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1.
Elife ; 122023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37843188

RESUMO

The role of myelination for axonal conduction is well-established in projection neurons but little is known about its significance in GABAergic interneurons. Myelination is discontinuous along interneuron axons and the mechanisms controlling myelin patterning and segregation of ion channels at the nodes of Ranvier have not been elucidated. Protein 4.1B is implicated in the organization of the nodes of Ranvier as a linker between paranodal and juxtaparanodal membrane proteins to the spectrin cytoskeleton. In the present study, 4.1B KO mice are used as a genetic model to analyze the functional role of myelin in Lhx6-positive parvalbumin (PV) and somatostatin (SST) neurons, two major classes of GABAergic neurons in the hippocampus. We show that 4.1B-deficiency induces disruption of juxtaparanodal K+ channel clustering and mislocalization of nodal or heminodal Na+ channels. Strikingly, 4.1B-deficiency causes loss of myelin in GABAergic axons in the hippocampus. In particular, stratum oriens SST cells display severe axonal dysmyelination and a reduced excitability. This reduced excitability is associated with a decrease in occurrence probability of small amplitude synaptic inhibitory events on pyramidal cells. In contrast, stratum pyramidale fast-spiking PV cells do not appear affected. In conclusion, our results indicate a class-specific effect of dysmyelination on the excitability of hippocampal interneurons associated with a functional alteration of inhibitory drive.


Assuntos
Hipocampo , Interneurônios , Camundongos , Animais , Interneurônios/fisiologia , Hipocampo/metabolismo , Células Piramidais/metabolismo , Axônios/fisiologia , Neurônios GABAérgicos/metabolismo , Parvalbuminas/metabolismo
2.
Life (Basel) ; 11(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374190

RESUMO

The precise axonal distribution of specific potassium channels is known to secure the shape and frequency of action potentials in myelinated fibers. The low-threshold voltage-gated Kv1 channels located at the axon initial segment have a significant influence on spike initiation and waveform. Their role remains partially understood at the juxtaparanodes where they are trapped under the compact myelin bordering the nodes of Ranvier in physiological conditions. However, the exposure of Kv1 channels in de- or dys-myelinating neuropathy results in alteration of saltatory conduction. Moreover, cell adhesion molecules associated with the Kv1 complex, including Caspr2, Contactin2, and LGI1, are target antigens in autoimmune diseases associated with hyperexcitability such as encephalitis, neuromyotonia, or neuropathic pain. The clustering of Kv1.1/Kv1.2 channels at the axon initial segment and juxtaparanodes is based on interactions with cell adhesion molecules and cytoskeletal linkers. This review will focus on the trafficking and assembly of the axonal Kv1 complex in the peripheral and central nervous system (PNS and CNS), during development, and in health and disease.

3.
Biochim Biophys Acta Biomembr ; 1862(5): 183211, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32032590

RESUMO

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.


Assuntos
Axônios/fisiologia , Fibras Nervosas Mielinizadas/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/fisiologia , Humanos , Junções Intercelulares/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Nós Neurofibrosos/fisiologia , Vertebrados/metabolismo , Vertebrados/fisiologia
4.
Front Cell Neurosci ; 13: 222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31164806

RESUMO

In myelinated fibers, the voltage-gated sodium channels Nav1 are concentrated at the nodal gap to ensure the saltatory propagation of action potentials. The voltage-gated potassium channels Kv1 are segregated at the juxtaparanodes under the compact myelin sheath and may stabilize axonal conduction. It has been recently reported that hippocampal GABAergic neurons display high density of Nav1 channels remarkably in clusters along the axon before myelination (Freeman et al., 2015). In inhibitory neurons, the Nav1 channels are trapped by the ankyrinG scaffold at the axon initial segment (AIS) as observed in pyramidal and granule neurons, but are also forming "pre-nodes," which may accelerate conduction velocity in pre-myelinated axons. However, the distribution of the Kv1 channels along the pre-myelinated inhibitory axons is still unknown. In the present study, we show that two subtypes of hippocampal GABAergic neurons, namely the somatostatin and parvalbumin positive cells, display a selective high expression of Kv1 channels at the AIS and all along the unmyelinated axons. These inhibitory axons are also highly enriched in molecules belonging to the juxtaparanodal Kv1 complex, including the cell adhesion molecules (CAMs) TAG-1, Caspr2, and ADAM22 and the scaffolding protein 4.1B. Here, taking advantage of hippocampal cultures from 4.1B and TAG-1 knock-out mice, we observed that 4.1B is required for the proper positioning of Caspr2 and TAG-1 along the distal axon, and that TAG-1 deficiency induces alterations in the axonal distribution of Caspr2. However, the axonal expression of Kv1 channels and clustering of ankyrinG were not modified. In conclusion, this study allowed the analysis of the hierarchy between channels, CAMs and scaffolding proteins for their expression along hippocampal inhibitory axons before myelination. The early steps of channel compartmentalization preceding myelination may be crucial for stabilizing nerve impulses switching from a continuous to saltatory conduction during network development.

5.
J Cell Sci ; 132(2)2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30598502

RESUMO

The distribution of the voltage-gated Kv1 K+ channels at the axon initial segment (AIS) influences neuronal intrinsic excitability. The Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) subunits are associated with cell adhesion molecules (CAMs), including Caspr2 (also known as CNTNAP2) and LGI1, which are implicated in autoimmune and genetic neurological diseases with seizures. In particular, mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). Here, by using rat hippocampal neurons in culture, we showed that LGI1 is recruited to the AIS where it colocalizes with ADAM22 and Kv1 channels. Strikingly, the missense mutations S473L and R474Q of LGI1 identified in ADLTE prevent its association with ADAM22 and enrichment at the AIS. Moreover, we observed that ADAM22 and ADAM23 modulate the trafficking of LGI1, and promote its ER export and expression at the overall neuronal cell surface. Live-cell imaging indicated that LGI1 is co-transported in axonal vesicles with ADAM22 and ADAM23. Finally, we showed that ADAM22 and ADAM23 also associate with Caspr2 and TAG-1 (also known as CNTN2) to be selectively targeted to different axonal sub-regions. Hence, the combinatorial expression of Kv1-associated CAMs may be critical to tune intrinsic excitability in physiological and epileptogenic contexts.


Assuntos
Proteínas ADAM/metabolismo , Axônios/metabolismo , Epilepsia do Lobo Frontal/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mutação de Sentido Incorreto , Transtornos do Sono-Vigília/metabolismo , Proteínas ADAM/genética , Substituição de Aminoácidos , Animais , Axônios/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Epilepsia do Lobo Frontal/genética , Epilepsia do Lobo Frontal/patologia , Células HEK293 , Hipocampo , Humanos , Transporte Proteico/genética , Ratos , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Transtornos do Sono-Vigília/genética , Transtornos do Sono-Vigília/patologia
6.
Elife ; 72018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30311906

RESUMO

Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.


Assuntos
Cerebelo/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Polissacarídeos/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/deficiência , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Animais , Orientação de Axônios , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Cerebelo/embriologia , Grânulos Citoplasmáticos/metabolismo , Deleção de Genes , Glicosilação , Imunoglobulinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos Knockout , Atividade Motora , Mutação/genética , Vias Neurais/metabolismo , Proteômica , Células de Purkinje/metabolismo , Reprodutibilidade dos Testes , Resposta a Proteínas não Dobradas
7.
Hum Mol Genet ; 27(11): 1941-1954, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29788201

RESUMO

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 Proteica
8.
Glia ; 66(4): 874-888, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29285794

RESUMO

Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. We developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. We then analyzed the consequences of ND23 knockdown in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.


Assuntos
Proteínas de Drosophila/deficiência , Metabolismo dos Lipídeos/fisiologia , Doenças Mitocondriais/patologia , NADH Desidrogenase/deficiência , Doenças Neurodegenerativas/patologia , Neuroglia/patologia , Trifosfato de Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Feminino , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Homeostase/fisiologia , Humanos , Doenças Mitocondriais/metabolismo , Atividade Motora/fisiologia , NADH Desidrogenase/genética , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Interferência de RNA , RNA Mensageiro/metabolismo
9.
Sci Rep ; 7(1): 14411, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29089585

RESUMO

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a heterogeneous disease in which diverse autoantibodies have been described but systematic screening has never been performed. Detection of CIDP-specific antibodies may be clinically useful. We developed a screening protocol to uncover novel reactivities in CIDP. Sixty-five CIDP patients and 28 controls were included in our study. Three patients (4.6%) had antibodies against neurofascin 155, four (6.2%) against contactin-1 and one (1.5%) against the contactin-1/contactin-associated protein-1 complex. Eleven (18.6%) patients showed anti-ganglioside antibodies, and one (1.6%) antibodies against peripheral myelin protein 2. No antibodies against myelin protein zero, contactin-2/contactin-associated protein-2 complex, neuronal cell adhesion molecule, gliomedin or the voltage-gated sodium channel were detected. In IgG experiments, three patients (5.3%) showed a weak reactivity against motor neurons; 14 (24.6%) reacted against DRG neurons, four of them strongly (7.0%), and seven (12.3%) reacted against Schwann cells, three of them strongly (5.3%). In IgM experiments, six patients (10.7%) reacted against DRG neurons, while three (5.4%) reacted against Schwann cells. However, results were not statistically significant when compared to controls. Immunoprecipitation experiments identified CD9 and L1CAM as potential antigens, but reactivity could not be confirmed with cell-based assays. In summary, we describe a diverse autoantibody repertoire in CIDP patients, reinforcing the hypothesis of CIDP's pathophysiological heterogeneity.


Assuntos
Autoanticorpos/imunologia , Proteínas do Tecido Nervoso/imunologia , Sistema Nervoso Periférico/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Animais , Autoanticorpos/análise , Células Cultivadas , Estudos de Coortes , Feminino , Gânglios Espinais/imunologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Neurônios/imunologia , Ratos , Células de Schwann/imunologia
10.
J Cell Sci ; 130(13): 2209-2220, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28533267

RESUMO

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/fisiologia
11.
Curr Biol ; 27(7): 1068-1073, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28318976

RESUMO

Nodes of Ranvier in the axons of myelinated neurons are exemplars of the specialized cell surface domains typical of polarized cells. They are rich in voltage-gated sodium channels (Nav) and thus underpin rapid nerve impulse conduction in the vertebrate nervous system [1]. Although nodal proteins cluster in response to myelination, how myelin-forming glia influence nodal assembly is poorly understood. An axoglial adhesion complex comprising glial Neurofascin155 and axonal Caspr/Contactin flanks mature nodes [2]. We have shown that assembly of this adhesion complex at the extremities of migrating oligodendroglial processes promotes process convergence along the axon during central nervous system (CNS) node assembly [3]. Here we show that anchorage of this axoglial complex to the axon cytoskeleton is essential for efficient CNS node formation. When anchorage is disrupted, both the adaptor Protein 4.1B and the cytoskeleton protein ßII spectrin are mislocalized in the axon, and assembly of the node of Ranvier is significantly delayed. Nodal proteins and migrating oligodendroglial processes are no longer juxtaposed, and single detached nodal complexes replace the symmetrical heminodes found in both the CNS and peripheral nervous system (PNS) during development. We propose that axoglial adhesion complexes contribute to the formation of an interface between cytoskeletal elements enriched in Protein 4.1B and ßII spectrin and those enriched in nodal ankyrinG and ßIV spectrin. This clusters nascent nodal complexes at heminodes and promotes their timely coalescence to form the mature node of Ranvier. These data demonstrate a role for the axon cytoskeleton in the assembly of a critical neuronal domain, the node of Ranvier.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Nós Neurofibrosos/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo
12.
Glia ; 64(5): 840-52, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26840208

RESUMO

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 Wistar
13.
Front Cell Neurosci ; 9: 265, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26217189

RESUMO

Contactin-associated protein-like 2 (Caspr2), also known as CNTNAP2, is a cell adhesion molecule that clusters voltage-gated potassium channels (Kv1.1/1.2) at the juxtaparanodes of myelinated axons and may regulate axonal excitability. As a component of the Kv1 complex, Caspr2 has been identified as a target in neuromyotonia and Morvan syndrome, but also in some cases of autoimmune limbic encephalitis (LE). How anti-Caspr2 autoimmunity is linked with the central neurological symptoms is still elusive. In the present study, using anti-Caspr2 antibodies from seven patients affected by pure LE, we determined that IgGs in the cerebrospinal fluid of four out seven patients were selectively directed against the N-terminal Discoïdin and LamininG1 modules of Caspr2. Using live immunolabeling of cultured hippocampal neurons, we determined that serum IgGs in all patients strongly targeted inhibitory interneurons. Caspr2 was highly detected on GAD65-positive axons that are surrounding the cell bodies and at the VGAT-positive inhibitory presynaptic contacts. Functional assays indicated that LE autoantibodies may induce alteration of Gephyrin clusters at inhibitory synaptic contacts. Next, we generated a Caspr2-Fc chimera to reveal Caspr2 receptors on hippocampal neurons localized at the somato-dendritic compartment and post-synapse. Caspr2-Fc binding was strongly increased on TAG-1-transfected neurons and conversely, Caspr2-Fc did not bind hippocampal neurons from TAG-1-deficient mice. Our data indicate that Caspr2 may participate as a cell recognition molecule in the dynamics of inhibitory networks. This study provides new insight into the potential pathogenic effect of anti-Caspr2 autoantibodies in central hyperexcitability that may be related with perturbation of inhibitory interneuron activity.

14.
Neuron ; 81(4): 814-29, 2014 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-24559674

RESUMO

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.


Assuntos
Axônios/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Contactina 2/metabolismo , Neuropeptídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Camundongos , Neurogênese/fisiologia
15.
J Biol Chem ; 289(11): 7907-18, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24497634

RESUMO

Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.


Assuntos
Moléculas de Adesão Celular/química , Contactinas/química , Fatores de Crescimento Neural/química , Doenças do Sistema Nervoso Periférico/metabolismo , Polissacarídeos/química , Animais , Autoanticorpos/química , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cricetulus , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Ratos
16.
Front Cell Neurosci ; 7: 196, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24194699

RESUMO

Specific cell adhesion molecules (CAMs) are dedicated to the formation of axo-glial contacts at the nodes of Ranvier of myelinated axons. They play a central role in the organization and maintenance of the axonal domains: the node, paranode, and juxtaparanode. In particular, CAMs are essential for the accumulation of voltage-gated sodium channels at the nodal gap that ensures the rapid and saltatory propagation of the action potentials (APs). The mechanisms regulating node formation are distinct in the central and peripheral nervous systems, and recent studies have highlighted the relative contribution of paranodal junctions and nodal extracellular matrix. In addition, CAMs at the juxtaparanodal domains mediate the clustering of voltage-gated potassium channels which regulate the axonal excitability. In several human pathologies, the axo-glial contacts are altered leading to disruption of the nodes of Ranvier or mis-localization of the ion channels along the axons. Node alterations and the failure of APs to propagate correctly from nodes to nodes along the axons both contribute to the disabilities in demyelinating diseases. This article reviews the mechanisms regulating the association of the axo-glial complexes and the role of CAMs in inherited and acquired neurological diseases.

17.
J Biol Chem ; 286(49): 42426-42434, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22009740

RESUMO

The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.


Assuntos
Axônios/metabolismo , Moléculas de Adesão Celular/química , Fibronectinas/química , Fatores de Crescimento Neural/química , Moléculas de Adesão de Célula Nervosa/química , Neuroglia/metabolismo , Nós Neurofibrosos/metabolismo , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas de Membrana , Bainha de Mielina/química , Proteínas do Tecido Nervoso , Ligação Proteica , Estrutura Terciária de Proteína , Células de Schwann/metabolismo , Ressonância de Plasmônio de Superfície
18.
J Neurosci ; 30(14): 4868-76, 2010 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-20371806

RESUMO

The formation of paranodal axo-glial junctions is critical for the rapid and efficient propagation of nerve impulses. Genetic ablation of genes encoding the critical paranodal proteins Caspr, contactin (Cont), and the myelinating glia-specific isoform of Neurofascin (Nfasc(NF155)) results in the disruption of the paranodal axo-glial junctions, loss of ion channel segregation, and impaired nerve conduction, but the mechanisms regulating their interactions remain elusive. Here, we report that loss of immunoglobulin (Ig) domains 5 and 6 in Nfasc(NF155) in mice phenocopies complete ablation of Nfasc(NF155). The mutant mice lack paranodal septate junctions, resulting in the diffusion of Caspr and Cont from the paranodes, and redistribution of the juxtaparanodal potassium channels toward the nodes. Although critical for Nfasc(NF155) function, we find that Ig5-6 are dispensable for nodal Nfasc(NF186) function. Moreover, in vitro binding assays using Ig5-6 deletion constructs reveal their importance for the association of Nfasc(NF155) with Cont. These findings provide the first molecular evidence demonstrating domain-specific requirements controlling the association of the paranodal tripartite complex in vivo. Our studies further emphasize that in vivo structure/function analysis is necessary to define the unique protein-protein interactions that differentially regulate the functions of Neurofascins during axonal domain organization.


Assuntos
Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/fisiologia , Deleção de Genes , Imunoglobulinas/deficiência , Fibras Nervosas Mielinizadas/metabolismo , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/fisiologia , Animais , Axônios/metabolismo , Axônios/patologia , Células CHO , Moléculas de Adesão Celular/química , Cricetinae , Cricetulus , Humanos , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fibras Nervosas Mielinizadas/patologia , Fatores de Crescimento Neural/química , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/fisiologia , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Ratos
19.
FEBS Lett ; 584(9): 1787-92, 2010 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-19703450

RESUMO

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.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Proteínas de Membrana/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Axônios/química , Axônios/metabolismo , Transporte Biológico/fisiologia , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Modelos Biológicos , Nós Neurofibrosos/fisiologia
20.
J Cell Sci ; 122(Pt 18): 3403-13, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19706678

RESUMO

Contactin-associated protein 2 (Caspr2) is a neuronal membrane protein that is mutated in autism and related disorders. Although it is highly enriched at juxtaparanodes of Ranvier where it is essential for Shaker-type K(+) channel clustering, little is known about its function and regulation. In the present study, we examined the polarized expression of Caspr2 in hippocampal neurons using extracellular hemagglutinin (HA)-tagged Caspr2 constructs. We found that Caspr2 was targeted to the axonal surface, but colocalized with early endosomes in the somatodendritic compartment. The inhibition of endocytosis using a Dynamin-1 mutant or treatment with Dynasore prevented Caspr2 internalization from the dendrites and cell body. We identified a short sequence included into the 4.1B-binding domain that is required for the endocytosis of Caspr2. This sequence contains a protein kinase C (PKC) substrate motif on Thr1292, and point mutation of this residue or treatment with a PKC inhibitor prevented the somatodendritic internalization of Caspr2. Thus, the PKC-dependent trafficking of Caspr2 underlies its polarized expression in hippocampal neurons.


Assuntos
Axônios/metabolismo , Dendritos/metabolismo , Endocitose , Hipocampo/citologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Compartimento Celular , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Vesículas Citoplasmáticas/metabolismo , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Fosforilação , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Transdução de Sinais
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