A single intracerebral microinjection of platelet-derived growth factor (PDGF) accelerates the rate of remyelination in vivo.

We had demonstrated that platelet-derived growth factor (PDGF) enhanced the reconstruction of myelin-like membranes after their disruption by lysophosphatidylcholine (LPC) in vitro. To investigate its role in vivo, a demyelinating lesion of the corpus callosum was induced in adult Wistar rats by a stereotaxic microinjection of 1 microl LPC, then 63 pairs of rats received either 1 microg PDGF, or its vehicle buffer which were injected above LPC. The effects of PDGF were significant after 2 weeks: the number of oligodendrocytes (OL) expressing 2',3'-cyclic nucleotide 3'-phosphodiesterase in the lesion increased by 49%, mature OL labelled by in situ hybridization for myelin basic protein-mRNA increased by 27% (P<10(-2)), and the total volume of demyelination decreased by 60% compared to controls. The proliferation of cells of the OL lineage was also enhanced up to 67% by PDGF compared to LPC controls (P<2.5 x 10(-2)). Ultrastructural studies confirmed this dramatic improvement, and the ratio of remyelinated to demyelinated axons, determined at the maximal demyelination site, in the centre of the lesion, increased by 10-fold (P<2.5 x 10(-3)) in animals treated with PDGF. Remyelination was complete after 3 months for both treatments. Neither exacerbation of gliosis nor glial tumoural transformation were observed. Mechanisms through which PDGF improves remyelination could involve proliferation of OL progenitors, and/or of already differentiated surviving OLs, and a chemotactic effect, which had been identified in vitro.

Hepatocyte-derived cell lines express a functional receptor for cardiotrophin-1.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study, we show that CT-1 binds to hepatocyte-derived cell lines of rat and human origin with high (Kd = 600-800 pM) and low (Kd approximately 3-6 nM) binding affinities. Treatment of HepG2 cells with CT-1 resulted in the induction of tyrosine phosphorylation of both transducing receptor subunits, gp130 and LIF receptor, and this phosphorylation was completely inhibited by a neutralizing anti-gp130 mAb. Addition of CT-1 to HepG2 or H35 cell cultures induced a dose-dependent production of several acute phase proteins (haptoglobin, fibrinogen, alpha1-acid glycoprotein, alpha2-macroglobulin). Moreover, the use of a neutralizing mAb to gp130 in cultures of HepG2 cells grown in the presence of CT-1, inhibited the induction of acute phase protein secretion, indicating an absolute requirement of gp130 in the formation of a functional CT-1 receptor. Altogether, these results suggest that CT-1 could play an important role in the regulation of hepatocyte metabolism in inflammatory responses.

Signaling of type II oncostatin M receptor.

Oncostatin M (OSM) mediates its bioactivities through two different heterodimer receptors. They both involve the gp130-transducing receptor, which dimerizes with either leukemia inhibitory receptor beta or with OSM receptor beta (OSMRbeta) to generate, respectively, type I and type II OSM receptors. Co-precipitation of gp130-associated proteins, flow cytometry, polymerase chain reaction, and tyrosine phosphorylation analyses allowed the characterization of both types of OSM receptors expressed on the surface of different cell lines. It also allowed the detection of a large size protein, p250, that specifically associates to the type II OSM receptor components and that is tyrosine-phosphorylated after the activation peak of the gp130.OSMRbeta heterocomplex. The restricted expression of type I OSM receptor by the JAR choriocarcinoma cell line, and type II receptor by the A375 melanoma cell line, permitted the characterization of their signaling machineries. Both type I and type II OSM receptors activated Jak1, Jak2, and Tyk2 receptor-associated tyrosine kinases. The information is next relayed to the nucleus by the STAT3 transcriptional activator, which is recruited by both types of OSM receptors. In addition, STAT5b was specifically activated through the gp130.OSMRbeta type II heterocomplex. The signaling pathway differences observed between the common type I LIF/OSM receptor and the specific type II OSM receptor might explain some of the bioactivities specifically displayed by OSM.

Signaling of the cardiotrophin-1 receptor. Evidence for a third receptor component.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study we show that CT-1 activates its receptor expressed at the surface of a human neural cell line by recruiting gp130 and gp190/leukemia inhibitory factor receptor beta, as shown by analyzing their tyrosine phosphorylation level. Neutralizing antibody directed against gp130 and reconstitution experiments performed in the COS-7 cell line demonstrate that gp130-gp190 heterocomplex formation is essential for CT-1 signaling. Analysis of the subsequent activation events revealed that CT-1 induces and utilizes Jak1-, Jak2-, and Tyk2-associated tyrosine kinases, which are in turn relayed by STAT-3 transcription factor. Cross-linking of iodinated CT-1 to the cell surface led to the identification of a third alpha component in addition to gp130 and gp190, with an apparent molecular mass of 80 kDa. Removal of N-linked carbohydrates from the protein backbone of the alpha component resulted in a protein of 45 kDa. Our results provide evidence that the CT-1 receptor is composed of a tripartite complex, a situation similar to the high affinity receptor for ciliary neurotrophic factor.

Alanine substitution for Thr268 and Asp269 of soluble ciliary neurotrophic factor (CNTF) receptor alpha component defines a specific antagonist for the CNTF response.

Ciliary neurotrophic factor (CNTF) associates with an alpha subunit (CNTFRalpha) of the receptor complex to initiate signal transduction by facilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) beta. CNTFRalpha is anchored to the membrane by a glycosylphosphatidylinositol linkage; however, a soluble form of the alpha subunit can still bind CNTF to recruit the signal transducing components of the receptor complex. In the present study we show that alanine substitution for residues Thr268 and Asp269 of the CNTFRalpha subunit results in a mutated receptor subunit (R3), which can bind CNTF with an affinity similar to that of the wild type CNTFRalpha but, when expressed as a soluble receptor subunit, lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3alpha inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFRalpha but not by IL-6 or oncostatin M. Similarly, CNTFR3alpha specifically antagonizes the induction of gp130 and LIFRbeta tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFRalpha in the HepG2 hepatoma cell line, as well as the subsequent events leading to haptoglobin synthesis. Positions 268 and 269 of CNTFRalpha appear to be critical for its interaction with gp130 and LIFRbeta, whereby alanine substitution of the residues at these positions results in antagonism of the CNTF-induced response.

Interleukin-6 family of cytokines induced activation of different functional sites expressed by gp130 transducing protein.

Gp130 transducing protein was shown to be involved in the formation of the high affinity receptors for interleukin 6 (IL-6), interleukin-11 (IL-11), leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1. In the present study we have characterized the functional properties of antibodies directed against this protein and identified a group of monoclonal antibodies able to antagonize the biological activities of all the cytokines belonging to the IL-6 cytokine family. The B-R3 pan-blocking antibody weakly interfered with the binding of the radiolabeled ligands (with the exception of OSM, whose binding was abrogated in the presence of B-R3 monoclonal antibody) but inhibited the gp130 homodimerization or its association with gp190/leukemia inhibitory factor receptor, as well as the subsequent tyrosine phosphorylation events. In addition we identified antibodies that were able to neutralize only one single cytokine of the IL-6 family. This was the case for the B-K5 antibody, which antagonized the binding of OSM to gp130 but did not interfere with the signals provided by the related cytokines triggering the proliferation of the TF1 erythroleukemia cell line or the induction of haptoglobin synthesis in the HepG2 hepatoma cell line. Similarly, we also characterized two additional antibodies B-P8 and B-P4, which inhibited the TF1 cell proliferation observed in the presence of CNTF and IL-11, respectively. B-P8 antibody only faintly interfered with the binding of the gp130-ligands and might modulate the signal transduction pathways. This study indicates that in addition to functional site(s) required by the whole family of IL-6 type cytokines to transduce the signal insight the cell, specific cognate functional sites were recruited by OSM, CNTF, or IL-11.

gp130 transducing receptor cross-linking is sufficient to induce interleukin-6 type responses.

gP130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XGI hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells.

Platelet-derived growth factor partly prevents chemically induced oligodendrocyte death and improves myelin-like membranes repair in vitro.

We have previously shown that pure oligodendrocyte (OL) secondary cultures derived from newborn rat brain, in which cells form myelin-like membranes, can be used as a model to investigate the putative role of growth factors in myelin repair. After disruption of these membranes by lysophosphatidylcholine (LPC), a 3 day treatment with 10 ng/ml basic fibroblast growth factor (bFGF) induced reconstruction of myelin figures, albeit less compacted than in untreated controls. Here we show that in LPC treated cultures: 1) bFGF can not prevent OL from LPC-induced cell death; 2) platelet-derived growth factor (PDGF) pretreatment although preventing some cell death does not improve recovery compared to delayed treatment; 3) PDGF is as potent as bFGF in terms of O-2A progenitor proliferation; 4) PDGF is far more effective than bFGF, inducing the reappearance of more myelin-like structures with a better compaction; 5) there is no potentiation between these growth factors; and 6) after withdrawal of bFGF the compaction of myelin figures partly increases. These results indicate that PDGF, probably by inducing O-2A progenitors to proliferate and then allowing them to differentiate into mature myelinating OL, is a better candidate than bFGF to participate in myelin repair mechanisms in the central nervous system.

Fate and biocompatibility of three types of microspheres implanted into the brain.

The implantation of polymer devices in the brain that release neuroactive drugs locally and in a controlled manner is gaining increasing interest. The fates and tissue reactions of poly(epsilon-caprolactone), ethylcellulose, and polystyrene microspheres, prepared by the solvent evaporation method, radiosterilized by gamma-irradiation, and stereotactically implanted in rat brain have been studied by routine staining and immunohistochemistry. During the first few days after implantation, a nonspecific astrocytic brain tissue reaction was observed along with a macrophagous-microglial cell reaction typically found following any damage in the central nervous system, except in the presence of certain foreign body giant cells. Nine months into the experiment, microspheres appeared to be engulfed by histiocytic cells. The microsphere cluster was surrounded by a sheath composed of collagen and astrocytic cells. No necrosis was observed, suggesting the absence of toxicity. In some animals, however, an hydrocephalus developed as a result of obstruction of the medial ventricle by some microspheres.

Lipopolysaccharide intracerebral administration induces minimal inflammatory reaction in rat brain.

An inflammatory reaction, essential for defence against infection and for wound repair, may also induce irreversible tissue damage. It appears that the central nervous system has developed its own immunosuppressive strategy in order to limit the destructive effects of inflammation. To clarify this point, we have characterized in one unique model of inflammation induced in the rat by intracerebral lipopolysaccharide injection the kinetics of the inflammatory reaction, the participation of immunitary and glial cells and of three growth factors. Among these molecules, brain-derived neurotrophic factor mRNA expression was found decreased following LPS injection. No striking differences were observed in the brain parenchyma after stab lesion or inflammatory lesion apart from an increase in the number of monocytes/macrophages recruited early to the lesion area. Macrophages were later accumulated around the lesion when astroglia and microglia reactions occurred. Some of the macrophages and microglia expressed major histocompatibility complex class II antigens on their surface whereas no T or B lymphocytes were observed in the brain parenchyma. However, a subpopulation of CD3- and CD4-negative CD8-positive cells, likely natural killer cells, was observed around the lesion site; this recruitment was inhibited by the highest dose of LPS. This study therefore supports the hypothesis of a suppression of some aspects of cell-mediated immunity in the brain, mechanisms which need to be further characterized.

[Ultrastructural and immunohistochemical study of the olfactory mucosa in Alzheimer's disease]. / Etude ultrastructurale et immunohistochimique de la muqueuse olfactive dans la maladie d'Alzheimer.

The discovery of reliable peripheral markers would be of great interest for the diagnosis of Alzheimer's disease. Interestingly specific lesions of Alzheimer's disease were found in the olfactory areas of the brain. The loss of the detection and the identification capabilities in olfaction suggest a defect in the olfactory mucosa. The ultrastructural study of biopsy of olfactory mucosa from patients suspected to suffer of Alzheimer's disease revealed a degeneration of sustentacullar and olfactory cells as well as an architectural disorganization. No neuropathological characteristic lesion such as Paired Helicoidal Filaments or amyloid fibrils could be evidenced. Immunohistochemical analysis achieved with a polyclonal antiserum raised against A4 protein (fragment 1-28) showed a specific staining in the sustencellular and of the outer part of the olfactory epithelium corresponding to the mucus area, whereas appropriate controls were negative. Our study raised the question of the amyloid protein origin in the olfactory mucosa. Analysis of some more patients will enable us to determine the diagnostic value of this study in Alzheimer's disease.

Autoantibodies against H- and M-subunits of neurofilaments are induced by PC12 cell grafts or lesions into different sites of rat brain.

Since autoantibodies against neurofilaments (NF) were frequently found in neurodegenerative disorders, this work is an attempt to investigate whether the same phenomenon occurs after intracerebral grafting or lesioning. We have thus either grafted PC12 cells or injected culture medium alone into three sites of rat central nervous system (CNS): olfactory bulb (OB), olfactory anterior nucleus (OAN) and hippocampus (HC), all three sites being impaired in Alzheimer's disease. At day 15, rat sera were collected and tested against NF by western blotting. Sera from grafted rats recognized the H- and M-subunits of NF; we have then quantified the autoantibody response by using an ELISA technique. We show that, in all cases of grafts, the autoantibody response against NF significantly increased when compared to controls (normal rats without grafts or lesions) for total immunoglobulin (Ig) amount. In contrast, concerning the Ig isotypes, some differences appeared depending on the implantation site: for grafts into OB, the immune response was of both the IgG and IgM isotypes, into OAN it was mainly of the IgM isotype and into HC, the isotype of antibodies against NF was mostly IgG. In the case of lesions alone into OAN and HC, no significant enhancement of autoantibody response was observed; in contrast, lesions into OB induced an increase in autoantibody response against NF which significantly differed from controls for all Ig isotypes tested. These data point out the diversity of the autoantibody responses following lesions or grafts according to the rat brain areas.

n-6 polyunsaturated fatty acids increase the neurite length of PC12 cells and embryonic chick motoneurons.

We have tested the action of three n-6 polyunsaturated fatty acids, either free or in the form of ethyl esters, on the neurite outgrowth in two neuronal models: a rat pheochromocytoma cell line (PC12) and embryonic chick motoneurons, after 7 days in culture. An inverted microscope coupled with the 'VIDS 4' software was used for measuring the neurite length. Free fatty acids were found to be cytotoxic at 10(-3) M and the maximal increase of the neurite length was obtained at 10(-5) M. In contrast, fatty acids in the form of ethylesters were not cytotoxic and at 10(-3) M induced the maximal increase in the neurite length. This increase (1.2 to 2 fold) significantly differed from the control and was dose-dependent. These results were discussed in relation to the action of fatty acids on enzyme activation and membrane fluidity.

Biodegradation and brain tissue reaction to poly(D,L-lactide-co-glycolide) microspheres.

The therapeutic application of neuroactive molecules in neuroscience is limited, due to the problems posed by the administration of these drugs (peripheral metabolism, systemic effect and passage of the blood-brain barrier). One solution is the implantation in the brain of biodegradable polymer devices with controlled release of a neuroactive drug. The biodegradation and tissue reaction of the copolymer poly(D,L-lactide-co-glycolide) microspheres prepared by the solvent evaporation method, radiosterilized and stereotactically implanted in the rat brain were studied by routine staining, immunohistochemistry and transmission electronic microscopy. The brain tissue reaction observed was a non-specific astrocytic proliferation and a macrophagous-microglial cell reaction, typically found following damage to the central nervous system. Some foreign-body giant cells were observed and the inflammatory and macrophagous reaction decreased dramatically after 1 month and almost ended after 2 months when the microspheres were totally biodegraded. The copolymer poly(D,L-lactide-co-glycolide) microspheres may be considered biocompatible to the brain tissue.

Differential behaviour of PC12 cells grafted into rat hippocampus and striatum.

To investigate the mechanisms involved in graft survival, a rat cell line (PC12) that differentiates into sympathetic-like neurons by exposure to trophic factors has been grafted into rat striatum and hippocampus, two structures which differ in their amounts of trophic factors. Our results show that grafted PC12 cells behave differently depending on the area of implantation; they display a differentiated morphology in the hippocampus and proliferate as a tumor in the striatum. A qualitatively similar immunological reaction occurs in both structures, characterized by the invasion of T and B lymphocytes, macrophage-like cells and by the expression of major histocompatibility complex class I and II antigens around the graft.

Abnormal expression of actin in lymphocytes of Alzheimer's disease and Down's syndrome patients.

Alzheimer's disease (AD) is a degenerative disorder of the central nervous system accompanied by several immunological disturbances and a number of common features exist between AD and Down's syndrome (DS). High resolution two-dimensional electrophoresis of lymphocyte proteins demonstrates an actin abnormality in AD and DS: a double actin spot instead of the single spot observed in controls. This dual form was studied by pulse-chase experiments and seems to be related to extracellular factors which influence the post-translational modification of actin. These results agree with the immunological disturbances observed in AD and DS, and with the well established hypothesis that AD is a systemic as well as cerebral disease.

Pure Schwann cell suspension grafts promote regeneration of the lesioned septo-hippocampal cholinergic pathway.

Regeneration of central nervous system (CNS) axons has been studied in the cholinergic septo-hippocampal system using various 'bridges' able to support fiber growth. In this study, a pure Schwann cell (Sc) suspension labeled with bisbenzimide (Hoechst 33342) was grafted in the lesioned septo-hippocampal pathway. At 2 weeks post-grafting, acetyl-cholinesterase (AChE)-positive fibers invaded the graft and grew in association with the Hoechst-labeled Sc, some of which expressed the low-affinity nerve growth factor receptor (NGF-R). At 2 months and 4 months post-grafting, the dorsal hippocampus was reinnervated with an apparently normal innervation pattern. Analysis of fiber growth in the hippocampus at four months post-grafting revealed a significant increase of reinnervation in the grafted animals (2 mm) compared to the non-grafted ones. No difference was observed in the number of cholinergic septal neurons expressing the NGF-R. These results demonstrate that a Sc suspension grafted into the lesioned septo-hippocampal system, integrates well into the host tissue, and supports axonal CNS outgrowth, implying that Sc by themselves provide an adequate environment for regeneration to occur.

[Intracerebral grafts in Parkinson's disease]. / Les greffes intracérébrales dans la maladie de Parkinson.

Although the beginnings of neural transplantation can be traced back to the last century, this technique was not fully developed until the 1970's. Following an experimental stage, considered insufficient by some authors, the first neural grafts on humans were performed in Parkinsonians in 1982. One must, in fact, distinguish between two types of operation, each with its own ethical and scientific problems. The first operation is human embryonic neural transplants the effects of which are related to a real reinnervation of the striatum. The other operation consists of autografts of adrenal medulla, which still have hypothetical modes of action. The first results obtained in man with both types of operation are rather disappointing. Even though neuronal grafting is unquestionably a technique of the future, much caution must be exerted since intracerebral grafting in Parkinson's disease remains at the experimental stage.

Aging and Alzheimer's disease: protease inhibitors in cerebrospinal fluid.

Recent advances suggested that proteases and their inhibitors could be implicated in the genesis and/or maturation of insoluble deposits associated with Alzheimer's disease (AD). This study was designed to measure the level of alpha 1-antichymotrypsin (ACT) and alpha 1-antitrypsin (AT) in cerebrospinal fluid (CSF) of patients with AD and nondemented humans at various ages. Our analysis failed to demonstrate a significant relationship between inhibitor content and disease. However, a positive correlation was observed between age and the ACT level for the normal control group. Such observation suggests a specific association of ACT with the mechanisms of brain aging.

[Olfactory mucosa and Alzheimer's disease. Technique for biopsy and ultrastructural study]. / Muqueuse olfactive et maladie d'Alzheimer. Technique biopsique et étude ultrastructurale.

The discovery of a reliable peripheral marker would be of a great interest for the early diagnosis of the Alzheimer's Disease. The olfactory deficit and the major histologic changes of the olfactory-related areas of the brain occurring during this disease raised the possibility that the olfactory epithelia could be one of the way of entry of a possible process that still has to be identified. We have developed an instrument and a technique of biopsy of the human olfactory mucosa to search for the presence of characteristics lesions on patients suffering of an Alzheimer's Disease. These small specimens have been prepared for electronic microscopy. The ultrastructural study of a sample of olfactory mucosa has been realised in 9 cases (5 Alzheimer's-4 controls) revealing in 4 patients suspected of an Alzheimer's Disease a complete architectural disorganisation with a destruction of the dendrite of the olfactory cells and a severe degeneration of the sustentacular cells. We did not find any characteristic changes such as Paired Helicoidal Filaments or amyloid fibrils. These results do not presuppose of their eventual presence at a precocious stage of the disease. Further ultrastructural and immunochemical studies carried out with patients at various stages of the disease are necessary in order to confirm this hypothesis.