Joan P. Schwartz

Protective role of reactive astrocytes in brain ischemia

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP−/−Vim−/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP−/−Vim−/− than in wild-type (WT) mice; GFAP−/−, Vim−/− and WT mice had the same infarct volume. Endothelin B receptor (ETBR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP−/−Vim−/− astrocytes. In WT astrocytes, ETBR colocalized extensively with bundles of IFs. GFAP−/−Vim−/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP−/−Vim−/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ETBR-mediated control of gap junctions, and PAI-1 expression.

INVOLVEMENT OF CYTOKINES IN NORMAL CNS DEVELOPMENT AND NEUROLOGICAL DISEASES : RECENT PROGRESS AND PERSPECTIVES

Cytokines have been recognized to play an important role both in normal development of the brain, when they act as neurotrophic factors, as well as following injury. While both the cytokines and their receptors are synthesized and expressed in the brain normally (albeit at low levels), it has become clear that elevated levels are associated with many neurological disorders. In this review, we have chosen to present the data for only a few of the cytokines, including interleukin‐1β, interleukin‐3, interleukin‐6, interferon‐γ, transforming growth factor‐β, and tumor necrosis factor‐α. Data are presented that suggest roles they may play in human disorders, including stroke, multiple sclerosis, Alzheimers disease, and several psychiatric disorders. The results in human disease are compared with results obtained in a variety of transgenic animal models. The mouse models have very different disorders depending on whether a cytokine is overexpressed either peripherally or in either astrocytes or neurons. The potential significance of this to the understanding of human disease is discussed. J. Neurosci. Res. 52:7–16, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Effect of exposure to anti-NGF on sensory neurons of adult rats and guinea pigs

Nerve growth factor (NGF) deprivation was produced in adult rats and guinea pigs by immunization against mouse NGF. Exposure of adult animals to anti-NGF had no effect on sensory ganglion neuronal number or size-frequency histograms. However, the substance P content of sensory ganglia, spinal cord and hind paw skin decreased to as great an extent as was seen in animals exposed to anti-NGF in utero. We conclude that NGF has two separate effects on sensory neurons: adult sensory neurons require NGF for normal function whereas developing neurons require NGF for both survival and transmitter expression.

Expression of inducible nitric oxide synthase by neurones following exposure to endotoxin and cytokine

In the CNS, nitric oxide (NO) has been implicated as both a mediator of neurotoxicity and a neuromodulator. The inducible NO synthase (iNOS), thought to mediate toxic effects of NO, has been attributed to glial cells in the CNS. We now report that cerebellar granule cell neurones can be stimulated by lipopolysaccharide and interferon‐γ to express iNOS in vitro, as demonstrated by reverse transcription‐polymerase chain reaction and fluorescent in situ hybridisation. The expression of both constitutive NO synthase (cNOS) and iNOS by neurones suggests that NO has diverse functions in the brain, and supports the possibility that iNOS plays a role in neuronal damage and inflammation following activation of brain microglia and production of cytokines.

Neurotrophic factor gene expression in astrocytes during development and following injury.

Astrocyte cultures were utilized to examine regulation of the expression of several trophic factor genes. Regulation by the beta-adrenergic receptor was demonstrated by exposure of striatal and cortical astrocytes to isoproterenol, which resulted in increased content of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor, and proenkephalin (PE), as well as NGF and Met-enkephalin. Developmental regulation was analyzed by preparing cortical astrocytes from animals of four different ages--embryonic day 20, postnatal days 3 and 8, and adult. Because both the PE and NGF genes showed developmental downregulation, we asked whether we could prepare reactive astrocytes from lesioned adult brain and see expression turned back on. Astrocytes prepared from 6-hydroxydopamine-lesioned rat striatum or MPTP-lesioned mouse striatum contained increased GFAP and NGF mRNA. Comparable changes in GFAP and NGF could be achieved by treatment of control cultures with interferon-gamma or interleukin-1 beta. These results suggest that locus coeruleus neurons could control astrocyte synthesis of neurotrophic factors through release of of norepinephrine, but that in injured brain other factors, such as cytokines, may become equally important.

Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture

Abstract: Pigment epithelium‐derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y‐79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose‐response relationship, with a larger effect in chemically defined medium than in serum‐containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum‐containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme‐linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.

Cell culture models for reactive gliosis: new perspectives.

Reactive gliosis, which occurs in response to any damage or disturbance to the central nervous system, has been recognized for many years, but is still not completely understood. The hallmark is the increased expression of glial fibrillary acidic protein (GFAP), yet studies in GFAP knockout mice suggest that GFAP may not be required for an astrocyte to become hypertrophic. In this review, we describe a series of tissue culture models that have been established in order to address: 1) the biochemical phenotype of reactive astrocytes; 2) the factor and/or cell responsible for induction of gliosis; 3) the mechanisms by which one might block the induction. These models range from cultures of astrocytes, both neonatal and adult, to co‐cultures of astrocytes with either neurons or microglia, to organ cultures. None is ideal: each addresses a different set of questions, but taken together, they are beginning to provide useful information which should allow a better understanding of the plasticity response of astrocytes to brain injury. J. Neurosci. Res. 51:675–681, 1998.

NFκB Activation Is Required for the Neuroprotective Effects of Pigment Epithelium-derived Factor (PEDF) on Cerebellar Granule Neurons

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1–3 days in vitro) against induced apoptosis and mature cells (5+ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFκB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFκB activation. PEDF induced an increased phosphorylation of IκBα, decreased levels of IκB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFκB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IκB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IκB, orN-acetyl-Leu-Leu-norleucinal, which blocks proteosome degradation of IκB, demonstrating that NFκB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFκB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.

We have shown previously that pigment epithelium‐derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed “natural” apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum‐ and 25 mM KCl‐containing medium to serum‐free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine‐protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis. J. Neurosci. Res. 53:7–15, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity.

Abstract: Pigment epithelium‐derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 µM glutamate, glutamate‐induced neuronal death was significantly reduced. The protective effect of rPEDF was dose‐dependent in the range from 0.023 to 7.0 nM (1–500 ng/ml), with a half‐maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate‐induced neurotoxicity, possibly via a calcium‐related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.