Jerry Silver

Regeneration beyond the glial scar

After injury to the adult central nervous system (CNS), injured axons cannot regenerate past the lesion. In this review, we present evidence that this is due to the formation of a glial scar. Chondroitin and keratan sulphate proteoglycans are among the main inhibitory extracellular matrix molecules that are produced by reactive astrocytes in the glial scar, and they are believed to play a crucial part in regeneration failure. We will focus on this role, as well as considering the behaviour of regenerating neurons in the environment of CNS injury.

Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro

In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratin sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories. To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached. Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alteration with lanes of the growth-promoting molecule laminin (LN). Neurite outgrowth was abundant along stripes of LN. In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border. The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing. We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present. A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG. These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth. Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycoaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition. These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.

Regeneration of adult axons in white matter tracts of the central nervous system

It is widely accepted that the adult mammalian central nervous system (CNS) is unable to regenerate axons. In addition to physical or molecular barriers presented by glial scarring at the lesion site, it has been suggested that the normal myelinated CNS environment contains potent growth inhibitors, or lacks growth-promoting molecules,. Here we investigate whether adult CNS white matter can support long-distance regeneration of adult axons in the absence of glial scarring, by using a microtransplantation technique that minimizes scarring to inject minute volumes of dissociated adult rat dorsal root ganglia directly into adult rat CNS pathways. This atraumatic injection procedure allowed considerable numbers of regenerating adult axons immediate access to the host glial terrain, where we found that they rapidly extended for long distances in white matter, eventually invading grey matter. Abortive regeneration correlated precisely with increased levels of proteoglycans within the extracellular matrix at the transplant interface, whereas successfully regenerating transplants were associated with minimal upregulation of these molecules. Our results demonstrate, to our knowledge for the first time, that reactive glial extracellular matrix at the lesion site is directly associated with failure of axon regrowth in vivo, and that adult myelinated white matter tracts beyond the glial scar can be highly permissive for regeneration.

CNS Injury, Glial Scars, and Inflammation: Inhibitory extracellular matrices and regeneration failure

Spinal cord and brain injuries lead to complex cellular and molecular interactions within the central nervous system in an attempt to repair the initial tissue damage. Many studies have illustrated the importance of the glial cell response to injury, and the influences of inflammation and wound healing processes on the overall morbidity and permanent disability that result. The abortive attempts of neuronal regeneration after spinal cord injury are influenced by inflammatory cell activation, reactive astrogliosis and the production of both growth promoting and inhibitory extracellular molecules. Despite the historical perspective that the glial scar was a mechanical barrier to regeneration, inhibitory molecules in the forming scar and methods to overcome them have suggested molecular modification strategies to allow neuronal growth and functional regeneration. Unlike myelin associated inhibitory molecules, which remain at largely static levels before and after central nervous system trauma, inhibitory extracellular matrix molecules are dramatically upregulated during the inflammatory stages after injury providing a window of opportunity for the delivery of candidate therapeutic interventions. While high dose methylprednisolone steroid therapy alone has not proved to be the solution to this difficult clinical problem, other strategies for modulating inflammation and changing the make up of inhibitory molecules in the extracellular matrix are providing robust evidence that rehabilitation after spinal cord and brain injury has the potential to significantly change the outcome for what was once thought to be permanent disability.

Injury-Induced Proteoglycans Inhibit the Potential for Laminin-Mediated Axon Growth on Astrocytic Scars

Following injury to the adult CNS, the expression of a number of extracellular matrix molecules increases in regions of reactive gliosis. This glial matrix includes certain chondroitin sulfate proteoglycans (CS-PGs) which have been correlated with an inhibition of axon outgrowth. In order to test the influence of glial associated CS-PGs on neurite elongation directly, we sought to determine whether enzymatic modification of injury-induced CS-PGs could enhance neurite outgrowth across the surface of intact glial scars formed in vivo after implanting nitrocellulose filters into the cortex of adult rats. This gliotic tissue was subsequently explanted in vitro and used as a substrate for growing embryonic retinal neurons. Treatment of adult explants with chondroitinase ABC led to a significant increase in mean neurite length over the scar surface. Heparitinase treatment caused a much smaller, although significant, increase in neurite outgrowth. This suggested that more than one type of PG was present or that a single PG with both CS and HS side chains was upregulated. Western analysis revealed that a PG(s) with a core protein between 180 and 400 kDa was found to be relatively more abundant in areas of reactive gliosis induced to form in adult rather than neonatal animals. Simultaneous treatment of adult glial scars with chondroitinase and antibodies to the beta 1, beta 2 chain of laminin partially reversed the growth-enhancing effect of enzymatic digestion alone. These data demonstrate that the increase in neurite outgrowth along the surface of reactive astrocytes following enzymatic modification of injury-induced PGs was due, in part, to the presence of laminin. Thus, in this model of gliosis, particular PGs may act as inhibitors of neurite outgrowth by attenuating the potential for axon elongation that could occur due to the concomitant expression of growth-promoting molecules in regions of reactive gliosis.

PTPσ Is a Receptor for Chondroitin Sulfate Proteoglycan, an Inhibitor of Neural Regeneration

Toward Neuronal Regeneration Neurons in the central nervous system that are severed or crushed do not regenerate well. Part of the problem derives from the glial scars left behind after such damage. The scar tissue contains sulfated proteoglycans that seem to inhibit axon regeneration. Shen et al. (p. 592, published online 15 October) have now identified a protein tyrosine phosphatase (PTP) in mouse neuronal membranes that functions as a receptor for the proteoglycans. Neurons that lacked this particular PTP showed improved regeneration. Regeneration remained incomplete, presumably due to other inhibitory factors in the way of complete axon regeneration. Mouse neurons that lack a receptor for inhibitory proteoglycans show improved regeneration. Chondroitin sulfate proteoglycans (CSPGs) present a barrier to axon regeneration. However, no specific receptor for the inhibitory effect of CSPGs has been identified. We showed that a transmembrane protein tyrosine phosphatase, PTPσ, binds with high affinity to neural CSPGs. Binding involves the chondroitin sulfate chains and a specific site on the first immunoglobulin-like domain of PTPσ. In culture, PTPσ–/– neurons show reduced inhibition by CSPG. A PTPσ fusion protein probe can detect cognate ligands that are up-regulated specifically at neural lesion sites. After spinal cord injury, PTPσ gene disruption enhanced the ability of axons to penetrate regions containing CSPG. These results indicate that PTPσ can act as a receptor for CSPGs and may provide new therapeutic approaches to neural regeneration.

Pet-1 ETS Gene Plays a Critical Role in 5-HT Neuron Development and Is Required for Normal Anxiety-like and Aggressive Behavior

The central serotonin (5-HT) neurotransmitter system is an important modulator of diverse physiological processes and behaviors; however, the transcriptional mechanisms controlling its development are largely unknown. The Pet-1 ETS factor is a precise marker of developing and adult 5-HT neurons and is expressed shortly before 5-HT appears in the hindbrain. Here we show that in mice lacking Pet-1, the majority of 5-HT neurons fail to differentiate. Remaining ones show deficient expression of genes required for 5-HT synthesis, uptake, and storage. Significantly, defective development of the 5-HT system is followed by heightened anxiety-like and aggressive behavior in adults. These findings indicate that Pet-1 is a critical determinant of 5-HT neuron identity and implicate a Pet-1-dependent program in serotonergic modulation of behavior.

The role of extracellular matrix in CNS regeneration.

Chondroitin sulfate proteoglycans are the principal inhibitory component of glial scars, which form after damage to the adult central nervous system and act as a barrier to regenerating axons. Recent findings have furthered our understanding of the mechanisms that result in a failure of regeneration after spinal cord injury and suggest that a multipartite approach will be required to facilitate long-distance regeneration and functional recovery.

Guidance of optic axons in vivo by a preformed adhesive pathway on neuroepithelial endfeet

Antibodies against the neural cell adhesion molecule (NCAM) were used in vivo both to localize NCAM antigenic determinants in developing tissues of the chicken visual system and to perturb cell-cell adhesion during growth of optic fibers to the tectum. The immunohistochemical studies revealed a staining pattern on neuroepithelial cells which coincided with certain regions of the presumptive route for optic axons, not only with respect to the overall pathway from the eye to the tectum, but also in the preferential distribution of the antigen on the marginal endfeet which are contacted by optic axon growth cones. The antibody-perturbation studies, which involved intraocular injection of anti-NCAM Fab at embryonic Day 3.5, demonstrated that inhibition of NCAM-mediated adhesion results in a dramatic distortion of growth cone-neuroepithelial cell relationships and consequently of the optic pathway. Together, these studies suggest that guidance of optic axons along the margin of the brain is at least in part influenced by a preformed adhesive pathway on neuroepithelial cells associated with NCAM antigens.

Inhibition of neurite outgrowth on astroglial scars in vitro

Traumatic injury to the adult mammalian CNS results in the formation of an astroglial-mesenchymal scar that seals the wound site but blocks axonal regeneration in the process. The mechanism that leads to this inhibition of axon outgrowth has been proposed to be either a physical barrier blocking the advancement of the growth cone or chemical factors actively inhibiting axon outgrowth. At present, it is unknown whether one or both of these mechanisms are responsible for the inhibitory nature of the glial scar in vivo. Using a model of CNS trauma that allows for removal of an adult rat glial scar intact on a nitrocellulose support and placement in vitro with the upper surface exposed, we addressed the question of whether the inhibitory effects could be accounted for by chemical components at the scar surface. A purified population of rat hippocampal neurons was seeded onto the scar explants as well as onto explants taken from neonatal rat cerebral cortex, and the extent of neurite outgrowth was compared. We found that the glial scar, at best, stimulates only minimal neurite outgrowth over its surface when compared to the immature environment explanted in the same manner. This growth-inhibitory state cannot merely be explained by neuronotoxic factors or fibroblasts preventing astrocyte-mediated neurite outgrowth. The inhibition is more probably due to the expression of molecules on the surface of the adult scar that either directly inhibit growth cones or inhibit them indirectly by occluding neurite-promoting factors in the extracellular matrix or on the astrocyte surface.