RESUMEN
Axon diameter influences the conduction properties of myelinated axons, both directly, and indirectly through effects on myelin. However, we have limited understanding of mechanisms controlling axon diameter growth in the central nervous system, preventing systematic dissection of how manipulating diameter affects myelination and conduction along individual axons. Here we establish zebrafish to study axon diameter. We find that importin 13b is required for axon diameter growth, but does not affect cell body size or axon length. Using neuron-specific ipo13b mutants, we assess how reduced axon diameter affects myelination and conduction, and find no changes to myelin thickness, precision of action potential propagation, or ability to sustain high frequency firing. However, increases in conduction speed that occur along single myelinated axons with development are tightly linked to their growth in diameter. This suggests that axon diameter growth is a major driver of increases in conduction speeds along myelinated axons over time.
Asunto(s)
Axones , Pez Cebra , Animales , Axones/fisiología , Vaina de Mielina/fisiología , Sistema Nervioso Central , NeuronasRESUMEN
The mechanisms regulating myelin repair in the adult central nervous system (CNS) are unclear. Here, we identify DNA hydroxymethylation, catalyzed by the Ten-Eleven-Translocation (TET) enzyme TET1, as necessary for myelin repair in young adults and defective in old mice. Constitutive and inducible oligodendrocyte lineage-specific ablation of Tet1 (but not of Tet2), recapitulate this age-related decline in repair of demyelinated lesions. DNA hydroxymethylation and transcriptomic analyses identify TET1-target in adult oligodendrocytes, as genes regulating neuro-glial communication, including the solute carrier (Slc) gene family. Among them, we show that the expression levels of the Na+/K+/Cl- transporter, SLC12A2, are higher in Tet1 overexpressing cells and lower in old or Tet1 knockout. Both aged mice and Tet1 mutants also present inefficient myelin repair and axo-myelinic swellings. Zebrafish mutants for slc12a2b also display swellings of CNS myelinated axons. Our findings suggest that TET1 is required for adult myelin repair and regulation of the axon-myelin interface.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica/métodos , Vaina de Mielina/genética , Proteínas Proto-Oncogénicas/genética , Remielinización/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Ratones Noqueados , Ratones Transgénicos , Mutación , Vaina de Mielina/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , RNA-Seq/métodos , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Pez Cebra/genéticaRESUMEN
Through a genetic screen in zebrafish, we identified a mutant with disruption to myelin in both the CNS and PNS caused by a mutation in a previously uncharacterized gene, slc12a2b, predicted to encode a Na+, K+, and Cl- (NKCC) cotransporter, NKCC1b. slc12a2b/NKCC1b mutants exhibited a severe and progressive pathology in the PNS, characterized by dysmyelination and swelling of the periaxonal space at the axon-myelin interface. Cell-type-specific loss of slc12a2b/NKCC1b in either neurons or myelinating Schwann cells recapitulated these pathologies. Given that NKCC1 is critical for ion homeostasis, we asked whether the disruption to myelinated axons in slc12a2b/NKCC1b mutants is affected by neuronal activity. Strikingly, we found that blocking neuronal activity completely prevented and could even rescue the pathology in slc12a2b/NKCC1b mutants. Together, our data indicate that NKCC1b is required to maintain neuronal activity-related solute homeostasis at the axon-myelin interface, and the integrity of myelinated axons.
Asunto(s)
Axones/metabolismo , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Células de Schwann/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Proteínas de Pez Cebra/genética , Potenciales de Acción , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Axones/efectos de los fármacos , Axones/ultraestructura , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Humanos , Mutación , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/patología , Células de Schwann/efectos de los fármacos , Células de Schwann/ultraestructura , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Bloqueadores de los Canales de Sodio/toxicidad , Miembro 2 de la Familia de Transportadores de Soluto 12/deficiencia , Tetrodotoxina/toxicidad , Pez Cebra , Proteínas de Pez Cebra/deficienciaRESUMEN
Selection of the correct targets for myelination and regulation of myelin sheath growth are essential for central nervous system (CNS) formation and function. Through a genetic screen in zebrafish and complementary analyses in mice, we find that loss of oligodendrocyte Neurofascin leads to mistargeting of myelin to cell bodies, without affecting targeting to axons. In addition, loss of Neurofascin reduces CNS myelination by impairing myelin sheath growth. Time-lapse imaging reveals that the distinct myelinating processes of individual oligodendrocytes can engage in target selection and sheath growth at the same time and that Neurofascin concomitantly regulates targeting and growth. Disruption to Caspr, the neuronal binding partner of oligodendrocyte Neurofascin, also impairs myelin sheath growth, likely reflecting its association in an adhesion complex at the axon-glial interface with Neurofascin. Caspr does not, however, affect myelin targeting, further indicating that Neurofascin independently regulates distinct aspects of CNS myelination by individual oligodendrocytes in vivo.
Asunto(s)
Sistema Nervioso Central/citología , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Oligodendroglía/citología , Animales , Axones/metabolismo , Cuerpo Celular/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neurogénesis/fisiología , Neuroglía/metabolismo , Pez Cebra/metabolismoRESUMEN
Zebrafish are now well established as the preeminent vertebrate model with which to carry out gene discovery/forward genetic screens to identify the molecular genetic basis of biological processes. Gene discovery screens in zebrafish have already provided novel insight into mechanisms of glial cell development and function. The vast majority of genetic screens in zebrafish are based around a three generation screen that starts with the random induction of mutations in adult males using the chemical mutagen ENU. Here we outline the methods that underlie this type of screen, detailing each step, from ENU mutagenesis, through the breeding schemes required to recover homozygous mutant animals in subsequent generations, the screening procedure itself, with a focus on the analysis of myelinating glia, and the subsequent confirmation of mutant phenotypes.
Asunto(s)
Etilnitrosourea/efectos adversos , Pruebas Genéticas/métodos , Vaina de Mielina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Cruzamiento , Femenino , Homocigoto , Masculino , Modelos Animales , Mutación , FenotipoRESUMEN
The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/metabolismo , Vaina de Mielina/metabolismo , Filogenia , Proteínas ADAM/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Axones/metabolismo , Secuencia Conservada , Prueba de Complementación Genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Sistema Nervioso Periférico/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Relación Estructura-Actividad , Pez CebraRESUMEN
The development and function of the vertebrate nervous system depend on specific interactions between different cell types. Two examples of such interactions are synaptic transmission and myelination. LGI1-4 (leucine-rich glioma inactivated proteins) play important roles in these processes. They are secreted proteins consisting of an LRR (leucine-rich repeat) domain and a so-called epilepsy-associated or EPTP (epitempin) domain. Both domains are thought to function in protein-protein interactions. The first LGI gene to be identified, LGI1, was found at a chromosomal translocation breakpoint in a glioma cell line. It was subsequently found mutated in ADLTE (autosomal dominant lateral temporal (lobe) epilepsy) also referred to as ADPEAF (autosomal dominant partial epilepsy with auditory features). LGI1 protein appears to act at synapses and antibodies against LGI1 may cause the autoimmune disorder limbic encephalitis. A similar function in synaptic remodelling has been suggested for LGI2, which is mutated in canine Benign Familial Juvenile Epilepsy. LGI4 is required for proliferation of glia in the peripheral nervous system and binds to a neuronal receptor, ADAM22, to foster ensheathment and myelination of axons by Schwann cells. Thus, LGI proteins play crucial roles in nervous system development and function and their study is highly important, both to understand their biological functions and for their therapeutic potential. Here, we review our current knowledge about this important family of proteins, and the progress made towards understanding their functions.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Fenómenos Fisiológicos del Sistema Nervioso/genética , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Evolución Biológica , Perros , Glioma/metabolismo , Humanos , Encefalitis Límbica/genética , Encefalitis Límbica/metabolismo , Datos de Secuencia Molecular , Mutación/fisiología , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/crecimiento & desarrollo , Neoplasias del Sistema Nervioso/metabolismo , Sistema Nervioso Periférico/fisiología , Conformación Proteica , Procesamiento Proteico-Postraduccional/genética , Ratas , Pez Cebra , Proteínas de Pez CebraRESUMEN
Products of the Doublecortin Like Kinase (DCLK) gene are implicated in cortical migration and hippocampal maturation during embryogenesis. However, one of its splice variants, called CaMK Related Peptide (CARP), is expressed during adulthood in response to neurological stimuli, such as kainic acid-induced seizures and BDNF-LTP. The function of this transcript of the DCLK gene is poorly understood. To elucidate its function during adulthood we have created transgenic mice with over-expression of CARP in the brain. To study potential functions of CARP in the hippocampus we performed an electrophysiological characterization of the CA3/CA1 network of transgenic and wild-type mice and showed that field excitatory post synaptic potentials (fEPSPs) are highly increased in transgenic mice, while population spike amplitudes (PSAs) remained equal between genotypes. Consequently, hippocampal CA3/CA1 network excitability was decreased in transgenic mice. In addition we show a 2-fold up-regulation of the Ca(2+)-binding protein calretinin and a down-regulation of Rapgef4, a guanine exchange factor for Rap1, in the hippocampus. Given previously established conditions during which CARP is induced and our current data, we propose that this DCLK gene product affects glutamatergic neuronal transmission in response to neurological stimuli.
Asunto(s)
Encéfalo/fisiología , Hipocampo/fisiología , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Animales , Animales Modificados Genéticamente , Calbindina 2 , Quinasas Similares a Doblecortina , Estimulación Eléctrica , Epilepsia/inducido químicamente , Potenciales Postsinápticos Excitadores/fisiología , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hipocampo/efectos de los fármacos , Ácido Kaínico/efectos adversos , Ratones , Proteínas Musculares , Neuronas/fisiología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteína G de Unión al Calcio S100/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Transcripción GenéticaRESUMEN
The segregation and myelination of axons in the developing PNS, results from a complex series of cellular and molecular interactions between Schwann cells and axons. Previously we identified the Lgi4 gene (leucine-rich glioma-inactivated4) as an important regulator of myelination in the PNS, and its dysfunction results in arthrogryposis as observed in claw paw mice. Lgi4 is a secreted protein and a member of a small family of proteins that are predominantly expressed in the nervous system. Their mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins, in particular Adam22. We found that Lgi4 and Adam22 are both expressed in Schwann cells as well as in sensory neurons and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors. To determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action we performed heterotypic Schwann cell sensory neuron cultures and cell type-specific ablation of Lgi4 and Adam22 in mice. We show that Schwann cells are the principal cellular source of Lgi4 in the developing nerve and that Adam22 is required on axons. Our results thus reveal a novel paracrine signaling axis in peripheral nerve myelination in which Schwann cell secreted Lgi4 functions through binding of axonal Adam22 to drive the differentiation of Schwann cells.
Asunto(s)
Proteínas ADAM/fisiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/fisiología , Células de Schwann/fisiología , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/genética , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Animales , Animales Recién Nacidos , Línea Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/fisiología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Vaina de Mielina/genética , Vaina de Mielina/fisiología , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/genética , Ratas , Células de Schwann/metabolismo , Células de Schwann/ultraestructura , Células Receptoras Sensoriales/fisiología , Células Receptoras Sensoriales/ultraestructuraRESUMEN
Successful regeneration of damaged neurons depends on the coordinated expression of neuron-intrinsic genes. At present however, there is no comprehensive view of the transcriptional regulatory mechanisms underlying neuronal regeneration. We used high-content cellular screening to investigate the functional contribution of 62 transcription factors to regenerative neuron outgrowth. Ten transcription factors are identified that either increase or decrease neurite outgrowth. One of these, NFIL3, is specifically upregulated during successful regeneration in vivo. Paradoxically however, knockdown of NFIL3 and overexpression of dominant-negative NFIL3 both increase neurite outgrowth. Our data show that NFIL3, together with CREB, forms an incoherent feedforward transcriptional regulatory loop in which NFIL3 acts as a negative regulator of CREB-induced regeneration-associated genes.