axons. Both the glial architecture and the CSPG distribution form as a consequence of the invasion of the first optic axons, shown by the developmental sequence of each, and by the fact that these glial and molecular features fail to form in the absence of optic axons. The data suggest a model in which the gradient of CSPGs across the depth of the tract contributes to the formation of the chronotopic fiber reordering by providing a relatively unfavorable environment for subsequent axonal growth. The CSPGs may do so by interfering with adhesion molecules on optic axons that normally promote elongation. J. Comp. Neurol. 380:355–372, 1997. © 1997 Wiley-Liss, Inc.