RESUMEN

Etoposide-induced death comprises such nuclear events as the formation of topoisomerase II-DNA cleavable complex and cytosolic events including caspase activation. By first establishing the temporospatial death sequence triggered by etoposide in a neuronal cell line, MN9D overexpressing Bcl-X(L) (MN9D/Bcl-X(L)) or control vector (MN9D/Neo), we examined whether formation of this complex is primarily responsible for cell death and at which strategic points and how Bcl-X(L) blocks etoposide-induced neuronal death. Etoposide induced death that was dependent on caspase, cycloheximide, and calpain in MN9D/Neo cells. Etoposide also induced death in enucleated MN9D/Neo cells, although this was less severe. The level of topoisomerase II-DNA cleavable complex reached at a maximum of 2 hr after etoposide treatment was identical in MN9D/Neo and MN9D/Bcl-X(L) cells. In MN9D/Neo cells, cytochrome c release into the cytosol and caspase activation occurred as early as 2 hr and 3-6 hr after etoposide treatment, respectively. Etoposide-induced DNA laddering potentially via caspase appeared as early as 12 hr after drug treatment, followed by nuclear swelling in MN9D/Neo cells (>18-20 hr). Subsequently, nuclear condensation started by 24-28 hr and became apparent thereafter. All of these events except for nuclear swelling were substantially blocked in MN9D/Bcl-X(L). At the later stage of cell death (<32-36 hr), a specific cleavage of Bax and fodrin appeared that was completely blocked by calpain inhibitor or by Bcl-X(L). Taken together, our data suggest that Bcl-X(L) prevents etoposide-induced neuronal death by exerting its anticaspase and anticalpain effect on cellular events after the formation of topoisomerase II-DNA cleavable complex that may not be a major contributor to cell death.

Asunto(s)

Apoptosis/fisiología , Sistema Nervioso Central/enzimología , ADN/antagonistas & inhibidores , Etopósido/antagonistas & inhibidores , Neuronas/enzimología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inhibidores de Topoisomerasa II , Apoptosis/efectos de los fármacos , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Sistema Nervioso Central/citología , Sistema Nervioso Central/efectos de los fármacos , Grupo Citocromo c/efectos de los fármacos , Grupo Citocromo c/metabolismo , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de Microfilamentos/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Proteína X Asociada a bcl-2 , Proteína bcl-X

RESUMEN

An MN9D dopaminergic neuronal cell line overexpressing calbindin-D28K (MN9D/Calbindin) was established in order to investigate directly the potential role of calcium-binding protein in neuronal differentiation. Overexpression of calbindin-D28K in MN9D cells resulted in significant increases in the number of neurites, the length of primary neurites, and the total extent of neurites. This robust neurite outgrowth occurred without cessation of cell division. Analysis of immunoblots revealed that this morphological differentiation was accompanied by increased expression of such markers of maturation as the synaptosomal protein SNAP-25. During calbindin-D28K-evoked neurite outgrowth in MN9D cells, phosphorylation of p38 mitogen-activated protein kinase (MAPK) dramatically increased while the levels and extent of phosphorylation of such other MAPKs as c-Jun N-terminal kinase (JNK) or extracellular response kinase (ERK) were not altered. Consequently, calbindin-D28K-induced neurite outgrowth was largely abolished by treatment with a p38 inhibitor, PD 169316, while the level of SNAP-25 in MN9D/Calbindin cells was not altered by this treatment. These data support an idea that calbindin-D28K and its associated p38 signaling pathway play a role in dopaminergic neuronal differentiation.

Asunto(s)

Dopamina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Proteína G de Unión al Calcio S100/biosíntesis , Animales , Calbindina 1 , Calbindinas , Diferenciación Celular , División Celular , Pollos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuroblastoma/metabolismo , Neuronas/citología , Neuronas/enzimología , Fosforilación , Unión Proteica , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 microM N-methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP(+) but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP(+)-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP(+), overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP(+)-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP(+)-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP(+)-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP(+)-induced dopaminergic neuronal death.

Asunto(s)

1-Metil-4-fenilpiridinio/farmacología , Dopaminérgicos/farmacología , Mitocondrias/enzimología , Mitocondrias/genética , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Animales , Muerte Celular , Línea Celular , ADN Mitocondrial/genética , Regulación hacia Abajo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , ATPasas de Translocación de Protón Mitocondriales , Neuronas/efectos de los fármacos , Oxidopamina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacos , Transfección

RESUMEN

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.

Asunto(s)

Apoptosis/fisiología , Degeneración Nerviosa/fisiopatología , Neuroglía/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Etiquetado Corte-Fin in Situ , Masculino , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/patología , Neuroglía/inmunología , Neuroglía/patología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/patología , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.

Asunto(s)

Ácidos/metabolismo , Astrocitos/enzimología , Calcio/metabolismo , Calpaína/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Concentración de Iones de Hidrógeno , Secuencia de Aminoácidos , Animales , Anticuerpos , Astrocitos/citología , Astrocitos/inmunología , Calcimicina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Células Cultivadas , Quelantes/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Diltiazem/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/química , Glicoproteínas/farmacología , Técnicas In Vitro , Ionóforos/farmacología , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/farmacología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Nifedipino/farmacología , Prosencéfalo/citología , Ratas , Ratas Endogámicas F344 , Especificidad por Sustrato , Verapamilo/farmacología

RESUMEN

Oxidative stress has been implicated as a primary cause of neuronal death in certain neurodegenerative disorders and in aging brains. Natural products have been used in Asian societies for centuries for treating such neurodegenerative disorders as senile dementia. In an effort to identify active neuroprotective compounds from these products, we have employed cultures of rat cortical neurons as our screening system. A methanolic extract from dried roots of Cynanchum wilfordii Hemsley (Asclepiadaceae) significantly mitigated the neurotoxicity induced by H2O2 in this screening system. Activity-guided fractionation using several chromatographic techniques resulted in the isolation of the neuroprotective compound, cynandione A, a biacetophenone. At a concentration of 50 microM, cynandione A significantly reduced neurotoxicity induced by H2O2. Cynandione A significantly attenuated decreases in levels of glutathione, superoxide dismutase, and other enzymes that participate in the cellular defense against oxidative stress. Furthermore, cynandione A alleviated neurotoxicity induced by the excitotoxic neurotransmitter, L-glutamate, the neurotoxicity induced by kainate, but not that mediated by N-methyl-D-aspartate. Cynandione A was demonstrated to be a natural antioxidant as it facilitated the breakdown of hydrogen peroxide in vitro; however, no mechanism was uncovered to explain its neuroprotectant effects against glutamate and kainate. Therefore, cynandione A may be efficacious in protecting neurons from oxidative stress mediated via activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptors since it exerted significant neuroprotective effects on cultured cortical neurons.

Asunto(s)

Compuestos de Bifenilo/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Glutámico/toxicidad , Peróxido de Hidrógeno/toxicidad , Ácido Kaínico/toxicidad , Neuronas/citología , Fármacos Neuroprotectores/farmacología , Oxidantes/toxicidad , Animales , Antioxidantes/farmacología , Compuestos de Bifenilo/aislamiento & purificación , Células Cultivadas , Corteza Cerebral/citología , Feto/citología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Neuroglía/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Raíces de Plantas/química , Plantas Medicinales/química , Ratas , Ratas Sprague-Dawley

RESUMEN

While screening extracts of natural products in search of anticholinesterase activity, we found that a total methanolic extract of the tuber of Corydalis ternata (Papaveraceae) showed significant inhibitory effects on acetylcholinesterase. Further fractionation of this extract using acetylcholinesterase inhibition as the parameter screened resulted in the isolation and purification of an alkaloid, protopine. Protopine inhibited acetylcholinesterase activity in a dose-dependent manner. The concentration required for 50% inhibition was 50 microM. The anti-acetylcholinesterase activity of protopine was specific reversible and competitive in manner. Furthermore, when mice were pretreated with protopine, the alkaloid significantly alleviated scopolamine-induced memory impairment. In fact, protopine had an efficacy almost identical to that of velnacrine, a tacrine derivative developed by a major drug manufacturer to treat Alzheimer's disease, at an identical therapeutic concentration. We suggest, therefore, that protopine has both anti-acetylcholinesterase and antiamnesic properties that may ultimately hold significant therapeutic value in alleviating certain memory impairments observed in dementia.

Asunto(s)

Alcaloides/farmacología , Amnesia/tratamiento farmacológico , Alcaloides de Berberina , Inhibidores de la Colinesterasa/farmacología , Nootrópicos/farmacología , Papaver/química , Plantas Medicinales , Alcaloides/uso terapéutico , Animales , Benzofenantridinas , Inhibidores de la Colinesterasa/uso terapéutico , Demencia/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos ICR , Nootrópicos/uso terapéutico

RESUMEN

Buffering extracellular pH at the site of a spinal cord crush-injury may stimulate axonal regeneration in rats (1; Guth et al., Exp. Neurol. 88: 44-55, 1985). We demonstrated in cultured astrocytes that acidic pH initiates a rapid increase in immunoreactivity for GFAP (GFAP-IR), a hallmark of reactive gliosis (2; Oh et al., Glia 13: 319-322, 1995). We extended these studies by investigating the effects of certain treatments on reactive gliosis developing in situ in a rat spinal cord injury model. A significant reactive gliosis was observed within 2 days of cord lesion in untreated crush or vehicle-treated, crush control animals as evidenced by increased GFAP-IR and hypertrophy of astrocytes. By contrast, infusion of Pipes buffer (pH 7.4) into the lesion site significantly reduced this increase. The increased GFAP-IR appeared to be linked to Ca2+ influx since infusion of a blocker of L-type calcium channels, nifedipine, reduced the ensuing reactive gliosis significantly. While Ca2+ modulates many signaling pathways within cells, its effect on reactive gliosis appeared to result from an activation of calpain I. Calpain inhibitor I, a selective inhibitor of mu-calpain, also significantly reduced reactive gliosis. However, calpain inhibitor II, a close structural analog which blocks m-calpain, had no salutary effect. We suggest, therefore, that the initial reactive gliosis seen in vivo may result from the activation of a neutral, Ca2+-dependent protease, calpain I, through calcium influx.

Asunto(s)

Calcio/metabolismo , Calpaína/metabolismo , Gliosis/etiología , Traumatismos de la Médula Espinal/complicaciones , Heridas no Penetrantes/complicaciones , Enfermedad Aguda , Animales , Astrocitos/patología , Bloqueadores de los Canales de Calcio/farmacología , Activación Enzimática/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Hipertrofia , Inmunohistoquímica , Masculino , Compresión Nerviosa , Nifedipino/farmacología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología

RESUMEN

Certain natural products and Asian herbal remedies have been used in Asia to attenuate neurodegenerative diseases, including senile dementia. We have examined derivatives of several natural products for potential neuroprotective activity in an in vitro test system. In the present study, we assayed a number of compounds that were isolated from Panax ginseng C.A. Meyer (Araliaceae) for an ability to protect rat cortical cell cultures from the deleterious effects of the neurotoxicant, glutamate. We found that ginsenosides Rb1 and Rg3 significantly attenuated glutamate-induced neurotoxicity. Brief exposure of cultures to excess glutamate caused extensive neuronal death. Glutamate-induced neuronal cell damage was reduced significantly by pretreatment with Rb1 and Rg3. Ginsenosides Rb1 and Rg3 inhibited the overproduction of nitric oxide, which routinely follows glutamate neurotoxicity, and preserved the level of superoxide dismutase in glutamate-treated cells. Furthermore, in cultures treated with glutamate, these ginsenosides inhibited the formation of malondialdehyde, a compound that is produced during lipid peroxidation, and diminished the influx of calcium. These results show that ginsenosides Rb1 and Rg3 exerted significant neuroprotective effects on cultured cortical cells. Therefore, these compounds may be efficacious in protecting neurons from oxidative damage that is produced by exposure to excess glutamate.

Asunto(s)

Corteza Cerebral/citología , Ácido Glutámico/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Saponinas/farmacología , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Feto , Ginsenósidos , Malondialdehído/análisis , Degeneración Nerviosa , Neuronas/citología , Neuronas/fisiología , Panax , Plantas Medicinales , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo

RESUMEN

Neuroepithelial progenitor cells from forebrains of newborn rat pups develop into "mature" astrocytes in an epidermal growth factor-containing medium free of serum (Von Visger et al: Exp Neurol 128:34, 1994). Eight-week-old "mature" astrocyte cultures on poly-L-lysine-coated dishes were exposed to an acidic medium (pH 5.8-6.0) for 2-6 h. Immunoreactivity for glial fibrillary acidic protein (GFAP) dramatically and rapidly increased; this immediate increase was not affected by pretreatment with cycloheximide. In further experiments we found that the increase in GFAP was undiminished for 24-48 h after the acid-treated astrocytes were returned to normal growth medium. The Ca2+ channel antagonists nifedipine and diltiazem attenuated the increase in GFAP immunoreactivity. These results suggest that extracellular acidosis may produce a rapid increase in GFAP immunoreactivity in astrocytes independent of de novo protein synthesis, possibly by increasing intracellular levels of free Ca2+ ions.

Asunto(s)

Astrocitos/metabolismo , Concentración de Iones de Hidrógeno , Prosencéfalo/metabolismo , Acidosis , Animales , Calcio/metabolismo , Células Cultivadas , Inmunohistoquímica , Neuroglía/inmunología , Neuroglía/metabolismo , Prosencéfalo/citología , Ratas , Ratas Endogámicas F344

RESUMEN

We have studied the reactive responses of both astrocytes and microglia to dopaminergic denervation of the striatum by MPTP. Following MPTP treatment, increased GFAP immunoreactivity reached a peak at 2 days and persisted for at least 6 weeks. Immunoreactivity to vimentin was also markedly increased in astrocytes 48 h after MPTP treatment. Striatal laminin immunoreactivity, however, appeared to be unaffected by drug treatment. GFAP protein levels increased to 196% and 321% of control 24 and 48 hours after MPTP treatment, respectively. Concomitantly, GFAP mRNA levels increased to 560% and 1620% of control, respectively. These reactive changes in striatal astrocytes in response to MPTP treatment were also accompanied by a reactive microglial response as evidenced by increased immunohistochemical visualization of striatal microglia using antibodies to Mac-1. Our results and those reported previously by O'Callaghan et al., strongly suggest that MPTP-induced reactive gliosis in mouse striatum is associated with reactive microglia, albeit without increased interleukin-1 beta.

Asunto(s)

Intoxicación por MPTP , Neostriado/citología , Neuroglía/efectos de los fármacos , Animales , Antígenos de Superficie/inmunología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Northern Blotting , Western Blotting , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Laminina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neostriado/efectos de los fármacos , Neostriado/enzimología , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/enzimología , Neuroglía/metabolismo , ARN/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Vimentina/metabolismo

RESUMEN

Neuroepithelial progenitor cells from striata of adult mice develop into either astrocytes or neurons when cultured in the presence of epidermal growth factor (B. A. Reynolds, and S. Weiss, Science 255: 1707-1710, 1992). We instituted primary cultures of such progenitor cells from forebrains of newborn rat pups in an epidermal growth factor-containing medium free of serum in order to study the development of astrocytes in culture. At 4-6 days, primary cultures consisted of floating clusters of proliferating cells which expressed nestin, a marker for neuroepithelial progenitor cells, the ganglioside GD3, and vimentin. When clusters were transferred to polylysine-coated dishes, cells attached to the substrate and began to express antigens characteristic of particular differentiated neurons, astrocytes, or oligodendrocytes within 2 weeks. In 4- to 6-week-old secondary cultures, levels of vimentin expression appeared to decrease within maturing astrocytes which had increased levels of glial fibrillary acidic protein. These results suggest that multipotential epidermal growth factor-progenitor cells can give rise to both neurons and macroglia of the adult central nervous system, and that maturation of the astrocytes in vitro may be occurring in a pattern similar to that seen in vivo. Furthermore, no glial fibrillary acidic protein-positive cells expressed the A2B5 antigen in the same cell indicating an absence of type-2 astrocytes.

Asunto(s)

Astrocitos/citología , Astrocitos/fisiología , Cuerpo Estriado/citología , Células Madre/citología , Animales , Animales Recién Nacidos , Diferenciación Celular , Células Cultivadas , Senescencia Celular , Células Epiteliales , Ratas , Ratas Sprague-Dawley

RESUMEN

Recent evidence suggests that interleukin (IL)-1 and tumor necrosis factor (TNF) may play a role in astrogliosis following injury to the CNS. The short-term biochemical effects of these immune-related cytokines were determined on cultured rat polygonal and process-bearing astrocytes. Both IL-1 and TNF stimulated the rate of thymidine incorporation in polygonal astrocytes up to 137% and 215%, respectively, over the level observed in untreated controls. By contrast, thymidine incorporation was relatively unaffected by these cytokines in process-bearing astrocytes. The cytokines did not significantly affect the level of glial fibrillary acidic protein (GFAP) within polygonal astrocytes, even though they appeared to downregulate the expression of GFAP mRNA by as much as 62%. Both cytokines increased the intracellular expression of transferrin (Tf) within some polygonal astrocytes. In untreated control cultures, fewer than than 2% of polygonal astrocytes were immunoreactive for Tf. By contrast, approximately 30% of polygonal astrocytes treated with IL-1 or TNF-alpha became strongly immunoreactive for Tf. Neither IL-2 nor a number of other known growth factors appeared to alter the level of immunoreactive Tf in these cells. Process-bearing astrocytes were negative for Tf, regardless of the treatment used. Northern blot analysis demonstrated that the level of Tf mRNA in cultures of polygonal astrocytes increased 148% above the level observed in untreated controls following treatment with either IL-1 or TNF, whereas no change was observed following treatment with IL-2. These results suggest that increased levels of particular cytokines known to be present in injured CNS can produce pronounced biochemical alterations within a subtype of cultured astrocytes.

Asunto(s)

Astrocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/biosíntesis , Interleucina-1/farmacología , Transferrina/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Animales , Astrocitos/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Estimulación Química , Transferrina/genética

RESUMEN

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.

Asunto(s)

Astrocitos/metabolismo , Benzodiazepinas/metabolismo , Interleucina-1/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Astrocitos/química , Astrocitos/ultraestructura , Benzodiazepinas/análisis , Benzodiazepinonas/metabolismo , Sitios de Unión , Células Cultivadas , Convulsivantes/metabolismo , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Ratas , Ratas Endogámicas , Factores de Tiempo

RESUMEN

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Asunto(s)

Músculos/fisiología , Factores de Crecimiento Nervioso , Neuronas/efectos de los fármacos , Péptidos/farmacología , Médula Espinal/citología , Acetilcolinesterasa/metabolismo , Animales , Carbocianinas , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Colina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ganglios Simpáticos/citología , Ganglios Simpáticos/metabolismo , Peroxidasa de Rábano Silvestre , Inmunohistoquímica , Metrizamida/farmacología , Neuronas Motoras/metabolismo , Músculos/metabolismo , Neuronas/enzimología , Péptidos/metabolismo , Médula Espinal/efectos de los fármacos , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de Trigo

RESUMEN

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.

Asunto(s)

Astrocitos/enzimología , Malato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Oligodendroglía/enzimología , Animales , Anticuerpos Monoclonales/inmunología , Astrocitos/ultraestructura , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/inmunología , Inmunohistoquímica/métodos , Microscopía Electrónica , Mitocondrias/ultraestructura , Proteína Básica de Mielina/inmunología , Oligodendroglía/ultraestructura , Oxidación-Reducción , Ratas , Ratas Endogámicas , Rodamina 123 , Rodaminas

RESUMEN

The development and survival of spinal motor neurons depends upon muscle-derived trophic factors. Some circumstantial evidence suggested to us that the regulatory subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAMP-dPK)-type II might be involved in neuritic outgrowth from spinal neurons. In the present study, we tested a commercial preparation of cAMP-dPK for neurite-promoting activity. Commercial cAMP-dPK-type II from skeletal and cardiac muscles elicited a significant neurite outgrowth from cultured embryonic chicken neurons when the enzyme preparation was bound to polylysine-coated substrata; type I cAMP-dPK from skeletal muscle was ineffective. Neither cAMP-dPK-type I nor -type II had a significant effect on the survival of spinal neurons in culture. Type II cAMP-dPK also stimulated neurite outgrowth from chicken cerebral hemisphere neurons, dorsal root ganglionic neurons, ciliary ganglionic neurons, and rat sympathetic ganglionic neurons in culture. The neurite-promoting activity appears to reside in a contaminant of the preparation since neither the purified regulatory nor catalytic subunits of cAMP-dPK-type II had an effect on neurite outgrowth per se from cultured neurons and since neurite-promoting activity did not correlate with [3H]cAMP binding or cAMP-dependent kinase activity. The neurite-promoting protein was then partially purified from commercial cAMP-dPK-type II by gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DE-52 cellulose. Sodium dodecyl sulfate gel electrophoresis of the active protein peak revealed a major protein band (MW 50 kDa) and several minor bands (e.g., MW 200 kDa, 52 kDa, 45 kDa). Also, immunoblot analysis and immunoprecipitation revealed that the partially purified neurite-promoting protein was distinct from laminin, heparan sulfate proteoglycan, nerve growth factor, neural cell adhesion molecule, and fibronectin. Furthermore, the neurite-promoting activity was not diminished by treatment with heparinase nor was it bound to heparin conjugated to Sepharose. Our results demonstrate that a protein unrelated to laminin or its associated macromolecules and which copurifies with the type II cAMP-dPK of striated muscle stimulates neurite outgrowth from neurons of the central and peripheral nervous systems.

Asunto(s)

Axones/fisiología , AMP Cíclico/farmacología , Músculos/análisis , Neuronas/ultraestructura , Proteínas Quinasas/metabolismo , Proteínas/farmacología , Animales , Axones/efectos de los fármacos , Encéfalo/citología , Células Cultivadas , Embrión de Pollo , Electroforesis en Gel de Poliacrilamida , Ganglios Parasimpáticos/citología , Ganglios Espinales/citología , Ganglios Simpáticos/citología , Inmunoensayo , Peso Molecular , Proteínas Quinasas/aislamiento & purificación , Proteínas/aislamiento & purificación , Ratas

RESUMEN

To investigate whether immunocytochemical localization of muscle-specific aldolase can be used for fiber phenotype determination, we produced specific antibodies against the enzyme and studied its distribution in adult chicken skeletal muscles by indirect immunofluorescence microscopy. Monoclonal antibodies against the myosin heavy chains of fast-twitch (MF-14) and slow-tonic (ALD-58) muscle fibers were also used to correlate aldolase levels with the fiber phenotype. The goat anti-aldolase antibody was found to be specific for the A form of aldolase, as evidenced by sodium dodecyl sulfate gel electrophoresis, immunotitration experiments, and immunoblot analysis. The antibody reacted strongly with the fast-twitch myofibers of normal pectoralis and posterior latissimus dorsi muscles; the phenotype of these muscle fibers was confirmed by a positive immunofluorescent reaction after incubation with MF-14 antibody. By contrast, the slow-tonic myofibers of normal anterior latissimus dorsi, which react positively with ALD-58 antibody, reacted weakly with anti-aldolase antibodies. In denervated chicken muscles, reaction to anti-aldolase antibodies was markedly reduced in fast-twitch fibers, although reaction to MF-14 was not diminished. By contrast, in dystrophic muscle, fast-twitch fibers showed reduced reactivity to anti-aldolase and marked to moderate reduction in MF-14 reactivity. Our results show that: (a) in normal muscles, reactivity to anti-aldolase matches the phenotype obtained by using anti-fast or anti-slow myosin heavy chain antibodies, and therefore can serve to identify mature fibers as fast or slow; and (b) in denervated or dystrophic muscles, the intracellular expressions of aldolase and fast-twitch myosin heavy chains are regulated independently.

Asunto(s)

Fructosa-Bifosfato Aldolasa/metabolismo , Distrofia Muscular Animal/enzimología , Animales , Pollos , Técnica del Anticuerpo Fluorescente , Técnicas Inmunológicas , Contracción Muscular , Desnervación Muscular , Músculos/enzimología , Miosinas/metabolismo

RESUMEN

Transferrin accumulates within neurons of the developing nervous system of humans, sheep, pigs and chickens. To assess the relationship of this accumulation with the ontogeny of oxidative metabolism, we studied the immunocytochemical localization of transferrin (Tf) and the mitochondrial form of malate dehydrogenase (mMDH) in developing neural tissues by the peroxidase-antiperoxidase method. Rabbit anti-rat Tf was obtained commercially and gave a single band of reaction product (MW = 80 kd) on Western blots. Antibodies to porcine heart mMDH were elicited in a rabbit. Western blot analysis showed that this anti-porcine mMDH antibody reacted with the mMDH from porcine, rat or avian tissue but not with the cytosolic MDH from pigs. Tf was first detected in rat brain neurons at about the 18th embryonic day and reached a peak at about the 6th postnatal day. All neurons were immunoreactive with large neurons throughout the brain showing a strong reaction for Tf. From this time onward, the level in brain neurons gradually decreased until adulthood. However, Tf immunoreactivity still remained strongly evident in capillary endothelial cells. The localization of Tf within rat spinal cord neurons peaked as early as the 1st postnatal day and remained elevated to the 6th postnatal day. By contrast, reactivity for Tf within dorsal root ganglia neurons was intense as early as the 18th embryonic day and diminished only gradually. Mitochondrial MDH, a marker for oxidative metabolism, appeared to reach a peak after the crest of intraneuronal Tf had been observed. For example, brain and spinal cord MDH immunoreactivity increased with intense staining in the cell bodies and fibers of neurons from the 6th to the 13th postnatal day; immunoreactivity gradually diminished into adulthood. The gradient of reactivity was low in some areas of the brain but more intense in areas containing large neuronal cell bodies such as the red nucleus. This occurred after the peak of intraneuronal Tf at day 6 and suggested a precursor-product relationship. By contrast, immunoreactivity for neuron-specific enolase, a glycolytic enzyme, showed a developmental pattern that differed from either Tf or MDH in that reactivity appeared later in development and was less intense. These data suggest that as cerebral metabolic rates begin to increase as early as 5-6 days after birth in the rat, an increase in mMDH occurs coincident with the onset of oxidative metabolism. Furthermore, this rise in intraneuronal mMDH follows the peak of intraneuronal Tf and suggests that Tf supplies the iron required for the synthesis of other mitochondrial ferroproteins.

Asunto(s)

Envejecimiento/metabolismo , Sistema Nervioso Central/metabolismo , Desarrollo Embrionario y Fetal , Malato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Transferrina/metabolismo , Animales , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Inmunohistoquímica , Malato Deshidrogenasa/fisiología , Mitocondrias/fisiología , Ratas , Ratas Endogámicas , Transferrina/fisiología

RESUMEN

Transferrin is one of several serum proteins localized within neurons during development of the nervous system. The expression of transferrin receptors appears to precede the active accumulation of transferrin by neurons. The first cells immunoreactive for transferrin appear adjacent to the ventricles or to the central canal of the spinal cord. These cells then appear to migrate from this site. These neurons become progressively more immunoreactive for transferrin, attain a peak of reactivity and then lose their reaction to antitransferrin antibodies. Thus, a "window" of transferrin immunoreactivity is found. As neurons lose their reactivity to antitransferrin antibodies, glia and the walls of capillaries become positive. In the rat nervous system, the gradual decrease in intraneuronal transferrin is accompanied by an increase in mitochondrial malate dehydrogenase, an enzyme of the tricarboxylic acid cycle. Thus, the accumulation of transferrin appears to closely precede the ontogeny of oxidative metabolism in the brain. As transferrin appears transiently in all neurons, this protein may be involved in a number of other important developmental events such as the expression of dopamine D2 receptors and the period of "programmed" cell death in the spinal cord.

Asunto(s)

Sistema Nervioso/análisis , Transferrina/análisis , Animales , Embrión de Pollo , Sistema Nervioso/crecimiento & desarrollo , Ratas