RESUMEN
Carbon nanotubes are of huge biotechnological interest because they can penetrate most biological barriers and, inside cells, can biomimetically interact with the cytoskeletal filaments, triggering anti-proliferative and cytotoxic effects in highly dividing cells. Unfortunately, their intrinsic properties and bio-persistence represent a putative hazard that relapses their application as therapies against cancer. Here we investigate mild oxidation treatments to improve the intracellular enzymatic digestion of MWCNTs, but preserving their morphology, responsible for their intrinsic cytotoxic properties. Cell imaging techniques and confocal Raman spectroscopic signature analysis revealed that cultured macrophages can degrade bundles of oxidized MWCNTs (o-MWCNTs) in a few days. The isolation of nanotubes from these phagocytes 96 hours after exposure confirmed a significant reduction of approximately 30% in the total length of these filaments compared to the control o-MWCNTs extracted from the cell culture medium, or the intracellular pristine MWCNTs. More interestingly, in vivo single intratumoral injections of o-MWCNTs triggered ca. 30% solid melanoma tumour growth-inhibitory effects while displaying significant signs of biodegradation at the tumoral/peri-tumoral tissues a week after the therapy has had the effect. These results support the potential use of o-MWCNTs as antitumoral agents and reveal interesting clues of how to enhance the efficient clearance of in vivo carbon nanotubes.
Asunto(s)
Macrófagos/metabolismo , Melanoma Experimental/tratamiento farmacológico , Nanotubos de Carbono , Células Cultivadas , Células HeLa , Humanos , Oxidación-Reducción , Espectrometría RamanRESUMEN
Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.
Asunto(s)
Microglía/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Diferenciación Celular , Línea Celular , Humanos , Interferones/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Microglía/metabolismo , Chaperonas Moleculares , Regulación hacia ArribaRESUMEN
This unit describes various protocols for the isolation and purification of the main constituents of microtubules, chiefly alpha- and beta-tubulin, and the most significant microtubule associated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We include a classical isolation method for soluble tubulin heterodimer as the first basic purification protocol. In addition, we show how to analyze the tubulin and MAPs obtained after a phosphocellulose chromatography purification procedure. This unit also details a powerful and simple method to determine the native state of the purified tubulin based on one-dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last protocol describes the application of a new technique that allows visualizing the quality of polymerized microtubules based on atomic force microscopy (AFM).
Asunto(s)
Electroforesis/métodos , Proteínas de Microtúbulos/aislamiento & purificación , Microtúbulos/química , Cromatografía , Microscopía de Fuerza Atómica , Proteínas de Microtúbulos/análisis , Proteínas de Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Paclitaxel/análisisRESUMEN
Tubulin folding cofactors B (TBCB) and E (TBCE) are alpha-tubulin binding proteins that, together with Arl2 and cofactors D (TBCD), A (TBCA or p14) and C (TBCC), participate in tubulin biogenesis. TBCD and TBCE have also been implicated in microtubule dynamics through regulation of tubulin heterodimer dissociation. Understanding the in vivo function of these proteins will shed light on the Kenny-Caffey/Sanjad-Sakati syndrome, an important human disorder associated with TBCE. Here we show that, when overexpressed, TBCB depolymerizes microtubules. We found that this function is based on the ability of TBCB to form a binary complex with TBCE that greatly enhances the efficiency of this cofactor to dissociate tubulin in vivo and in vitro. We also show that TBCE, TBCB and alpha-tubulin form a ternary complex after heterodimer dissociation, whereas the free beta-tubulin subunit is recovered by TBCA. These complexes might serve to escort alpha-tubulin towards degradation or recycling, depending on the cell requirements.
Asunto(s)
Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/metabolismo , Chaperonas Moleculares/fisiología , Tubulina (Proteína)/metabolismo , Cromatografía en Gel , Dimerización , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Paclitaxel/farmacología , Estructura Terciaria de Proteína , Transfección , Moduladores de Tubulina/farmacologíaRESUMEN
Tubulin-folding cofactor E (TBCE) is an alpha-tubulin-binding protein involved in the formation of the tubulin dimer and in microtubule dynamics, through the regulation of tubulin heterodimer dissociation. TBCE has also been implicated in two important related human disorders, the Kenny-Caffey and Sanjad-Sakati syndromes. The expression of TBCE as a recombinant protein in bacteria results in the formation of insoluble inclusion bodies in the absence of denaturing agents. Although the active protein can be obtained from mammalian tissues, biochemical studies of TBCE present a special challenge. To express and purify native TBCE, a recombinant baculovirus expression system was used. Native wild-type TBCE purified from Sf9 extracts was sequentially purified chromatographically through cation exchange, hydrophobic interaction, and high-resolution gel-filtration columns. Mass spectrometric analysis identified 30% of the sequence of human TBCE. A stoichiometric excess of purified TBCE dissociated tubulin heterodimers. This reaction produced a highly unstable TBCE-alpha-tubulin complex, which formed aggregates. To distinguish between the aggregation of tubulin dimers induced by TBCE and tubulin dissociation, TBCE and tubulin were incubated with tubulin-folding cofactor A (TBCA). This cofactor captures the beta-tubulin released from the heterodimer with a stoichiometry of 1:1, as previously demonstrated. The beta-tubulin polypeptide was recovered as TBCA-beta-tubulin complexes, as demonstrated by non-denaturing gel electrophoresis and specific antibodies directed against beta-tubulin and TBCA.
Asunto(s)
Baculoviridae , Chaperonas Moleculares/química , Chaperonas Moleculares/aislamiento & purificación , Tubulina (Proteína)/química , Animales , Línea Celular , Cromatografía Liquida , Dimerización , Enfermedades Genéticas Congénitas/genética , Humanos , Chaperonas Moleculares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Spodoptera/citología , Síndrome , Tubulina (Proteína)/genética , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the alphabeta-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627-632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422-9426; Campo et al. (1994) FEBS Lett. 353, 162-166; Lewis et al. (1996) J. Cell Biol. 132, 1-4; Lewis et al. (1997) Trends Cell Biol. 7, 479-484; Tian et al. (1997) J. Cell Biol. 138, 821-823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327-332; Hirata et al. (1998) EMBO J. 17, 658-666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243-254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule-destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe-inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361-363; McNally (1999) Curr. Biol. 9, R274-R276]. Here we show that the tubulin folding cofactor D, although it does not co-polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering beta-tubulin from GTP-bound alphabeta-heterodimers.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Células 3T3 , Animales , Bovinos , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.
Asunto(s)
Proteínas/genética , Espermatogénesis/genética , Animales , Chaperoninas , Dimerización , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Proteínas/análisis , Proteínas/fisiología , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , Epitelio Seminífero/química , Epitelio Seminífero/citología , Testículo/metabolismo , Distribución Tisular , Tubulina (Proteína)/químicaRESUMEN
INTRODUCTION: Neurons and oligodendrocytes are terminally differentiated cells. This means that once they have differentiated from their precursor cells, they cannot proliferate. A direct consequence of this type of differentiation is that cell repair is impossible in areas where neurodegenerative disease have caused the death of neurons and oligodendroglia. Recently multipotential neuroepithelial precursors of the central nervous system (CNS) have been isolated and characterized in vitro. DEVELOPMENT: In this study we review the capacity for nervous repair of these neuroepithelial precursors from the neurobiological point of view. These cells, known as neuro-spheres can be cultivated, amplified and cryopreserved for subsequent transplanting. Already there are many studies showing how neuro-spheres maintain their capacity for differentiation in vivo and that they can reach certain localized areas of the CNS. CONCLUSIONS: From this review we conclude that through the study and manipulation of these neuro-spheres, new goals in CNS repair may be achieved.
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Encéfalo/fisiología , Regeneración Nerviosa/fisiología , Neuronas/fisiología , Oligodendroglía/fisiología , Muerte Celular/fisiología , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Humanos , Degeneración Nerviosa , Factor de Crecimiento Derivado de Plaquetas/fisiologíaRESUMEN
Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and posttranslational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III beta-tubulin (also known as beta6-tubulin). Both the upregulation and the post-translational processing of class-III beta-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III beta-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-beta6, specific to unphosphorylated class-III beta-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III beta-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta6 bound unphosphorylated soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-beta6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-beta6 +ve/TuJ-1-ve cells); a differentiation stage (U-beta6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III beta-tubulin immunostaining levels. These investigations also conclude that U-beta6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.
Asunto(s)
Células Epiteliales/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Biomarcadores , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Sistema Nervioso Central/citología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/química , Células Epiteliales/citología , Expresión Génica/fisiología , Ratones , Datos de Secuencia Molecular , Neuronas/química , Neuronas/citología , Sistema Nervioso Periférico/citología , Fosforilación , ARN Mensajero/análisis , Conejos , Nervios Espinales/citología , Telencéfalo/citología , Transcripción Genética/fisiología , Tubulina (Proteína)/análisis , Tubulina (Proteína)/inmunologíaAsunto(s)
Anticuerpos Monoclonales/análisis , Especificidad de Anticuerpos , Tubulina (Proteína)/genética , Tubulina (Proteína)/inmunología , Secuencia de Aminoácidos , Animales , Electroforesis/métodos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Pliegue de Proteína , Tubulina (Proteína)/químicaRESUMEN
Current studies support the morphological classification of oligodendrocytes proposed by Del Rio Hortega ([1922] Bol. R. Soc. Esp. Hist. Nat. 10:25-29; [1924] C.R. Soc. Biol. 91:818-820), in which cells either myelinate multiple internodes that are associated with small axons, or they myelinate restricted/single internodes of large-diameter axons. The reasons why an oligodendrocyte myelinates a particular calibre of axon are unknown. Because progenitors are generated in restricted, subventricular zones, an intrinsic program would imply that germinal centres contain a mixture of cells, each committed to myelinate axons of a particular size. Conversely, each cell could have the potential ability to myelinate any size axon. We tested this latter hypothesis that oligodendrocyte progenitors are uncommitted in their ability to myelinate a particular axon size. We introduced oligodendrocyte lineage cells from the optic nerve, which normally encounter only small-diameter axons, to a myelin-deficient environment containing a large range of axon sizes. Dissociated, mixed glial cells from the optic nerve were characterised immunocytochemically and were grafted into the spinal cord ventral column of neonatal, myelin-deficient rat mutants. Examination of the patches of myelin produced by these cells at different times after transplantation revealed that optic nerve oligodendrocytes were capable of producing a widespread, nonselective myelination of axons that were destined to have both small or large calibres. Thus, an axonal or local signal, and not an intrinsic program, is probably responsible for the previously described oligodendrocyte diversity.
Asunto(s)
Axones/fisiología , Trasplante de Tejido Encefálico , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Nervio Óptico/trasplante , Animales , Axones/ultraestructura , Linaje de la Célula/fisiología , Masculino , Microscopía Electrónica , Proteínas de la Mielina/deficiencia , Proteínas de la Mielina/genética , Vaina de Mielina/ultraestructura , Oligodendroglía/ultraestructura , Nervio Óptico/citología , Nervio Óptico/fisiología , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Médula Espinal/cirugíaRESUMEN
To understand the developmental role of Hoxb-8, this relatively 5' Hoxb gene was ectopically expressed in embryonic regions where only more 3' Hox genes are normally expressed. Hoxb-8 coding sequences driven by a retinoic acid receptor beta2 promoter fragment were introduced in the mouse germ line by pronuclear injection. The promoter was chosen with the aim to extend rostrally the expression domain of the gene in neurectoderm and mesoderm at the time of development when Hox gene expression domains are being established. Embryos developing from DNA-injected zygotes, and from transgenic mouse lines were analyzed. Pattern alterations were observed in transgenic embryos, some of which involved the peripheral nervous system. Spinal ganglia in the mouse are first detectable around embryonic day 9.5. By day 11.5, the first of these ganglia (C1, Froriep's ganglion) has degenerated in the mouse and other amniotes. In contrast, this first ganglion did persist in the Hoxb-8 gain-of-function transgenic mice. We have started to take advantage of the phenotype of transgenic versus wild-type embryos to understand the mechanisms underlying the ontogeny and degeneration of Froriep's ganglion in wild-type mice, and the role of Hoxb-8 in C1 maintenance in transgenic embryos. The present work describes a morphological, histological and immunocytological analysis of both the degenerating and the permanent C1, and a preliminary characterization of the axonal extensions from the transgenic C1. We discuss the methodology of generating gain-of-function transgenic mice to study the genetics of pattern formation along the antero-posterior axis, and the usefulness of analyzing these particular Hoxb-8 transgenic embryos to understand some aspects of the ontogenesis and development of the upper cervical dorsal root ganglia.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Sistema Nervioso Periférico/embriología , Animales , Axones/ultraestructura , Ectodermo/fisiología , Desarrollo Embrionario y Fetal/genética , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Morfogénesis/genética , Neuronas Aferentes/metabolismo , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/genética , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismo , TransgenesRESUMEN
Oligodendrocytes originate in different neural tube domains, within boundaries of expression of a series of patterning genes which condition the diverse morphogenetic programme of each area. Although neuronal and astrocyte heterogeneity are widely accepted, and despite accumulating evidence for oligodendrocyte heterogeneity in vivo, oligodendrocytes in vitro are currently considered as a homogeneous cell population. The present investigation demonstrates that oligodendrocyte diversity can be detected in vitro and characterizes a novel morphological class of O4-positive oligodendrocyte which is consistently identifiable in rat central nervous system cultures. These cells have a very characteristic epithelioid, unbranched and often lobulated morphology which enables their identification within 2 h of plating. Immunostaining shows that this morphological type is sometimes positive for GD3, A2B5 and vimentin, and most of the time positive for Ranscht antibody, O1 and Rip but negative for glial fibrillary acidic protein, OX-42, neuron-specific enolase, nestin and erbB2. The apparent levels and/or distributions of (i) microtubules, (ii) surface glycolipids recognized by O4, O1 and Ranscht antibody, and (iii) the less specific marker carbonic anhydrase II, typically differ from those of nearby classical, branched oligodendrocytes. Cells with this epithelioid morphology also express myelin basic protein and O10 (a proteolipid protein epitope), both of which are markers for mature oligodendrocytes. Conversely, O4+/O1- cells with this membranous appearance were also seen. Although these atypical oligodendrocytes were most abundant in spinal cord cultures (representing >10% of the O4+ population), they were not exclusive to this region and occurred at a low frequency in neonatal optic nerve cultures.
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Oligodendroglía/clasificación , Oligodendroglía/citología , Médula Espinal/citología , Animales , Biomarcadores , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Morfogénesis , Ratas , Ratas Sprague-Dawley , Vimentina/análisisRESUMEN
DM-20, a product of the proteolipid protein (plp) gene, has been demonstrated in the spinal cord of the mouse embryo as early as embryonic day 12 (E12) in certain cells, some of which are identifiable as oligodendrocyte progenitors. The present work uses optic pathways of rat and mouse as well-characterized systems for the study of gliogenesis. plp gene expression was monitored with a combination of reverse transcriptase polymerase chain reaction, in situ hybridization, and immunostaining with antibodies to different PLP peptide sequences, combined with O-2A lineage markers. In tissue sections, hybridizing cells were detected initially in the proximal optic tracts between E18 and birth and thereafter progressively in the chiasm and optic nerves. Small unbranched cells expressing DM-20 but not myelin basic protein (MBP) and probably representing progenitors were detectable by immunostaining in similar locations. With increasing postnatal ages, cells representing maturing oligodendrocytes which co-label for PLP and MBP are present in the optic pathways. In vitro analysis of freshly dissociated cells from premyelinated optic nerve demonstrated that the plp gene is expressed in some O-2A progenitor cells as well as mature oligodendrocytes. We also present evidence that increase in expression of the plp gene along the O-2A lineage differentiation is not progressive but that downregulation at the proligodendroblast (O4+/O1-) stage probably occurs. We suggest that progenitors express the dm-20 isoform while oligodendrocytes express predominantly the plp isoform. Not all progenitors express the plp gene at the times studied, indicating that the presence of DM-20 is either transitory in individual cells or that only a sub-population is involved. The function of DM-20 at this early stage of the oligodendrocyte lineage has yet to be determined.
Asunto(s)
Oligodendroglía/metabolismo , Nervio Óptico/crecimiento & desarrollo , Nervio Óptico/metabolismo , Proteolípidos/metabolismo , Animales , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos , RatasRESUMEN
Oligodendrocyte progenitors, originating in the ventral ventricular zone of the embryonic rodent spinal cord, migrate and differentiate into the oligodendrocytes myelinating the future white matter. Transcripts for the dm-20 isoform of the proteolipid protein (plp) gene are detectable initially in cells of the ventral ventricular region of the embryonic central canal and subsequently throughout the white matter. The dm-20+ cells are present several days before oligodendrocytes or myelin sheaths are detectable. The purpose of the present study was to determine if DM-20 protein is present and whether DM-20+ cells can be linked to the oligodendrocyte lineage in the mouse spinal cord. Expression of plp and dm-20 transcripts and product was monitored using reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization and immunostaining of cryosections and associated cultures. Cell identification was performed using antigenic markers characterizing different stages of oligodendrocyte differentiation. We show a temporal and spatial progression of cells expressing dm-20 transcripts and product from the ventral ventricular zone at embryonic day 13 (E13.0), via the lateral borders of the floor plate to the ventral pia and white matter. The cells, initially devoid of myelin basic protein (MBP) and PLP, co-express these myelin proteins at approximately E16.5/17.0. Some DM-20+ cells co-label with definitive markers of the early oligodendrocyte lineage, are capable of mitosis and subsequently differentiate into oligodendrocytes. Other DM-20+ cells may represent earlier precursor cells. The expression of DM-20 in oligodendrocyte progenitors is consistent with a postulated role in glial cell development.
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Proteína Proteolipídica de la Mielina/biosíntesis , Proteínas del Tejido Nervioso , Oligodendroglía/fisiología , Médula Espinal/embriología , Células Madre/fisiología , Animales , Linaje de la Célula , Células Cultivadas , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/embriología , Ventrículos Cerebrales/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C3H , Proteína Proteolipídica de la Mielina/genética , Oligodendroglía/ultraestructura , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificación , ARN/metabolismo , Sondas ARN , Médula Espinal/ultraestructura , Células Madre/ultraestructuraRESUMEN
In a previous study we demonstrated that differentiation and development of mouse oligodendrocytes is similar to that of the rat after the stage at which O4 is acquired. In this present study we compare directly the early differentiation of oligodendrocytes in the mouse and rat post natal optic nerve and show that the two species differ at the O-2A progenitor and proligodendroblast stages. Mouse progenitors show a variety of morphologies compared to the typical bipolar appearance in the rat. Many murine cells fail to immunolabel with A2B5, GD3, O4, and RmAb, classical markers for rat progenitors, proligodendroblasts, and immature oligodendrocytes. We find that these "unlabeled" cells stain for GAP-43 and that expression of GAP-43 overlaps A2B5 and GD3 in the earlier progenitors and O4, RmAb, and O1 in the later proligodendroblasts and immature oligodendrocytes. Our data suggest that in the development of the mouse O-2A progenitor cells there is a developmental discontinuity between the earlier markers such as A2B5 and GD3 and the later marker O4, which can be filled by GAP-43. We therefore consider that GAP-43 could be used in the mouse, in addition to the classical O-2A markers, for the study of the early oligodendrocyte lineage as it labels an otherwise undetectable O-2A population.
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Antígenos/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Nervio Óptico/citología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Antígenos/inmunología , Biomarcadores , Recuento de Células , Células Cultivadas , Técnica del Anticuerpo Fluorescente Directa , Ratones , Proteínas del Tejido Nervioso/inmunología , Oligodendroglía/fisiología , Nervio Óptico/crecimiento & desarrollo , Nervio Óptico/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Células Madre/metabolismoRESUMEN
The myelin mutants have been extensively used as tools to study the complex process of myelination in the central and peripheral nervous system. A multidisciplinary approach to the study of these models ultimately allows a correlation to be made between phenotype and genotype. This correlation may then lead to the formation of new hypotheses about the functions of the products of genes involved in myelination. This review presents a number of new myelin mutants which have recently been described. The species involved include mouse, rat, rabbit, hamster, and dog models. The genetic defect has not been elucidated in all of these animals, but most have been characterized clinically and pathologically, and, in some cases, biochemically. In addition, a better known myelin mutant, the trembler mouse, is discussed. Recent molecular findings have brought this fascinating mutant to the forefront of the field of peripheral nervous system research. The range of abnormalities in the mutants described in this review includes defects in specific myelin proteins, suspected abnormalities in membrane formation, and apparent defects of the oligodendrocyte cytoskeleton. These findings underscore the complexity of the myelination process and highlight the numerous ways in which it can be disrupted.
Asunto(s)
Mutación , Vaina de Mielina/patología , Animales , Cricetinae , Perros , Mesocricetus , Ratones , Proteína Básica de Mielina/análisis , Proteína Proteolipídica de la Mielina/análisis , Proteína Proteolipídica de la Mielina/genética , Vaina de Mielina/química , Vaina de Mielina/fisiología , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
The jimpy rumpshaker (jprsh) mutation is an amino acid substitution in exon 4 (Ile186-->Thr) of the proteolipid protein (PLP) gene on the X chromosome. Affected mice show moderate hypomyelination of the central nervous system (CNS) with increased numbers of oligodendrocytes in the white matter of the spinal cord, a feature distinguishing them from other PLP mutations such as jp, in which premature cell death occurs with reduced numbers of oligodendrocytes. Myelin sheaths of jprsh immunostain for myelin basic protein (MBP) and DM-20, but very few contain PLP. This study examines the differentiation of oligodendrocytes cultured from the spinal cords of young mutant and wild type mice using various surface and cytoplasmic antigenic markers to define the stage of development. The majority of oligodendrocytes from mutant mice progress normally to express MBP; approximately 30%, relative to wild type, contain DM-20 at the in vivo age of 16 days, but very few immunostain for PLP or the O10 and O11 markers. The morphology of mutant cells in respect to membrane sheets and processes appears similar to normal. The jprsh oligodendrocyte is, therefore, characterized by a failure to express the markers indicative of the most mature cell; however, it is probably able to achieve a normal period of survival. These data, taken in conjunction with previous results, suggest that the PLP gene has at least two functions; one, probably involving PLP, is concerned with a structural role in normal myelin compaction; the other, perhaps related to DM-20 (or another lower molecular weight proteolipid), is essential for cell survival. The mutation in jprsh at residue 186 suggests that this region, which is common to PLP and DM-20, is not critical for this latter function.
Asunto(s)
Diferenciación Celular/genética , Ratones Jimpy/genética , Mutación , Proteínas de la Mielina/genética , Proteínas del Tejido Nervioso , Oligodendroglía/citología , Animales , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Ratones Mutantes , Proteínas de la Mielina/análisis , Proteína Proteolipídica de la Mielina , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/fisiología , Proteolípidos/análisis , Médula Espinal/citología , Médula Espinal/metabolismo , Cromosoma XRESUMEN
Rumpshaker (rsh) is an X-linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (jp). The mutation (known as jprsh) differs in several respects from other X-linked myelin mutants, including jp, in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10-20%, respectively, of age-matched wild type mice. DM-20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the number of PLP mRNA-expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM-20 mRNA.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/fisiología , Enfermedades Desmielinizantes/genética , Regulación de la Expresión Génica , Proteínas de la Mielina/genética , Médula Espinal/fisiología , Cromosoma X , Alelos , Animales , Northern Blotting , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Mutantes , Proteína Básica de Mielina/análisis , Proteína Básica de Mielina/genética , Biosíntesis de Proteínas , Proteolípidos/análisis , Proteolípidos/genética , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/metabolismo , Médula Espinal/crecimiento & desarrollo , Médula Espinal/fisiopatologíaRESUMEN
The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.