Biomedical subjects

S Fedoroff

Publications and source records attributed to S Fedoroff.

At least 19 recordsLinked to original sources

PubMed1999

Expression of colony stimulating factor-1 receptor (CSF-1R) by CNS neurons in mice.

We report that neurons in the central nervous system express colony stimulating factor-1 receptor (CSF-1R) mRNA and protein and that the expression has regional specificity. The presence of CSF-1R in neurons was demonstrated by the use of four different types of antibodies to CSF-1R and the presence of CSF-1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF-1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF-1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF-1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF-1 binds to its receptor (CSF-1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF-1 dose dependent. We propose that CSF-1/CSF-1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes.

Animals↗

PubMed1999

Expression of stem cell factor and c-kit receptor in neural cells after brain injury.

Previously it has been shown that c-kit receptor (c-kitR) and its ligand, stem cell factor (SCF), are expressed in the central nervous system. We have reported that SCF in cultures regulates mouse microglial function. Here we demonstrate that SCF/c-kitR signaling also takes place in situ. We used a penetrating stab wound injury as a model and analyzed the SCF and c-kitR expression in neural cells by immunohistochemistry and in situ hybridization. We found that microglia activated by injury up-regulated c-kitR expression, whereas some astrocytes in the vicinity of the wound expressed SCF mRNA in addition to neurons. This observation suggests that SCF/c-kitR signaling between neurons, astrocytes and microglia also occurs in situ.

Animals↗

PubMed1998

Modulation of microglia by stem cell factor.

We reported previously that stem cell factor (SCF) is produced mainly by neurons and that its receptor (c-kitR), encoded by the protooncogene c-kit, is expressed in microglia, suggesting that SCF/c-kitR signaling may be involved in neuron-microglia interactions. We now report that SCF supports microglial survival in cultures, maintains them in process-bearing morphology, and inhibits microglial proliferation induced by colony stimulating factor-1. SCF potentiates microglial expression of the mRNAs of nerve growth factor, brain-derived neurotrophic factor and ciliary neurotrophic factor, and downregulates microglial expression of the inflammation-associated cytokines, tumor necrosis factor-a (TNF-alpha), and interleukin-1beta (IL-1beta). SCF potentiates lipopolysaccharide-stimulated, but attenuates interferon-gamma TFNalpha mediated expression of the mRNAs of IL-1beta and TNF-alpha. The SCF-induced expression of neurotrophin mRNAs is enhanced by the addition of lipopolysaccharide (LPS) but is reduced by IFNgamma. The interactions between SCF and LPS or IFNgamma in the regulation of inflammation-associated cytokine gene expression are accompanied by the differential regulation of c-kitR in microglia. These observations suggest that SCF/c-kitR signaling modulates microglial activity.

Animals↗

PubMed1998

Lipopolysaccharide induction of MARCKS-related protein and cytokine secretion are differentially impaired in microglia from LPS-nonresponsive (C3H/HeJ) mice.

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (> or = 100 ng/ml) than in normal (C3H/OuJ) microglia (< or = 10 ng/ml). By contrast, TNF alpha production was not significantly increased in C3H/HeJ microglia even at 1 microgram LPS/ml. Microglia expressed PKC isoforms alpha, beta, delta, and zeta (but not gamma and epsilon); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.

Animals↗

PubMed1997

Microglia and astroglia have a common progenitor cell.

Disaggregated neopallial cells from newborn C3H/HeJ mice were cloned in Grenier hybridoma tissue culture dishes, and culture wells that contained only one cell were marked. After 8-10 days of culturing, the cultures were fixed and double immunolabeled for microglia with Mac-1 antibody and for astroglia with antibody to GFAP. Each marked well containing a clone was identified as either a microglia, astroglia, mixed microglia-astroglia, or an unlabeled clone. The effect of LM cell line conditioned medium (LM-CM), which contains colony-stimulating factor-1, on the development of mixed microglia-astroglia clones was determined. Formation of mixed clones was dose dependent (P < 0.0001). We concluded that microglia and astroglia have a common progenitor cell and that the development of mixed clones is LM-CM dependent.

Animals↗

PubMed1997

Upregulation of F-actin and alpha-actinin in reactive astrocytes.

We have shown previously that in tissue culture stellate astrocytes downregulate F-actin and actin binding proteins (ABPs) (Abd-El-Basset et al.: J Neurosci Res 30:1-17, 1991), whereas the reactive-like astrocytes upregulate their F-actin (Fedoroff et al.: Neuroscience 22:255-266, 1987). In the present study we report that in normal brain, as in tissue culture, neither F-actin nor alpha-actinin (an ABP) could be detected in stellate astrocytes. When a stab wound was made in brain, F-actin and alpha-actinin were upregulated in reactive astrocytes. We also demonstrated that reactive-like astrocytes in tissue culture express alpha-actinin, which has a "dotted" appearance when immunostained, and is colocalized with F-actin in a specific arrangement.

Actinin↗

PubMed1997

Cellular localization of stem cell factor and c-kit receptor in the mouse nervous system.

We have characterized the cellular localization of stem cell factor (SCF) and c-kit receptor (c-kitR) in the adult mouse nervous system in situ and in culture by using immunocytochemistry. We found that SCF is largely confined to the neuronal population in normal brain, whereas c-kitR is expressed by glial cells as well as some neurons. We also found that astroglia at an early stage of culture (7 days in vitro) are strongly SCF positive and weakly c-kitR positive. Microglia in cultures express both SCF and c-kitR, but the immunostaining of SCF is weak and diffuse when microglia are cultured in the presence of colony stimulating factor-1. Northern blot analysis confirmed the expression of mRNAs of c-kit and SCF in cultured neurons, astroglia, and microglia. The addition of recombinant SCF to astroglia in culture upregulates the expression of mRNAs of nerve growth factor, brain derived neurotrophic factor, and ciliary neurotrophic factor. These observations suggest that SCF/c-kitR signaling is involved in neuron-neuron as well as neuron-glia interactions.

Animals↗

PubMed1997

Role of colony stimulating factor-1 in brain damage caused by ischemia.

A marked effect of colony stimulating factor-1(CSF-1) on microglial response and neuron survival in cerebral cortex ischemic damage was observed. In osteopetrotic op/op mice, which lack systemically functional CSF-1 microglia do not respond to ischemic damage to the cerebral cortex, and the infarcts are considerably larger than in CSF-1 producing mice with similar vascular impairment. Delivery of extraneous CSF-1 to op/op mice alleviates the functional deficiency of the microglia and potentiates neuron survival in ischemic lesion. Delivery of extraneous recombinant CSF-1 to normal CSF-1 producing mice does not increase either the number or degree of activation of microglia, but does further potentiate neuronal survival. We found that neurons in the cerebral cortex have active CSF-1 receptors, and we therefore propose that neuronal rescue in cerebral cortex ischemic damage is linked to activation of the CSF-1 receptor on neurons.

Animals↗

PubMed1996

Cell cycle time of murine neopallial cells in vitro.

Disaggregated glial cells from newborn CD1 mouse neopallia were cultured in low concentration (4.2 x 10(3) cells/cm2) for 72 hr and then either pulse labeled with BrdU by one 2-hr pulse at various times of culturing or continuously labeled for various lengths of time. At the end of incubation, the cells were fixed and immunoreacted with BrdU. All BrdU+ and BrdU- cell nuclei were counted in an area of 4.84 cm2. A three-compartment model for interpretation of the experimental data was developed consisting of active proliferating cells, non-active cells with proliferating potential, and nonproliferating cells. The model is based on assumptions of time invariance of culture conditions, random re-entry of cells into cell cycle and random exit from the proliferating pool. Furthermore, it is assumed that average values are representative for describing the numbers of cells in specific compartments as functions of time. A set of relationships representing the numbers of labeled cells for pulse labeling and continuous labeling assays is derived from these assumptions and the generally accepted representation of cell progress through the cell cycle, i.e., a genetically predetermined sequence of post-mitosis rest phase, S-phase, pre-mitosis rest phase, and mitosis. These relationships are used to evaluate the S-phase time tau s and cell cycle time tau c of proliferating cells. Under our particular conditions, we obtain approximately tau s = 8 hr and tau c = 16 hr, respectively. The applicability of the model and possible distorting factors are discussed.

Animals↗

PubMed1996

Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia.

Microglia rapidly respond to lipoplysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immuno-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes. We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alaninerich C kinase substrate) and MRP (MARCKS-related protein). Within 6 hours of addition, LPS induced a twofold increase in [3H]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP. The differential effect of LPS on expression of MRP vs. MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS. TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription. CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS. Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1.

Animals↗

PubMed1996

The hematopoietic cytokine colony stimulating factor 1 is also a growth factor in the CNS: (II). Microencapsulated CSF-1 and LM-10 cells as delivery systems.

The aim of this study was to develop delivery systems for administration of CSF-1 to remedy the systemic deficiency of this cytokine in osteopetrotic op/op mice and to study the microglial response and neuronal survival in op/op mice following cerebral cortex ischemic lesion. Unilateral cerebral cortex ischemic lesions were produced in homozygous op/op mice and either microencapsulated rhCSF-1 or LM-10 fibroblast-like cells producing CSF-1 were administered either locally, at the site and time of the lesioning, or into the peritoneum 2 weeks before the lesion was made. Physical properties (shape, size, integrity) and kinetics of rhCSF-1 release were assessed prior to the experiments in situ. Depending on the characteristics of the biodegradable polymer (e.g., chitosan with different densities or poly-L-lactic-poly-glycolic acid), remarkable differences in survival of encapsulated cells were observed. Cellular integrity following encapsulation and metabolic activity was regularly assessed for a period of 1 month. The best level of viability was achieved with highly viscous chitosan (311). The results from these studies demonstrate that: (1) rhCSF-1 incorporated into biodegradable spheres can be released and retain its biological activity; (2) microencapsulated LM-10 cells which produce CSF-1 can survive and constitutively release CSF-1 in alginate-chitosan spheres for different lengths of time depending on the physical properties of the chitosan used; and (3) CSF-1 is an important growth factor in the central nervous system where it can both strongly alter morphological changes of microglia and enhance survival of neurons in injured brain.

Animals↗

PubMed1996

Neuron-microglia interactions in vitro.

We observed that soluble factor(s) in microglia-conditioned medium supported the survival of cerebral cortical neurons from E15 mouse in a dose-dependent manner. In mixed neuron-microglia cultures, neurons possessed long neurites with extensive arborization and could survive for up to 4 weeks. In such cultures, neurons had an up-regulated level of phosphotyrosine immunoreactivity as compared to those in pure neuron cultures. In mixed cultures, microglia extended cytoplasmic processes toward the growing neurites, and when they contacted neurites, the microglia changed morphology by flattening and rounding up by extending thin cytoplasmic processes. Microglia survived longer in mixed cultures than in pure microglia cultures, with or without neuron-conditioned medium. Under all these culture conditions, microglia were phagocytic as evaluated by Fc receptor-mediated phagocytosis of opsinized sheep erythrocytes, suggesting that the phagocytic activity of microglia does not impair their capacity to support neuronal survival. However, lipopolysaccharide treated microglia did impede neuronal survival in mixed cultures. These observations indicate that, in vitro, microglia can be either neurotrophic or neurotoxic depending upon the microenvironment. The mixed neuron-microglia cultures described provide a valuable in vitro model systems for studying the direct interactions between neurons and microglia.

Animals↗

PubMed1996

Colony stimulating factor-1 potentiates neuronal survival in cerebral cortex ischemic lesion.

The effect of the cytokine, colony stimulating factor-1 (CSF-1), on neuronal survival in cerebral cortex ischemic lesion was determined. Ischemic lesions were made in C3H/HeJ mice by disrupting blood vessels that penetrate the cerebral cortex from the pial-vascular plexus. Recombinant human colony stimulating factor 1 (rhCSF-1) was delivered in chitosan microcapsules that were either implanted intraperitoneally 2 weeks before surgery or at the site of the lesion at the time of surgery. Neuronal survival was twofold greater and the size of the infarct was considerably smaller in animals that received rhCSF-1-containing microcapsules. There was no significant difference whether the microcapsules were implanted intraperitoneally or at the site of the lesion. We found that CSF-1 receptor (c-fms) was upregulated in neurons at the site of the lesion and we propose that neuron rescue in ischemic damage is potentiated by CSF-1 signaling through CSF-1 receptor in the neurons.

Animals↗

PubMed1995

Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures.

We examined the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the morphology, cell motility, cytoskeletal organization, and phagocytic activity of microglia in tissue cultures initiated from neopallia of newborn C3H/OuJ mice. Normally, the microglia in our cultures are non-migratory and Mac-1 positive, have ameboid cell morphology, no polarity, many short processes that extend into lamellipodia in opposing directions, and undulating cell membrane projections. When 1-5 micrograms/ml LPS is added to such cultures, some cells acquire polarity by forming a large lamellipodium and begin to migrate. Two hours later migration ceases; the membrane undulations stop; and the cells become non-polar, assume a large, round, flat shape, and gradually develop many microspikes all over the cell body. Those cells that do not transform into large, round, flat cells enlarge and extend numerous lamellipodia in opposing directions. We found that the cytoskeleton of microglia is composed of actin, vimentin-containing intermediate filaments (IF) and microtubules (MT). Vimentin-containing IF and MT form dense networks that radiate into the cell periphery, whereas F-actin is diffusely arranged throughout the cytoplasm. The LPS-treated cells show changes in the organization of the main components of the cytoskeleton. F-actin is reorganized by the formation of bundles underneath and parallel to the cell membrane and other bundles projecting into the cores of the microspikes. The vimentin-containing IF dense network reorganizes into two condensed rings, with fine strands of IF extended between the two rings and the MT networks become less dense and extend throughout the cytoplasm. The LPS treatment potentiates the phagocytic activity of the microglia. However, approximately 30% of microglia lose the expression of MHC class II antigens.

Actins↗

PubMed1995

Microglia in colony-stimulating factor 1-deficient op/op mice.

Mice that are homozygous for the autosomal recessive mutation osteopetrosis (op) suffer from a general skeletal sclerosis, and the numbers of macrophages in various tissues are significantly decreased. We report that microglia in op/op mice are not affected by the mutation. They have normal morphology and are present in the CNS in normal frequency. In cultures, disaggregated cells of neopallia can form microglia, but such cells from neopallia of op/op mice form microglia only when colony-stimulating factor 1 (CSF-1) is added to the culture medium. The addition of granulocyte/macrophage (GM)-CSF or interleukin (IL)-3 to the culture medium does not stimulate production of microglia. Microglia that form in op/op neopallial cell cultures, in the presence of CSF-1, are capable of Fc-receptor-mediated phagocytosis. Based on our experiments, it seems that microglia are CSF-1 dependent but in op/op mice (in which CSF-1 is absent) microglia may use other locally produced factors.

Animals↗

PubMed1994

Astrocyte proliferation in culture following exposure to potassium ion.

Following brain injury astrocytes swell acutely and proliferate thereafter. To investigate the direct relationship between swelling and proliferation, cultured astroglial cells were exposed to 60 mM K+ ion or hypo-osmolar (205 mOsm) electrolyte solution for 1 h to cause transient swelling. Proliferation was assessed by bromodeoxyuridine (BrdU) uptake 24 and 96 h later. The cells were double-labelled with antibodies to glial fibrillary acidic protein (GFAP) and BrdU. At 96 h the GFAP immunoreactive cells exposed to K+ exhibited 44.9% uptake versus 27.0% and 21.1% for control and hypo-osmolar exposed cells, respectively. We conclude that swelling of astroglia in vitro does not induce cell proliferation but transient depolarization following exposure to high K+ does.

Animals↗

PubMed1994

Development of microglia in mouse neopallial cell cultures.

Microglia develop in cultures initiated from disaggregated neopallial cells of newborn C3H/HeJ mice when the cultures are subjected to nutritional deprivation for 10 or more days (Hao et al: Int J Dev Neurosci 9:1-14, 1991). In the present experiments, the cultures were pulsed with BrdU for 3 hours at different times during incubation and then the cells were immunoreacted with antibodies against BrdU, GFAP, and CR3 receptor. The dividing cells (BrdU+) were found to be either GFAP+ or GFAP-, but not Mac-1+/BrdU+. Infection of proliferating cells after 2 or more days of incubation with replication-deficient retroviral vector containing E. coli lacZ reporter gene resulted in many labeled astroglia cell clones but no labeled microglia. However, when cells were infected right after disaggregation of neopallium, labeled Mac-1+ microglia were found. When Mac-1+ cells in a suspension of disaggregated neopallial cells were killed using complement mediated lysis before setting up the cultures, Mac-1+ microglia developed, in spite of the treatment. We conclude that in cultures initiated from mouse neopallium there are MAC-1-/GFAP- microglia progenitor cells which do not divide in nutritionally deprived cultures but can transform into Mac-1+ microglia under the influence of astroglia-derived trophic factors. Microglia, which become Mac-1+ (i.e., express CR3 receptor), proliferate extensively in the presence of CSF-1 (which is produced by astroglia).

Animals↗

PubMed1994

Dynamics of actin filaments in microglia during Fc receptor-mediated phagocytosis.

The phagocytic ability of mouse microglia during their differentiation in culture and after stimulation with bacterial wall lipopolysaccharide (LPS) has been investigated using Fc receptor-mediated phagocytosis of immunoglobulin (IgG)-coated sheep erythrocytes (SRBCs). We observed that in 10-14 day-confluent neopallial cell cultures some immature microglia are not phagocytic but, on further culturing, they do become phagocytic. LPS-stimulated microglia are able to phagocytose larger numbers of IgG-coated SRBCs and at a faster rate than non-stimulated microglia. Within 5-10 min of phagocytosis the actin filaments of the LPS-stimulated microglia become depolymerized, leaving only bundles of actin filaments around the phagocytosed SRBCs (phagosome cups). At 30 min after the start of phagocytosis the actin filaments of the LPS-stimulated microglia begin to polymerize, and within 2 h the original pre-phagocytosis pattern of the actin filament network is re-established. The non-LPS-stimulated microglia exhibit actin filament depolymerization in only a few lamellipodia and polymerization of actin filaments around engulfed particles, but much later during phagocytosis.

Actins↗