RESUMO
Organization of neuronal connections into topographic maps is essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotectal mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum, leading to the sharpening of the retinotopic map along the antero-posterior axis. Finally, using a CRISPR-mediated mutagenesis approach, we demonstrate that the refinement of nasal retinal projections requires the adhesion molecule Contactin 2. Altogether, our study provides the first analysis of a topographic map maturing in real time in a live animal and opens new strategies for dissecting the molecular mechanisms underlying precise topographic mapping in vertebrates.
Assuntos
Axônios/metabolismo , Contactina 2/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Contactina 2/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.
Assuntos
Ventrículos do Coração/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese/fisiologia , Molécula de Adesão de Leucócito Ativado , Animais , Moléculas de Adesão Celular/metabolismo , Contactina 2/metabolismo , Expressão Gênica , Coração , Sistema de Condução Cardíaco/metabolismo , Camundongos , Camundongos Knockout , Ácidos Siálicos , SialiltransferasesRESUMO
OBJECTIVE: The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. METHODS: IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70µm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. RESULTS: Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. INTERPRETATION: IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.
Assuntos
Autoanticorpos , Encefalite , Proteínas de Membrana , Proteínas do Tecido Nervoso , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Autoanticorpos/imunologia , Contactina 2/imunologia , Encefalite/imunologia , Humanos , Imunoglobulina G/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismoRESUMO
Mammalian adult neurons of the central nervous system (CNS) display limited ability to regrow axons after trauma. The developmental decline in their regenerative ability has been attributed to both intrinsic and extrinsic factors, including postnatal suppression of transcription factors and non-neuronal inhibitory components, respectively. The cell adhesion molecule Contactin 2 (CNTN2) is expressed in neurons and oligodendrocytes in the CNS. Neuronal CNTN2 is highly regulated during development and plays critical roles in axon growth and guidance and neuronal migration. On the other hand, CNTN2 expressed by oligodendrocytes interferes with the myelination process, with its ablation resulting in hypomyelination. In the current study, we investigate the role of CNTN2 in neuronal survival and axon regeneration after trauma, in the murine optic nerve crush (ONC) model. We unveil distinct roles for neuronal and glial CNTN2 in regenerative responses. Surprisingly, our data show a conflicting role of neuronal and glial CNTN2 in axon regeneration. Although glial CNTN2 as well as hypomyelination are dispensable for both neuronal survival and axon regeneration following ONC, the neuronal counterpart comprises a negative regulator of regeneration. Specifically, we reveal a novel mechanism of action for neuronal CNTN2, implicating the inhibition of Akt signalling pathway. The in vitro analysis indicates a BDNF-independent mode of action and biochemical data suggest the implication of the truncated form of TrkB neurotrophin receptor. In conclusion, CNTN2 expressed in CNS neurons serves as an inhibitor of axon regeneration after trauma and its mechanism of action involves the neutralization of Akt-mediated neuroprotective effects.
Assuntos
Axônios , Traumatismos do Nervo Óptico , Animais , Contactina 2 , Camundongos , Regeneração Nervosa , Neurônios , Nervo ÓpticoRESUMO
The CNTNAP2 gene, coding for the cell adhesion glycoprotein Caspr2, is thought to be one of the major susceptibility genes for autism spectrum disorder (ASD). A large number of rare heterozygous missense CNTNAP2 variants have been identified in ASD patients. However, most of them are inherited from an unaffected parent, questioning their clinical significance. In the present study, we evaluate their impact on neurodevelopmental functions of Caspr2 in a heterozygous genetic background. Performing cortical neuron cultures from mouse embryos, we demonstrate that Caspr2 plays a dose-dependent role in axon growth in vitro. Loss of one Cntnap2 allele is sufficient to elicit axonal growth alteration, revealing a situation that may be relevant for CNTNAP2 heterozygosity in ASD patients. Then, we show that the two ASD variants I869T and G731S, which present impaired binding to Contactin2/TAG-1, do not rescue axonal growth deficits. We find that the variant R1119H leading to protein trafficking defects and retention in the endoplasmic reticulum has a dominant-negative effect on heterozygous Cntnap2 cortical neuron axon growth, through oligomerization with wild-type Caspr2. Finally, we identify an additional variant (N407S) with a dominant-negative effect on axon growth although it is well-localized at the membrane and properly binds to Contactin2. Thus, our data identify a new neurodevelopmental function for Caspr2, the dysregulation of which may contribute to clinical manifestations of ASD, and provide evidence that CNTNAP2 heterozygous missense variants may contribute to pathogenicity in ASD, through selective mechanisms.
Assuntos
Transtorno do Espectro Autista/genética , Contactina 2/genética , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Alelos , Animais , Transtorno do Espectro Autista/fisiopatologia , Axônios/metabolismo , Axônios/patologia , Variação Genética , Heterozigoto , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Humanos , Camundongos , Mutação de Sentido Incorreto , Neurônios/metabolismo , Neurônios/patologia , Ligação ProteicaRESUMO
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Conexinas/genética , Conexinas/metabolismo , Contactina 2/genética , Contactina 2/metabolismo , Estimulação Elétrica , Expressão Gênica , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/fisiologia , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Cultura Primária de Células , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Troponina I/genética , Troponina I/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.
Assuntos
Biomarcadores/metabolismo , Membrana Celular/metabolismo , Separação Celular/métodos , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/citologia , Doença de Parkinson/terapia , Animais , Diferenciação Celular , Contactina 2/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Doença de Parkinson/patologia , Proteômica , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismoRESUMO
Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.
Assuntos
Segmento Inicial do Axônio/metabolismo , Contactina 2/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Superfamília Shaker de Canais de Potássio/genética , Axônios/metabolismo , Axônios/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Células HEK293 , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.
Assuntos
Autoanticorpos/metabolismo , Membrana Celular/metabolismo , Contactina 2/metabolismo , Encefalite/imunologia , Doença de Hashimoto/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Contactina 2/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Ratos , Agregação de Receptores , Superfamília Shaker de Canais de Potássio/genética , Regulação para CimaRESUMO
OBJECTIVE: To determine the pathogenic mechanisms of autoantibodies to the cell adhesion molecule Caspr2 in acquired neuromyotonia and autoimmune encephalitis. METHODS: Caspr2-positive samples were confirmed using a cell-based assay, and their IgG subtypes were determined by enzyme-linked immunosorbent assay and cell-based assay. A solid phase binding assay quantified the binding of Caspr2 to contactin-2 in the presence of Caspr2 autoantibodies. Living cultures of primary rat hippocampal neurons were incubated with Caspr2-positive or control sera, and the distribution of Caspr2-positive immunofluorescent puncta and total surface Caspr2 was quantified. HEK cells transfected to express Caspr2 were incubated with Caspr2-positive or control samples, and cell-surface biotinylation and Western blot were used to assess total, internalized, and surface levels of Caspr2. RESULTS: We confirmed 6 samples with strong Caspr2 reactivity. IgG4 Caspr2 antibodies were present in all 6 cases. Caspr2 interacted with another cell adhesion molecule, contactin-2, with nanomolar affinity in the solid phase assay, and Caspr2 autoantibodies inhibited this interaction. Caspr2 autoantibodies did not affect the surface expression of Caspr2 in rat primary hippocampal neurons or transfected HEK cells. INTERPRETATION: Caspr2 autoantibodies inhibit the interaction of Caspr2 with contactin-2 but do not cause internalization of Caspr2. Functional blocking of cell adhesion molecule interactions represents a potential mechanism with therapeutic implications for IgG4 autoantibodies to cell adhesion molecules in neurological diseases. Ann Neurol 2018;83:40-51.
Assuntos
Autoanticorpos/imunologia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Síndrome de Isaacs/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Animais , Biotinilação , Contactina 2/imunologia , Contactina 2/metabolismo , Feminino , Humanos , Imunoglobulina G/análise , Masculino , Neurônios/imunologia , Neurônios/metabolismo , RatosRESUMO
The oligodendrocyte maturation process and the transition from the pre-myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG-1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin-gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin-regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de- and remyelination. However during remyelination, a novel, CNTN2-independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.
Assuntos
Contactina 2/metabolismo , Doenças Desmielinizantes/metabolismo , Oligodendroglia/metabolismo , Animais , Axônios/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Canais de Cálcio/metabolismo , Células Cultivadas , Contactina 2/genética , Cuprizona , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Condução Nervosa/fisiologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Técnicas de Cultura de TecidosRESUMO
The olfactory system provides mammals with the abilities to investigate, communicate and interact with their environment. These functions are achieved through a finely organized circuit starting from the nasal cavity, passing through the olfactory bulb and ending in various cortical areas. We show that the absence of transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice results in a significant and selective defect in the number of the main projection neurons in the olfactory bulb, namely the mitral cells. A subpopulation of these projection neurons is reduced in Tag1-deficient mice as a result of impaired migration. We demonstrate that the detected alterations in the number of mitral cells are well correlated with diminished odor discrimination ability and social long-term memory formation. Reduced neuronal activation in the olfactory bulb and the corresponding olfactory cortex suggest that Tag1 is crucial for the olfactory circuit formation in mice. Our results underpin the significance of a numerical defect in the mitral cell layer in the processing and integration of odorant information and subsequently in animal behavior.
Assuntos
Movimento Celular , Contactina 2/deficiência , Bulbo Olfatório/patologia , Bulbo Olfatório/fisiopatologia , Animais , Contagem de Células , Contactina 2/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Bulbo Olfatório/embriologia , Bulbo Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Neurônios Receptores Olfatórios/patologiaRESUMO
Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.
Assuntos
Contactina 2/genética , MicroRNAs/fisiologia , Traumatismos dos Nervos Periféricos/genética , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Animais , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Humanos , MicroRNAs/genética , Traumatismos dos Nervos Periféricos/metabolismo , Ratos Sprague-DawleyRESUMO
Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule.
Assuntos
Contactina 2/química , Proteínas de Membrana/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Contactina 2/genética , Contactina 2/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of VN is not fully understood. In this study, we analyzed the effects of VN loss on the proliferation and differentiation of CGCPs in VN knockout (VNKO) mice in vivo. First, immunohistochemistry showed that VN was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of VN suppressed the ratio of NeuN, a neuronal differentiation marker, to positive cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of VN suppresses the differentiation into CGCs. However, the loss of VN did not significantly affect the proliferation of CGCPs. Next, the effect of VN loss on the initial differentiation stage of CGCPs was examined. The loss of VN increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that VN promotes the progress of the initial differentiation stage of CGCPs.
Assuntos
Diferenciação Celular/fisiologia , Cerebelo/metabolismo , Neurônios/metabolismo , Vitronectina/metabolismo , Animais , Proliferação de Células/fisiologia , Cerebelo/citologia , Contactina 2/genética , Contactina 2/metabolismo , Proteínas de Ligação a DNA , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Vitronectina/genéticaRESUMO
Multiple sclerosis (MS) is a chronic central nervous system (CNS) inflammation. Efficient drug delivery to brain is however hampered by blood-brain barrier (BBB). In order to have highly efficient and safe delivery of drugs to brain, solid lipid nanoparticles (SLNs) have indicated promising potentials as smart carriers that can pass the blood-brain barrier and deliver therapeutic biomolecules to the brain. In this study, PEGylated SLNs surface modified using anti-Contactin-2 or anti-Neurofascin, two axo-glial-glycoprotein antigens located in node of Ranvier, were prepared. These targeting moieties are considered as the main targets of autoimmune reaction in MS. The targeted SLNs were then characterized and their in vitro release profile together with their cell viability and uptake were studied. Their brain uptakes were also probed following injections in MS-induced mice. It was found that the targeted PEGylated SLNs had no significant cytotoxicity on U87MG cells although their cellular uptake was increased 4- and 8-fold when surface modified with anti-Contactin-2 or anti-Neurofascin, respectively, compared to control. Brain uptake results demonstrated higher uptake of surface-modified SLNs in the brain tissue compared with the PEGylated SLNs. The results of this report will help scientist to design more efficient nanocarriers for treatment of MS.
Assuntos
Anti-Inflamatórios/administração & dosagem , Encéfalo/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Contactina 2/antagonistas & inibidores , Portadores de Fármacos/química , Metilprednisolona/administração & dosagem , Nanopartículas/química , Fatores de Crescimento Neural/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/química , Encéfalo/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Humanos , Metilprednisolona/farmacocinética , Metilprednisolona/uso terapêutico , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Propriedades de SuperfícieRESUMO
The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination.
Assuntos
Gânglios Espinais/citologia , Canal de Potássio Kv1.1/metabolismo , Proteínas dos Microfilamentos/metabolismo , Nós Neurofibrosos/metabolismo , Células de Schwann/metabolismo , Animais , Células Cultivadas , Contactina 2/metabolismo , Venenos Elapídicos/farmacocinética , Embrião de Mamíferos , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.2/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Ratos , Ratos WistarRESUMO
OBJECTIVE: In autoimmune encephalitis the etiologic role of neuronal cell-surface antibodies is clear; patients diagnosed and treated early have better outcomes. Neuronal antibodies have also been described in patients with pediatric epilepsy without encephalitis. The aim was to assess whether antibody presence had any effect on long-term outcomes in these patients. METHODS: Patients (n = 178) were recruited between 1988 and 1992 as part of the prospective Dutch Study of Epilepsy in Childhood; none received immunotherapy. Healthy age-matched bone-marrow donors served as controls (n = 112). All sera were tested for serum N-methyl-d-aspartate receptor (NMDAR), alpha amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, leucine rich glioma inactivated 1, contactin associated protein like 2 (CASPR2), contactin-2, glutamic acid decarboxylase, and voltage gated potassium channel (VGKC)-complex antibodies by standard techniques. No cerebrospinal fluid (CSF) samples were available. Results were correlated with clinical data collected over 15 years. RESULTS: Seventeen patients (9.5%) were positive for VGKC complex (n = 3), NMDAR (n = 7), CASPR2 (n = 4), and contactin-2 (n = 3), compared to three (3/112; 2.6%) healthy controls (VGKC complex [n = 1], NMDAR [n = 2]; p = 0.03; Fisher's exact test). Titers were relatively low (≤1:100 for cell-surface antibodies), but 8 (47%) of the 17 positive samples bound to the surface of live hippocampal neurons consistent with a potential pathogenic antibody. Preexisting cognitive impairment was more frequent in antibody-positive patients (9/17 vs. 33/161; p = 0.01). Fourteen antibody-positive patients were treated with standard antiepileptic drugs (AEDs); three (17%) became intractable but this was not different from the 16 (10%) of 161 antibody-negative patients. In 96 patients with available follow-up samples at 6 and/or 12 months, 6 of 7 positive antibodies had disappeared and, conversely, antibodies had appeared for the first time in a further 7 patients. SIGNIFICANCE: Neuronal antibodies were found at low levels in 9.5% of patients with new-onset pediatric epilepsy but did not necessarily persist over time, and the development of antibodies de novo in later samples suggests they could be due to a secondary response to neuronal damage or inflammation. Moreover, as the response to standard AEDs and the long-term outcome did not differ from those of antibody-negative pediatric patients, these findings suggest that routine neuronal antibody testing is unlikely to be helpful in pediatric epilepsy. However, the higher incidence of preexisting cognitive problems in the antibody-positive group, the CASPR2 and contactin-2 antibodies in 7 of 17 patients, and the binding of 8 of 17 of serum samples to live hippocampal neurons suggest that neuronal antibodies, even if secondary, could contribute to the comorbidities of pediatric epilepsy.
Assuntos
Autoanticorpos/sangue , Epilepsia/sangue , Epilepsia/diagnóstico , Proteínas do Tecido Nervoso/imunologia , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Contactina 2/imunologia , Epilepsia/classificação , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/imunologia , Países Baixos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Receptores de N-Metil-D-Aspartato/imunologiaRESUMO
Myelinated fibers are divided into discrete subdomains around the Nav-enriched nodes of Ranvier: the paranodes, where axoglial interactions occur, the juxtaparanodes, where voltage-gated potassium channels (VGKCs) are aggregated, and the internode. Perinodal changes have been reported in Multiple Sclerosis (MS) with functional consequences for the axon. Here we report on alterations of the juxtaparanodal proteins TAG-1, Caspr2 and VGKCs in normal appearing white matter (NAWM), perilesion and chronic lesion areas in post-mortem white matter tissue from MS patients compared to control white matter. We show that the molecular organization and maintenance of juxtaparanodes is affected in lesions, perilesions and NAWM in chronic MS through protein and mRNA expression as well as immunohistochemistry. The three molecules analyzed were differentially altered. TAG-1 clustering at juxtaparanodes was reduced in NAWM; TAG-1 and Caspr2 are diffused in perilesions and absent in lesion areas. VGKCs were no longer enriched at juxtaparanodes either at the NAWM or the perilesion and demyelinated plaques. While the protein levels of the three molecules showed only a tendency of reduction in the plaques, there was a significant upregulation of Caspr2 mRNA in the lesions accompanied by a transcriptional increase of paranodal Caspr, indicating an axonal homeostatic mechanism.
Assuntos
Esclerose Múltipla/metabolismo , Nós Neurofibrosos/metabolismo , Substância Branca/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Axônios/metabolismo , Estudos de Casos e Controles , Contactina 2/genética , Contactina 2/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nós Neurofibrosos/patologia , Substância Branca/patologiaRESUMO
Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.