A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis.

Early signs of inflammatory demyelination include entry of fibrin(ogen) into the central nervous system (CNS), which is normally excluded by the blood-brain barrier, and up-regulation of components of the plasminogen activator system. Using mice deficient in tissue-type plasminogen activator (tPA-/-) and urokinase plasminogen activator receptor (uPAR-/-), we investigated the involvement of the PA system on the clinical and pathological features of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. tPA-/- mice suffered an early and a more severe acute disease characterized by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of plasminogen activator inhibitor-1. This correlated with fibrin accumulation, which co-localized with nonphosphorylated neurofilament on thickened axons in experimental allergic encephalomyelitis tissue. In contrast, uPAR-/- mice had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells. These animals developed chronic disease as a result of steadily increased inflammation, increased levels of urokinase-type plasminogen activator (uPA), and greater degree of demyelination. Thus, the plasminogen activator system can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA and uPAR on fibrinolysis and cell adhesion/migration, manipulation of which may have therapeutic implications for multiple sclerosis.

tPA receptors and the fibrinolytic response in multiple sclerosis lesions.

Axonal damage in multiple sclerosis (MS) lesions is associated with failure of fibrinolysis because of the inhibition of the plasminogen activator system. Plasma membrane receptors for tissue plasminogen activator (tPA) and plasminogen concentrate proteolytic activity on the cell surface and provide protection from inhibitors that in turn may locally enhance the fibrinolytic response. Therefore, we have investigated expression of two of these receptors in MS lesions, annexin II tetramer (AIIt) and low-density lipoprotein receptor-related protein (LRP). In acute MS lesions both AIIt and LRP were immunolocalized on macrophages and astrocytes while LRP was additionally found on neuronal cells in cortical gray matter. Western blot analysis confirmed a significant increase in AIIt in MS lesions and in a proportion of normal-appearing white matter samples, with a highly significant correlation between annexin II levels and factors associated with impeded fibrinolysis, such as plasminogen activator inhibitor-1. Immunoblotting analysis of plasmin(ogen) revealed increased levels of lysine-plasminogen in samples expressing high AIIt protein levels. Our results suggest that limited availability of tPA in MS lesions because of formation of tPA-plasminogen activator inhibitor-1 complexes reduces capability of tPA receptors to generate plasmin, which further diminishes fibrinolytic capacity in active MS lesions and possibly leads to axonal damage.

Sodium channels contribute to microglia/macrophage activation and function in EAE and MS.

Loss of axons is a major contributor to nonremitting deficits in the inflammatory demyelinating disease multiple sclerosis (MS). Based on biophysical studies showing that activity of axonal sodium channels can trigger axonal degeneration, recent studies have tested sodium channel-blocking drugs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and have demonstrated a protective effect on axons. However, it is possible that, in addition to a direct effect on axons, sodium channel blockers may also interfere with inflammatory mechanisms. We therefore examined the novel hypothesis that sodium channels contribute to activation of microglia and macrophages in EAE and acute MS lesions. In this study, we demonstrate a robust increase of sodium channel Nav1.6 expression in activated microglia and macrophages in EAE and MS. We further demonstrate that treatment with the sodium channel blocker phenytoin ameliorates the inflammatory cell infiltrate in EAE by 75%. Supporting a role for sodium channels in microglial activation, we show that tetrodotoxin, a specific sodium channel blocker, reduces the phagocytic function of activated rat microglia by 40%. To further confirm a role of Nav1.6 in microglial activation, we examined the phagocytic capacity of microglia from med mice, which lack Nav1.6 channels, and show a 65% reduction in phagocytic capacity compared with microglia from wildtype mice. Our findings indicate that sodium channels are important for activation and phagocytosis of microglia and macrophages in EAE and MS and suggest that, in addition to a direct neuroprotective effect on axons, sodium channel blockade may ameliorate neuroinflammatory disorders via anti-inflammatory mechanisms.

Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture.

Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS). Demyelination-associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies. The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism. In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor 1 knockout mice with wildtype counterparts. Aggregate formation and neurofilament/myelin basic protein accumulation were dependent on the age of foetal dissection and species used. Following treatment with interferon-gamma, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro. Cytokine treatment resulted in significant loss of the neuronal marker neurofilament-H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective. Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death. Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage. Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis.

Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger.

Although voltage-gated sodium channels are known to be deployed along experimentally demyelinated axons, the molecular identities of the sodium channels expressed along axons in human demyelinating diseases such as multiple sclerosis (MS) have not been determined. Here we demonstrate changes in the expression of sodium channels in demyelinated axons in MS, with Nav1.6 confined to nodes of Ranvier in controls but with diffuse distribution of Nav1.2 and Nav1.6 along extensive regions of demyelinated axons within acute MS plaques. Using triple-labeled fluorescent immunocytochemistry, we also show that Nav1.6, which is known to produce a persistent sodium current, and the Na+/Ca2+ exchanger, which can be driven by persistent sodium current to import damaging levels of calcium into axons, are colocalized with beta-amyloid precursor protein, a marker of axonal injury, in acute MS lesions. Our results demonstrate the molecular identities of the sodium channels expressed along demyelinated and degenerating axons in MS and suggest that coexpression of Nav1.6 and Na+/Ca2+ exchanger is associated with axonal degeneration in MS.

Remyelination of cytokine- or antibody-demyelinated CNS aggregate cultures is inhibited by macrophage supplementation.

Remyelination in CNS aggregate cultures is determined both by macrophage enrichment and the mode of demyelination. Despite the same degree of myelin loss, accumulation of MBP in anti-MOG antibody-demyelinated aggregates overtakes that of controls, while recovery is significantly delayed following IFN-gamma-induced demyelination. In antibody-treated cultures, remyelination was associated with a significant increase in culture supernatant levels of TGF-beta1, FGF-2, and PDGF-AA as well as an induction of TNF-alpha immediately following removal of the demyelinating insult. The impaired recovery in IFN-gamma-treated cultures, denoted by a significant reduction in TGF-beta1, was reversed by treatment with hrTGF-beta1. Macrophage supplementation of the cultures prior to the addition of either demyelinating agent induced a greater degree of myelin loss followed by incomplete remyelination in both cases. This failure to remyelinate was associated in both groups with a several-fold elevation in TNF-alpha and with modest increases in PDGF-AA and FGF-2 in the antibody-treated cultures. In contrast, macrophage supplementation to mature cultures in the absence of any demyelinating treatment resulted in enhanced accumulation of MBP associated with a promyelinative growth factor and TNF-alpha profile similar to that in aggregates enriched with macrophages at the outset of the culture period. Hence, effector elements of the adaptive immune response appear to override promyelinogenic in favor of proinflammatory macrophage factors in mature CNS aggregates, counteracting the potential for myelin repair.

Role for TGF-beta1, FGF-2 and PDGF-AA in a myelination of CNS aggregate cultures enriched with macrophages.

The increase in myelin basic protein (MBP) synthesis observed in brain aggregate cultures supplemented with macrophages is reflected in elevated supernatant protein levels of the key promoters of oligodendrocyte proliferation, fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA), during the premyelinating phase. Although supernatant levels of transforming growth factor-beta1 (TGF-beta1), the most abundant growth factor produced at the transcriptional and translational levels by phagocytic macrophages, were reduced at this stage, it was the only growth factor for which mRNA expression was increased significantly in macrophage-enriched cultures. TGF-beta1, which supports oligodendrocyte differentiation, was increased in the supernatant of macrophage-enriched cultures only after the onset of myelinogenesis. Hence, standard cultures treated with TGF-beta1 during the premyelinating period reproduced effects of macrophage supplementation, inducing an increase in MBP synthesis and in PDGF-AA and FGF-2 bioavailability. A similar increase in MBP synthesis in PDGF-AA treated cultures emphasises its central role in oligodendrocyte progenitor proliferation. In contrast, FGF-2 blocked MBP synthesis in the cultures. In cultures treated with anti-TGF-beta1 antibody before or after the first detection of MBP, supernatant levels of TGF-beta1, FGF-2, and PDGF-AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF-beta1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF-beta1 in oligodendrocyte development and, as a source of TGF-beta1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.

Cannabinoids inhibit neurodegeneration in models of multiple sclerosis.

Multiple sclerosis is increasingly being recognized as a neurodegenerative disease that is triggered by inflammatory attack of the CNS. As yet there is no satisfactory treatment. Using experimental allergic encephalo myelitis (EAE), an animal model of multiple sclerosis, we demonstrate that the cannabinoid system is neuroprotective during EAE. Mice deficient in the cannabinoid receptor CB1 tolerate inflammatory and excitotoxic insults poorly and develop substantial neurodegeneration following immune attack in EAE. In addition, exogenous CB1 agonists can provide significant neuroprotection from the consequences of inflammatory CNS disease in an experimental allergic uveitis model. Therefore, in addition to symptom management, cannabis may also slow the neurodegenerative processes that ultimately lead to chronic disability in multiple sclerosis and probably other diseases.

Interleukin-12 induces mild experimental allergic encephalomyelitis following local central nervous system injury in the Lewis rat.

The major pathological feature in the central nervous system (CNS) following traumatic brain injury is activation of microglia both around and distant from the injury site. Intraperitoneal administration of interleukin-12 (IL-12) after brain injury resulted in a 7% weight loss, clinical signs of mild EAE and significant myelin basic protein (MBP)-specific splenic cell proliferation. The extent of pathology, in terms of the number of inflammatory perivascular cuffs and activation of microglia was greatest if IL-12 was administered immediately compared to a week following brain injury, whether at one or two sites. Specifically immunostaining for MHC class II and iNOS on macrophages and microglia, ICAM-1 on endothelial cells and macrophages was observed around the site of injury. A degree of myelin processing was apparent from immunostaining of MBP in inflammatory cells distant from the lesion. Inflammatory cuffs comprising macrophages, activated microglia, CD4(+) T cells and iNOS(+) cells were also detected distant to the injury site in the medulla and spinal cord of animals treated with IL-12. These results suggest that immune-mediated events in which IL-12 production is stimulated as for example viral infection, superimposed on a brain injury, could provide a trigger for a MS-like pathology.

Impaired fibrinolysis in multiple sclerosis: a role for tissue plasminogen activator inhibitors.

Tissue plasminogen activator (tPA), a neuronal as well as the key fibrinolytic enzyme, is found concentrated on demyelinated axons in multiple sclerosis lesions together with fibrin(ogen) deposits. The decreased tPA activity in normal-appearing white and grey matter and lesions of multiple sclerosis is reflected in diminished fibrinolysis as measured by a clot lysis assay. Nonetheless, peptide products of fibrin, including D-dimer, accumulate on demyelinated axons-the result of fibrinogen entry through a compromised blood-brain barrier (BBB). Analysis of tissue samples on reducing and non-reducing polyacrylamide gels demonstrates complexes of tPA with plasminogen activator inhibitor-1 (PAI-1) but not with neuroserpin, a tPA-specific inhibitor concentrated in grey matter. As total tPA protein remains unchanged in acute lesions and the concentration of PAI-1 rises several fold, complex formation is a probable cause of the impaired fibrinolysis. Although the tPA-plasmin cascade promotes neurodegeneration in excitotoxin-induced neuronal death, in inflammatory conditions with BBB disruption it has been demonstrated to have a protective role in removing fibrin, which exacerbates axonal injury. The impaired fibrinolytic capacity resulting from increased PAI-1 synthesis and complex formation with tPA, which is detectable prior to lesion formation, therefore has the potential to contribute to axonal damage in multiple sclerosis.

Annexin II/p11 is up-regulated in Purkinje cells in EAE and MS.

The sensory neuron specific sodium channel Na(v)1.8 is normally detectable at only very low levels within cerebellar Purkinje cells. Annexin II light chain (p11) binds to the amino terminus of Na(v)1.8 and facilitates its functional expression within the cell membrane. We previously demonstrated that expression of Na(v)1.8 is up-regulated in cerebellar Purkinje cells in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS). In this study we demonstrate that expression of p11 is significantly up-regulated in Purkinje cells in EAE (71 +/- 9.0% vs 21.3 +/- 4.9% in controls) and in MS(65.5 +/- 1.6% vs 21.8 +/- 6.2% in controls). We also demonstrate a high degree of co-expression of p11 and Na(v)1.8 (84.8 +/- 8.9%). Together with earlier results which show that experimental expression of Na(v)1.8 within Purkinje cells perturbs the temporal pattern of impulse generation in these cells, our results extend the evidence for an acquired channelopathy which interferes with cerebellar function in MS.

A role for caspase-1 and -3 in the pathology of experimental allergic encephalomyelitis : inflammation versus degeneration.

Axonal loss, already present in the acute and first relapse phases of experimental allergic encephalomyelitis (EAE) in the ABH mouse, only becomes apparent in the third relapse in the interleukin-12 model of relapsing EAE in the Lewis rat. Caspase-1 immunostaining in the spinal cord of Lewis rats was mainly localized to inflammatory cuffs with the greatest proportion of active caspase-1-positive cells detected during the first and second relapses, correlating with enzyme activity and protein on Western blots. However, in the spinal cord of ABH mice during acute EAE, caspase-1 immunostaining was localized both on inflammatory and neuronal cells, again correlating with enzyme activity and protein production. In contrast, caspase-3 expression in the spinal cord of Lewis rats did not increase significantly until the third relapse when inflammatory and neuronal cells and axons became positive in line with a significant increase in caspase activity. In ABH mice active caspase-3 was already immunolocalized on axons and apoptotic neurons in the spinal cord during the acute stage of EAE. Because caspase-3 is a downstream cell death signal it may be possible to reduce apoptosis by selectively blocking caspase-3 and therefore provide a therapeutic target for EAE and potentially, multiple sclerosis.

Extracellular proteolysis in brain injury and inflammation: role for plasminogen activators and matrix metalloproteinases.

The role of intracellular proteases (e.g., calpains and caspases) in the pathophysiology of neuronal cell death has been extensively investigated. More recently, accumulating data have suggested that extracellular proteolysis also plays a critical role. The two major systems that modify the extracellular matrix in brain are the plasminogen activator (PA) and matrix metalloproteinase (MMP) axes. This Mini-Review delineates major pathways of PA and MMP action after stroke, brain trauma, and chronic inflammation. Deleterious effects include the disruption of blood-brain barrier integrity, amplification of inflammatory infiltrates, demyelination, and possibly interruption of cell-cell and cell-matrix interactions that may trigger cell death. In contrast, PA-MMP actions may contribute to extracellular proteolysis that mediates parenchymal and angiogenic recovery after brain injury. As the mechanisms of deleterious vs. potentially beneficial PA and MMP actions become better defined, it is hoped that new therapeutic targets will emerge for ameliorating the sequelae of brain injury and inflammation.