Antibodies to neuronal surface proteins in Tourette Syndrome: Lack of evidence in a European paediatric cohort.

In Tourette Syndrome (TS) a role for autoantibodies directed against neuronal proteins has long been suspected, but so far results are still inconsistent. The aim of this study was to look for antibodies to specific or undefined neuronal proteins that could be involved in the aetiology of the disease. Sera from children with Tourette Syndrome or another chronic tic disorder (TS/TD), collected as part of the longitudinal European Multicenter Tics in Children Study, were investigated. Participants included 30 siblings of patients with TS/TD prior to developing tics (preclinical stage) and the same children after the first tic onset (onset), and 158 patients in the chronic phase undergoing an acute relapse (exacerbation). Presence of antibodies binding to rodent brain tissue was assessed by immunohistology on rat brain sections and by immunofluorescent staining of live hippocampal neurons. Live cell-based assays were used to screen for antibodies to NMDAR, CASPR2, LGI1, AMPAR and GABAAR. Immunohistology indicated evidence of antibodies reactive with brain tissue, binding mainly to the hippocampus, the basal ganglia or the cerebellum in 26/218 (12%), with 8% of the preclinical or onset sera binding to the dentate gyrus/CA3 region or cerebellum. Only two individuals (one pre-clinical, one chronic) had antibodies binding the NMDAR and the binding was only weakly positive. No other specific antibodies were detected. Despite some immunoreactivity towards neuronal antigens on brain tissue, this was not mirrored by antibodies binding to live neurons, suggesting the presence of non-specific antibodies or those that bind non-pathogenic intracellular epitopes. NMDAR or the other neuronal surface antibodies tested were very infrequent in these patients. The evidence for pathogenic antibodies that could be causative of TS is weak.

Radiofrequency radiation (900 MHz) induces Egr-1 gene expression and affects cell-cycle control in human neuroblastoma cells.

Many environmental signals, including ionizing radiation and UV rays, induce activation of Egr-1 gene, thus affecting cell growth and apoptosis. The paucity and the controversial knowledge about the effect of electromagnetic fields (EMF) exposure of nerve cells prompted us to investigate the bioeffects of radiofrequency (RF) radiation on SH-SY5Y neuroblastoma cells. The effect of a modulated RF field of 900 MHz, generated by a wire patch cell (WPC) antenna exposure system on Egr-1 gene expression, was studied as a function of time. Short-term exposures induced a transient increase in Egr-1 mRNA level paralleled with activation of the MAPK subtypes ERK1/2 and SAPK/JNK. The effects of RF radiations on cell growth rate and apoptosis were also studied. Exposure to RF radiation had an anti-proliferative activity in SH-SY5Y cells with a significant effect observed at 24 h. RF radiation impaired cell cycle progression, reaching a significant G2-M arrest. In addition, the appearance of the sub-G1 peak, a hallmark of apoptosis, was highlighted after a 24-h exposure, together with a significant decrease in mRNA levels of Bcl-2 and survivin genes, both interfering with signaling between G2-M arrest and apoptosis. Our results provide evidence that exposure to a 900 MHz-modulated RF radiation affect both Egr-1 gene expression and cell regulatory functions, involving apoptosis inhibitors like Bcl-2 and survivin, thus providing important insights into a potentially broad mechanism for controlling in vitro cell viability.

Behaviour of SH-SY5Y neuroblastoma cell line grown in different media and on different chemically modified substrates.

Among the parameters that can be tested in experiments on neuronal cell culture the use of different culture media and substrates represents a powerful assay to influence cell adhesion and differentiation. In this work, plasma-enhanced-chemical vapour depositions (PE-CVD) from acrylic acid and allylamine vapours have been performed to deposit coatings bearing oxygen (O)- and nitrogen (N)-containing functional groups on polyethylenetherephtalate (PET) surface. Human neuroblastoma SH-SY5Y cells were grown on plasma modified substrates and in presence of media containing different amount of fetal calf serum (FCS) or in serum-free medium containing cAMP. Our results showed that N-containing substrates improved cell adhesion, while the neurites sprouting was influenced by cell culture media. Interestingly, the presence of carboxylic groups on the modified surface can influence the expression of a differentiation marker, neurofilament-200 (NF-H), in cells grown in serum-containing media.

Transgenic mice expressing F3/contactin from the transient axonal glycoprotein promoter undergo developmentally regulated deficits of the cerebellar function.

We have shown that transgenic transient axonal glycoprotein (TAG)/F3 mice, in which the mouse axonal glycoprotein F3/contactin was misexpressed from a regulatory region of the gene encoding the transient axonal glycoprotein TAG-1, exhibit a transient disruption of cerebellar granule and Purkinje cell development [Development 130 (2003) 29]. In the present study we explore the neurobehavioural consequences of this mutation. We report on assays of reproductive parameters (gestation length, litter size and offspring viability) and on somatic and neurobehavioural end-points (sensorimotor development, homing performance, motor activity, motor coordination and motor learning). Compared with wild-type littermates, TAG/F3 mice display delayed sensorimotor development, reduced exploratory activity and impaired motor activity, motor coordination and motor learning. The latter parameters, in particular, were affected also in adult mice, despite the apparent recovery of cerebellar morphology, suggesting that subtle changes of neuronal circuitry persist in these animals after development is complete. These behavioural deficits indicate that the finely coordinated expression of immunoglobulin-like cell adhesion molecules such as TAG-1 and F3/contactin is of key relevance to the functional, as well as morphological maturation of the cerebellum.

Alternative promoters drive the expression of the gene encoding the mouse axonal glycoprotein F3/contactin.

F3/Contactin is a neuronal glycoprotein which mediates axonal growth control via complex interactions with a number of cell surface or matrix components. As part of this developmental role, its expression undergoes differential regulation during the maturation of definite neuronal populations within the central and peripheral nervous tissue. To elucidate the underlying molecular mechanisms we study here the organization of the regulatory region of the mouse F3/Contactin gene. We show that this region displays peculiar features in that it spans more than 80 kb, bears very large introns and includes four untranslated exons which undergo complex splicing events leading to 11 potential arrangements of the F3/Contactin mRNA 5' end. Within this region we identify three alternative neurospecific promoters which, as deduced from the developmental profile of the associated 5' exons (A1,C1,0), drive two different patterns of F3/Contactin gene expression. The activity of the A1 exon-associated promoter displays only minor developmental changes and is likely to contribute to the basal level of the F3/Contactin gene expression; by contrast, the activities of the exon C1- and exon 0-associated promoters are significantly upregulated at the end of the first postnatal week. The data indicate that differential regulation of the F3/Contactin expression during development may depend upon alternative utilization of distinct promoter elements and may involve complex splicing events of the 5' untranslated exons. Several consensuses for homeogene transcription factors are scattered within the identified regulatory region, in agreement with the general assumption of homeotic gene regulation of neural morphoregulatory molecules.

A functional interaction between the neuronal adhesion molecules TAG-1 and F3 modulates neurite outgrowth and fasciculation of cerebellar granule cells.

F3 and TAG-1 are two closely related adhesion glycoproteins of the Ig superfamily that are both expressed by the axons of cerebellar granule cells. In an in vitro system in which cerebellar granule cells were cultured on monolayers of transfected Chinese hamster ovary (CHO) cells, we show that F3 and TAG-1 interact functionally. F3 transfectants have been shown to inhibit outgrowth and induce fasciculation of granule cell neurites. By contrast TAG-1 transfectants have no effect on these events. However, when TAG-1 is coexpressed with F3, the inhibitory effect of F3 is blocked. Two possible mechanisms may account for this functional interaction: (1) either TAG-1 and F3 compete for the same neuronal receptor, and in favor of this we observed that binding sites for microspheres conjugated with F3 and TAG-1 are colocalized on the granule cell growth cones, (2) or alternatively, F3 and TAG-1 associate in a multimolecular complex after their binding to independent receptors. Extensive co-clustering of F3 with TAG-1 can in fact be achieved by anti-TAG-1 antibody-mediated cross-linking in double-transfected CHO cells. Moreover, F3 coimmunoprecipitates with TAG-1 in Triton X-100-insoluble microdomains purified from newborn brain. These data strongly suggest that F3 and TAG-1 may associate under physiological conditions to modulate neurite outgrowth and fasciculation of the cerebellar granule cells.

Functional organization of the promoter region of the mouse F3 axonal glycoprotein gene.

F3 is a developmentally regulated adhesive glycoprotein expressed by subpopulations of central and peripheral neurons which mediates neurite growth and fasciculation via cis- and trans-interactions with cell-surface or matrix components. We previously reported on the characterization of the F3 gene 5' flanking region in which we identified promoter and enhancer elements. Here, we report on the functional organization of the F3 gene regulatory regions. We show that the F3 promoter is built of linearly arranged positive and negative elements scattered through the 5' flanking region of the F3 gene and the 1st exon (exon 0). Neural- and cell type-specific expression of F3 appears to be governed by elements located in the most proximal promoter region which includes a neural-specific enhancer. In retardation assays, all these cis-acting elements bind nuclear proteins, three of which interact with the identified enhancer element while a single species interacts with sequences located within exon 0. Some of these proteins are also specifically expressed within the brain, indicating that they could correspond to neural-specific trans-acting factors. Elements located immediately upstream of the cell type-specific enhancer and within exon 0 are responsible for regulation of F3 expression by cAMP and retinoic acid.

Use of chimeric F3-NCAM molecules to explore the properties of VASE exon in modulating polysialylation and neurite outgrowth.

Differential splicing of VASE exon in the fourth immunoglobulin (Ig) domain and attachment to the fifth Ig domain of alpha 2-8 linked sialic acid (PSA) both dramatically change, in opposite manner, Neural Cell Adhesion Molecule (NCAM) functional properties. Reciprocal patterns of VASE and PSA expression suggest that they might be mutually exclusive. Here, we tested whether informations conferring polysialylation reside in NCAM-Ig domains 4 and 5 and the influence of the VASE exon encoded sequence on this process. We also examined if the VASE sequence was still able to inhibit neurite outgrowth when presented out of its normal NCAM context. Constructs have been prepared encoding NCAM-Ig domains 4 (with or without the VASE exon) and 5 fused to the F3 molecule. Stable clones expressing the chimeric molecules or wild type F3 were then obtained in the AtT-20 cell line. Although the chimeric molecules were expressed on the cell surface none of them was bearing PSA. Thus, polysialylation cannot be conferred to proteins by addition of the NCAM-Ig domains 4 and 5 modular motif and in this molecular context, the VASE sequence is not influencing the process. These chimeric molecules, either expressed at the surface of RIN or COS cells or presented as soluble forms, were examined for their effect on neurite outgrowth. In all cases, the length of neurites of sensory neurons was significantly reduced when grown in presence of the VASE containing chimera by comparison with the chimera without VASE or wild type F3. When neurons from NCAM knock-out mice were used for the assay, the VASE inhibition could not be detected. Thus VASE is able to act as a modular motif and NCAM expressed on neurons participates in transducing its effect.

F3 neuronal adhesion molecule controls outgrowth and fasciculation of cerebellar granule cell neurites: a cell-type-specific effect mediated by the Ig-like domains.

F3 is a glycane phosphatidylinositol-anchored neuronal adhesion glycoprotein which consists of immunoglobulin (Ig) domains and fibronectin type III repeats. Here we showed that total F3 or F3-Ig domains when presented as membrane components of CHO transfected cells influenced growth cone morphology, strongly inhibited outgrowth, and induced fasciculation of cerebellar granule cell axons. An F3-Ig-Fc chimera induced neurite fasciculation from cerebellar neuron aggregates when used as a coated substrate but not in the soluble form. The F3 effect on neurite elongation is highly specific for neuronal cell types since under the same experimental conditions it did not modify neurite outgrowth of hippocampal neurons and was shown to stimulate elongation of neurites from sensory neurons in both membrane-anchored and soluble form. Our results provide evidence to extend the proposed role of F3 and strongly suggest that axonal-growth-controlling molecules may quite generally exert dual actions which are likely to depend on the receptor repertoire of the responding neuron.

Characterization of the 5' and promoter regions of the gene encoding the mouse neuronal cell adhesion molecule F3.

F3 is a 135 kDa neuronal cell surface adhesive glycoprotein belonging to the immunoglobulin supergene family (IgSF) which mediates heterophilic contact formation among neural cells and is involved in the control of neurite growth. F3 expression is regulated, during critical developmental periods, on neuronal subpopulations thus suggesting that control of F3 gene expression could be of morphogenetic relevance. To shed light on the mechanism involved in the control of F3 gene expression we isolated clones covering about 50 kilobases of the F3 gene which also included the promoter region. The study of F3 gene exon/intron organization revealed that, like other neural IgSF molecules, each of the first two F3 C2 domains is encoded by two exons while the N-terminus, the signal peptide and the 5' untranslated region are each encoded by distinct exons. A single transcription start site was identified, surrounded by a short 114 bp sequence able to direct reporter gene expression in both F3-expressing and -non-expressing cells. In addition, a cell type-specific enhancer, only active in F3-expressing cells, was found immediately upstream to it. Structural analysis of the promoter region revealed consensus sequences for binding transcription factors involved in cell type-specific and/or developmental regulations. Most of them are homeobox containing transcription factors thus suggesting that regulation of F3 gene expression could be part of a large developmental program.

Different domains of the F3 neuronal adhesion molecule are involved in adhesion and neurite outgrowth promotion.

The mouse F3 cell surface protein is preferentially expressed on axons of subpopulations of neurons and is anchored to the membrane by a glycosyl-phosphatidylinositol group. It consists of six immunoglobulin-like domains and four fibronectin type III homologous repeats, and can be found both in membrane-anchored and soluble forms. We have previously established that F3 fulfills the operational criteria of a cell adhesion molecule when anchored to the plasma membrane and that its soluble form stimulates neurite initiation and neurite outgrowth. To further characterize F3-mediated adhesion and to investigate whether adhesion and neurite outgrowth promoting activities are displayed by different parts of the molecule, we (i) selected F3 transfected CHO cells expressing increasing levels of F3 at their surface and (ii) prepared transfectants expressing an F3 molecule with its fibronectin type III repeats deleted. We show that the F3 molecule mediates divalent-cation-independent, temperature-dependent binding. The levels of aggregation of F3 transfectants are proportional to the level of F3 expression. Transfectants expressing F3 deleted of the fibronectin type III repeats lose their adhesive properties; conversely, cells expressing wild-type F3 and treated with collagenase, specifically removing the immunoglobulin-like domains, are still able to aggregate. Therefore, in this model adhesion site(s) mapped to the fibronectin type III repeats. By contrast, transfectants expressing deleted F3, as well as the soluble forms of this F3 deleted molecule, were able to stimulate neurite outgrowth of sensory neurons similarly to wild-type F3. Our data indicate that F3 is a multifunctional molecule and that adhesion and neurite outgrowth promoting properties are expressed by distinct and independent domains.

A case of Landau-Kleffner syndrome secondary to inflammatory demyelinating disease.

A 6-year old girl developed acquired aphasia with epilepsy and a paroxysmal EEG (Landau-Kleffner syndrome). Isoelectric CSF focusing showed oligoclonal IgG bands. Small lesions were visualized in periventricular left frontal white matter and right parietal lobe centrum semiovale with magnetic resonance imaging (MRI). After a week of ACTH therapy, the EEG paroxysmal activity disappeared; during the next few months, the language disorder improved. Further MRI examination showed a decrease in size and signal of the left frontal lesions, with localized white matter atrophy, dilatation of the subarachnoidal spaces, and disappearance of the right parietal lesion. The clinical and neuroradiologic features and the laboratory data suggest an acute disseminated encephalomyelitis.

Antiepileptic drugs in pregnancy: late effects on the children's cognitive abilities. Preliminary data.

The authors report preliminary data on cognitive development of 57 children, perspectively followed, who were exposed to antiepileptic drugs in utero for maternal epilepsy. Cognitive impairments are associated with other risk factors in 5 cases, so that a direct AEDs responsibility is not easy to prove.

Multiple sclerosis in childhood. Longitudinal study in 14 cases.

The Authors review the literature on the occurrence of multiple sclerosis in children and add 14 personal cases below the age of 15.5 years, the youngest being 6.4 years old. Modality of onset, clinical course, clinical classification according to the criteria proposed by McDonald and Halliday, paraclinical evidence of lesions and disability grade at the last control are widely discussed. Eleven cases were scheduled as clinically definite multiple sclerosis. In the youngest children the recovery may be often complete or the disability grade may be low.