Axonal regeneration is compromised in NFH-LacZ transgenic mice but not in NFH-GFP mice.

To investigate neurofilament (NF) dynamics during the cytoskeleton reorganization in regenerating axons, and their electrophysiological and histological consequences, we used two transgenic lines of mice: neurofilament high (NFH)-LacZ and NFH-green fluorescent protein (GFP). In NFH-LacZ mice, NFs are retained in cell bodies and deficient in axons (Eyer and Peterson, 1994), while in NFH-GFP mice the fluorescent fusion protein is normally transported along axons (Letournel et al., 2006). Following a crush of the sciatic nerve, conduction recovery in NFH-GFP mice is similar to wild-type (wt) mice, but it is reduced in NFH-LacZ mice. Moreover, changes of axonal calibres following regeneration are similar between NFH-GFP and wt mice, but they are systematically reduced in NFH-LacZ mice. Finally, the axonal transport of NFH-GFP fusion protein and NFs is re-initiated after the crush as evidenced by the fluorescent and immunolabelling of axons distal from the crushed point, but NFs and the fusion protein are not transported along axons during regeneration in NFH-LacZ mice. Together, these results argue that the absence of axonal NFs in NFH-LacZ mice compromises the axonal regeneration, and that the NFH-GFP reporter fusion protein represents an efficient model to evaluate the NF dynamics during axonal regeneration.

[Preanalytical guidelines for clinical proteomics investigation of biological fluids]. / Recommandations préanalytiques pour les analyses de protéomique clinique des fluides biologiques.

Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.

An autopsy case of acute multiple sclerosis (Marburg's type) during pregnancy.

We report a case of a 9-month pregnant woman who presented acute psychiatric and neurological symptoms with extensive involvement of the white matter on MRI and no oligoclonal bands on CSF examination. Despite high doses of intravenous steroids, plasmapheresis and immunosuppressive drugs, a fatal outcome (coma) was noted 8 months later. Neuropathological examination confirmed the diagnosis of Marburg's type of multiple sclerosis showing sharp-edged lesions of demyelination, giant astrocytes, numerous macrophages and little perivascular inflammation. We discuss the definition and limits of the Marburg entity with reference to acute disseminated encephalomyelitis, impact of pregnancy, unusual MRI features, neuropathology and treatment.

Neurofilament high molecular weight-green fluorescent protein fusion is normally expressed in neurons and transported in axons: a neuronal marker to investigate the biology of neurofilaments.

The carboxy-terminal side arm of the neurofilament high subunit consists of a highly phosphorylated domain and a negatively charged region. Multiple evidences suggested that these domains are essential for the axonal phosphorylation and transport of neurofilaments and play a role in their abnormal accumulation following chemical intoxication or during neurodegenerative disorders such as amyotrophic lateral sclerosis. In order to investigate the consequences of altering this side arm of neurofilament high subunit we used a fusion protein (neurofilament high subunit-green fluorescent protein) between the mouse neurofilament high subunit missing a major part of the C-terminal domain and the reporter green fluorescent protein. In cell culture and in transgenic mice this fusion protein co-assembles and co-distributes with the endogenous intermediate filament network. Conditions known to disturb the cytoskeleton were also found to alter the distribution of the fusion protein in cell cultures. In transgenic mice the expression of the transgene evaluated by its fluorescent properties was found to be restricted to neurons, where the neurofilament high subunit-green fluorescent protein fusion protein is axonally transported. Biochemical approaches showed that the fusion protein is phosphorylated and co-purified with neurofilaments. Despite the presence of such an neurofilament high subunit-green fluorescent protein fusion protein, the axonal cytoskeletal density and the axonal caliber were not altered. Together these data show that removal of this portion of neurofilament high subunit does not affect the capacity of neurofilament high subunit to assemble and to be transported into axons, suggesting that this sequence is involved in another function. Moreover, the fluorescent properties of this fusion protein represent a useful marker.

[Malignant non-Hodgkin lymphoma presenting with Garcin's syndrome]. / Syndrome de Garcin révélant un lymphome malin non hodgkinien.

INTRODUCTION: R Garcin described progressive unilateral cranial nerve palsy in 1926. Garcin syndrome is characterized by progressive involvement of the cranial nerves culminating in total unilateral paralysis of all cranial nerves. Carcinoma of the skull base or ENT regions is the most common etiology. CASE REPORT: A 74-year-old man developed signs involving the left Vth (V2 and V3) cranial nerve then the VIth, VIIth and VIIIth cranial nerves and finally the IXth and Xth. MRI showed involvement of these cranial nerves with gadolinium uptake and involvement of the pons at the terminal phase. Careful ENT explorations failed to reveal a cause. The lymphocyte count was elevated in the cerebrospinal fluid. The patient died one year after diagnosis and the general autopsy was normal. The neuropathological studies led to the post-mortem diagnosis of type B non-Hodgkin lymphoma. CONCLUSION: In patients with Garcin syndrome, lymphoma is a possible diagnosis when carcinoma of the ENT regions or of the skull bases are not present.

Stable tubule only polypeptides (STOP) proteins co-aggregate with spheroid neurofilaments in amyotrophic lateral sclerosis.

A major cytopathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of axonal spheroids containing abnormally accumulated neurofilaments. The mechanism of their formation, their contribution to the disease, and the possibility of other co-aggregated components are still enigmatic. Here we analyze the composition of such lesions with special reference to stable tubule only polypeptide (STOP), a protein responsible for microtubule cold stabilization. In normal human brain and spinal cord, the distribution of STOP proteins is uniform between the cytoplasm and neurites of neurons. However, all the neurofilament-rich spheroids present in the tissues of affected patients are intensely labeled with 3 different anti-STOP antibodies. Moreover, when neurofilaments and microtubules are isolated from spinal cord and brain, STOP proteins are systematically co-purified with neurofilaments. By SDS-PAGE analysis, no alteration of the migration profile of STOP proteins is observed in pathological samples. Other microtubular proteins, like tubulin or kinesin, are inconstantly present in spheroids, suggesting that a microtubule destabilizing process may be involved in the pathogenesis of ALS. These results indicate that the selective co-aggregation of neurofilament and STOP proteins represent a new cytopathological marker for spheroids.

Two clinicopathological cases of a dominantly inherited, adult onset orthochromatic leucodystrophy.

Leucodystrophies of orthochromatic type are a heterogeneous group that occur mainly in childhood and have no known enzyme deficiency. We report here the clinicopathological features of a new family of orthochromatic leucodystrophy with three main characteristics: a probably autosomal dominant inheritance; two phenotypes based on age of onset; and very few abnormalities of white matter on MRI findings in one case. The first patient, aged 58 years, had frontal dementia and epilepsy; the second, aged 38 years, had motor signs and dementia, but no epilepsy. The histopathological features of our two cases were leucodystrophy of orthochromatic subtype. However, the radiological features (MRI and mostly FLAIR sequences) of the first case did not suggest leucodystrophy.

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.

Central sympathetic dysregulation and immunological abnormalities in a case of progressive facial hemiatrophy (Parry-Romberg disease).

A case of hemifacial atrophy (Parry-Romberg disease) is discussed. Electrophysiological and immunological studies were performed. Electromyography, blink reflex and trigeminal evoked potential abnormalities indicate that the brain stem may be implicated in the aetiology of the disease. Immunological evidence favoured this possibility and demonstrated possible involvement of the noradrenergic system. Hyperactivity of the brain stem sympathetic centres, possibly caused by an autoimmune process, may be the primary cause of the cutaneous and subcutaneous atrophy in Parry-Romberg disease.

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.