Spinal cord injury leads to a devastating cascade of secondary complications that eventually results in the formation of scar tissue many times the size of the original insult. Inflammation plays a very important role towards the development of such scar, but paradoxically, at the same time it has neuroprotective properties. Only recently have we understood enough about the relevant events to make the repair of injured spinal cords a reachable goal. Over the past decade, researchers have designed and tested numerous innovative therapeutic strategies, and many of such involve manipulation of the immune response. Interestingly, both immuno-stimulatory and immuno-suppressive interventions have shown positive results, which include the prevention of further tissue damage, prevention of secondary cell death and axonal degeneration, promotion of remyelination, stimulation of axonal regeneration, and facilitation of sensorimotor function recovery.
Inhibition of Rho-kinase (ROCK) with Y27632 stimulates sprouting by injured corticospinal tract and dorsal column tract axons, and accelerates functional recovery. However, regeneration of these axons across the glial scar was not observed. Here we examined the effects of Y27632 treatment on chondroitin sulfate proteoglycan (CSPG) expression by astrocytes, which are a key component of the reactive gliosis inhibiting axonal regeneration. In vivo, rats underwent a dorsal column transection and were treated with Y27632 via intrathecal pump infusion. Compared with controls, Y27632-treated injury sites displayed exaggerated upregulation of glial fibrillary acid protein and neurocan immunoreactivity along the lesion edge. In vitro, astrocytes assumed a reactive morphology (stellate shape) and increased their expression of CSPGs after Y27632 treatment. Neurite growth by dissociated cortical neurons decreased when cultured on the extracellular matrix (ECM) derived from Y27632-treated astrocytes. This decrease in neurite growth was reversed with chondroitinase-ABC (ChABC) digestion, indicating that the inhibition was due to CSPG depositions within the ECM. Interestingly, conditioned medium (CM) from untreated astrocytes was inhibitory to neurite growth, which was overcome by ChABC digestion. Such inhibitory activity was not found in the CM of Y27632-treated astrocytes. Taken together, these data support a model where ROCK inhibition by Y27632 modifies astrocytic processing of CSPGs, and increases the presence of CSPGs within the ECM while reduces CSPGs in the CM (cerebrospinal fluid in vivo). This increased expression of inhibitory CSPGs in the ECM of the glial scar may counteract the growth promoting effects of ROCK inhibition on axonal growth cones.
Aggrecan is one of the major chondroitin sulfate proteoglycans (CSPGs) expressed in the central nervous system. The signaling pathways activated downstream of cell interaction with aggrecan and with CSPGs in general and the importance of chondroitin sulfate-glycosaminoglycan side chains in their inhibition are unclear. Therefore, to analyze the effect of different components of aggrecan in inhibiting neurite growth, neurite outgrowth was quantified in an in vitro model in which chick dorsal root ganglion (DRG) explants were grown on substrates containing aggrecan bound to hyaluronan and link protein as a macromolecular aggregate, aggrecan monomers, hyaluronan, or ChABC-treated aggrecan. Aggrecan aggregate, aggrecan monomer, and hyaluronan inhibited neurite outgrowth from nerve growth factor (NGF)- and neurotrophin-3 (NT3)-responsive DRG neurons. Aggrecan inhibition was dependent on its chondroitin sulfate-glycosaminoglycans, as ChABC digestion alleviated neurite inhibition because of aggrecan. Growth cones displayed full or partial collapse on aggrecan aggregate, hyaluronan, and ChABC-treated aggrecan. Inhibition of Rho kinase (ROCK) with Y27632 increased neurite growth on some but not all of the aggrecan components tested. With NGF in the culture medium, Y27632 increased neurite outgrowth on aggrecan aggregate, monomers, and ChABC-treated aggrecan, but not on hyaluronan. The ROCK inhibitor also increased NT3-responsive outgrowth on aggrecan aggregate and hyaluronan, but not on ChABC-treated aggrecan. This study showed that the matrix proteoglycan aggrecan and its components have multiple effects on neurite outgrowth and that some of these effects involve the Rho/ROCK pathway.
Glial cells, including astrocytes and macrophages/microglia, are thought to modulate pathological states following spinal cord injury (SCI). In the present study, we evaluated the therapeutic effects of interferon-γ (IFN-γ), which is one of the cytokines regulating glial function, in a mouse contusive SCI model. We found that intraperitoneal injection of IFN-γ significantly facilitated locomotor improvement following SCI. Immunohistochemistry demonstrated that IFN-γ decreased the accumulation of chondroitin sulfate proteoglycans (CSPGs), which are critical axon outgrowth inhibitors produced by reactive astrocytes in the injured central nervous system (CNS). Quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting demonstrated that neurocan, one of several CSPGs, was reduced in the spinal cords of IFN-γ-treated mice compared to vehicle-treated mice. Consistently, IFN-γ inhibited the production of neurocan from activated astrocytes in vitro. In addition, IFN-γ treatment enhanced the number of serotonin-positive nerve fibers and myelinated nerve fibers around the lesion epicenter. We also found that glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor-1 (IGF-1) were upregulated post-SCI following IFN-γ treatment. Our results indicate that IFN-γ exhibits therapeutic effects in mouse contusive SCI, presumably by reducing CSPG expression from reactive astrocytes and increasing the expression of neurotrophic factors.
Recent research into neurodegenerative disorders found that their pathogeneses have a link to the inositol 1,4,5-trisphosphate receptors (IP3R). This is encouraging, because despite extensive efforts, researchers have not fully understood the pathophysiologies of those disorders, and have yet to find the cure. The IP3R provides a possible point of convergence that new therapeutic drugs can target. This review highlights patents that manipulate activities of the IP3R. They generally involve the use of peptides designed from the amino acid sequences of IP3R-binding proteins, and of buffers that limit the availability of its ligand, IP3. Additionally, one of them details the use of a chromophore-conjugated small synthetic molecule to directly inhibit the IP3R in a highly spatiotemporally specific manner. Although many of them have only been tested in vitro or are in the early stages of in vivo application, more research-effective therapies for neurodegenerative diseases can hopefully be developed.
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