Diffuse traumatic axonal injury in mice induces complex behavioural alterations that are normalized by neutralization of interleukin-1ß.

Widespread traumatic axonal injury (TAI) results in brain network dysfunction, which commonly leads to persisting cognitive and behavioural impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro-inflammatory cytokine interleukin (IL)-1ß has not been established in TAI. An IL-1ß-neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Mice subjected to moderate cFPI (n = 41) were compared with sham-injured controls (n = 20) and untreated, naive mice (n = 9). The anti-IL-1ß antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30 µg/brain tissue) at 24 h post-injury in both cFPI (n = 5) and sham-injured (n = 3) mice, with lower concentrations at 72 h post-injury (up to ~18 µg/g brain tissue in three cFPI mice). Functional outcome was analysed with the multivariate concentric square field (MCSF) test at 2 and 9 days post-injury, and the Morris water maze (MWM) at 14-21 days post-injury. Following TAI, the IL-1ß-neutralizing antibody resulted in an improved behavioural outcome, including normalized behavioural profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL-1ß-neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL-1ß is an important contributor to the processes causing complex cognitive and behavioural disturbances following TAI.

The dual S1PR1/S1PR5 drug BAF312 (Siponimod) attenuates demyelination in organotypic slice cultures.

BACKGROUND: BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination. METHODS: Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H. RESULTS: BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca(2+) signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312. CONCLUSIONS: Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS.

Fractalkine shedding is mediated by p38 and the ADAM10 protease under pro-inflammatory conditions in human astrocytes.

BACKGROUND: The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes. METHODS: The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5. RESULTS: A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1ß-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1ß, TNF-α or IFN-γ. In addition, an IKKß inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1ß or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway. CONCLUSIONS: In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.

The selective anti-IL17A monoclonal antibody secukinumab (AIN457) attenuates IL17A-induced levels of IL6 in human astrocytes.

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1ß in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes.

Neutralization of interleukin-1ß reduces cerebral edema and tissue loss and improves late cognitive outcome following traumatic brain injury in mice.

Increasing evidence suggests that interleukin-1ß (IL-1ß) is a key mediator of the inflammatory response following traumatic brain injury (TBI). Recently, we showed that intracerebroventricular administration of an IL-1ß-neutralizing antibody was neuroprotective following TBI in mice. In the present study, an anti-IL-1ß antibody or control antibody was administered intraperitoneally following controlled cortical injury (CCI) TBI or sham injury in 105 mice and we extended our histological, immunological and behavioral analysis. First, we demonstrated that the treatment antibody reached target brain regions of brain-injured animals in high concentrations (> 11 nm) remaining up to 8 days post-TBI. At 48 h post-injury, the anti-IL-1ß treatment attenuated the TBI-induced hemispheric edema (P < 0.05) but not the memory deficits evaluated using the Morris water maze (MWM). Neutralization of IL-1ß did not influence the TBI-induced increases (P < 0.05) in the gene expression of the Ccl3 and Ccr2 chemokines, IL-6 or Gfap. Up to 20 days post-injury, neutralization of IL-1ß was associated with improved visuospatial learning in the MWM, reduced loss of hemispheric tissue and attenuation of the microglial activation caused by TBI (P < 0.05). Motor function using the rotarod and cylinder tests was not affected by the anti-IL-1ß treatment. Our results suggest an important negative role for IL-1ß in TBI. The improved histological and behavioral outcome following anti-IL-1ß treatment also implies that further exploration of IL-1ß-neutralizing compounds as a treatment option for TBI patients is warranted.

Neutralization of interleukin-1beta modifies the inflammatory response and improves histological and cognitive outcome following traumatic brain injury in mice.

Interleukin-1beta (IL-1beta) may play a central role in the inflammatory response following traumatic brain injury (TBI). We subjected 91 mice to controlled cortical impact (CCI) brain injury or sham injury. Beginning 5 min post-injury, the IL-1beta neutralizing antibody IgG2a/k (1.5 microg/mL) or control antibody was infused at a rate of 0.25 microL/h into the contralateral ventricle for up to 14 days using osmotic minipumps. Neutrophil and T-cell infiltration and microglial activation was evaluated at days 1-7 post-injury. Cognition was assessed using Morris water maze, and motor function using rotarod and cylinder tests. Lesion volume and hemispheric tissue loss were evaluated at 18 days post-injury. Using this treatment strategy, cortical and hippocampal tissue levels of IgG2a/k reached 50 ng/mL, sufficient to effectively inhibit IL-1betain vitro. IL-1beta neutralization attenuated the CCI-induced cortical and hippocampal microglial activation (P < 0.05 at post-injury days 3 and 7), and cortical infiltration of neutrophils (P < 0.05 at post-injury day 7). There was only a minimal cortical infiltration of activated T-cells, attenuated by IL-1beta neutralization (P < 0.05 at post-injury day 7). CCI induced a significant deficit in neurological motor and cognitive function, and caused a loss of hemispheric tissue (P < 0.05). In brain-injured animals, IL-1beta neutralizing treatment resulted in reduced lesion volume, hemispheric tissue loss and attenuated cognitive deficits (P < 0.05) without influencing neurological motor function. Our results indicate that IL-1beta is a central component in the post-injury inflammatory response that, in view of the observed positive neuroprotective and cognitive effects, may be a suitable pharmacological target for the treatment of TBI.

Nogo-A inhibition induces recovery from neglect in rats.

Neglect is a complex human cognitive spatial disorder typically induced by damage to prefrontal or posterior parietal association cortices. Behavioral treatments for neglect rarely generalize outside of the therapeutic context or across tasks within the same therapeutic context. Recovery, when it occurs, is spontaneous over the course of weeks to months, but often it is incomplete. A number of studies have indicated that anti-Nogo-A antibodies can be used to enhance plasticity and behavioral recovery following damage to motor cortex, and spinal cord. In the present studies the anti-Nogo-A antibodies IN-1, 7B12, or 11C7 were applied intraventricularly to adult rats demonstrating severe neglect produced by unilateral medial agranular cortex lesions in rats. The three separate anti-Nogo-A antibody groups were treated immediately following the medial agranular cortex lesions. Each of the three antibodies induced dramatic significant behavioral recovery from neglect relative to controls. Severing the corpus callosum to destroy inputs from the contralesional hemisphere resulted in reinstatement of severe neglect, pointing to a possible role of interhemispheric mechanisms in behavioral recovery from neglect.

Cognitive outcome following brain injury and treatment with an inhibitor of Nogo-A in association with an attenuated downregulation of hippocampal growth-associated protein-43 expression.

OBJECT: Central nervous system axons regenerate poorly after traumatic brain injury (TBI), partly due to inhibitors such as the protein Nogo-A present in myelin. The authors evaluated the efficacy of anti-Nogo-A monoclonal antibody (mAb) 7B12 administration on the neurobehavioral and cognitive outcome of rats following lateral fluid-percussion brain injury, characterized the penetration of the 7B12 or control antibodies into target brain regions, and evaluated the effects of Nogo-A inhibition on hemispheric tissue loss and sprouting of uninjured motor tracts in the cervical cord. To elucidate a potential molecular response to Nogo-A inhibition, we evaluated the effects of 7B12 on hippocampal GAP-43 expression. METHODS: Beginning 24 hours after lateral fluid-percussion brain injury or sham injury in rats, the mAb 7B12 or control antibody was infused intracerebroventricularly over 14 days, and behavior was assessed over 4 weeks. RESULTS: Immunoreactivity for 7B12 or immunoglobulin G was detected in widespread brain regions at 1 and 3 weeks postinjury. The brain-injured animals treated with 7B12 showed improvement in cognitive function (p < 0.05) at 4 weeks but no improvement in neurological motor function from 1 to 4 weeks postinjury compared with brain-injured, vehicle-treated controls. The enhanced cognitive function following inhibition of Nogo-A was correlated with an attenuated postinjury downregulation of hippocampal GAP-43 expression (p < 0.05). CONCLUSIONS: Increased GAP-43 expression may be a novel molecular mechanism of the enhanced cognitive recovery mediated by Nogo-A inhibition after TBI in rats.

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157.

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model.

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1-/-) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1-/- mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1-/- mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1-/- mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1-/- mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1-/- mice. However, these effects on macrophages and astrocytes in MCP-1-/- mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1-/- mice produced significantly less interleukin-1beta in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.

Anti-Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spontaneously hypertensive rats.

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti-Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 +/- 1.5% after PCI (control group: 1.1 +/- 0.5%) and 4.5 +/- 2.2% after MCAO in SHR rats (control group: 1.8 +/- 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti-Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.

Fingolimod attenuates splenocyte-induced demyelination in cerebellar slice cultures.

The family of sphingosine-1-phosphate receptors (S1PRs) is G-protein-coupled, comprised of subtypes S1PR1-S1PR5 and activated by the endogenous ligand S1P. The phosphorylated version of Fingolimod (pFTY720), an oral therapy for multiple sclerosis (MS), induces S1PR1 internalisation in T cells, subsequent insensitivity to S1P gradients and sequestering of these cells within lymphoid organs, thus limiting immune response. S1PRs are also expressed in neuronal and glial cells where pFTY720 is suggested to directly protect against lysolecithin-induced deficits in myelination state in organotypic cerebellar slices. Of note, the effect of pFTY720 on immune cells already migrated into the CNS, prior to treatment, has not been well established. We have previously found that organotypic slice cultures do contain immune cells, which, in principle, could also be regulated by pFTY720 to maintain levels of myelin. Here, a mouse organotypic cerebellar slice and splenocyte co-culture model was thus used to investigate the effects of pFTY720 on splenocyte-induced demyelination. Spleen cells isolated from myelin oligodendrocyte glycoprotein immunised mice (MOG-splenocytes) or from 2D2 transgenic mice (2D2-splenocytes) both induced demyelination when co-cultured with mouse organotypic cerebellar slices, to a similar extent as lysolecithin. As expected, in vivo treatment of MOG-immunised mice with FTY720 inhibited demyelination induced by MOG-splenocytes. Importantly, in vitro treatment of MOG- and 2D2-splenocytes with pFTY720 also attenuated demyelination caused by these cells. In addition, while in vitro treatment of 2D2-splenocytes with pFTY720 did not alter cell phenotype, pFTY720 inhibited the release of the pro-inflammatory cytokines such as interferon gamma (IFNγ) and interleukin 6 (IL6) from these cells. This work suggests that treatment of splenocytes by pFTY720 attenuates demyelination and reduces pro-inflammatory cytokine release, which likely contributes to enhanced myelination state induced by pFTY720 in organotypic cerebellar slices.

Delayed anti-nogo-a antibody application after spinal cord injury shows progressive loss of responsiveness.

Blocking the function of the myelin protein Nogo-A or its signaling pathway is a promising method to overcome an important neurite growth inhibitory factor of the adult central nervous system (CNS), and to enhance axonal regeneration and plasticity after brain or spinal cord injuries. Several studies have shown increased axonal regeneration and enhanced compensatory sprouting, along with substantially improved functional recovery after treatment with anti-Nogo-A antibodies, Nogo-receptor antagonists, or inhibition of the downstream mediator RhoA/ROCK in adult rodents. Proof-of-concept studies in spinal cord-injured macaque monkeys with anti-Nogo-A antibodies have replicated these findings; recently, clinical trials in spinal cord-injured patients have begun. However, the optimal time window for successful Nogo-A function blocking treatments has not yet been determined. We studied the effect of acute as well as 1- or 2-weeks delayed intrathecal anti-Nogo-A antibody infusions on the regeneration of corticospinal tract (CST) axons and the recovery of motor function after large but anatomically incomplete thoracic spinal cord injuries in adult rats. We found that lesioned CST fibers regenerated over several millimeters after acute or 1-week-delayed treatments, but not when the antibody treatment was started with a delay of 2 weeks. Swimming and narrow beam crossing recovered well in rats treated acutely or with a 1-week delay with anti-Nogo-A antibodies, but not in the 2-week-delayed group. These results show that the time frame for treatment of spinal cord lesions with anti-Nogo-A antibodies is restricted to less than 2 weeks in adult rodents.

Intrathecal treatment with anti-Nogo-A antibody improves functional recovery in adult rats after stroke.

Stroke often results in devastating neurological disabilities with no specific treatment available to improve functional recovery. Neurite growth inhibitory proteins such as Nogo-A play a critical role in impeding regain of function after stroke. We have reported that treatment with anti-Nogo-A antibody using the intracerebroventricular route resulted in improvement of function and neuroplasticity in adult or aged rats after stroke. This present study tested a more clinically accessible route for applying anti-Nogo-A antibodies, the intrathecal route. Anti-Nogo-A or control antibody was administered intrathecally at lower lumbar levels 1 week after middle cerebral artery occlusion in adult rats. Our results show that anti-Nogo-A antibody delivered by this intrathecal route for 2 weeks penetrated into brain parenchyma and bound to myelin-enriched structures such as the corpus callosum and striatal white matter. Animals receiving anti-Nogo-A antibody treatment significantly improved recovery of function on the skilled forelimb reaching task as compared to stroke only and stroke/control antibody animals. These findings show that anti-Nogo-A antibody delivered through the intrathecal route is as effective in restoring lost functions after stroke as the intracerebroventricular route. This is of great importance for the future application of anti-Nogo-A immunotherapy for ischemic stroke treatment.

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.

Recovery and brain reorganization after stroke in adult and aged rats.

Stroke is a prevalent and devastating disorder, and no treatment is currently available to restore lost neuronal function after stroke. One unique therapy that improves recovery after stroke is neutralization of the neurite inhibitory protein Nogo-A. Here, we show, in a clinically relevant model, improved functional recovery and brain reorganization in the aged and adult rat when delayed anti-Nogo-A therapy is given after ischemic injury. These results support the efficacy of Nogo-A neutralization as treatment for ischemic stroke, even in the aged animal and after a 1-week delay, and implicate neuronal plasticity from unlesioned areas of the central nervous system as a mechanism for recovery.

Targeting monocyte chemoattractant protein-1 signalling in disease.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated in many inflammatory and autoimmune diseases. The G-protein-coupled receptor CCR-2B is probably the most important MCP-1 receptor in vivo, and loss of MCP-1 effector function alone is sufficient to impair monocytic trafficking in inflammation models. MCP-1 signalling appears to be a relevant target, especially in rheumatoid arthritis (RA). In RA patients, MCP-1 is produced by synovial cells and infiltrating monocytes, plasma MCP-1 concentrations correlate with swollen joint count, and elevated serum MCP-1 concentrations were found in juvenile RA in patients with active disease. Modulation of MCP-1 signalling in experimental RA showed beneficial effects on inflammation and joint destruction. With respect to chronic neuroinflammation, a critical role for MCP-1 has been established in animal models for multiple sclerosis. In acute neuroinflammation, experimental evidence for a detrimental role of MCP-1 in stroke and excitotoxic injury has been found. Several selective small molecular weight CCR-2B antagonists and MCP-1-blocking antibodies have been described. The proof for the validity of targeting MCP-1 signalling in disease, however, has yet to be established in clinical trials.

Similar time-course of interleukin-1 beta production and extracellular-signal-regulated kinase (ERK) activation in permanent focal brain ischemic injury.

The present study investigated the activation of extracellular-signal-regulated kinase (ERK) and the potential role of interleukin-1 beta (IL-1beta) in the brain's response to focal brain ischemia in the permanent middle cerebral artery occlusion (pMCAO) model. Phosphorylated ERK p44 and p42 were increased time-dependently and significantly 18- and 28-fold, respectively, at 24-h post-pMCAO. Similarly, IL-1beta protein levels were significantly increased with the peak at 24 h in the lesioned core of the ischemic hemisphere compared to the contralateral side. Previous studies using various stimuli have shown ERK-dependent IL-1 induction. The results from our study suggest that this relation may also exist in vivo in ischemic brain tissue. Based on the progressive nature of IL-1 induction, we hypothetized that inhibition of interleukin-converting enzyme (ICE) could provide an extended time-window for neuroprotection. Therefore, we applied N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD x fmk), an ICE blocker 3 or 6 h after pMCAO. Reductions of infarct volume, however, were not observed. Taken together with previous results, where we showed protective activity of zVAD x fmk when given immediately after pMCAO, we conclude that the time window for zVAD x fmk is less than 3 h.

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.