Showing papers in "Glia in 1993"

Cytotoxicity of microglia

Abstract: The most characteristic property of microglia is their swift activation in response to neuronal stress and their capacity for site-directed phagocytosis. The transformation of microglia into intrinsic brain macrophages appears to be under strict control and takes place if neuronal and/or terminal degeneration occurs in response to nerve lesion. The differentiation of microglia into brain macrophages is accompanied by the release of several secretory products, e.g., proteinases, cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates. Interference with the microglial activation or the productions of cytotoxic metabolites by microglia may thus offer new therapeutic opportunities for the prevention of neuronal cell death in CNS disease.

Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer's disease

Abstract: Microglia are associated with central nervous system (CNS) pathology of both Alzheimer's disease (AD) and the acquired immunodeficiency syndrome (AIDS). In AD, microglia, especially those associated with amyloid deposits, have a phenotype that is consistent with a state of activation, including immunoreactivity with antibodies to class II major histocompatibility antigens and to inflammatory cytokines (interleukin-1-beta and tumor necrosis factor-alpha). Evidence from other studies in rodents indicate that microglia can be activated by neuronal degeneration. These results suggest that microglial activation in AD may be secondary to neurodegeneration and that, once activated, microglia may participate in a local inflammatory cascade that promotes tissue damage and contributes to amyloid formation. In AIDS, microglia are the primary target of retroviral infection. Both ramified and ameboid microglia, in addition to multinucleated giant cells, are infected by the human immunodeficiency virus (HIV-1). The mechanism of microglial infection is not known since microglia lack CD4, the HIV-1 receptor. Microglia display high affinity receptors for immunoglobulins, which makes antibody-mediated viral uptake a possible mechanism of infection. In AIDS, the extent of active viral infection and cytokine production may be critically dependent upon other factors, such as the presence of coinfecting agents. In the latter circumstance, very severe CNS pathology may emerge, including necrotizing lesions. In other circumstances, HIV infection of microglia probably leads to CNS pathology by indirect mechanisms, including release of viral proteins (gp120) and toxic cytokines. Such a mechanism is the best hypothesis for the pathogenesis of vacuolar myelopathy in adults and the diffuse gliosis that characterizes pediatric AIDS, in which very little viral antigen can be detected.

The origin and nature of ramified and amoeboid microglia: a historical review and current concepts.

Abstract: The origin of ramified microglia has been a longstanding controversial issue, with 4 major schools of thought, which state that they are derived (1) from invasion of mesodermal pial elements, (2) from neuroectodermal matrix cells together with the macroglia, (3) from pericytes, and (4) from invasion of monocytes in early development. This paper is in support of the last-mentioned hypothesis. It is known that ramified microglial cells do not divide under normal circumstances, and since our studies in the corpus callosum have shown that these cells do not appear until the fifth postnatal day, it is reasoned that they must be derived from some preexisting mitotically active cells. The putative precursor is the preponderant amoeboid microglia in the same region. Our experimental studies with the carbon labelling technique have demonstrated for the first time that blood monocytes invade into the early postnatal brain to become amoeboid microglia, which then differentiate into ramified microglia. Just like other tissue macrophages, the monocyte-derived amoeboid microglia exhibit features indicative of phagocytic activities. These include the content of hydrolytic enzymes, uptake of carbon, and a characteristic surface morphology, as seen by scanning electron microscopy. The transformation of amoeboid microglia into ramified microglia, which occurs between the second and third postnatal week, is considered to be a regressive phenomenon, as shown by the diminution of their content of hydrolytic enzymes and the downregulation of membrane antigen. Apart from their primary role as active phagocytes, their involvement in Alzheimer's disease (AD) is evidenced recently by the fact that the cells are specifically marked by antibodies present in the cerebrospinal fluid of AD patients. In conclusion, ramified microglial cells are derived from monocytes, but through an intermediate amoeboid microglia stage as active macrophages in the perinatal period.

Microglia in degenerative neurological disease

Abstract: Microglia express many leukocyte surface antigens which are upregulated in such chronic degenerative neurological diseases as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). These surface antigens include leukocyte common antigen, immunoglobulin Fc receptors, MHC class I and class II glycoproteins, β2-integrins, and the vitronectin receptor. Ligands for these receptors are also found. They include immunoglobulins, complement proteins of the classical pathway, T lymphocytes of the cytotoxic/suppressor and helper/inducer classes, and vitronectin. T lymphocytes marginate along capillary venules, with some penetrating into the tissue matrix. Immunoglobulins and complement proteins are synthesized locally in brain, although they may also come from the bloodstream if the blood-brain barrier is compromised. The membrane attack complex, which is formed from C5b-9, the terminal components of complement, has been identified in AD and multiple sclerosis brain tissue. In addition, proteins designed to defend against bystander lysis caused by the membrane attack complex, including protectin, C8 binding protein, clusterin, and vitronectin, are associated with damaged neuronal processes in AD. Autodestruction may play a prominent part in these 2 diseases.

Altered antigen expression of microglia in the aged rodent CNS

Abstract: Microglia, the resident macrophages of the central nervous system, are characterised by a highly specialized morphology and unusual antigenic phenotype. Microglia appear to be downregulated by their microenvironment when compared to other tissue macrophages. We have studied the microglia in brains of healthy, aged rats with a panel of monoclonal antibodies. We have found that microglia in the brains of these aged rats express antigens that are downregulated or absent from microglia of juvenile rats. The stimuli which give rise to this upregulated phenotype are not known. Age related changes in the phenotype of microglia should be taken into account when considering the possible role of microglia in neuropathological conditions.

Bone marrow derived elements and resident microglia in brain inflammation

Abstract: Infection of the central nervous (CNS) system by the human immunodeficiency virus (HIV) depends on the migration of infected hematogenous cells into the brain. We thus used quantitative light and electron microscopic immunocytochemistry to study the homing and turnover of bone marrow derived cells in the CNS in radiation bone marrow chimeras under normal conditions and in experimental autoimmune encephalomyelitis (EAE) as an experimental model of brain inflammation. Our studies suggest the following conclusions. First, the central nervous system is continuously patrolled by a small number of T-lymphocytes and monocytes. Meningeal and perivascular monocytes are slowly replaced by hematogenous cells under normal conditions, and this turnover is accelerated in the course of inflammation. In contrast, resident microglia represent a very stable cell pool, which in adult animals is only exceptionally replaced by hematogenous cells, even after recovery from severe brain inflammation. Second, although in bone-marrow-chimeric animals resident microglia, astrocytes, and ependymal cells are not able to present antigen to Lewis T-lymphocytes, the inflammatory reaction in EAE is qualitatively and quantitatively similar in these animals compared to fully histocompatible Lewis rats. Finally, resident microglia express the macrophage activation antigen ED1. Thus, microglia cells appear to function as effector cells in EAE lesions.

Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain.

Abstract: The nature and function of previously described perineuronal nets are still obscure. In the present study their polyanionic components were demonstrated in the rat brain using colloidal iron hydroxide (CIH) staining. In subcortical regions, such as the red nucleus, cerebellar, and vestibular nuclei, most neurons were ensheathed by CIH-binding material. In the cerebral cortex perineuronal nets were seen around numerous nonpyramidal neurons. Biotinylated hyaluronectin revealed that hyaluronan occurs in perineuronal nets. Two plant lectins [Wisteria floribunda agglutinin (WFA) and Vicia villosa agglutinin (VVA)] with affinity for N-acetylgalactosamine visualized perineuronal nets similar to those rich in anionic components. Glutamic acid decarboxylase (GAD)-immunoreactive synaptic boutons were shown to occupy numerous meshes of perineuronal VVA-positive nets. Electron microscopically, VVA binding sites were scattered throughout perisynaptic profiles, but accumulated at membranes and in the extracellular space except not in synaptic clefts. To investigate the spatial relationship between glial cell processes and perineuronal nets, two astrocytic markers (S100-protein and glutamine synthetase) were visualized at the light and electron microscopic level. Two methods to detect microglia by the use of Griffonia simplicifolia agglutinin (GSA I-B4) and the monoclonal antibody, OX-42, were also applied. Labelled structures forming perineuronal nets were observed with both astrocytic, but not with microglial, markers. It is concluded that perineuronal nets are composed of a specialized type of glia-associated extracellular matrix rich in polyanionic groups and N-acetylgalactosamine. The net-like appearance is due to perisynaptic arrangement of the astrocytic processes and these extracellular components. Similar to the ensheathment of nodes of Ranvier, perineuronal nets may provide a special ion buffering capacity required around various, perhaps highly active, types of neurons.

Reactive glia as rivals in regulating neuronal survival.

Abstract: Reactive gliosis is a response noted after nearly every type of CNS injury and involves both activated microglia and astroglia. Although many investigators believe that reactive glia in some way regulate the survival of injured neurons, the influence of glial elements upon damaged neural tissues remains uncertain. To examine relationships between reactive glia and neurons, secretion products from both microglia and astroglia are tested for their effects upon the survival of cultured neurons. Microglia are found to secrete neurotoxic agents, while astroglia are a source of neuronotrophic factors. Similar patterns of soluble factor production are noted for astroglia-rich or microglia-rich regions of rat neocortex damaged by ischemia. These observations suggest that microglia and astroglia compete for control of neuronal survival. Importantly, microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.

Development and differentiation of glial precursor cells in the rat cerebellum

Abstract: The development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin-sulfate proteoglycan, a cell-surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes. Cells expressing the NG2 antigen appeared in the cerebellum during the last 3-4 days of embryonic life. Over the first 10 days of postnatal life, the NG2-labeled cells incorporated 3H-thymidine into their nuclei and their total number increased. At all ages examined, the NG2-labeled cells did not contain either vimentin-like or glial fibrillary acidic protein (GFAP)-like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2-labeled cells of embryonic animals expressed GD3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2-positive cells of postnatal animals did not express GD3 immunoreactivity. Nevertheless, the NG2-labeled cells of the nascent white matter expressed oligodendrocyte-specific marker antigens. Cells lying outside of the white matter continued to express the NG2 antigen. In adult animals, the NG2-labeled cells incorporated 3H-thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.

Characterization of microglia and macrophages in the central nervous system of rats: Definition of the differential expression of molecules using standard and novel monoclonal antibodies in normal CNS and in four models of parenchymal reaction

Abstract: This report describes the development of a new panel of monoclonal antibodies produced following immunization of mice with cultured rat microglial cells. Using these new reagents and previously defined antibodies that bind to microglia or macrophages, the responses of parenchymal microglia, perivascular "microglial" cells, and infiltrating macrophage/monocytes were examined in 4 divergent models of central nervous system reaction. These were brain abscess, experimental allergic encephalomyelitis, Wallerian degeneration, and stab wound. No single new antibody was specific only for microglia; all antibodies positively staining microglial cells also labeled various subsets of macrophage/monocytic cells in normal tissues of the immune system. Moreover, the results indicate that microglia are capable of different levels and a variety of types of response, as defined by the molecules they elaborate. These findings suggest that these CNS resident cells belong to the extended monocyte/macrophage/dendritic cell family and that they do not respond in a stereotypic manner to all forms of CNS insult.

Inducible nitric oxide synthase activity of cloned murine microglial cells

Abstract: Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M phi) could be induced to synthesize nitrite (NO2-), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M phi, these clones were found to release high levels of NO2- in response to recombinant interferon-gamma (rIFN-gamma) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-alpha (rTNF-alpha). As previously demonstrated for M phi, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO2- production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M phi-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Abstract: Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca2+]i) that was communicated to surrounding cells. Following propagation of the Ca2+ wave, many cells showed asynchronous oscillations in [Ca2+]i. Dantrolene sodium (10 microM) inhibited the increase in [Ca2+]i associated with this Ca2+ wave by 60-80%, and prevented subsequent Ca2+ oscillations. Despite the markedly decreased magnitude of the increase in [Ca2+]i, the rate of propagation and the extent of communication of the Ca2+ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 microM) induced an initial increase in [Ca2+]i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca2+]i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of Ca2+ wave from the stimulated cell to adjacent cells. Glutamate (50 microM) induced an initial increase in [Ca2+]i in most cells that was followed by sustained oscillations in [Ca2+]i in some cells. Dantrolene (10 microM) inhibited this initial [Ca2+]i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 micron) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca2+ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca2+ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP3) mediates the propagation of Ca2+ waves whereas Ca(2+)-induced Ca2+ release amplifies Ca2+ waves and generates subsequent Ca2+ oscillations.

Electrophysiological behavior of microglia.

Abstract: The present knowledge of voltage- and ligand-activated ion channels of cultured microglial cells is described and its relevance is discussed. All microglial cells cultured from rat or mouse brain express an inward rectifying K+ channel but no outward currents. This expression is not changed by the length of the cultivation period, nor is it different in freshly isolated cells. It makes the microglial cells distinct from peritoneal macrophages, which possess an outward rectifying K+ channel. In bone marrow, 2 populations of cells could be distinguished electrophysiologically, one with the channel pattern of macrophages and one with that of microglial cells. This finding is interesting in light of the fact that it is presently hypothesized that the differentiation of monocytes into microglia takes place exclusively during embryonic development but not in the adult. The available data thus support the hypothesis that within the bone marrow a population of macrophage precursor cells exists with a possible lineage relationship to brain macrophages. The lack of outward currents in the microglial cells has the functional consequence that even a small inward current leads to a large membrane depolarization, since K+ outward currents are not activated with the depolarization. The microglial cell is thus very sensitive to depolarizing events. We found that ATP induced an inward current and an increase in the conductance, whereas ADP, AMP, and adenosine did not. These relative potencies indicate that microglia possess a P2 purinoceptor linked to an ion channel. The amplitude of the inward current elicited by ATP is about 80 pA and is sufficient to depolarize microglial cells close to 0 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of microglial and perivascular cell populations: Insights gained from the facial nucleus paradigm

Abstract: We reflect here on the development of a neuroimmunological concept which has been formulated over the past 5 years through studying microglial cell responses in the facial nerve system. A simple axotomy of the adult rat facial nerve which causes regeneration of facial motor neurons and little, if any, cell death can activate microglial cells just as easily as a full-blown degeneration of the entire nucleus induced by toxic ricin. In both instances, the prompt microglial reaction is characterized by a series of structural and phenotypic changes which are in many ways similar to an immune response, e.g., there is cell proliferation and upregulation of MHC antigens. However, since white blood cells do not participate in the retrograde response of facial motor neurons, we have adopted a notion which views microglia as a CNS-wide network of immunocompetent cells whose morphological dissimilarities from leukocytes are a result of their unique adaptation to the CNS architecture. We have continued our in vivo investigations of the phagocytic and immunophenotypic properties of microglial and perivascular cells during the retrograde reaction of facial motor neurons by using intra-neural injections of fluorogold (FG) and ricin followed by lectin and immunostaining for microglia. Two new findings can be added to the microglial neuroimmune network: (1) Microglia take up FG only after motor neuron degeneration, whereas perivascular cells may take up FG under nondegenerating conditions. (2) Immunologically important molecules, such as MHC class II, CD4, and leukocyte common antigens, are expressed by different microglial subpopulations. Thus there is functional and phenotypic heterogeneity among immunocompetent cells of the CNS.

Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus.

Abstract: By taking advantage of the specific neuronal and connective organization of the hippocampus and the different susceptibility of hippocampal neurons to transient cerebral ischemia or intraventricular injections of kainic acid (KA), we examined the microglial reactions to different types of neuronal injury. In all areas with neuronal or axonal degeneration, the microglial cells reacted by specific degeneration-related morphological transformations and expression of class I major histocompatibility complex (MHC) antigen. Subpopulations of microglial cells also expressed class II MHC antigen and leukocyte common antigen (LCA) in relation to (1) degenerating nerve cell bodies in the dentate hilus and the CA1 and CA3 pyramidal cell layers, (2) postischemic degeneration of dendrites in the stratum radiatum of CA1, and (3) combined dendritic and axonal degeneration in the stratum radiatum of the KA-lesioned CA3. MHC II and LCA expression was not observed in relation to degeneration of the CA3-derived Schaffer collaterals in CA1 after KA-induced CA3 lesions. In the case of ischemia the degeneration-related reactions were preceded by an early, generalized microglial reaction, which also included areas without subsequent signs of neural degeneration. This reaction, which was transient and characterized by subtle morphological changes and induction of class I MHC antigen only, was presumably triggered by a general postischemic pertubation of the cerebral microenvironment, and not by actual neural degeneration. In conclusion, we found that microglial expression of class I MHC antigen was a sensitive marker of both the general pertubation after ischemia and axonal degeneration distant from the areas of actual nerve cell death. Induction of microglial LCA and class II MHC antigen expression, together with protracted expression of class I, was, characteristic of a protracted reaction, only found in areas with degeneration of nerve cell bodies and dendrites.

Distinct effects of bFGF and PDGF on oligodendrocyte progenitor cells

Abstract: We have compared the effects of platelet-derived (PDGF) and basic fibroblast (bFGF) growth factors on the shape, migration, and differentiation of oligodendrocyte progenitor cells, the precursors of myelin-forming cells in the CNS. In the presence of bFGF, oligodendrocyte progenitors purified from rat neonatal brain cultures were stellate, non-motile, and had a morphological complexity of 1.26 +/- 0.03 as measured by fractal dimension (D). These cells expressed transcripts encoding the POU-homeodomain transcription factor Oct-6, but not myelin genes. Upon addition of PDGF, bFGF-treated cells became motile and twofold less complex in shape (D = 1.19 +/- 0.03). These changes occurred within 6 +/- 4 h and were dependent on de novo transcription and translation, but not DNA synthesis. Upon removal of PDGF the cells reverted to their stellate shape (D = 1.26). Removal of both bFGF and PDGF resulted in oligodendrocyte differentiation after 3 days, with a fourfold increase in complexity of shape (D = 1.55 +/- 0.08), loss of Oct-6 transcripts, and gain of myelin transcripts. Thus PDGF is both necessary and sufficient to induce a motile state in progenitor cells growing in the presence of bFGF. Together with our previous data (McKinnon et al.: Neuron 5:603, 1990), our results suggest that bFGF and PDGF may control distinct phases of proliferation and migration of oligodendrocyte progenitor cells in vivo.

Early and rapid de novo synthesis of Alzheimer beta A4-amyloid precursor protein (APP) in activated microglia.

Abstract: Upon acute activation, microglia, the immuneffector cells of the brain parenchyma, express the amyloid precursor protein (APP) that is otherwise prominent in pathological structures related to Alzheimer's disease. In this disease complex amyloidbearing neuritic plaques contain βA4-amyloid protein, the APP, and numerous inflammatory proteins. The accompanying activation of microglia has mostly been viewed as a secondary reaction to amyloid deposits. Activation of microglia was performed in a graded fashion. Transection of peripheral nerves such as the facial or sciatic nerve causes a microglial reaction within hours in the nucleus of origin or in projection areas of the CNS. A predominantly glial up-regulation of APP mRNA and protein could be detected as early as 6 h post lesion not only at the site of affected neuronal cell bodies but also in corresponding projection areas. Its time course suggests rapid transneuronal signalling to glial cells in the projection area. Light and electron microscopy demonstrate that microglia, which are cells of mononuclear phagocyte lineage and comprise up to 20% of all glial cells, are the dominant source for non-neuronal APP expression. Ultrastructurally, brain perivascular cells within the basal lamina constitutively express APP and thus are a possible source of vascular amyloid. Additionally, microglia express leukocyte-derived (L)-APP mRNA and protein that have recently been described in mononuclear cells of the immune system. Increased L-APP expression may serve as a potential marker for glial/microglial activation. Such immune-mediated amyloidogenesis initiated by microglia might have implications for the treatment of neurodegenerative diseases. © 1993 Wiley-Liss, Inc.

Intracellular pH regulation in single cultured astrocytes from rat forebrain.

Abstract: We used the fluorescent pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3− -free medium buffered to pH 7.40 with HEPES at 37°C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3− caused the steady-state pHi to increase by an average of 0.35, suggesting the presence of a HCO3− -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3−-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pHi to fall to a value substantially below the initial one. pHi spontaneously recovered from this acid load, stabilizing at a value ∼ 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na+ caused pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na+-dependent/HCO3−-independent acid-extrusion mechanism; the final pHi after returning Na+ was ∼ 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Increasing [K+]− from 5 to 25 mM caused pHi to increase reversibly by ∼ 0.2 in nominally CO2/HCO3−-free solutions, and by ∼ 0.1 in CO2/HCO3−-containing solutions, although the initial pHi was ∼ 0.17 higher in the presence of CO2/HCO3-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO3− dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pHi is sensitive to changes in either membrane voltage or [K+]0 per se. © 1993 Wiley-Liss, Inc.

Expression of Janusin (J1-160/180) in the retina and optic nerve of the developing and adult mouse

Abstract: We have analyzed the expression of the oligodendrocyte-derived extra-cellular matrix molecule janusin (previously termed J1-160/180) in the retina and optic nerve of developing and adult mice using indirect light and electron microscopic immunocytochemistry, immunoblot analysis, and enzyme-linked immunosorbent assay. In the optic nerve, janusin is not detectable in neonatal and only weakly detectable in 7-day-old animals. Expression is at a peak in 2- or 3-week-old animals and subsequently decreases with increasing age. In the retina, expression increases until the third postnatal week and then remains at a constant level. In immunocytochemical investigations at the light microscopic level, janusin was found in the myelinated regions of the nerve with spots of increased immunoreactivity possibly corresponding to an accumulation of the molecule at the nodes of Ranvier. At the electron microscopic level, contact sites between unmyelinated axons, between axons and glial cells, and between axons and processes of myelinating oligodendrocytes were immunoreactive. Cell surfaces of astrocytes at the periphery of the nerve and forming the glial-limiting membrane, in contrast, were only weakly immunopositive or negative. In cell cultures of young postnatal mouse or rat optic nerves, oligodendrocytes and type-2 astrocytes, but not type-1 astrocytes were stained by janusin antibodies. In the oligodendrocyte-free retina, janusin was detectable in association with neuronal cell surfaces, but not with cell surfaces of Muller cells or retinal astrocytes. Our observations indicate that expression of janusin in the optic nerve and in the retina is developmentally differentially regulated and that other cell types, in addition to oligodendrocytes, express the molecule. Since the time course of janusin expression in the optic nerve coincides with the appearance of oligodendrocytes and myelin and since janusin is associated with cell surfaces of oligodendrocytes and outer aspects of myelin sheaths and is concentrated at nodes of Ranvier, we suggest that janusin is functionally involved in the process of myelination.

Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L.

Abstract: The activation and differentiation of microglia is a prominent pathophysiological process in numerous inflammatory and demyelinating diseases of the central nervous system, including Alzheimer's disease and the AIDS encephalopathy. The tissue damage during these diseases has partly been attributed to lipid peroxidating reactive oxygen intermediates for which activated microglia are a major source. The destruction of tissue may also involve the release of proteolytic enzymes, such as the lysosomal cysteine proteinases cathepsin B and L, which are present notably in phagocytic cells. The cathepsins B and L are endopeptidases with a substrate specificity including important proteins, like myelin basic protein, extracellular matrix components, or the class II major histocompatibility complex. Because of this pathophysiological relevance the cathepsins B and L were chosen for histochemical demonstration in isolated and cultured rat microglia and measurement by a new flow cytometric method. Cathepsin B/L activity was measured flow cytometrically in single viable cells by the intracellular cleavage of non-fluorescent (Z-Phe-Arg)2-rhodamine 110 to the green fluorescent monoamide Z-Phe-Arg-rhodamine 110 and rhodamine 110. In microglia we measured a cathepsin B/L activity that was 2.5 times higher than in thioglycolate-elicited, i.e., inflammatory peritoneal rat macrophages. In elicited peritoneal macrophages the formation of fluorescent product was 6.2 times higher than in unstimulated resident peritoneal macrophages, demonstrating that the activation and differentiation of mononuclear phagocytes is accompanied by an increased cathepsin B/L enzyme activity. The subcellular localization of cathepsin B/L activity in plated viable microglia was demonstrated histochemically by the use of Z-Ala-Arg-Arg-4-methoxy-2-naphthylamide. Its blue fluorescent cleavage product 4-methoxy-2-naphthylamide was found in lysosomes. Our study shows that activated microglia are an important potential source of cathepsin B/L. This is particularly interesting as enzymatically active cathepsins have recently been found extracellularly at high levels in the senile plaques of Alzheimer's disease, which are known to contain many activated microglia. The release of proteinases by microglia may play a crucial role in the pathomechanism of tissue-destructing diseases in the brain.

The molecular genetics of myelination: an update.

Abstract: Recent molecular genetic studies have provided new insights into the structure and function of 2 of the major integral membrane proteins of myelin--the proteolipid protein (PLP) and protein zero (P0)--and have uncovered a third such protein--PMP22/gas3 The rumpshaker mouse has been shown to carry a point mutation in the PLP gene that uncouples a deleterious effect on CNS myelin assembly, which these mice exhibit, from oligodendrocyte degeneration and cell death, which they do not The developmental importance of the P0 protein in PNS myelination has been dramatically demonstrated by the analysis of loss-of-function mutations engineered through the expression of antisense RNA and through the insertional inactivation of the P0 gene by homologous recombination in embryonic stem cells and the generation of P0-deficient mice The cloned promoter of the P0 gene has been shown to drive quantitative, Schwann cell-specific expression of heterologous genes in transgenic mice The PMP22/gas3 gene, previously cloned from fibroblast cell lines, has been found to encode an axonally regulated Schwann cell protein that is assembled into PNS myelin Importantly, this gene appears to be the target of mutations that result in the Trembler alleles in mice, and in Charcot-Marie-Tooth disease Type 1a, the most common inherited peripheral neuropathy in humans

Glial plasmalemmal vesicles: a subcellular fraction from rat hippocampal homogenate distinct from synaptosomes.

Abstract: By a Percoll density-gradient centrifugation of rat hippocampal homogenate, we found a novel subcellular fraction (specific gravity approximately 1.046 g/ml), besides synaptosomes (approximately 1.060 g/ml), which showed a high activity of Na(+)-dependent glutamate uptake. The initial rate of the glutamate uptake in this fraction was as high as twice that in synaptosomes. Activities of choline acetyltransferase and high affinity choline uptake were, on the other hand, much lower. gamma-Aminobutyric acid uptake activity was nearly equivalent in both fractions. Electron microscopic observations revealed that the fraction was morphologically different from synaptosomal or myelin fractions, but mainly consisted of two different types of empty membrane vesicles; irregular (0.3-0.8 micron in diameter) and spheroid type (0.2 micron). The immunoreactivity to glial fibrillary acidic protein was appreciably high in this fraction. The marker enzyme analysis showed the fraction was rich in plasma membranes. On the basis of these results, the fraction is termed glial plasmalemmal vesicles (GPV). We analyzed kinetically the reaction of Na(+)-dependent glutamate uptake by GPV comparing with that by synaptosomes. Km values for glutamate in GPV was 4.7 microM and Vmax was 33 nmol/mg/min, while in synaptosomes 11 microM and 17 nmol/mg/min, respectively. Hill coefficients of Na+ activation in GPV and synaptosomes were 1.1 and 2.0, respectively. Thus, the mechanism or transporter molecule in glial cells for Na(+)-dependent glutamate transport is likely to be different from that in neurons.

Monoamine oxidase-B in astrocytes.

Abstract: In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.

Characterization of the peripheral-type benzodiazepine receptors in cultured astrocytes: evidence for multiplicity.

Abstract: In mammalian brain peripheral benzodiazepine (PBZD) receptors are predominantly localized on astroglial cells. Previous studies utilizing whole membrane preparations from brain and peripheral organs of various species have indicated several distinctions between the drug-receptor interactions of the two prototypic PBZD receptor ligands, PK 11195 and Ro5-4864. The present study was undertaken to determine whether putative differences in the binding of PBZD receptor ligands in homogenates of primary astrocyte cultures can be interpreted as the labeling of PBZD receptor subtypes. Equilib-rium competition and saturation binding experiments in homogenate preparations of primary astrocytes from cerebral cortex of new born rats revealed that [3H]PK 11195 labels twice the number of [3H]Ro5-4864 binding sites. Unlabeled Ro5-4864 competes for [3H]PK 11195 binding in a manner suggesting the existence of multiple PK 11195 bind-ing sites. The competition binding experiments, using various benzodiazepines, indicate that one binding component of PK 11195 corresponds to Ro5-4864 binding sites, whereas the second is different. The latter binding site does not correspond to the central BZD receptor but displays the pharmacological properties of the PBZD receptor. Further differences between the binding of PK 11195 and Ro5-4864 in astrocytes were detected in the presence of ethanol which was more effective in inhibiting the binding of the latter. Subcellular distribution studies indicated, however, that the binding of both [3H]PK 11195 and [3H]Ro5-4864 is associated primarily with the mitochondrial fraction of astro-cytes. Taken together, the present study indicates the existence of non-overlapping PBZD binding sites in astrocytes and thus suggests the existence of PBZD receptor subtypes. It appears, however, that there is no distinction in the subcellular localization of the putative subtypes of the PBZD receptor. © 1993 Wiley-Liss, Inc.

Axons modulate the expression of transforming growth factor-betas in Schwann cells.

Abstract: We have investigated the expression of transforming growth factor (TGF)-beta 1,-beta 2, and -beta 3 in developing, degenerating, and regenerating rat peripheral nerve by immunohistochemistry and Northern blot analysis. In normal adult sciatic nerve, TGF-beta 1, -beta 2, and -beta 3 are detected in the cytoplasm of Schwann cells, and the levels of TGF-beta 1 and -beta 3 mRNAs are constant during post-natal development. When sciatic nerves are transected to cause axonal degeneration and prevent axonal regeneration, the level of TGF-beta 1 mRNA in the distal nerve-stump increases markedly and remains elevated, whereas the level of TGF-beta 3 mRNA falls modestly and remains depressed. When sciatic nerves are crushed to cause axonal degeneration and allow axonal regeneration, the level of TGF-beta 1 mRNA initially increases as axons degenerate, and then falls as axons regenerate. TGF-beta 2 mRNA was not detected in developing or lesioned sciatic nerves at any time. Cultured Schwann cells have high levels of TGF-beta 1 mRNA, the amount of which is reduced by forskolin, which mimics the effect of axonal contact. These data demonstrate that Schwann cells express TGF-beta 1, -beta 2, and -beta 3, and that TGF-beta 1 and -beta 3 mRNA predominate over TGF-beta 2 mRNA in peripheral nerve. Axonal contact and forskolin decrease the expression of TGF-beta 1 in Schwann cells.

Effects of transforming growth factor-beta 1 on the extracellular matrix and cytoskeleton of cultured astrocytes.

Abstract: The present study was performed on primary cultures and subcultures of cerebellar astrocytes in order to investigate the effects of transforming growth factor-beta 1 (TGF beta 1) on proliferation, extracellular matrix (ECM) components, and cytoskeletal structures in relation to morphological changes. The expression and cellular distribution of the ECM components laminin and fibronectin and the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and actin were investigated by immunoblotting, immunocytochemistry, and phalloidin staining. The proliferation of primary cultures was strongly inhibited by TGF beta 1. Treated cells became enlarged and spread onto the substratum. TGF beta 1 promoted the appearance of actin stress fibers and increased the cell actin content. It elicited a slight increase in GFAP expression and induced dispersion of thin filaments of GFAP. TGF beta 1 also stimulated the production of laminin and fibronectin and their incorporation into the ECM of primary cultures grown in medium with or without serum. Astrocytes grown in serum-containing medium for 1 day after subculturing responded strongly to TGF beta 1. Changes promoted by TGF beta 1 in cell shape, cytoskeleton, and ECM production of cultured astrocytes may have relevance for understanding the mechanisms of action of TGF beta 1 during brain development.

Transforming growth factor‐βs inhibit mitogen‐stimulated proliferation of astrocytes

Abstract: We have studied the influence of three members of the transforming growth factor-beta (TGF-beta) family of multifunctional growth factors on the proliferation of cultured astrocytes isolated from newborn mouse cerebral cortex. Although TGF-beta s 1, 2, and 3 cause only a small reduction in the low level of astrocyte proliferation occurring in chemically defined medium, they each inhibit the effects of five astrocyte mitogens (bFGF, EGF, PDGF, IL-1 alpha, and IL-2). Inhibition is observed when astrocytes are exposed to mitogen and TGF-beta at the same time and when they are exposed to TGF-beta prior to, and separately from, mitogen. This latter effect appears to be due to the binding of TGF-beta s to astrocyte-secreted extracellular matrix. These findings raise the possibility that TGF-beta may co-operate with other growth factors to control astrocyte proliferation in vivo.

Single-channel characteristics of the large-conductance anion channel in rat cortical astrocytes in primary culture.

Abstract: Cultured rat cortical astrocytes, in addition to a variety of voltage-sensitive potassium channels, also express anion channels. However, the behavior and regulation of these anion channels have been far less studied. This paper describes a patch-clamp study on a voltage-sensitive 200–300 pS high-conductance single-channel anion current, which seems to possess at least five different open sublevels or, alternatively, be formed froin five or more small-conductance ion channels linked together. This channel is voltage dependent, showing a bell-shaped open probability curve with highest open probability close to the reversal potential (zero-current). Although potassium channels are commonly detected in astrocytes in cell-attached and excised patches with both normal osmolarity and hypoosmotic solutions, the occurrence of the anion channel is clearly increased in isolated patches when hypoosmotic bath solutions are used. Also, cell aging in culture and the preparation of secondary cell cultures by trypsinization seem to increase the rate of occurrence of the anion channel. Though this channel is more routinely seen when a membrane patch is excised from the cell, occasionally cell-attached configurations with instant channel activity can be formed. While the modulation of this anion channel was being studied, it was found to be blocked by an anion transport inhibitor, L-644,711, reported to affect cell volume regulation in astrocytes. © 1993 Wiley-Liss, Inc.

Differential activation of microglia and astrocytes in aniso- and isomorphic gliotic tissue

Abstract: Reactive astrocytes and microglial cells are both involved in the formation of gliotic tissue Using immunohistochemical markers, we have compared the response of both these cell types after two different kinds of damage in the brain: traumatic injury (anisomorphic gliosis) and neurotoxic induced lesion (isomorphic gliosis), in two distinct regions of the brain, the cortex and the hippocampus We show that the time course and the relative contribution of astrocytes and microglial cells differ greatly in the two kinds of lesions While in anisomorphic gliosis there is little activation of endogenous microglial cells independently of the brain region damaged, these cells contribute in large measure and for prolonged periods of time to the formation of isomorphic gliotic tissue Astrocytes are quickly activated at the border of anisomorphic lesions, and after 3 days they already occupy an extensive portion of the brain parenchyma However, after 1 month, they are found restricted to a thin strip at the lesion boundary In contrast, after an isomorphic lesion, astrocytes become reactive around the site of neuronal cell loss but not at the site of the lesion itself Only after 2 weeks do they totally invade the damaged region, persisting for at least 1 month Such differences are observed independently of the brain region damaged These results suggest that the cellular, and therefore the molecular, composition of gliotic tissue depends on the type of insult the CNS has suffered © 1993 Wiley-Liss, Inc