Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI. METHODS: In a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions. RESULTS: We provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ. CONCLUSIONS: We have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.

Neuregulin-1 elicits a regulatory immune response following traumatic spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) triggers a robust neuroinflammatory response that governs secondary injury mechanisms with both degenerative and pro-regenerative effects. Identifying new immunomodulatory therapies to promote the supportive aspect of immune response is critically needed for the treatment of SCI. We previously demonstrated that SCI results in acute and permanent depletion of the neuronally derived Neuregulin-1 (Nrg-1) in the spinal cord. Increasing the dysregulated level of Nrg-1 through acute intrathecal Nrg-1 treatment enhanced endogenous cell replacement and promoted white matter preservation and functional recovery in rat SCI. Moreover, we identified a neuroprotective role for Nrg-1 in moderating the activity of resident astrocytes and microglia following injury. To date, the impact of Nrg-1 on immune response in SCI has not yet been investigated. In this study, we elucidated the effect of systemic Nrg-1 therapy on the recruitment and function of macrophages, T cells, and B cells, three major leukocyte populations involved in neuroinflammatory processes following SCI. METHODS: We utilized a clinically relevant model of moderately severe compressive SCI in female Sprague-Dawley rats. Nrg-1 (2 µg/day) or saline was delivered subcutaneously through osmotic mini-pumps starting 30 min after SCI. We conducted flow cytometry, quantitative real-time PCR, and immunohistochemistry at acute, subacute, and chronic stages of SCI to investigate the effects of Nrg-1 treatment on systemic and spinal cord immune response as well as cytokine, chemokine, and antibody production. RESULTS: We provide novel evidence that Nrg-1 promotes a pro-regenerative immune response after SCI. Bioavailability of Nrg-1 stimulated a regulatory phenotype in T and B cells and augmented the population of M2 macrophages in the spinal cord and blood during the acute and chronic stages of SCI. Importantly, Nrg-1 fostered a more balanced microenvironment in the injured spinal cord by attenuating antibody deposition and expression of pro-inflammatory cytokines and chemokines while upregulating pro-regenerative mediators. CONCLUSION: We provide the first evidence of a significant regulatory role for Nrg-1 in neuroinflammation after SCI. Importantly, the present study establishes the promise of systemic Nrg-1 treatment as a candidate immunotherapy for traumatic SCI and other CNS neuroinflammatory conditions.

Neuregulin-1 promotes remyelination and fosters a pro-regenerative inflammatory response in focal demyelinating lesions of the spinal cord.

Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1ß1 (rhNrg-1ß1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons.

Design and optimization of PLGA microparticles for controlled and local delivery of Neuregulin-1 in traumatic spinal cord injury.

Spinal cord injury (SCI) results in significant tissue damage that underlies functional impairments. Pharmacological interventions to confer neuroprotection and promote cell replacement are essential for SCI repair. We previously reported that Neuregulin-1 (Nrg-1) is acutely and permanently downregulated after SCI. Nrg-1 is a critical growth factor for differentiation of neural precursor cells (NPCs) into myelinating oligodendrocytes. We showed that intrathecal delivery of Nrg-1 enhances oligodendrocyte replacement following SCI. While an effective delivery system, intrathecal and systemic administration of growth factors with diverse biological targets may pose adverse off-target effects. Here, we have developed and optimized an injectable biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles system for sustained and prolonged intraspinal delivery of Nrg-1 in SCI. Recombinant human Nrg-1ß1 peptide was encapsulated into PLGA microparticles. Optimal Nrg-1 release rate and duration were achieved by manipulating the porosity and size of PLGA particles. Our in vitro analysis showed a direct correlation between particle size and porosity with Nrg-1 release rate, while Nrg-1 loading efficiency in PLGA microparticles was inversely correlated with particle porosity. In SCI, local intraspinal injection of PLGA-Nrg-1 microparticles maintained significantly higher tissue levels of Nrg-1 for a long-term duration compared to Nrg-1 delivered intrathecally by osmotic pumps. Bioactivity of Nrg-1 in PLGA microparticles was verified by promoting oligodendrocyte differentiation of NPCs in vitro, and preservation of oligodendrocytes and axons in SCI. PLGA-Nrg-1 also attenuated neuroinflammation and glial scarring following SCI. We show, for the first time, the feasibility, efficacy and safety of PLGA microparticle system for local and controlled administration of Nrg-1 in SCI.

Neuregulin-1 positively modulates glial response and improves neurological recovery following traumatic spinal cord injury.

Spinal cord injury (SCI) results in glial activation and neuroinflammation, which play pivotal roles in the secondary injury mechanisms with both pro- and antiregeneration effects. Presently, little is known about the endogenous molecular mechanisms that regulate glial functions in the injured spinal cord. We previously reported that the expression of neuregulin-1 (Nrg-1) is acutely and chronically declined following traumatic SCI. Here, we investigated the potential ramifications of Nrg-1 dysregulation on glial and immune cell reactivity following SCI. Using complementary in vitro approaches and a clinically-relevant model of severe compressive SCI in rats, we demonstrate that immediate delivery of Nrg-1 (500 ng/day) after injury enhances a neuroprotective phenotype in inflammatory cells associated with increased interleukin-10 and arginase-1 expression. We also found a decrease in proinflammatory factors including IL-1ß, TNF-α, matrix metalloproteinases (MMP-2 and 9) and nitric oxide after injury. In addition, Nrg-1 modulates astrogliosis and scar formation by reducing inhibitory chondroitin sulfate proteoglycans after SCI. Mechanistically, Nrg-1 effects on activated glia are mediated through ErbB2 tyrosine phosphorylation in an ErbB2/3 heterodimer complex. Furthermore, Nrg-1 exerts its effects through downregulation of MyD88, a downstream adaptor of Toll-like receptors, and increased phosphorylation of Erk1/2 and STAT3. Nrg-1 treatment with the therapeutic dosage of 1.5 µg/day significantly improves tissue preservation and functional recovery following SCI. Our findings for the first time provide novel insights into the role and mechanisms of Nrg-1 in acute SCI and suggest a positive immunomodulatory role for Nrg-1 that can harness the beneficial properties of activated glia and inflammatory cells in recovery following SCI.

Chondroitin Sulfate Proteoglycans Negatively Modulate Spinal Cord Neural Precursor Cells by Signaling Through LAR and RPTPσ and Modulation of the Rho/ROCK Pathway.

Multipotent adult neural precursor cells (NPCs) have tremendous intrinsic potential to repair the damaged spinal cord. However, evidence shows that the regenerative capabilities of endogenous and transplanted NPCs are limited in the microenvironment of spinal cord injury (SCI). We previously demonstrated that injury-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) restricts the survival, migration, integration, and differentiation of NPCs following SCI. CSPGs are long-lasting components of the astroglial scar that are formed around the lesion. Our recent in vivo studies demonstrated that removing CSPGs from the SCI environment enhances the potential of transplanted and endogenous adult NPCs for spinal cord repair; however, the mechanisms by which CSPGs regulate NPCs remain unclear. In this study, using in vitro models recapitulating the extracellular matrix of SCI, we investigated the direct role of CSPGs in modulating the properties of adult spinal cord NPCs. We show that CSPGs significantly decrease NPCs growth, attachment, survival, proliferation, and oligodendrocytes differentiation. Moreover, using genetic models, we show that CSPGs regulate NPCs by signaling on receptor protein tyrosine phosphate sigma (RPTPσ) and leukocyte common antigen-related phosphatase (LAR). Intracellularly, CSPGs inhibitory effects are mediated through Rho/ROCK pathway and inhibition of Akt and Erk1/2 phosphorylation. Downregulation of RPTPσ and LAR and blockade of ROCK in NPCs attenuates the inhibitory effects of CSPGS. Our work provide novel evidence uncovering how upregulation of CSPGs challenges the response of NPCs in their post-SCI niche and identifies new therapeutic targets for enhancing NPC-based therapies for SCI repair.