M. Louise Cuzner

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.

Expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.

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.

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.

Role for TGF-β1, 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‐β1 (TGF‐β1), 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‐β1, 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‐β1 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‐β1 antibody before or after the first detection of MBP, supernatant levels of TGF‐β1, FGF‐2, and PDGF‐AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF‐β1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF‐β1 in oligodendrocyte development and, as a source of TGF‐β1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.

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.

Temporal analysis of growth factor mRNA expression in myelinating rat brain aggregate cultures: Increments in CNTF, FGF‐2, IGF‐I, and PDGF‐AA mRNA are induced by antibody‐mediated demyelination

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet‐derived growth factor‐AA (PDGF‐AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor‐2 (FGF‐2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor β1 (TGF‐β1), insulin‐like growth factor‐I (IGF‐I), and neurotrophin‐3 (NT‐3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti‐myelin oligodendrocyte glycoprotein (anti‐MOG) antibody resulted in a significant increase in MBP peptide fragments with a C‐terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF‐2 and IGF‐I, and of PDGF‐AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration. GLIA 30:342–351, 2000.

Myelin/Axonal Pathology in Interleukin-12 Induced Serial Relapses of Experimental Allergic Encephalomyelitis in the Lewis Rat

Lewis rats, on recovery from monophasic clinical experimental allergic encephalomyelitis (EAE), can be induced to develop repeated paralytic relapses with a graded reduction in clinical severity following intraperitoneal administration of IL-12. By the time of the third relapse, the number and size of inflammatory cuffs in the spinal cord were reduced with the makeup of the cellular infiltrate shifting to a significantly increased number of B cells. Serum levels of myelin basic protein (MBP)-specific IgG1 and IgG2b were found to rise over time while MBP and MBP peptide-positive macrophages and microglia became evident in perivascular cuffs and in spinal cord parenchyma, indicative of myelin phagocytosis. Axonal death was observed in semithin and EM sections of spinal cord in third relapse animals in association with iNOS and tPA immunostaining throughout gray and white matter. These neurotoxic or excitotoxic agents may contribute to axonal damage directly or indirectly by activated microglia and macrophages, leading to limited damage of the axonal-myelin unit.

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‐γ‐induced demyelination. In antibody‐treated cultures, remyelination was associated with a significant increase in culture supernatant levels of TGF‐β1, FGF‐2, and PDGF‐AA as well as an induction of TNF‐α immediately following removal of the demyelinating insult. The impaired recovery in IFN‐γ‐treated cultures, denoted by a significant reduction in TGF‐β1, was reversed by treatment with hrTGF‐β1. 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‐α 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‐α 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.

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‐γ, 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.