Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury.

Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity. It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting. Protein tyrosine phosphatase σ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates. We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition. Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems. Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.

Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury.

NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell-cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents. Studies using varying ratios of CSPGs and adhesion molecules along with chondroitinase ABC, as well as purified adult cord-derived NG2 glia, demonstrate that CSPGs are involved in entrapping neurons. Once dystrophic axons become stabilized upon NG2+ cells, they form synaptic-like connections both in vitro and in vivo. In NG2 knock-out mice, sensory axons in the dorsal columns dieback further than their control counterparts. When axons are double conditioned to enhance their growth potential, some traverse the lesion core and express reduced amounts of synaptic proteins. Our studies suggest that proteoglycan-mediated entrapment upon NG2+ cells is an additional obstacle to CNS axon regeneration.

Multipotent adult progenitor cells prevent macrophage-mediated axonal dieback and promote regrowth after spinal cord injury.

Macrophage-mediated axonal dieback presents an additional challenge to regenerating axons after spinal cord injury. Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to ameliorate this detrimental inflammation-related process has not been investigated. Using an in vitro model of axonal dieback as well as an adult rat dorsal column crush model of spinal cord injury, we found that multipotent adult progenitor cells (MAPCs) can affect both macrophages and dystrophic neurons simultaneously. MAPCs significantly decrease MMP-9 (matrix metalloproteinase-9) release from macrophages, effectively preventing induction of axonal dieback. MAPCs also induce a shift in macrophages from an M1, or "classically activated" proinflammatory state, to an M2, or "alternatively activated" antiinflammatory state. In addition to these effects on macrophages, MAPCs promote sensory neurite outgrowth, induce sprouting, and further enable axons to overcome the negative effects of macrophages as well as inhibitory proteoglycans in their environment by increasing their intrinsic growth capacity. Our results demonstrate that MAPCs have therapeutic benefits after spinal cord injury and provide specific evidence that adult stem cells exert positive immunomodulatory and neurotrophic influences.

Adult NG2+ cells are permissive to neurite outgrowth and stabilize sensory axons during macrophage-induced axonal dieback after spinal cord injury.

We previously demonstrated that activated ED1+ macrophages induce extensive axonal dieback of dystrophic sensory axons in vivo and in vitro. Interestingly, after spinal cord injury, the regenerating front of axons is typically found in areas rich in ED1+ cells, but devoid of reactive astrocyte processes. These observations suggested that another cell type must be present in these areas to counteract deleterious effects of macrophages. Cells expressing the purportedly inhibitory chondroitin sulfate proteoglycan NG2 proliferate in the lesion and intermingle with macrophages, but their influence on regeneration is highly controversial. Our in vivo analysis of dorsal column crush lesions confirms the close association between NG2+ cells and injured axons. We hypothesized that NG2+ cells were growth promoting and thereby served to increase axonal stability following spinal cord injury. We observed that the interactions between dystrophic adult sensory neurons and primary NG2+ cells derived from the adult spinal cord can indeed stabilize the dystrophic growth cone during macrophage attack. NG2+ cells expressed high levels of laminin and fibronectin, which promote neurite outgrowth on the surface of these cells. Our data also demonstrate that NG2+ cells, but not astrocytes, use matrix metalloproteases to extend across a region of inhibitory proteoglycan, and provide a permissive bridge for adult sensory axons. These data support the hypothesis that NG2+ cells are not inhibitory to regenerating sensory axons and, in fact, they may provide a favorable substrate that can stabilize the regenerating front of dystrophic axons in the inhibitory environment of the glial scar.

Overcoming macrophage-mediated axonal dieback following CNS injury.

Trauma to the adult CNS initiates multiple processes including primary and secondary axotomy, inflammation, and glial scar formation that have devastating effects on neuronal regeneration. After spinal cord injury, the infiltration of phagocytic macrophages coincides with long-distance axonal retraction from the initial site of injury, a deleterious phenomenon known as axonal dieback. We have previously shown that activated macrophages directly induce long-distance retraction of dystrophic axons in an in vitro model of the glial scar. We hypothesized that treatments that are primarily thought to increase neuronal regeneration following spinal cord injury may in fact derive a portion of their beneficial effects from inhibition of macrophage-mediated axonal retraction. We analyzed the effects of protease inhibition, substrate modification, and neuronal preconditioning on macrophage-axon interactions using our established in vitro model. General inhibition of matrix metalloproteinases and specific inhibition of MMP-9 prevented macrophage-induced axonal retraction despite significant physical interactions between the two cell types, whereas inhibition of MMP-2 had no effect. Chondroitinase ABC-mediated digestion of the aggrecan substrate also prevented macrophage-induced axonal retraction in the presence of extensive macrophage-axon interactions. The use of a conditioning lesion to stimulate intrinsic neuronal growth potential in the absence of substrate modification likewise prevented macrophage-induced axonal retraction in vitro and in vivo following spinal cord injury. These data provide valuable insight into the cellular and molecular mechanisms underlying macrophage-mediated axonal retraction and demonstrate modifications that can alleviate the detrimental effects of this unfavorable phenomenon on the postlesion CNS.

Another barrier to regeneration in the CNS: activated macrophages induce extensive retraction of dystrophic axons through direct physical interactions.

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-1+ 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.

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.

Multipotent Adult Progenitor Cells, but Not Tissue Inhibitor of Matrix Metalloproteinase-3, Increase Tissue Sparing and Reduce Urological Complications following Spinal Cord Injury.

Following spinal cord injury (SCI), inflammation amplifies damage beyond the initial insult, providing an opportunity for targeted treatments. An ideal protective therapy would reduce both edema within the lesion area and the activation/infiltration of detrimental immune cells. Previous investigations demonstrated the efficacy of intravenous injection of multipotent adult progenitor cells (MAPC®) to modulate immune response following SCI, leading to significant improvements in tissue sparing, locomotor and urological functions. Separate studies have demonstrated that tissue inhibitor of matrix metalloproteinase-3 (TIMP3) reduces blood-brain barrier permeability following traumatic brain injury in a mouse model, leading to improved functional recovery. This study examined whether TIMP3, delivered alone or in concert with MAPC cells, improves functional recovery from a contusion SCI in a rat model. The results suggest that intravenous delivery of MAPC cell therapy 1 day following acute SCI significantly improves tissue sparing and impacts functional recovery. TIMP3 treatment provided no significant benefit, and further, when co-administered with MAPC cells, it abrogated the therapeutic effects of MAPC cell therapy. Importantly, this study demonstrated for the first time that acute treatment of SCI with MAPC cells can significantly reduce the incidence of urinary tract infection (UTI) and the use of antibiotics for UTI treatment.

Chronic enhancement of the intrinsic growth capacity of sensory neurons combined with the degradation of inhibitory proteoglycans allows functional regeneration of sensory axons through the dorsal root entry zone in the mammalian spinal cord.

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 zymosan-alone 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.

Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation, and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

Mesenchymal Stromal Cells as a Therapeutic Strategy to Support Islet Transplantation in Type 1 Diabetes Mellitus.

Type 1 diabetes is an autoimmune disorder that leads to destruction of pancreatic ß islet cells and is a growing global health issue. While insulin replacement remains the standard therapy for type 1 diabetes, exogenous insulin does not mimic the physiology of insulin secretion. Transplantation of pancreatic islets has the potential to cure this disease; however, there are several major limitations to widespread implementation of islet transplants. The use of mesenchymal stromal cells (MSCs) in the treatment of type 1 diabetes has been investigated as an adjunct therapy during islet graft administration to prevent initial islet loss and promote engraftment and revascularization of islets. In this review we will discuss the results of recent MSC studies in animal models of diabetes with a focus on islet transplantation and explore the potential for these findings to be extended to clinical use for the treatment of type 1 diabetes.

PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural 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, PTPsigma, binds with high affinity to neural CSPGs. Binding involves the chondroitin sulfate chains and a specific site on the first immunoglobulin-like domain of PTPsigma. In culture, PTPsigma(-/-) neurons show reduced inhibition by CSPG. A PTPsigma fusion protein probe can detect cognate ligands that are up-regulated specifically at neural lesion sites. After spinal cord injury, PTPsigma gene disruption enhanced the ability of axons to penetrate regions containing CSPG. These results indicate that PTPsigma can act as a receptor for CSPGs and may provide new therapeutic approaches to neural regeneration.