RESUMEN
In addition to androgen differences between males and females, there are genetic differences that are caused by unequal dosage of sex chromosome genes. Using the cuprizone-induced demyelination model, we recently showed that surgical gonadectomy of adult mice resulted in decreased normal myelination and remyelination compared to gonadally intact animals, suggesting a supporting role for sex hormones in the maintenance of myelination. However, inherent sex differences in normal myelination and remyelination persisted even after gonadectomy, with males consistently remyelinating to a lesser extent relative to normal myelination as assayed by axon conduction and immunohistochemistry. This suggests a potential role for the sex chromosome complement in mediating the differential rates of remyelination observed in males and females. The present study focuses on the impact that sex chromosomes might have on these myelination differences. Making use of the four core-genotype mice and cuprizone-diet induced demyelination/remyelination paradigm, our results demonstrate sex chromosome-mediated asymmetry between XX and XY mice. The rate of functional remyelination following cuprizone diet-induced callosal demyelination in four core-genotype mice is attenuated in XY compared to XX animals of both gonadal sexes. Importantly, this difference arises only in the absence of circulating sex hormones following gonadectomy and confirms the role of sex hormones in the remyelination process reported earlier by our group. Because a genotype-mediated difference only arises following gonadectomy, the chromosomal contribution to myelination and remyelination is subtle yet significant. To explain this difference, we propose a possible asymmetry in the expression of myelination-related genes in XX vs. XY mice that needs to be investigated in future studies.
Asunto(s)
Cuerpo Calloso/fisiología , Fibras Nerviosas Mielínicas/fisiología , Caracteres Sexuales , Cromosomas Sexuales/fisiología , Animales , Cuerpo Calloso/ultraestructura , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fibras Nerviosas Mielínicas/ultraestructuraRESUMEN
Axonal dysfunction as a result of persistent demyelination has been increasingly appreciated as a cause of functional deficit in demyelinating diseases such as multiple sclerosis. Therefore, it is crucial to understand the ultimate causes of ongoing axonal dysfunction and find effective measures to prevent axon loss. Our findings related to functional deficit and functional recovery of axons from a demyelinating insult are important preliminary steps towards understanding this issue. Cuprizone diet for 3-6 wks triggered extensive corpus callosum (CC) demyelination, reduced axon conduction, and resulted in loss of axon structural integrity including nodes of Ranvier. Replacing cuprizone diet with normal diet led to regeneration of myelin, but did not fully reverse the conduction and structural deficits. A shorter 1.5 wk cuprizone diet also caused demyelination of the CC, with minimal loss of axon structure and nodal organization. Switching to normal diet led to remyelination and restored callosal axon conduction to normal levels. Our findings suggest the existence of a critical window of time for remyelination, beyond which demyelinated axons become damaged beyond the point of repair and permanent functional loss follows. Moreover, initiating remyelination early within the critical period, before prolonged demyelination-induced axon damage ensues, will improve functional axon recovery and inhibit disease progression.
Asunto(s)
Axones/fisiología , Cuerpo Calloso/fisiología , Vaina de Mielina/fisiología , Potenciales de Acción , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Axones/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/metabolismo , Linaje de la Célula , Cuprizona/administración & dosificación , Dieta , Femenino , Canal de Potasio Kv.1.2/metabolismo , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/fisiología , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , Nódulos de Ranvier/efectos de los fármacos , Nódulos de Ranvier/ultraestructura , Regeneración , Canales de Sodio/metabolismoRESUMEN
Multiple sclerosis (MS) is characterized by demyelination of the CNS with associated neurological deficits. Remyelination can occur but is often incomplete. The process of myelin repair requires the proliferation and migration of oligodendrocyte progenitor cells (OPC) into the lesion from the neighboring areas. OPC migration is altered by several factors, including antibodies that bind to OPC surface proteins. We have previously reported elevated anti-OSP/claudin-11 antibodies in the cerebrospinal fluid (CSF) of MS patients and that anti-OSP/claudin-11 antibodies generated in rabbits can inhibit OPC migration. In the study presented here, we investigated the effect of CSF IgG from MS patients and controls on OPC migration in culture. Rat OPC cultured with CSF from MS patients tended to migrate more than those cultured with control CSF, but this did not reach statistical significance. To determine whether the IgG fraction in the CSF influenced migration, we removed it using protein-A sepharose. A dramatic decrease in OPC migration was found in both MS (45 +/- 24 vs.16 +/- 9) and control (40 +/- 19 vs. 22 +/- 13) samples after IgG was removed (P <.05). Anti-OSP/claudin-11 antibody concentration did not significantly correlate with OPC migration. These data demonstrate that CSF IgG promotes OPC migration. Identification of the specific IgG fraction responsible for this effect could lead to novel therapies to promote recovery in MS.
Asunto(s)
Movimiento Celular/inmunología , Inmunoglobulina G/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Oligodendroglía/citología , Células Madre/citología , Adulto , Animales , Femenino , Humanos , Inmunoglobulina G/fisiología , Masculino , Persona de Mediana Edad , Oligodendroglía/inmunología , Ratas , Células Madre/inmunologíaRESUMEN
Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.
Asunto(s)
División Celular/fisiología , Movimiento Celular/fisiología , Integrina beta1/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Northern Blotting , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Claudinas , Fibronectinas/metabolismo , Hibridación in Situ , Sustancias Macromoleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oligodendroglía/química , Oligodendroglía/citología , Oligodendroglía/ultraestructura , Alineación de Secuencia , Tetraspaninas , Uniones Estrechas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Oligodendrocyte-specific protein (OSP/claudin-11) is a major component of CNS myelin and has been recently added to the claudin family of tight junction proteins. In this study, the developmental expression of OSP/claudin-11 was determined using in situ hybridization, immunohistochemistry (IH), and Western blot analysis. OSP/claudin-11 mRNA was expressed in a bimodal fashion. During prenatal development, OSP/claudin-11 mRNA was abundant in developing meninges, in areas adjacent to cartilage, and in mesoderm. In postnatal animals, OSP/claudin-11 was expressed primarily in developing oligodendrocytes and to a lesser extent, in testes. Double-labeled IH using O2-A progenitor cells revealed that OSP/claudin-11 expression occurs from the early progenitor stage and continues in mature oligodendrocytes. Electron microscopic IH localized OSP/claudin-11 to laminar myelin in the adult CNS. Western blot analysis of OSP/claudin-11 in developing brain revealed the expression of two separate transcripts that were developmentally regulated. These data demonstrate that OSP/claudin-11 expression is highly regulated during development and, therefore, may play an important role in growth and differentiation of oligodendrocytes and other cells outside the CNS.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/crecimiento & desarrollo , Proteínas de la Membrana/biosíntesis , Proteínas del Tejido Nervioso , Animales , Western Blotting , Encéfalo/citología , Diferenciación Celular , Claudinas , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Embarazo , Ratas , Células Madre/fisiología , Testículo/crecimiento & desarrollo , Testículo/inervación , Testículo/metabolismoRESUMEN
Oligodendrocyte-specific protein (OSP/claudin-11) is a four transmembrane protein concentrated in central nervous system myelin. Recent evidence has emerged suggesting that OSP/claudin-11 is involved in membrane interactions at tight junctions and with the extracellular matrix. OSP/claudin-11 seems to modulate proliferation and migration of oligodendrocytes presumably through these interactions. Furthermore, evidence is presented implicating OSP/claudin-11 as an autoantigen in the development of autoimmune demyelinating disease.
Asunto(s)
Membrana Celular/metabolismo , Enfermedades Autoinmunes Desmielinizantes SNC/fisiopatología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Animales , ClaudinasRESUMEN
Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K(+) channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D. M. Papazian. 1997. Biophys. J. 72:1489-1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K(+) channels.
Asunto(s)
Mutación/fisiología , Canales de Potasio/genética , Canales de Potasio/fisiología , Animales , Electrofisiología , Potenciales de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , XenopusRESUMEN
In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.
Asunto(s)
Membrana Celular/química , Canales de Potasio/química , Canales de Potasio/metabolismo , Pliegue de Proteína , Animales , Electroforesis en Gel de Poliacrilamida , Electrofisiología , Expresión Génica/genética , Activación del Canal Iónico , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Canales de Potasio/genética , Estructura Secundaria de Proteína , Canales de Potasio de la Superfamilia Shaker , XenopusRESUMEN
Entry of Ca2+ through Ca2+ channels is thought to trigger the acrosome reaction of spermatozoa during fertilization. Antagonists of the L-type Ca2+ channel are known to prevent the intracellular Ca2+ (Ca2+) increase and inhibit acrosomal exocytosis in mammalian sperm. Planar bilayer recordings were used to study Ca2- channels incorporated from partially purified boar sperm plasma membranes. With symmetrical 50 mM NaCl and 100 mM BaCl2 on the cis side, single-channel events consistent with Ba2+ flux from cis to trans were observed. These channels were activated by the dihydropyridine agonist (+/-)BAY K 8644 and blocked by the antagonist nitrendipine. Sperm Ca2- channels did not require depolarization for activation and did not inactivate. The (+/-)BAY K 8644 and (S-)BAY K 8644 enantiomers increased apparent open time in a dose-dependent [half-maximal activity constant (K0.5) = 0.9 and 0.3 microM, respectively] manner. Dihydropyridine antagonists nitrendipine (K0.5 = 0.5 microM) and (R+)BAY K 8644 (K0.5 = 2.8 microM) decreased apparent open times. The channels described in this report share some properties with brain, cardiac, and skeletal muscle t tubule Ca2+ channels and may be involved in increasing Cai2+ before the acrosome reaction.