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Proteins are nanoscale molecular machines with a panoply of functions regimenting ubiquitous biological processes across life on earth. Functions of proteins range from nutrient transport, rate enhancement of chemical reactions, to making up structures that make up living things. From the most complex multicellular organisms such as animals or plants, to the more simplistic unicellular organisms such as E.coli, all living things on earth share defining cellular features of which perhaps the most striking is the cellular membrane. The cellular or “plasma” membrane, made up of a lipid bilayer separates the interior of a cell from the exterior environment. This interface serves as a hub which cells communicate with their environment and each other. Cellular membranes across different organisms are made up from distinct assortments of molecules. Such assortments of molecules have a long history under evolutionary pressure, which has led to the emergence of intricate molecular mechanisms controlling diverse biological processes. For formation of complex organisms, processes such as cellular recognition and communication are required and tightly controlled by intricate molecular mechanisms involving diverse molecules. In this dissertation, work on structural and biophysical characterization of vertebrate cell surface contactin proteins is presented. An integrative and multidisciplinary approach is pursued, using structural insights gained from experiments to inform on biological function of contactins in wiring the nervous system in vertebrates. Provided structures inform current knowledge in the molecular basis of signaling and adhesion mechanisms of contactin proteins as required for maintaining specific neuronal contacts and directing wiring between neuronal cells.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G&gt, A, p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

International audience; Septate-like junctions display characteristic ladder-like ultrastructure reminiscent of the invertebrate epithelial septate junctions and are present at the paranodes of myelinated axons. The paranodal junctions where the myelin loops attach to the axon at the borders of the node of Ranvier provide both a paracellular barrier to ion diffusion and a lateral fence along the axonal membrane. The septate-like junctions constrain the proper distribution of nodal Na + channels and juxtaparanodal K + channels, which are required for the safe propagation of the nerve influx and rapid saltatory conduction. The paranodal cell adhesion molecules have been identified as target antigens in peripheral demyelinating autoimmune diseases and the pathogenic mechanisms described. This review aims at presenting the recent knowledge on the molecular and structural organization of septate-like junctions, their formation and stabilization during development, and how they are involved in demyelinating diseases.

Synaptic degeneration is an early phenomenon in Parkinson&rsquo, s disease (PD) pathogenesis. We aimed to investigate whether levels of synaptic proteins contactin-1 and contactin-2 in cerebrospinal fluid (CSF) of PD patients are reduced compared to dementia with Lewy bodies (DLB) patients and controls and to evaluate their relationship with &alpha, synuclein aggregation. Contactin-1 and -2 were measured in CSF from PD patients (n = 58), DLB patients (n = 72) and age-matched controls (n = 90). Contactin concentration differences between diagnostic groups were assessed by general linear models adjusted for age and sex. Contactin immunoreactivity was characterized in postmortem substantia nigra, hippocampus and entorhinal cortex tissue of PD patients (n = 4) and controls (n = 4), and its relation to &alpha, syn aggregation was evaluated using confocal laser scanning microscopy. Contactin-1 levels were lower in PD patients (42 (36&ndash, 49) pg/mL) compared to controls (52 (44&ndash, 58) pg/mL, p = 0.003) and DLB patients (56 (46&ndash, 67) pg/mL, p = 0.001). Contactin-2 levels were similar across all diagnostic groups. Within the PD patient group, contactin-1 correlated with t-&alpha, syn, tTau and pTau (r = 0.30&ndash, 0.50, p &lt, 0.05), whereas contactin-2 only correlated with t-&alpha, syn (r = 0.34, p = 0.03). Contactin-1 and -2 were observed within nigral and cortical Lewy bodies and clustered within bulgy Lewy neurites in PD brains. A decrease in CSF contactin-1 may reflect synaptic degeneration underlying Lewy body pathology in PD.

Discovery of disease-associated autoantibodies has transformed the clinical management of a variety of neurological disorders. Detection of autoantibodies aids diagnosis and allows patient stratification resulting in treatment optimization. In the last years, a set of autoantibodies against proteins located at the node of Ranvier has been identified in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). These antibodies target neurofascin, contactin1, or contactin-associated protein 1, and we propose to name CIDP patients with these antibodies collectively as seropositive. They have unique clinical characteristics that differ from seronegative CIDP. Moreover, there is compelling evidence that autoantibodies are relevant for the pathogenesis. In this article, we review the current knowledge on the characteristics of autoantibodies against the node of Ranvier proteins and their clinical relevance in CIDP. We start with a description of the structure of the node of Ranvier followed by a summary of assays used to identify seropositive patients; and then, we describe clinical features and characteristics linked to seropositivity. We review knowledge on the role of these autoantibodies for the pathogenesis with relevance for the emerging concept of nodopathy/paranodopathy and summarize the treatment implications.

Background Contactin-1 and contactin-2 are important for the maintenance of axonal integrity. Objective To investigate the cerebrospinal fluid levels of contactin-1 and contactin-2 in multiple sclerosis patients and controls, and their potential use as prognostic markers for neurodegeneration. Methods Cerebrospinal fluid contactin-1 and contactin-2 were measured in relapsing–remitting multiple sclerosis ( n = 41), secondary progressive multiple sclerosis ( n = 26) and primary progressive multiple sclerosis patients ( n = 13) and controls ( n = 18), and in a second cohort with clinically isolated syndrome patients ( n = 88, median clinical follow-up period of 2.3 years) and controls ( n = 20). Correlations/linear regressions were analysed with other baseline cerebrospinal fluid axonal damage markers and cross-sectional/longitudinal magnetic resonance imaging features. Results Contactin-1 and contactin-2 levels were up to 1.4-fold reduced in relapsing–remitting multiple sclerosis (contactin-1: p = 0.01, contactin-2: p = 0.02) and secondary progressive multiple sclerosis (contactin-1: p = 0.05, contactin-2: p = 0.02) compared to controls. In clinically isolated syndrome patients, contactin-1 tended to increase when compared to controls ( p = 0.07). Both contactin-1 and contactin-2 correlated with neurofilament light, neurofilament heavy and magnetic resonance imaging metrics differently depending on the disease stage. In clinically isolated syndrome patients, baseline contactin-2 level (β = –0.42, p = 0.04) predicted the longitudinal decline in cortex volume. Conclusion Cerebrospinal fluid contactin-1 and contactin-2 reveal axonal dysfunction in various stages of multiple sclerosis and their inclusion to the biomarker panel may provide better insight into the extent of axonal damage/dysfunction.

International audience; Congenital hypomyelinating neuropathy is a rare neonatal syndrome responsible for hypotonia and weakness. Nerve microscopic examination shows amyelination or hypomyelination. Recently, mutations in CNTNAP1 have been described in a few patients. CNTNAP1 encodes contactin-associated protein 1 (caspr-1), which is an essential component of the paranodal junctions of the peripheral and central nervous systems, and is necessary for the establishment of transverse bands that stabilize paranodal axo-glial junctions. We present the results of nerve biopsy studies of three patients from two unrelated, non-consanguineous families with compound heterozygous CNTNAP1 mutations. The lesions were identical, characterized by a hypomyelinating process; on electron microscopy, we detected, in all nodes of Ranvier, subtle lesions that have never been previously described in human nerves. Transverse bands of the myelin loops were absent, with a loss of attachment between myelin and the axolemma; elongated Schwann cell processes sometimes dissociated the Schwann cell and axon membranes that bound the space between them. These lesions were observed in the area where caspr-1 is located and are reminiscent of the lesions reported in sciatic nerves of caspr-1 null mice. CNTNAP1 mutations appear to induce characteristic ultrastructural lesions of the paranodal region.

© 2016, American Society for Biochemistry and Molecular Biology Inc. All rights reserved. Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3-6 (CNTN3-6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling.

Disruption in the genes coding for the neural cell adhesion molecules contactin-4 (Cntn4), contactin-5 (Cntn5), and contactin-6 (Cntn6) contribute to an increased risk of neuropsychiatric disorders, particularly autism spectrum disorders (ASD). This indicates that the absence or dysfunction of these proteins may disrupt normal brain development. While Cntn4, Cntn5, and Cntn6 are expressed in distinct but overlapping brain regions in rodents, the gross neuroanatomy of the respective null-mutant mice is not affected. More detailed analyses of specific processes in neurodevelopment did demonstrate impairments in several neurobiological processes, including neurite outgrowth, synaptogenesis, neural survival, guiding projections and terminal branches of axons in forming neural circuits. Although studies so far indicate distinct neuroanatomical expression patterns and behavioral phenotypes in the respective null-mutant mice, very little is still known about the molecular mechanisms underlying the biological pathways in which these Cntns participate. The common structural architecture of the Cntn subgroup indicates the necessity of binding partners in order for them to facilitate any neurobiological functions. While Cntns are membrane-linked, but not membrane-spanning, the involved mechanisms of action must rely on a signalling function of the Cntn protein itself or the interaction with other membrane-spanning proteins. Abundant evidence for contactin-1 (Cntn1) and contactin-2 (Cntn2) show that cis- and trans-interaction with several membrane-spanning proteins is part of their repertoire. Therefore, the aim of this thesis is to unravel the neurobiological mechanisms of Cntn4, Cntn5, and Cntn6, with emphasis on Cntn6. Through proteomics novel and known interacting proteins were identified for these three contactins. The interaction of Cntn6 with the cell adhesion G protein-coupled receptor latrophilin-1 (Lphn1, aka CIRL, ADGRL1) was investigated in detail, showing that Cntn6 inhibited Lphn1-mediated apoptosis. These results suggest that Lphn1 functions as a dependence receptor, which induces neuronal apoptosis only in absence of Cntn6. Indeed, neuroanatomical analysis of the visual cortex of Cntn6 null-mutantmice displayed an increase in apoptosis, demonstrating the significant consequences in vivo. Furthermore, it provides a novel look into modes of action of Cntns and into the consequences for brain development. The identification of cell adhesion properties of Cntn6 in the cerebral cortex points to a broader function for Cntn6 in neurodevelopment than was previously shown. This was further emphasized by the ability of Cntn6 to interact with multiple membrane-spanning proteins with each having several interactions of their own, including the well-known and ASD-implicated Nrxn1-Nlgn1 pathway. This may represent the leading edge for integrated protein networks important for brain development, maturation and plasticity. It may provide valuable starting points for future studies to unravel how absence of Cntns impair neurobiological processes, and ultimately lead to ASD pathophysiology.

Disruption in the genes coding for the neural cell adhesion molecules contactin-4 (Cntn4), contactin-5 (Cntn5), and contactin-6 (Cntn6) contribute to an increased risk of neuropsychiatric disorders, particularly autism spectrum disorders (ASD). This indicates that the absence or dysfunction of these proteins may disrupt normal brain development. While Cntn4, Cntn5, and Cntn6 are expressed in distinct but overlapping brain regions in rodents, the gross neuroanatomy of the respective null-mutant mice is not affected. More detailed analyses of specific processes in neurodevelopment did demonstrate impairments in several neurobiological processes, including neurite outgrowth, synaptogenesis, neural survival, guiding projections and terminal branches of axons in forming neural circuits. Although studies so far indicate distinct neuroanatomical expression patterns and behavioral phenotypes in the respective null-mutant mice, very little is still known about the molecular mechanisms underlying the biological pathways in which these Cntns participate. The common structural architecture of the Cntn subgroup indicates the necessity of binding partners in order for them to facilitate any neurobiological functions. While Cntns are membrane-linked, but not membrane-spanning, the involved mechanisms of action must rely on a signalling function of the Cntn protein itself or the interaction with other membrane-spanning proteins. Abundant evidence for contactin-1 (Cntn1) and contactin-2 (Cntn2) show that cis- and trans-interaction with several membrane-spanning proteins is part of their repertoire. Therefore, the aim of this thesis is to unravel the neurobiological mechanisms of Cntn4, Cntn5, and Cntn6, with emphasis on Cntn6. Through proteomics novel and known interacting proteins were identified for these three contactins. The interaction of Cntn6 with the cell adhesion G protein-coupled receptor latrophilin-1 (Lphn1, aka CIRL, ADGRL1) was investigated in detail, showing that Cntn6 inhibited Lphn1-mediated apoptosis. These results suggest that Lphn1 functions as a dependence receptor, which induces neuronal apoptosis only in absence of Cntn6. Indeed, neuroanatomical analysis of the visual cortex of Cntn6 null-mutantmice displayed an increase in apoptosis, demonstrating the significant consequences in vivo. Furthermore, it provides a novel look into modes of action of Cntns and into the consequences for brain development. The identification of cell adhesion properties of Cntn6 in the cerebral cortex points to a broader function for Cntn6 in neurodevelopment than was previously shown. This was further emphasized by the ability of Cntn6 to interact with multiple membrane-spanning proteins with each having several interactions of their own, including the well-known and ASD-implicated Nrxn1-Nlgn1 pathway. This may represent the leading edge for integrated protein networks important for brain development, maturation and plasticity. It may provide valuable starting points for future studies to unravel how absence of Cntns impair neurobiological processes, and ultimately lead to ASD pathophysiology.

Contactins (Cntns) are a six-member subgroup of the immunoglobulin cell adhesion molecule superfamily (IgCAMs) with pronounced brain expression and function. Recent genetic studies of neuropsychiatric disorders have pinpointed contactin-4 (CNTN4), contactin-5 (CNTN5) and contactin-6 (CNTN6) as candidate genes in neurodevelopmental disorders, particularly in autism spectrum disorders (ASDs), but also in intellectual disability, schizophrenia (SCZ), attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BD), alcohol use disorder (AUD) and anorexia nervosa (AN). This suggests that they have important functions during neurodevelopment. This suggestion is supported by data showing that neurite outgrowth, cell survival and neural circuit formation can be affected by disruption of these genes. Here, we review the current genetic data about their involvement in neuropsychiatric disorders and explore studies on how null mutations affect mouse behavior. Finally, we highlight to role of protein-protein interactions in the potential mechanism of action of Cntn4, -5 and -6 and emphasize that complexes with other membrane proteins may play a role in neuronal developmental functions.

Computer simulations of protein unfolding substantially help to interpret force-extension curves measured in single-molecule atomic force microscope (AFM) experiments. Standard all-atom (AA) molecular dynamics simulations (MD) give a good qualitative mechanical unfolding picture but predict values too large for the maximum AFM forces with the common pulling speeds adopted here. Fine tuned coarse-grain MD computations (CG MD) offer quantitative agreement with experimental forces. In this paper we address an important methodological aspect of MD modeling, namely the impact of numerical noise generated by random assignments of bead velocities on maximum forces (Fmax) calculated within the CG MD approach. Distributions of CG forces from 2000 MD runs for several model proteins rich in β structures and having folds with increasing complexity are presented. It is shown that Fmax have nearly Gaussian distributions and that values of Fmax for each of those β-structures may vary from 93.2 ± 28.9 pN (neurexin) to 198.3 ± 25.2 pN (fibronectin). The CG unfolding spectra are compared with AA steered MD data and with results of our AFM experiments for modules present in contactin, fibronectin and neurexin. The stability of these proteins is critical for the proper functioning of neuronal synaptic clefts. Our results confirm that CG modeling of a single molecule unfolding is a good auxiliary tool in nanomechanics but large sets of data have to be collected before reliable comparisons of protein mechanical stabilities are made. Figure Computational strechnings of single protein modeules leads to broad distributions of unfolding forces

La descripción de correlaciones clínico-inmunológicas en las enfermedades autoinmunes es importante para caracterizar distintos fenotipos dentro de una enfermedad, establecer grupos diagnósticos y pronósticos y para escoger las terapias en función del perfil inmunológico de cada paciente. Las neuropatías inflamatorias constituyen un grupo de enfermedades raras del sistema nervioso periférico de probable etiología autoinmune. Este grupo incluye neuropatías agudas como el síndrome de Guillain-Barré (SGB) y neuropatías crónicas como la Poliradiculoneuropatía Inflamatoria Desmielinizante Crónica (CIDP). En todas ellas ha sido propuesto un papel prominente de los autoanticuerpos en su patogenia. Sin embargo, las reactividades antigénicas y los mecanismos patogénicos de estas enfermedades son desconocidos en su mayor parte. El SGB es una neuropatía aguda post-infecciosa en la que los anticuerpos antigangliósido juegan un papel importante. Existen diversos fenotipos dentro del SGB, incluyendo variantes sensitivo-motoras, atáxicas, sensitivas y variantes loco-regionales. Cada variante ha sido asociada a diferentes patrones de reactividad antigangliósido. El síndrome de Miller-Fisher (SMF) es una variante de SGB caracterizada por la presencia de ataxia, arreflexia y oftalmoparesia. Se asocia con la presencia de anticuerpos dirigidos contra gangliósidos que contienen el epítopo Neu NAcNeuNAcGal, como GQ1b y GT1a. En el SGB atáxico los pacientes presentan ataxia con signo de Romberg negativo y sin oftalmoparesia aparente. También se asocia a anticuerpos contra GQ1b. Por otro lado una forma sensitiva pura de SGB asociada a anticuerpos contra GD1b y conocida como neuropatía aguda sensitivo-atáxica (ASAN) puede presentarse también con ataxia de origen periférico. Distinguir el ASAN del SMF o el SGB atáxico puede ser difícil desde el punto de vista clínico. Además, la presencia de desmielinización, un criterio diagnóstico en el SGB sensitivo, es rara en ASAN y , por tanto, es controvertida su inclusión como variante de SGB. Considerando lo anterior, diseñamos un estudio para describir las características clínica e inmunológicas del ASAN. Todos los pacientes incluidos en una base de datos conteniendo los resultados de test rutinarios de anticuerpos antigangliósido y que cumplían criterios de ASAN fueron analizados en cuanto a sus características epidemiológicas, clínicas, electrofisiológicas y de reactividad anti-gangliósido. De los 12 pacientes que cumplían criterios en primer lugar, siete presentaban anticuerpos contra gangliósidos con epítopo disialosil (GQ1b, GD1b, GD2, GD3). Todos los pacientes con anticuerpos contra disialosil comenzaron con síntomas sensitivos e inestabilidad de la marcha y manifestaban haber presentado una infección de vías respiratorias altas en los días previos. Cinco de los 7 pacientes presentaron ptosis palpebral. Todos ellos tuvieron un buen pronóstico, bien tras tratamiento con IVIg o bien espontáneamente. Teniendo en cuenta lo anterior concluimos que el ASAN es, de hecho, una variante del SGB y que constituye un síndrome diferente al SMF o el SGB atáxico. La CIDP es una neuropatía crónica autoinmune en la que se ha propuesto un papel importante en su patogenia de los autoanticuerpos. En estudios previos habían sido descritos anticuerpos contra proteínas de la mielina mediante ELISA o western-blot, pero los resultados no se reprodujeron usando ensayos basados en células que mantuvieran la conformación de la proteína sin desnaturalizar y ninguno de esos autoanticuerpos se demostró útil como biomarcador. Más recientemente el descubrimiento en biopsias del nervio sural de pacientes con CIDP de que la estructura del nodo de Ranvier está desorganizada ha llevado a buscar antígenos candidatos en el nodo de Ranvier y estructuras relacionadas. Nosotros, usando una aproximación no sesgada, diseñamos un estudio que utilizaba técnicas de inmunocitoquímica en neuronas, inmunoprecipitación y confirmación de antígenos en células transfectadas para tratar de encontrar autoanticuerpos que pudieran servir de biomarcadores en la práctica clínica diaria. De 46 pacientes con una CIDP bien caracterizada, encontramos 7 que reaccionaban contra neuronas. Dos de estos pacientes precipitaron la proteína contactin-1 (CNTN1) y otro precipitó CNTN1 y contactin-associated protein-1 (CASPR1). La reactividad contra dichas proteínas fue confirmada con inmunocitoquímica, inmunohistoquímica y experimentos de absorción. Ninguno de los 104 controles fue positivo. Los tres pacientes que reaccionaban contra CNTN1 compartían un fenotipo específico que incluía avanzada edad, inicio agresivo, predominio motor, degeneración axonal marcada de inicio y respuesta pobre a las IVIg. Estas características clínicas definen un subgrupo de CIDP con fenotipo homogéneo. Por tanto, los anticuerpos contra CNTN1 son el primer biomarcador con utilidad clínica identificado en CIDP. En resumen, estos resultados refuerzan la noción de que las neuropatías autoinmunes constituyen un grupo muy heterogéneo de enfermedades en las que la descripción de autoanticuerpos y sus correlatos clínicos puede tener importantes implicaciones diagnósticas, pronósticas y terapéuticas., The description of clinical-immunological correlations in autoimmune diseases is of paramount importance to characterize disease phenotypes, diagnostic and prognostic subgroups and to choose therapies depending on the immunological profile of patients. Inflammatory neuropathies constitute a group of infrequent diseases of the peripheral nerves of presumed autoimmune pathogenesis. It includes acute neuropathies such as Guillain-Barré syndrome (GBS) and chronic neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In all of them it has been postulated a prominent role of autoantibodies in their pathogenesis. However, the antigenic reactivities and the pathogenic mechanisms of these diseases remain largely unknown. GBS is a post-infectious acute neuropathy in which antibodies against gangliosides play an important role. There are various phenotypes of GBS, including sensory-motor, ataxic, sensory-ataxic and loco-regional variants. Each variant has been associated to different antiganglioside antibody patterns. Miller-Fisher syndrome (MFS) is a GBS variant characterized by ataxia, arreflexia and ophtalmoparesis. It associates with antiganglioside antibodies bearing the NeuNAcNeuNAcGal epitope, such as GQ1b and GT1a. In ataxic GBS patients present with ataxia and negative Romberg sign and there is no apparent ophtalmoparesis. It is also associated to GQ1b antibodies. On the other hand, a pure sensory form of GBS associated to GD1b antibodies known as acute sensory-ataxic neuropathy (ASAN) can also present with ataxia of peripheral origin. Distinguishing ASAN, MFS and ataxic GBS can be difficult from a clinical perspective. Moreover, demyelinating features, a diagnostic criteria in sensory GBS, are usually absent in ASAN and the inclusion of this form in the GBS spectrum is controversial. We designed a study to describe the clinical and immunological features of ASAN. All patients from a database including sera sent for routine antiganglioside-antibody testing and fulfilling ASAN criteria were collected and their epidemiological, clinical and electrophysiological features analyzed. Of 12 patients fulfilling criteria for ASAN, 7 had antibodies against gangliosides bearing disialosyl epitopes (GQ1b, GD2, GD3, GD1b). All ASAN patients with positive disialosyl antibodies presented with gait unsteadiness and sensory symptoms and reported an upper respiratory tract infection before disease onset. Five of the 7 patients presented eyelid ptosis. All patients had a good recovery with IVIg treatment or spontaneously. Considering these features we concluded that ASAN is, in fact, a GBS variant, and constitutes a specific phenotype different from ataxic GBS or MFS. CIDP is a chronic inflammatory neuropathy in which antibodies are thought to play an important role. Antibodies against myelin proteins had been described using ELISA and western-blot assays, but results were not reproduced in cell-based assays preserving protein conformation and none proved useful as biomarker. Recently, studies on sural nerve biopsies of CIDP patients have shown disorganization of node of Ranvier and the search of autoantibodies focused on proteins of the node of Ranvier. We used an unbiased approach with neuron immunocytochemistry, immunoprecipitation and confirmation of relevant antigens with transfected cells to find autoantibodies that could be useful in everyday practice. Starting with 46 well-characterized CIDP patients we found 7 patients reacting against neurons. Two patients precipitated contactin-1 (CNTN1) and one CNTN1 and contactin-associated protein-1 (CASPR1). Reactivity was confirmed with immunocytochemistry, inmmunohistochemistry and absorption studies. None of the 104 controls tested positive. All three patients shared old age, aggressive onset, motor predominance, prominent axonal degeneration at onset and poor response to IVIg. These features identify a subgroup of CIDP with homogeneous phenotype. Anti-CNTN1 antibodies are the first biomarker identified in CIDP with diagnostic, prognostic and therapeutic implications. These results strengthen the idea that autoimmune neuropathies are a very heterogeneous group of diseases in which autoantibody discovery and the definition of clinical-immunological correlations can have diagnostic, prognostic and therapeutic implications

Summary The neural cell-adhesion molecules contactin 4, contactin 5 and contactin 6 are involved in brain development, and disruptions in contactin genes may confer increased risk for autism spectrum disorders (ASD). We describe a co-culture of rat cortical neurons and HEK293 cells overexpressing and delivering the secreted forms of rat contactin 4–6. We quantified their effects on the length and branching of neurites. Contactin 4–6 effects were different depending on the contactin member and duration of co-culture. At 4 days in culture, contactin 4 and -6 increased the length of neurites, while contactin 5 increased the number of roots. Up to 8 days in culture, contactin 6 progressively increased the length of neurites while contactin 5 was more efficient on neurite branching. We studied the molecular sites of interaction between human contactin 4, -5 or -6 and the human Protein Tyrosine Phosphatase Receptor Gamma (PTPRG), a contactin partner, by modeling their 3D structures. As compared to contactin 4, we observed differences in the Ig2 and Ig3 domains of contactin 5 and -6 with the appearance of an omega loop that could adopt three distinct conformations. However, interactive residues between human contactin 4–6 and PTPRG were strictly conserved. We did not observe any differences in PTPRG binding on contactin 5 and -6 either. Our data suggest that the differential contactin effects on neurite outgrowth do not result from distinct interactions with PTPRG. A better understanding of the contactin cellular properties should help elucidate their roles in ASD.

Contactins are modular extracellular cell matrix proteins that are present in the brain, and they are responsible for the proper development and functioning of neurons. They contain six immunoglobulin-like IgC2 domains and four fibronectin type III repeats. The interactions of contactin with other proteins are poorly understood. The mechanical properties of all IgC2 domains of human contactin 4 were studied using a steered molecular dynamics approach and CHARMM force field with an explicit TIP3P water environment on a 10-ns timescale. Force spectra of all domains were determined computationally and the nanomechanical unfolding process is described. The domains show different mechanical stabilities. The calculated maxima of the unfolding force are in the range of 900–1700 pN at a loading rate of 7 N/s. Our data indicate that critical regions of IgC2 domains 2 and 3, which are responsible for interactions with tyrosine phosphatases and are important in nervous system development, are affected by even weak mechanical stretching. Thus, tensions present in the cell may modulate cellular activities related to contactin function. The present data should facilitate the interpretation of atomic force microscope single-molecule spectra of numerous proteins with similar IgC2 motives. Figure The general fold of IgC2 domains of contactin 4 protein. Vectors show directions of pulling forces applied in mechanical unfolding computer experiments. Electronic supplementary material The online version of this article (doi:10.1007/s00894-011-1010-y) contains supplementary material, which is available to authorized users.

International audience; Contactin and TAG-1 are glycan phosphatidyl inositol (GPI)-anchored cell adhesion molecules that play a crucial role in the organization of axonal subdomains at the node of Ranvier of myelinating fibers. Contactin and TAG-1 mediate axo-glial selective interactions in association with Caspr-family molecules at paranodes and juxtaparanodes, respectively. How membrane proteins can be confined in these neighbouring domains along the axon has been the subject of intense investigations. This review will specifically examine the properties conferred by the lipid microenvironment to regulate trafficking and selective association of these axo-glial complexes. Increasing evidences from genetic and neuropathological models point to a role of lipid rafts in the formation or stabilization of the paranodal junctions.