Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Expression of extracellular matrix degrading enzymes during migration of xenografted brain cells.

Proteolytic enzymes, postulated to create an avenue for cell migration by digestion of host extracellular matrix molecules, have been implicated in neoplastic glial cell migration. A similar process is likely to occur in the developing brain. Fetal rabbit brain fragments transplanted into the striatum of the neonatal Shiverer mouse give rise to cells which migrate from the graft site and differentiate into astrocytes and oligodendrocytes. Proteinase expression by transplanted brain cells was studied using immunohistochemistry and in situ hybridization. Immature donor cells expressed the mRNAs for matrix metalloproteinases (MMP) 1 (collagenase) and 3 (stromelysin). Northern blot analysis of rabbit brain showed that MMP-1 in particular is expressed in the immature rabbit cerebrum and down-regulated during maturation. Immature donor cells exhibited immunoreactivity for urokinase plasminogen activator. However, immunoreactivity was also present in maturing neurons. Donor and host astroglia in the vicinity of grafts were immunoreactive for MMP-2 and tissue-type plasminogen activator. This expression may represent a reactive phenomenon, not specifically related to cell migration, by mature astrocytes. Based upon our findings, MMP-1 appears to be a candidate for involvement in migration of immature brain cells in the cerebrum.

Expression of TNF alpha in central neurons of Lewis rat spinal cord after EAE induction.

Experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), is a demyelinating autoimmune disease of the central nervous system (CNS). The proinflammatory cytokine TNF alpha, as an endogenous mediator of inflammation, plays an important role in the pathogenesis of EAE disease. In this study, we demonstrate the presence of TNF alpha in spinal cord of Lewis rats, during the critical phase of EAE. The expression of TNF alpha is observed mainly in the gray matter of thoracic and lumbar levels of the spinal cord, in the motoneurons and interneurons of the ventral horn. Surprisingly, one month after recovery, we still found an intense TNF alpha-neuronal expression, including in the cervical region, and this positivity lasted up to 40 days after recovery, with, however, a decrease in its intensity. These results suggest that central neurons respond directly to massive infiltration of lymphocytes and macrophages after the breakdown of the blood-brain barrier (BBB), by producing TNF alpha cytokine. In addition, neuronal-TNF alpha detection in the recovery stage of EAE may suggest a role other than its classical action in promoting inflammatory processes.

Widespread neuronal expression of c-Fos throughout the brain and local expression in glia following a hippocampal injury.

Fos oncoprotein is an immediate early gene product and a marker of cell activation following a variety of insults. We have previously shown that a mechanical lesion to the hippocampus of adult mice induces a neuronal expression of the cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF alpha) whereas a lesion to the striatum does not. The role of these inflammatory cytokines in the pathophysiology of central neurons is still unclear. The present work was undertaken to study a possible correlation between the central expression patterns of c-Fos on the one hand and IL-1alpha and TNF alpha on the other hand. We show that Fos is expressed in a majority of brain neurons after a unilateral lesion to the hippocampus whereas it is confined to the site of injury when applied to the striatum, as previously described for the expression of the cytokines.

Gene transfer to the central nervous system by transplantation of cerebral endothelial cells.

A cerebral endothelial immortalized cell line was used in transplantation experiments to deliver gene products to the adult rat brain. Survival of grafted cells was observed for at least 1 year, without any sign of tumor formation. When genetically modified to express bacterial beta-galactosidase and transplanted into the striatum, these cells were shown, by light and electron microscope analysis, to integrate into the host brain parenchyma and microvasculature. Following implantation into the striatum and nucleus basalis of adult rats, endothelial cells engineered to secrete mouse beta-nerve growth factor (NGF) induced the formation of a dense network of low-affinity NGF receptor-expressing fibers near the implantation sites. This biological response was observed from 3 to 8 weeks after engraftment. The present study establishes the cerebral endothelial cell as an efficient vector for gene transfer to the central nervous system.

Calcium-dependent release specificities of various cell lines loaded with different transmitters.

By loading cells in culture with acetylcholine (ACh) we have characterized a calcium-dependent release mechanism and shown that it was expressed independently of synthesis or storage of ACh. (Israël et al., 1994, Neurochemistry International 37, 1475-1483; Falk-Vairant et al., 1996a, Proc. Natl. Acad. Sci. U.S.A. 93, 5203-5207; Falk-Vairant et al., 1996b, Neuroscience 75, 353-360; Falk-Vairant et al., 1996c, Journal of Neuroscience Research 45, 195-201). The transmitter loading procedure was applied to two other transmitters, gamma-aminobutyric acid (GABA) and glutamate (Glu). We could then study the specificity of the release mechanism for the three transmitters in a variety of cell lines, including neural-derived cells. Four different calcium-dependent release phenotypes were identified: two were specific for ACh or GABA, and two co-released two transmitters ACh and GABA but not Glu, or ACh and Glu but not GABA. We conclude that release mechanisms having different specificities are expressed by the cell lines studied, they become functional after loading the cells with the relevant transmitters. These observations will help the identification of proteins controlling the specificity of release, and provide an interesting model for pharmacological studies.

Identification and topography of neuronal cell populations expressing TNF alpha and IL-1 alpha in response to hippocampal lesion.

In previous studies, we have shown that a traumatic lesion to the hippocampus of adult mice induces the transitory expression of TNF alpha and IL-1 alpha by neurons of different brain areas and also by glial cells at the site of injury. The aim of the present study was to establish whether the expression of TNF alpha and IL-1 alpha is restricted to defined subpopulations, or else is common to most of the central neuronal populations. Using polyclonal anti-GAD 67, anti-TH and monoclonal anti-ChAT, and anti-5-HT antibodies in a double-labeling immunohistochemical procedure in combination with murine anti-TNF alpha and anti-IL-1 alpha polyclonal antibodies, we show that most GABAergic, catecholaminergic, and serotoninergic neurons, and a subgroup of the cholinergic neurons, express these cytokines. Although not immunohistochemically characterized, neurons in some glutamatergic structures such as the hippocampus and the prefrontal cortex also express these cytokines. Thus, we conclude that the capacity of central neurons to express cytokines like TNF alpha and IL-1 alpha in reaction to a brain injury is not restricted to peculiar neuronal subtypes, but could include most of the neuronal populations of the brain.

Differential oligodendroglial expression of the tumor necrosis factor receptors in vivo and in vitro.

The cytokine tumor necrosis factor-alpha (TNF alpha) has been proposed to play a key role in the degenerative processes observed in demyelinating diseases such as multiple sclerosis (MS). In the immune system the cellular responses to TNF are mediated by two different receptors: TNF-RI, which is involved in cell death, and TNF-RII, which has been shown to mediate cell proliferation. We investigated the oligodendroglial expression of TNF-RI and -RII. In vivo, in normal adult rodent brain, oligodendrocytes express TNF-RII but not TNF-RI. However, after 3 days in culture, both types of receptors were expressed by mature oligodendrocytes, purified from 4-week-old rats, suggesting that expression of TNF-RI was induced by either the isolation process or the culture conditions. This inducibility of TNF-RI may explain the differences in oligodendrocyte cell death reported in various experimental conditions and in the pathology of MS lesions.

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

[New concepts on the role of cytokines in the central nervous system]. / Nouveaux concepts sur le rôle des cytokines dans le système nerveux central.

Initially described as modulatory molecules in the peripheral immune system and during haematopoiesis, several cytokines also play a role in the brain. Their synthesis in the central nervous system (CNS) is not due solely to glial cell activation or invading immune cells. On the one hand, several functions of central neurons are modulated by cytokines such as IL-1, TNF alpha, IL-2 and IL-6. Thus, IL-1 and TNF alpha modulate the synthesis of several neuromediators and modify ion influxes. IL-2 regulates the effects of central dopaminergic neurons on cholinergic, noradrenergic, serotoninergic and glutamatergic functions. On the other hand, neurons have recently been shown to be able to synthesize some of these cytokines under specific traumatic conditions. For example, a lesion to the hippocampus induces neuronal synthesis of IL-1 alpha and TNF alpha. This induction through neuronal circuits may operate at a distance in contrast to the glial reaction operating only locally. The recent demonstration of the expression by central neurons of receptors specific for these cytokines support a potentially crucial role for these molecules in brain function. Some data emerge in the literature demonstrating a potent expression of cytokines in the central nervous system in numerous pathological situations. Then, it appears that, at the interface between nervous and immune systems, cytokines may bear a pivotal role in the development of specific symptoms in neuroimmune diseases.

[Cytokine synthesis: a new form of response of central neurons]. / Synthèse de cytokines: une nouvelle forme de réponse des neurones centraux.

This report provides the immunohistochemical demonstration of the appearance of tumor necrosis factor alpha and interleukin-1 alpha in neuronal cells of different regions of the brain after a surgical injury to the hippocampus. We also demonstrate, by an in situ hybridization technique using a digoxigenin-labeled probe, the induction of TNF alpha mRNA in these neurons. Control lesions in brain areas such as the striatum, the parietal cortex or the cerebellum do not induce the neuronal expression of these cytokines. We hypothesize that cytokines production by neurons may be related with neurotrophin synthesis and restoration of homeostasis in an attempt to promote survival and regeneration of specific neuronal cell populations after a lesion to the hippocampus.

TNF alpha gene expression is induced in neurones after a hippocampal lesion.

The tumour necrosis factor alpha (TNF alpha) protein is normally absent in the brain. Its production in the nervous tissue during pathological processes is commonly attributed to cells of the macrophage or astroglial lineages. However, an immunoreactivity for TNF alpha has been observed recently in adult mouse brain after a lesion to the hippocampus. The identification, in the present study, of the cells responsible for this synthesis demonstrates a neuronal localization of the TNF alpha messenger RNA. We propose that neurone-produced TNF alpha acts as a modulatory effector in post-traumatic regenerative attempts of the brain.

Localization of TNF alpha and IL-1 alpha immunoreactivities in striatal neurons after surgical injury to the hippocampus.

Since the inflammatory process develops after transplantation to the brain, we sought to determine the presence of cytokines following a surgical trauma to the brain of an adult mouse. We report the early and marked presence of tumor necrosis factor-alpha and interleukin-1 alpha in neuronal somata of the striatum following a surgical injury to the hippocampus. The expression of cytokines later extends to neuronal cells of the hippocampus, thalamus, cerebral cortex, brain stem, and cerebellum and to glial cells of the corpus callosum. By contrast, these cytokines are not expressed by neuronal cells following injury to other regions, such as the striatum, cerebellum, and cortex. This study suggests a possible role for certain neurons in the brain's early reaction to a penetrating injury.

In situ transformation of striatal glia into cerebellar-like glia after brain transplantation.

Transplants of striatum from rabbit embryo were implanted into the colliculus posterior of newborn mice. After 4 weeks, astroglial cells derived from the transplant had migrated into the cerebellum of the host. Whenever they had settled in the cerebellum they presented forms similar to local glia. Some migrated glial cells were found to transform into forms of glia, such as radial-like glia, which are not present in the striatum. This observation confirms that glial precursor cells are highly plastic. It is an in vivo demonstration that local conditions alone define the morphology of glial cells. After grafting in an heterotopic location they take on forms that they were not destined to express in the region of origin.