Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-β in terms of its biological activity. The assay used to assess the activity of TGF-β is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-β1, -β2, and -β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-α, platelet-derived growth factor-AB, insulin-like growth factor-I, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-β superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF-β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-β1/β2/β3. TGF-β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10⁻⁵ M ). These data suggest that TGF-β is stored in chromaffin granules and can be released by exocytosis.     

Evidence of Type I and Type II Transforming Growth Factor-β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia

Abstract: The peptides of the transforming growth factor-β (TGF-β) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-β receptors and their potential ligands (TGF-β1, TGF-β2, and TGF-β3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-β isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-βs and their receptors. We show that the three TGF-β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-β2. We have demonstrated that both type I and type II TGF-β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-β1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-β receptor was not altered by the ischemic insult, the pattern of the type II TGF-β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.     

3α-Hydroxysteroid Oxidoreductase in Rat Brain

Abstract: We describe a simple procedure for the microassay of 3α-hydroxysteroid oxidoreductase in homogenates of rat brain. This enzyme converts dihydrotestosterone to 3α-androstandiol. We have mapped the distribution of the enzymatic activity in 14 regions of the rat brain. The highest activities were observed in homogenates of olfactory bulb (51/nmol/mg protein/h) and olfactory tubercle (29 nmol/mg protein/h). Substantially lower values were seen in the other brain regions, including thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, and preoptic area (6–20 nmol/mg protein/ h).     

Tumor Necrosis Factor-α and Basic Fibroblast Growth Factor Decrease Glial Fibrillary Acidic Protein and Its Encoding mRNA in Astrocyte Cultures and Glioblastoma Cells

Abstract: Tumor necrosis factor-α is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-α. Treatment with tumor necrosis factor-α for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-α treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-α, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-α induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-α. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-α treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.     

Regulation of μ-Opioid Receptor mRNA in Rat Globus Pallidus: Effects of Enkephalin Increases Induced by Short- and Long-Term Haloperidol Administration

Abstract: The mRNA encoding μ-opioid receptors is expressed in neurons of the globus pallidus, a region of the basal ganglia that receives a dense enkephalinergic innervation from the striatum. The regulation of the mRNAs encoding the opioid peptide enkephalin in the striatum and the μ-opioid receptor in the globus pallidus was examined with in situ hybridization histochemistry following short- or long-term haloperidol treatments, which alter striatal enkephalin mRNA levels. Animals were administered haloperidol daily for 3 or 7 days (1 mg/kg, s.c.) or continuously for 8 months (1 mg/kg, depot followed by oral). Enkephalin and μ-opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, the enkephalin mRNA level was increased in the striatum, and μ-opioid receptor mRNA levels were markedly decreased in the globus pallidus after 7 days of haloperidol administration. Similar effects were observed in rats treated with haloperidol for 8 months. The results provide the first evidence of regulation of μ-opioid receptor mRNA in vivo.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

Interleukin 1 and Tumor Necrosis Factor-α Stimulate the Production of Colony-Stimulating Factor 1 by Murine Astrocytes

Astrocytes have the ability to secrete colony-stimulating factor 1 (CSF-1), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and interleukin 6 (IL-6) on the production of CSF-1 by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNF alpha and IL-1 increased CSF-1 mRNA and protein levels in the astrocytic cultures. In contrast, IL-6 was ineffective. The CSF-1 mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, increased CSF-1 mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines.     

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat α-enolase. In fact, PGn was found to bind to purified rat α-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat α-enolase antibody, and pretreatment with α-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α-enolase is present on the cell surface of a certain population of neurons. These results suggest that α-enolase may function as a PGn-binding molecule on the neuronal cell surface.     

Differential Effects of D1 and D2 Dopamine Receptor Antagonists on Acute Amphetamine- or Methamphetamine-Induced Up-Regulation of zif/268 mRNA Expression in Rat Forebrain

Abstract: This study investigated the hypothesis that D₁ and D₂ dopamine receptors interact to regulate the expression of zif/268 mRNA in rat forebrain after an acute injection of amphetamine or methamphetamine. Forty-five minutes and 3 h after a single injection of amphetamine (4 mg/kg i.p.) or methamphetamine (4 mg/kg i.p.), the mRNA expression of zif/268 in dorsal striatum and sensorimotor cortex was increased, as revealed by quantitative in situ hybridization histochemistry. Induction was more intense in striatal patches at 45 min than at 3 h, when a more homogeneous pattern of zif/268 mRNA induction was observed. SCH 23390, a selective D₁ receptor antagonist, suppressed, and eticlopride, a D₂ receptor antagonist, elevated, constitutive zif/268 mRNA levels in the striatum, but neither antagonist had a significant effect on the constitutive expression of zif/268 in sensorimotor cortex. Pretreatment with SCH 23390 completely blocked the stimulant-induced zif/268 expression in striatum and partially blocked the stimulant-induced zif/268 expression in cortex. Pretreatment with eticlopride augmented zif/268 mRNA expression in patch and matrix compartments of dorsal and ventral striatum 45 min after amphetamine or methamphetamine injection. However, at 3 h, eticlopride completely blocked amphetamine- and methamphetamine-stimulated zif/268 mRNA in dorsomedial, but not dorsolateral, striatum. In addition, eticlopride partially blocked cortical zif/268 induction by both amphetamines. Both antagonists prevented stimulant-induced hyperlocomotion and stereotypies. These results demonstrate that D₁ and D₂ receptors in mesolimbic/mesostriatal pathways both regulate amphetamine-and methamphetamine-stimulated behaviors and zif/268 mRNA expression. Furthermore, the effect of D₂ receptor blockade on zif/268 expression was found to be contingent on the time interval investigated after psychostimulant administration.     

创面愈合过程中EGFr基因表达的研究

应用原位分子杂交技术观测了大鼠创面愈合过程中表皮生长因于受体(Epidermalgrowth factor receptor,EGFr)的信使核糖核酸(mRNA)表达的变化.结果表明:伤后4天EGFrmRNA即有明显的表达,以后逐渐减弱,伤后16天EGFr mRNA已无明显表达.对EGFr基因表达的作用和机理进行了讨论.     

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental Hypoxic-Ischemic Injury

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.     

In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

p53 Induction by Tumor Necrosis Factor-α and Involvement of p53 in Cell Death of Human Oligodendrocytes

Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of OL cell injury. TNF-alpha is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF-alpha mediates cell death is through the activation of c-jun N-terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF-alpha leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF-alpha-induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF-alpha treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus-mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF-alpha-mediated cell death of human adult OLs.     

Nonneuronal Localization for Steroid Converting Enzyme: 3α-Hydroxysteroid Oxidoreductase in Olfactory Tubercle of Rat Brain

3 alpha-Hydroxysteroid oxidoreductase (EC 1.1.1.50) was localized in the rat brain by cryostat sectioning, microassay, and neurochemical lesions. Single 16-microns sections were cut, homogenized, and assayed. In the olfactory tubercle 3 alpha-hydroxysteroid oxidoreductase activity is high in the piaglial layer at the surface, 20-fold lower at a depth of 50 microns, and 50-fold lower at a depth of 200 microns. A similar pattern of activity was seen in the olfactory bulb, the interpeduncular nucleus, the frontal pole of the cortex, and the frontoparietal cortex. When kainic acid, a toxin that destroys neurons but leaves glia and axons of passage intact, was injected into the olfactory tubercle, 3 alpha-hydroxysteroid oxidoreductase activity was undiminished whereas glutamic acid decarboxylase activity was reduced by 80%. This laminar distribution and insensitivity to kainic acid are consistent with a nonneuronal localization. The high concentration of astrocytes in the piaglial layer, where 3 alpha-hydroxysteroid oxidoreductase activity is highest, lead us to suggest that this enzyme is localized to astrocytes. The presence of particular enzymes in some brain regions and not in others determines which products are synthesized and which are inactivated in those regions. Thus, the location of 3 alpha-hydroxysteroid oxidoreductase and other steroid converting enzymes can affect the activity of neuronal circuits and the behaviours regulated by those circuits.     

Cocaine and methamphetamine differentially affect opioid peptide mRNA expression in the striatum

In general, administration of methamphetamine and cocaine alters preprodynorphin and preproenkephalin mRNA levels in striatum. However, no study has directly compared the effects of these stimulants on opioid peptides in striatum. This study used in situ hybridization to compare directly the effects of cocaine and methamphetamine on preprodynorphin and preproenkephalin mRNAs in distinct striatal regions. Male Sprague-Dawley rats received a single administration of 15 mg/kg methamphetamine or 30 mg/kg cocaine and were killed 30 min or 3 h later. Methamphetamine and cocaine differentially affected preprodynorphin mRNA in striatum after 3 h. Densitometric analysis of film autoradiograms revealed that cocaine, but not methamphetamine, significantly increased preprodynorphin. This effect was seen throughout rostral striatum and dorsally in caudal striatum. However, specific analysis of "patches" in which preprodynorphin expression is high revealed a significantly greater effect of methamphetamine versus cocaine. In contrast, both cocaine and methamphetamine had similar effects on preproenkephalin mRNA, decreasing levels after 30 min in rostral striatum and in the core of nucleus accumbens. These data suggest that methamphetamine and cocaine have distinct postsynaptic consequences on striatal neurons.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.