Robert J. McKeon

Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury

Chondroitin sulfate proteoglycans (CSPGs) are a major class of axon growth inhibitors that are up-regulated after spinal cord injury (SCI) and contribute to regenerative failure. Chondroitinase ABC (chABC) digests glycosaminoglycan chains on CSPGs and can thereby overcome CSPG-mediated inhibition. But chABC loses its enzymatic activity rapidly at 37 °C, necessitating the use of repeated injections or local infusions for a period of days to weeks. These infusion systems are invasive, infection-prone, and clinically problematic. To overcome this limitation, we have thermostabilized chABC and developed a system for its sustained local delivery in vivo, obviating the need for chronically implanted catheters and pumps. Thermostabilized chABC remained active at 37 °C in vitro for up to 4 weeks. CSPG levels remained low in vivo up to 6 weeks post-SCI when thermostabilized chABC was delivered by a hydrogel-microtube scaffold system. Axonal growth and functional recovery following the sustained local release of thermostabilized chABC versus a single treatment of unstabilized chABC demonstrated significant differences in CSPG digestion. Animals treated with thermostabilized chABC in combination with sustained neurotrophin-3 delivery showed significant improvement in locomotor function and enhanced growth of cholera toxin B subunit–positive sensory axons and sprouting of serotonergic fibers. Therefore, improving chABC thermostability facilitates minimally invasive, sustained, local delivery of chABC that is potentially effective in overcoming CSPG-mediated regenerative failure. Combination therapy with thermostabilized chABC with neurotrophic factors enhances axonal regrowth, sprouting, and functional recovery after SCI.

Matrix Metalloproteinase-2 Facilitates Wound Healing Events That Promote Functional Recovery after Spinal Cord Injury

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in both injury and repair mechanisms in the CNS. Pharmacological blockade of MMPs, limited to the first several days after spinal cord injury, improves locomotor recovery. This beneficial response is, however, lost when treatment is extended beyond the acutely injured cord to include wound healing and tissue remodeling. This suggests that some MMPs play a beneficial role in wound healing. To test this hypothesis, we investigated the role of MMP-2, which is actively expressed during wound healing, in white matter sparing and axonal plasticity, the formation of a glial scar, and locomotor recovery after spinal cord injury. MMP-2 increased between 7 and 14 d after injury, where it was immunolocalized in reactive astrocytes bordering the lesion epicenter. There was reduced white matter sparing and fewer serotonergic fibers, caudal to the lesion in injured MMP-2 null animals. MMP-2 deficiency also resulted in increased immunoreactivity to chondroitin sulfate proteoglycans and a more extensive astrocytic scar. Most importantly, locomotion in an open field, performance on a rotarod, and grid walking were significantly impaired in injured MMP-2 null mice. Our findings suggest that MMP-2 promotes functional recovery after injury by regulating the formation of a glial scar and white matter sparing and/or axonal plasticity. Thus, strategies exploiting MMPs as therapeutic targets must balance these beneficial effects during wound healing with their adverse interactions in the acutely injured spinal cord.

CS-4,6 is differentially upregulated in glial scar and is a potent inhibitor of neurite extension

The precise contribution of different CS-GAGs to CSPG-mediated inhibition of axonal growth after CNS injury is unknown. Quantification of the CS-GAGs in uninjured and injured brain (scar tissue) using fluorophore-assisted carbohydrate electrophoresis (FACE) demonstrated that the dominant CS-GAG in the uninjured brain is CS-4 whereas, in glial scar, CS-2, CS-6, and CS-4,6 were over-expressed. To determine if the pattern of sulfation influenced neurite extension, we compared the effects of CS-GAGs with dominant CS-4, CS-6, or CS-4,6 sulfation to intact CSPG (aggrecan), chondroitin (CS-0), and hyaluronan on chick DRG neurite outgrowth. We report that CS-4,6 GAG, one of the upregulated CS-GAGs in astroglial scar, is potently inhibitory and is comparable to intact aggrecan, a CSPG with known inhibitory properties. Thus, a specific CS-GAG that is differentially over-expressed in astroglial scar is a potent inhibitor of neurite extension. These results may influence the design of more specific strategies to enhance CNS regeneration after injury.

Activation of Protease-Activated Receptor-1 Triggers Astrogliosis after Brain Injury

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

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.

TGF-β1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors

BackgroundThe adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-β) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-β1 regulates Ant1 gene expression in cultured primary rodent astrocytes.ResultsTranscription reporter analysis verified that TGF-β1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-β1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-β1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-β1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-β1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN)-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-β1.ConclusionThe specific regulation of Ant1 by TGF-β1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-β1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may represent a general mechanism that underlies the neuroprotective effects of TGF-β1.

Sustained Delivery of Activated Rho GTPases and BDNF Promotes Axon Growth in CSPG-Rich Regions Following Spinal Cord Injury

Background Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion. Methodology/Principal Findings We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site. Conclusion Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.

PAR-1 Deficiency Protects against Neuronal Damage and Neurologic Deficits after Unilateral Cerebral Hypoxia/Ischemia:

Cardiovascular and neurologic surgeries often involve a temporary reduction in cerebral blood flow. In these conditions, as well as during cerebral ischemia and traumatic brain injury, the temporary loss of oxygen and glucose initiates a cascade of cellular events that culminate in neuronal death and damage. Understanding the mechanisms that contribute to neuronal death after hypoxia/ischemia is critically important for treatment of such brain injury. Here, we use a model of combined cerebral hypoxia/ischemia (H/I) to examine the role of protease-activated receptor-1 (PAR-1) in hypoxic/ischemic neuronal damage. Our data show that PAR-1-deficient mice have smaller lesion volumes than wild-type controls after 45 minutes of H/I. The results of the genetic block of PAR-1 were corroborated using a PAR-1 antagonist, which decreased infarct volume in wild-type C57Bl6 mice. Examination of cellular responses to H/I reveals that PAR-1 -/- animals have less cellular death and diminished glial fibrillary acidic protein expression. Additionally, PAR-1 -/- mice exhibit less motor behavior impairment in rotorod and inverted wire-hang tests. These data suggest that PAR-1 contributes to hypoxic/ischemic brain injury and are consistent with other studies that implicate serine proteases and their receptors in neuropathology after cerebral insults.

Expression of full-length trkB receptors by reactive astrocytes after chronic CNS injury

Growth factors, including members of the neurotrophin family, are expressed by neuronal and glial elements following injury to the CNS. In order to assess the capacity for glial cells to respond to neurotrophins at sites of chronic injury, full-length trkB receptors were localized following implantation of a nitrocellulose filter into the cerebral cortex for 30 days. Northern analysis demonstrated that filter implants contained cells expressing transcripts for full-length and truncated trkB receptors, in contrast to the predominant expression of truncated trkB receptors by cultured astrocytes. In situ hybridization and immunohistochemistry using probes to the trkB kinase domain colocalized full-length receptors with GFAP-immunopositive reactive astrocytes adjacent to and within the filter implant. In contrast, OX-42-immunopositive microglia/macrophages were not stained for full-length trkB. These data indicate that reactive astrocytes can express functional trkB receptors following a chronic insult to the cerebral cortex and support the hypothesis that neurotrophins may regulate astrocytic responses to injury.

Targeted downregulation of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase significantly mitigates chondroitin sulfate proteoglycan-mediated inhibition.

Chondroitin sulfate‐4,6 (CS‐E) glycosaminoglycan (GAG) upregulation in astroglial scars is a major contributor to chondroitin sulfate proteoglycan (CSPG)‐mediated inhibition [Gilbert et al. ( 2005 ) Mol Cell Neurosci 29:545–558]. However, the role of N‐acetylgalactosamine 4‐sulfate 6‐O‐sulfotransferase (GalNAc4S6ST) catalyzed sulfation of CS‐E, and its contribution to CSPG‐mediated inhibition of CNS regeneration remains to be fully elucidated. Here, we used in situ hybridization to show localized upregulation of GalNAc4S6ST mRNA after CNS injury. Using in vitro spot assays with immobilized CS‐E, we demonstrate dose‐dependent inhibition of rat embryonic day 18 (E18) cortical neurons. To determine whether selective downregulation of CS‐E affected the overall inhibitory character of extracellular matrix produced by reactive astrocytes, single [against (chondroitin 4) sulfotransferase 11 (C4ST1) or GalNAc4S6ST mRNA] or double [against C4ST1 and GalNAc4S6ST mRNA] siRNA treatments were conducted and assayed using quantitative real‐time polymerase chain reaction and high‐performance liquid chromatography to confirm the specific downregulation of CS‐4S GAG (CS‐A) and CS‐E. Spot and Bonhoeffer stripe assays using astrocyte‐conditioned media from siRNA‐treated rat astrocytes showed a significant decrease in inhibition of neuronal attachment and neurite extensions when compared with untreated and TGFα‐treated astrocytes. These findings reveal that selective attenuation of CS‐E via siRNA targeting of GalNAc4S6ST significantly mitigates CSPG‐mediated inhibition of neurons, potentially offering a novel intervention strategy for CNS injury.