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.Recovery after central nervous system (CNS) injury is minimal, leading to substantial current interest in potential strategies to overcome this challenge (1-5). Chondroitin sulfate proteoglycans (CSPGs) show dramatic up-regulation after neural injury, within the extracellular matrix of scar tissue and in the perineuronal net within more-distant targets of the severed axons (6,7). The inhibitory nature of CSPGs is reflected not only in the formation of dystrophic axonal retraction bulbs that fail to regenerate through the lesion (8), but also in the limited capacity for collateral sprouting of spared fibers (8,9). This inhibition can be relieved by chondroitinase ABC digestion of the chondroitin sulfate (CS) side chains, which can promote regeneration and sprouting and restore lost function (10)(11)(12)(13)(14). It has been known for nearly two decades that sulfated proteoglycans are major contributors to the repulsive nature of the glial scar (15); however, the precise inhibitory mechanism remains poorly understood. Because the identification of specific neuronal receptors for CSPGs has been lacking, relatively nonspecific mechanisms brought about by arrays of negatively charged sulfate (16) or the occlusion of substrate adhesion molecules (17) have been suggested.Transmembrane protein tyrosine phosphatases (PTPs) form a large and diverse molecular family and have a structure typical of transmembrane cell-surface receptors (18,19). In previous work, we and others have found that PTPσ and other PTPs in the leukocyte antigen-related (LAR) subfamily can act as receptors for heparan sulfate proteoglycans (HSPGs) (20)(21)(22), and these PTPs are involved in axon guidance and synapse formation during development (18- ‡To whom correspondence should be addressed. flanagan@hms.harvard.edu. * These authors contributed equally to this work. † Present address: Motor Neuron Center, Columbia University, New York, NY 10032, USA. (Fig. 1A). Using a cell-free system with recombinant fusion proteins of the PTPσ extra-cellular domain with an immunoglobulin Fc tag (PTPσ-Fc) and neurocan with an alkaline phosphatase tag (Ncn-AP), a binding interaction was indeed identified (P < 0.001) (Fig. 1B). Genuine biological ligand-re...

Spinal cord injuries (SCI) often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. Unfortunately, the untoward effects of impaired breathing are a leading cause of SCI-related death, underscoring the importance of developing strategies to restore respiratory activity. Here we show that after cervical SCI, there is upregulation of the perineuronal net (PNN) associated chondroitin sulfate proteoglycans (CSPGs) around phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with Chondroitinase ABC (ChABC) can, by itself, promote plasticity of spared tracts and restore limited activity to the paralyzed diaphragm. However, when combined with application of a peripheral nerve autograft, ChABC treatment results in lengthy regeneration of serotonergic axons and other bulbospinal fibers with remarkable recovery of diaphragm function. Following recovery and initial transection of the bridge, there occurs an unusual, overall increased tonic diaphragmatic EMG activity, suggesting considerable remodeling of spinal cord circuitry after regeneration. This is followed by complete elimination of the restored activity proving that regeneration is critical for the return of function. Overall, these experiments present a way to profoundly restore function of a single muscle following debilitating CNS trauma, through both plasticity of spared tracts and regeneration of essential pathways.

Injured axons of the adult CNS undergo lengthy retraction from the initial site of axotomy after spinal cord injury. Macrophage infiltration correlates spatiotemporally with this deleterious phenomenon, but the direct involvement of these inflammatory cells has not been demonstrated. In the present study, we examined the role of macrophages in axonal retraction within the dorsal columns after spinal cord injury in vivo and found that retraction occurred between days 2 and 28 after lesion and that the ends of injured axons were associated with ED-1ϩ cells. Clodronate liposome-mediated depletion of infiltrating macrophages resulted in a significant reduction in axonal retraction; however, we saw no evidence of regeneration. We used time-lapse imaging of adult dorsal root ganglion neurons in an in vitro model of the glial scar to examine macrophage-axon interactions and observed that adhesive contacts and considerable physical interplay between macrophages and dystrophic axons led to extensive axonal retraction. The induction of retraction was dependent on both the growth state of the axon and the activation state of the macrophage. Only dystrophic adult axons were susceptible to macrophage "attack." Unlike intrinsically active cell line macrophages, both primary macrophages and microglia required activation to induce axonal retraction. Contact with astrocytes had no deleterious effect on adult dystrophic axons, suggesting that the induction of extensive retraction was specific to phagocytic cells. Our data are the first to indicate a direct role of activated macrophages in axonal retraction by physical cell-cell interactions with injured axons.

Peripherally conditioned sensory neurons have an increased capacity to regenerate their central processes. However, even conditioned axons struggle in the presence of a hostile CNS environment. We hypothesized that combining an aggressive conditioning strategy with modification of inhibitory reactive astroglial-associated extracellular matrix could enhance regeneration. We screened potential treatments using a model of the dorsal root entry zone (DREZ). In this assay, a gradient of inhibitory chondroitin sulfate proteoglycans (CSPGs) stimulates formation of dystrophic end bulbs on adult sensory axons, which mimics regeneration failure in vivo. Combining inflammation-induced preconditioning of dorsal root ganglia in vivo before harvest, with chondroitinase ABC (ChABC) digestion of proteoglycans in vitro allows for significant regeneration across a once potently inhibitory substrate. We then assessed regeneration through the DREZ after root crush in adult rats receiving the combination treatment, ChABC, or zymosan pretreatment alone or no treatment. Regeneration was never observed in untreated animals, and only minimal regeneration occurred in the ChABC-and zymosanalone groups. However, remarkable regeneration was observed in a majority of animals that received the combination treatment. Regenerated fibers established functional synapses, as demonstrated electrophysiologically by the presence of an H-reflex. Two different postlesion treatment paradigms in which the timing of both zymosan and ChABC administration were varied after injury were ineffective in promoting regeneration. Therefore, zymosan pretreatment, but not posttreatment, of the sensory ganglia, combined with ChABC modification of CSPGs, resulted in robust and functional regeneration of sensory axons through the DREZ after root injury.