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

Abstract

BackgroundTraumatic 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.MethodsIn 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (10.1186...

LAR and PTPσ receptors are negative regulators of oligodendrogenesis and oligodendrocyte integrity in spinal cord injury

“…Adult NPCs were isolated from the spinal cord of C57BL/6 mouse (8 weeks of age) as we described previously (Dyck et al, ; Dyck et al, ; Gauthier, Kosciuczyk, Tapley, & Karimi‐Abdolrezaee, ). Briefly, mice were deeply anesthetized by placing them in a bell jar saturated with a mixture of 40% isoflurane/60% propylene glycol.…”

“…Coating procedure was performed as described above for NPCs. Where appropriate, chondroitinase ABC (ChABC, 0.1 U/ml; Sigma‐Aldrich, Oakville, ON, Canada, C3667‐10UN) was added with laminin + CSPGs mixture during the coating step to degrade CSPGs as we described previously (Dyck et al, ). After 24 hr of plating, culture medium was changed to OPC differentiation medium containing DMEM (Sigma‐Aldrich, Oakville, ON, Canada), insulin (10 μg/ml), transferrin (50 μg/ml), sodium selenite (5.2 ng/ml), hydrocortisone (18 ng/ml), putrescine (16 μg/ml), progesterone (6.3 ng/ml), biotin (10 ng/ml), N‐acetyl‐ l ‐cysteine (5 μg/ml), BSA (0.1%), PSN (Invitrogen, LifeTechnologies Inc, ON, Canada), 1% FBS, and 5 μM of forskolin.…”

“…Following SCI, composition of the extracellular matrix (ECM) is altered profoundly by upregulation of chondroitin sulfate proteoglycans (CSPGs) (Cregg et al, ; Dyck & Karimi‐Abdolrezaee, ). CSPGs are shown to negatively influence several aspects of the secondary injury mechanisms including neuroinflammation, cell differentiation as well as axonal plasticity and regeneration (Alilain, Horn, Hu, Dick, & Silver, ; Barritt et al, ; Bradbury et al, ; Didangelos, Iberl, Vinsland, Bartus, & Bradbury, ; Dyck et al, ; Dyck et al, ; Dyck & Karimi‐Abdolrezaee, ; Galtrey, Asher, Nothias, & Fawcett, ; Karimi‐Abdolrezaee et al, ; Lang et al, ). We previously uncovered a negative role for CSPGs in restricting the regenerative properties of adult NPCs in vitro (Dyck et al, ) and in cell transplantation approaches for SCI (Karimi‐Abdolrezaee et al, ).…”

“…Our group and others have demonstrated that LAR and PTPσ can be efficiently manipulated in clinically relevant models of contusive and compressive SCI in the rat by two membrane permeable blocking peptides named intracellular LAR peptide (ILP) and intracellular sigma peptide (ISP), respectively (Dyck et al, ; Lang et al, ). These studies have identified that modulation of PTPσ promotes axon regeneration and remarkable serotonergic reinnervation below the level of injury that is associated with some improvement in functional recovery in rats with SCI (Fisher et al, ; Lang et al, ).…”

“…These studies have identified that modulation of PTPσ promotes axon regeneration and remarkable serotonergic reinnervation below the level of injury that is associated with some improvement in functional recovery in rats with SCI (Fisher et al, ; Lang et al, ). Moreover, our recent studies uncovered that co‐inhibition of LAR and PTPσ with ILP and ISP following SCI fosters a pro‐regenerative immune response characterized by an increase in the populations of M2 microglia/macrophages and regulatory T lymphocytes (Dyck et al, ).…”