RESUMEN

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15 percent increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52 percent compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Asunto(s)

Animales , Ratas , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , /efectos de los fármacos , Péptidos beta-Amiloides/farmacología , Apoptosis/fisiología , Diferenciación Celular , Proliferación Celular , Compuestos Ferrosos/farmacología , Nitrocompuestos/farmacología , Estrés Oxidativo/fisiología , Propionatos/farmacología , Transducción de Señal/fisiología , Estaurosporina/farmacología , /fisiología

RESUMEN

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid ß-peptide (Aß25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of ß-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer's and Huntington's diseases.

Asunto(s)

Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína Wnt3A/efectos de los fármacos , Péptidos beta-Amiloides/farmacología , Animales , Apoptosis/fisiología , Diferenciación Celular , Proliferación Celular , Compuestos Ferrosos/farmacología , Nitrocompuestos/farmacología , Estrés Oxidativo/fisiología , Células PC12 , Propionatos/farmacología , Ratas , Transducción de Señal/fisiología , Estaurosporina/farmacología , Proteína Wnt3A/fisiología

RESUMEN

Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) account for approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS). In this study, sequence analysis of exons 1-5 of SOD1 in a large German cohort with FALS was performed. Among 75 affected patients, who were not obviously related probands with a positive family history, nine had missense mutations in SOD1. Four of the nine probands carry the same R115G mutation in exon 4 of the SOD1 gene. Genotyping with markers from the SOD1 locus revealed a common haplotype and shared allelic characteristics in these patients. These findings suggest that the R115G mutation in the German population originates from a common founder.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Superóxido Dismutasa/genética , Adulto , Anciano , Análisis Mutacional de ADN , Exones/genética , Femenino , Genotipo , Alemania , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Superóxido Dismutasa-1

RESUMEN

Inhibitor-of-differentiation 2 (Id2) belongs to a family of transcriptional modulators that are characterized by a helix loop helix region but lack the basic amino acid domain. During development, Id2 antagonizes differentiation mediated by the retinoblastoma protein, probably by scavenging downstream E-box basic helix-loop-helix proteins. Here, using differential display RT-PCR, we identify Id2 as an induced gene during serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. Consistent with a biological role for induced Id2 messenger RNA and protein expression in neuronal cell death, expression of Id2 antisense RNA, or targeted deletion of the Id2 gene in neurons from Id2 knock-out mice, protect from apoptosis. Further, gene transfer- mediated overexpression of Id2 induces neuronal cell death both in high potassium and low potassium conditions. Thus, the present study defines a role for Id2 in the modulation of neuronal apoptosis.

Asunto(s)

Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Proteína 2 Inhibidora de la Diferenciación , Ratones , Ratones Endogámicos , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Plásmidos , Potasio/metabolismo , ARN sin Sentido/biosíntesis , ARN sin Sentido/genética , ARN sin Sentido/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transfección

RESUMEN

PURPOSE: Neuroborreliosis may cause various neuro-ophthalmological complications. We describe a case with a bilateral optic neuropathy. CASE REPORT: A 58-year-old female developed facial paresis six weeks after an insect bite. One week later she developed bilateral optic disc swelling with haemorrhages and nerve fibre bundle defects in the lower visual field of the left eye. In CSF and serum, raised IgM and IgG titres to Borrelia burgdorferi were found. Systemic antibiotic treatment led to improvement of the vision and facial paresis, but not all visual field defects resolved, probably due to ischemic lesions of the optic disc. DISCUSSION/CONCLUSIONS: In optic nerve lesions due to neuroborreliosis it is difficult to distinguish between inflammatory and ischemic lesions. This patient demonstrated features of an ischemic optic nerve lesion.

Asunto(s)

Borrelia burgdorferi/aislamiento & purificación , Neuroborreliosis de Lyme/complicaciones , Enfermedades del Nervio Óptico/etiología , Anticuerpos Antibacterianos/análisis , Borrelia burgdorferi/inmunología , Ceftriaxona/uso terapéutico , Cefalosporinas/uso terapéutico , Parálisis Facial/etiología , Femenino , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Infusiones Intravenosas , Neuroborreliosis de Lyme/diagnóstico , Neuroborreliosis de Lyme/tratamiento farmacológico , Persona de Mediana Edad , Enfermedades del Nervio Óptico/diagnóstico , Enfermedades del Nervio Óptico/tratamiento farmacológico , Papiledema/etiología , Hemorragia Retiniana/etiología , Campos Visuales

RESUMEN

BACKGROUND: Radio-gene therapy involves the delivery, to tumor cells, of a therapeutic transgene whose expression is controlled by irradiation. MATERIALS AND METHODS: Here, we sought to identify novel radio-inducible transcripts in U87MG human malignant glioma cells using suppression subtractive hybridization (SSH). RESULTS: Of 998 clones from a subtracted library of irradiated U87MG cells, 24 candidate clones were identified by dot blot and 3 clones were confirmed as having been induced by irradiation by Northern blot analysis. All three clones showed 99-100% homology to the cyclin-dependent kinase (cdk) inhibitor, p21(Waf/Cip1). A screening of 12 human malignant glioma cell lines revealed that irradiation increased p21 mRNA expression and p21 protein levels levels in all of the five cell lines retaining p53 wild-type activity in a p53 reporter assay, but in none of seven p53 reporter-negative cell lines. CONCLUSION: Irradiation induces p21 mRNA expression in a strictly p53-dependent manner and may only enhance the expression of a limited number of genes in glioma cells. We conclude that the identification of radio-inducible genomic sequences suitable for radio-gene therapy may turn out to be difficult.

Asunto(s)

Ciclinas/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Terapia Genética/métodos , Glioma/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Glioma/terapia , Humanos , Hibridación de Ácido Nucleico , Células Tumorales Cultivadas

RESUMEN

The cytokines SDF-1alpha and -1beta are two alternatively spliced variants of the CXC (alpha) chemokines that are highly conserved among species. SDF-1alpha was shown to function as a B-cell maturation factor, a ligand for the CXCR4 (LESTR/fusin) chemokine receptor, thereby inhibiting replication of T cell-tropic HIV-1 strains and inducing cell death in human neuronal cell lines. In this report the cloning of the rat SDF-1beta cDNA and a new SDF-1 isoform, SDF-1gamma, are presented. Using Northern blot analysis, the expression pattern of both isoforms was studied in different tissues and it is shown that during postnatal development of the central and peripheral nervous system SDF-1beta- and SDF-1gamma-mRNA expression is inversely regulated. Whilst SDF-1beta-mRNA is the predominant isoform in embryonic and early postnatal nerve tissue, SDF-1gamma-mRNA is expressed at higher levels in adulthood. After peripheral nerve lesion a transient increase in SDF-1beta-mRNA expression is observed. As revealed by in situ hybridization, neurons and Schwann cells are the main cellular sources of both SDF-1beta and SDF-1gamma mRNAs in the nervous system. Computer-assisted analysis revealed that both transcripts encode secreted peptides with putative proteolytic cleavage sites which might generate novel neuropeptides.

Asunto(s)

Empalme Alternativo/fisiología , Química Encefálica , Quimiocinas CXC/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Quimiocina CXCL12 , Clonación Molecular , Citocinas/genética , Hibridación in Situ , Datos de Secuencia Molecular , Neuronas/fisiología , ARN Mensajero/análisis , Ratas , Ratas Wistar , Células de Schwann/fisiología , Nervio Ciático/química , Nervio Ciático/citología , Nervio Ciático/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Transcripción Genética/fisiología

RESUMEN

Cerebellar granule neurons cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations but remain viable after serum deprivation alone. Here, we show that potassium deprivation is associated with the dephosphorylation of the BCL-2-related BAD protein. Exposure to insulin-like growth factor-1 (IGF-1) inhibits both apoptosis and dephosphorylation of BAD. Both effects of IGF-1 do not depend on protein synthesis but are nullified by the phosphatidylinositol-3 kinase inhibitors, wortmannin and LY294002. In contrast to the treatment with cycloheximde, IGF-1 does not block the translocation of cytochrome c from mitochondria to the cytosol. Further, dephosphorylation of BAD alone does not appear to be sufficient to trigger apoptosis, since inhibition of protein synthesis by cycloheximide prevents apoptosis, but not BAD dephosphorylation, after potassium deprivation. These results suggest the coexistence of two parallel pathways, protein synthesis-dependent cytochrome c translocation and protein synthesis-independent dephosphorylation of BAD, both of which have to be activated to induce neuronal apoptosis.

Asunto(s)

Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Cerebelo/metabolismo , Grupo Citocromo c/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Androstadienos/farmacología , Animales , Transporte Biológico , Cerebelo/citología , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Neuronas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Potasio/metabolismo , Ratas , Wortmanina , Proteína Letal Asociada a bcl

RESUMEN

In spite of modern antibiotic treatment, bacterial meningitis still has a dubious prognosis. Secondary complications are responsible for death or permanent neurologic deficits. The indication for imaging is 2-fold. Beside the search for a source of infection, an early detection of secondary complications is attempted.We report about a patient with hematogenous pneumococcal meningitis, who developed a subdural empyema and vascular stenoses of the basal cerebral arteries. These changes as well as the resulting infarctions were detected with MRI in an early state. Thus, an adequate therapy could be initiated. Especially FLAIR images, diffusion-weighted sequences and a time of flight MRA proved to be valuable in this setting.

RESUMEN

Although neurotrophins are best known for their trophic functions, growing evidence suggests that neurotrophins can also be neurotoxic, for instance by enhancing excitotoxic insults. We have shown recently that brain-derived neurotrophic factor (BDNF) limits its neuroprotective action on axotomized rat retinal ganglion cells (RGCs) by upregulating nitric oxide synthase (NOS) activity (Klöcker et al., 1998). The aim of the present study was to investigate this interaction of BDNF and NOS in the lesioned adult rat retina in more detail. We used NOS immunohistochemistry and NADPH-diaphorase (NADPH-d) reaction to characterize morphologically retinal NOS expression and activity. Using reverse transcription-PCR and Western blot analysis, we were able to identify the NOS isoforms being regulated. Six days after optic nerve lesion, we observed an increase in neuronal NOS (NOS-I) mRNA and protein expression in the inner retina. This did not lead to a marked increase in overall retinal NOS activity. Only RGC axons displayed strong de novo NADPH-d reactivity. In contrast, intraocular injection of BDNF resulted in a marked upregulation of NOS activity in NOS-I-immunoreactive structures, leaving the level of NOS-I expression unchanged. In addition, an induction of inducible NOS (NOS-II) was found after BDNF treatment. We identified microglial cells increasing in number and being activated by BDNF, which could serve as the cellular source of NOS-II. In summary, our data suggest that BDNF upregulates retinal NOS activity by both a post-translational regulation of NOS-I activity and an induction of NOS-II. These findings might be useful for developing pharmacological strategies to improve BDNF-mediated neuroprotection.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Fibras Nerviosas/enzimología , Óxido Nítrico Sintasa/genética , Nervio Óptico/fisiología , Retina/enzimología , Transcripción Genética , Animales , Axotomía , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Memantina/farmacología , Fibras Nerviosas/ultraestructura , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Retina/citología , Transcripción Genética/efectos de los fármacos

RESUMEN

BACKGROUND: POEMS occurs only in about 1% of plasmocytoma cases, but in more than 50% of the rare osteosclerotic subtypes and plasma cell dyscrasias. The estimated frequency is 20 cases per year in Germany. Swelling of the optic disks is an early sign of the syndrome. CASE REPORT: An osteosclerotic plasmocytoma type IgA lambda was known in a 63-year-old farmer for 4 years. The primary lesion in the left femur was irradiated at diagnosis. Half a year prior to our examination the patient experienced edema of the legs and recurrent diarrhea. A staging confirmed the earlier diagnosis of a POEMS syndrome. The patient was presented for ophthalmologic examination because of optic disk swelling and progressive visual field defects: raster perimetry revealed enlarged blind spots and increased thresholds. Neurologic examination revealed polyradiculitis, increased protein levels in the cerebrospinal fluid and intracranial hypertension. The patient was treated with oral steroids which entailed improvement of the visual fields and decrease of optic disk swelling. CONCLUSIONS: Ophthalmologists play an active role in the staging, in the ruling out of other causes, and in the treatment. Symptomatic treatment with oral steroids should be monitored with visual acuity, raster perimetry and fundus examinations.

Asunto(s)

Síndrome POEMS/diagnóstico , Papiledema/diagnóstico , Diagnóstico Diferencial , Humanos , Masculino , Persona de Mediana Edad , Grupo de Atención al Paciente , Campos Visuales

RESUMEN

Glutathione (GSH) levels are supposed to determine the vulnerability of many cells towards a wide array of insults. We investigated the effects of chronic inhibition of GSH synthesis and acute depletion of GSH on cerebellar granule neurons in vitro and determined cytoplasmic and mitochondrial GSH with relation to mitochondrial function and generation of reactive oxygen intermediates (ROI). l-buthionine sulfoximine (BSO), which irreversibly blocks gamma-glutamyl-cysteine synthase, led to a time- and concentration-dependent loss of cytoplasmic GSH, while mitochondrial GSH was relatively preserved. No increased generation of ROI was detected over 48 h and the mitochondrial membrane potential was largely maintained. Neuronal degeneration occurred when mitochondrial GSH levels had fallen below 50% of control after 24-36 h. In contrast, direct conjugation of mitochondrial and cytoplasmic GSH with etacrynic acid (EA), resulted in immediate loss of mitochondrial GSH, a large increase of ROI within 2 h, subsequent collapse of the mitochondrial membrane potential and complete cell death within 4-8 h. Electron microscopy studies revealed an as yet unknown change of the chromatin structure to a homogeneous granular pattern after BSO, while EA resulted in typical necrotic changes. No typical features of apoptosis, i.e., no chromatin condensation or DNA fragmentation were detected after GSH depletion after BSO or EA treatment.

Asunto(s)

Apoptosis/fisiología , Glutatión/metabolismo , Mitocondrias/metabolismo , Neuronas/citología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antimetabolitos/farmacología , Bisbenzimidazol , Butionina Sulfoximina/farmacología , Cerebelo/citología , Cicloheximida/farmacología , Fragmentación del ADN , Colorantes Fluorescentes , Glutatión/análogos & derivados , Glutatión/farmacología , Microscopía Electrónica , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley

RESUMEN

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

Asunto(s)

Adenoviridae , Apoptosis/fisiología , Técnicas de Transferencia de Gen , Neuronas/citología , Infecciones por Adenoviridae , Animales , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Cerebelo/citología , Inhibidores de Cisteína Proteinasa/genética , Fragmentación del ADN , Activación Enzimática/fisiología , Regulación Viral de la Expresión Génica , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Apoptosis , Neuronas/enzimología , Neurotoxinas/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Virales/genética

RESUMEN

Cerebellar granule cells (CGCs) can express the inducible isoform of nitric oxide synthase (iNOS) in response to inflammatory stimuli. We demonstrate that induction of iNOS in CGCs by bacterial lipopolysaccharide and pro-inflammatory cytokines results in cell death that was potentiated by excess L-arginine and inhibited by the selective iNOS inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. The NO-mediated cell death was accompanied by increased caspase-3-like activity, DNA fragmentation and positive terminal transferase dUTP nick end labeling (TUNEL), suggesting that apoptosis mediates CGC cell death. Incubation of CGCs with the non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen or indomethacin, or with 15-deoxy-delta12,14 prostaglandin J2 (PGJ2) downregulates iNOS expression and reduces subsequent cell death. Since in other cell types, both NSAIDs and PGJ2 can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma) and downregulate cytokine levels and iNOS expression, and since CGCs express PPARgamma in vivo and in vitro, our data suggest that activation of CGC PPARgamma mediates iNOS suppression and reduced cell death. Because PPARgamma is expressed in brains of Alzheimer's Disease (AD) patients, in which neuronal iNOS expression and apoptotic cell death have been described, these results may help explain the basis for the beneficial effects of NSAIDs in AD.

Asunto(s)

Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Citocinas/metabolismo , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Animales , Western Blotting , Caspasa 3 , Caspasas/biosíntesis , Supervivencia Celular/efectos de los fármacos , Cerebelo/citología , Cerebelo/enzimología , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Lipopolisacáridos/farmacología , Neuronas/enzimología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Prostaglandinas Sintéticas/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazinas/farmacología , Factores de Tiempo

RESUMEN

To test the hypothesis whether a failure to express neurotrophins or a neurotrophin receptor might underlie the pathology observed in mutant mice with degeneration of regionally distinct subpopulations of neurons, the expression of BDNF, NT-3, TrkB, TrkC and synaptophysin mRNA was examined in the cerebellum of mutant lurcher (lc/+) and weaver (wv/+)/(wv/wv) mice. To identify the expression patterns of individual neurons, we used in situ hybridization with digoxigenin labeled ribonucleotide probes. RT-PCR of cerebellar mRNA for BDNF, NT-3, TrkB and TrkC (GAPDH as internal standard) was performed in parallel. Although especially in homozygous (wv/wv) weaver mice the normal anatomical order and number of the cerebellar neurons is grossly disturbed, residual Purkinje and granule neurons of both mutants displayed a normal expression pattern of the neurotrophins examined. Thus, the affected animals showed no significant signal decrease compared to healthy littermates or C3H mice. Our results suggest that the loss of specific neuron populations in the cerebellum of either mutant occurs via mechanisms either independent or downstream of the neurotrophins examined in this study.

Asunto(s)

Cerebelo/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Transcripción Genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Cerebelo/patología , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C3H , Ratones Mutantes Neurológicos , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/patología , Neurotrofina 3 , Células de Purkinje/metabolismo , Células de Purkinje/patología , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Factor Neurotrófico Ciliar , Receptor trkC , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Bcl-2 family proteins are principal regulators of cell death during normal development as well as in many disease states. Differentiated cerebellar granule neurons are protected from apoptosis by depolarizing concentrations of potassium. Further, these cells acquire resistance to glutamate-mediated excitotoxicity when pre-exposed to subtoxic concentrations of the glutamate receptor agonist, N-methyl-D-aspartate. Here, we report that the expression of bcl-2, bcl-xL, bcl-xS, bax and bad mRNA as well as of Bcl-2, Bax, Bcl-XL, Bcl-XS and Bag-1 proteins is not modulated in these two paradigms of neuronal cell death. However, mitochondrial release of cytochrome c, which is thought to be controlled by Bcl-2 family proteins, is detected 5 h after switching the neurons to low potassium conditions. Thus, there appears to be regulation of Bcl-2 family protein bioactivity in the absence of altered protein expression during potassium deprivation-induced apoptosis of cerebellar granule neurons.

Asunto(s)

Apoptosis , Cerebelo/citología , Grupo Citocromo c/metabolismo , Neuronas/citología , Potasio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Células Cultivadas , Cerebelo/metabolismo , Medios de Cultivo , Regulación de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , N-Metilaspartato/farmacología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , Ratas

RESUMEN

Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differentiated PC12 cells was associated with cell death characterized by features of apoptosis. The NOS inhibitors N-monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl prevented TNF-alpha/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-alpha/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of L-arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-alpha/LPS-induced loss of viability.

Asunto(s)

Apoptosis/fisiología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/efectos de los fármacos , Arginina/farmacología , Biotina , Diferenciación Celular/fisiología , Fragmentación del ADN , Nucleótidos de Desoxiuracil , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Guanidinas/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Células PC12 , ARN Mensajero/análisis , Ratas , Coloración y Etiquetado , Tiazinas/farmacología , Transcripción Genética/fisiología , omega-N-Metilarginina/farmacología

RESUMEN

Here, we report on the expression of the small chondroitin/dermatan sulfate proteoglycan decorin in the developing postnatal rat brain. Northern analysis of brain RNA demonstrated decorin transcripts with peak expression on postnatal day 3 followed by a slow decline to the lower adult level. In situ hybridization and immunohistochemistry revealed postnatal decorin expression in the grey matter of neocortex, hippocampus and thalamus, in myelinated fibre tracts and in several mesenchymal tissues (blood vessels, pia mater and the choroid plexus). In the neocortex, decorin is expressed in a specific laminar pattern with intense staining of the cortical plate and its derivatives, which differs remarkably from the distributions observed for other proteoglycans [B. Miller, A.M. Sheppard, A.R. Bicknese, A.L. Pearlman, Chondroitin sulfate proteoglycans in the developing cerebral cortex: the distribution of neurocan distinguishes forming afferent and efferent axonal pathways, J. Comp. Neurol. 355 (1995) 615-28]. Thus, decorin seems to serve yet unknown functions in the developing rat brain parenchyma in addition to its well-established role as a constituent of the mesenchymal extracellular matrix.

Asunto(s)

Animales Recién Nacidos/crecimiento & desarrollo , Encéfalo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteoglicanos/biosíntesis , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/fisiología , Encéfalo/metabolismo , Decorina , Proteínas de la Matriz Extracelular , Proteínas del Tejido Nervioso/genética , Proteoglicanos/genética , ARN Mensajero/biosíntesis , Ratas

RESUMEN

Four connexin32 (Cx32) cDNA clones isolated from a rat sciatic nerve cDNA library differ in the nucleotide sequence of their 5' untranslated region (UTR) from the corresponding Cx32 cDNA clones previously characterized from liver. The new Cx32 5'UTR sequence detected in the sciatic nerve cDNA clones is identical to one previously found in the 6.5 kb intron of the murine Cx32 gene. Using primer extension and S1 nuclease protection analysis, we determined the transcriptional starting point of this new alternative Cx32 transcript expressed in the sciatic nerve. This starting point is located 444 bp (409 bp) upstream of exon2 in a region previously described as an intron of the Cx32 gene in the rat (and mouse) genome, respectively. The alternative exon1B comprises 99 bp in rat, but 97 bp in the mouse genome, and is spliced to the same exon2 acceptor site also used for splicing of exon1 in liver. Both transcripts are likely to code for the same Cx32 protein whose reading frame is located in exon2. The putative promoter region, upstream of the alternative exon1B, contains a TATAAA motif and has been sequenced and noticed before by Miller et al. (Biosci. Rep. 8, 455-464, (1988)). The alternative exon1B transcript is highly expressed in the sciatic nerve, (i.e. Schwann cells) and very low in liver (i.e. hepatocytes). Its expression is regulated after sciatic nerve injury. The time course of expression was similar to previously established myelin genes and, therefore, we suggest that the expression of the alternative exon1B Cx32 transcript is related to the process of myelination. Very recently, we have characterized another alternative Cx32 exon1A which is transcribed in mouse embryonic stem cells but not in the sciatic nerve (Dahl et al., submitted for publication, 1995). Thus, the murine Cx32 gene is likely to be regulated by three alternative promoters that appear to be activated in a cell type-specific manner.

Asunto(s)

Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Células de Schwann/metabolismo , Nervio Ciático/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Conexinas/genética , Cartilla de ADN , ADN Complementario , Exones , Expresión Génica , Biblioteca de Genes , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Células de Schwann/citología , Nervio Ciático/citología , Nervio Ciático/lesiones , Análisis de Secuencia , Homología de Secuencia de Ácido Nucleico , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Proteína beta1 de Unión Comunicante

RESUMEN

We have isolated a 1.476 bp cDNA (NTII11) representing a transcript that is differntially expressed during sciatic nerve development and regeneration in the rat. Nucleotide sequence comparison indicates partial identity with a recently isolated plasmolipin cDNA. However, our clone extends the published sequence by 234 bp at the 5' end and predicts a protein that contains an additional 25 amino acids at th N-terminus. The open reading frame of th NTII11 transcript encodes a 19.4 kDa protein with four putative transmembrane domains. Northern blot analyses revealed a tissue-specific expression was confirmed by in situ hybridization, and cellular localization of plasmolipin mRNA was demonstrated in Schwann cells of the sciatic nerve and in glial cells of myelinated brain structures. The steady-state levels of plasmolipin mRNA were markedly altered (i) during development of sciatic nerve and brain. (ii) after sciatic nerve injury, and (ii) in cured Schwann cells maintained under different conditions of cell growth and arrest. Our data indicate a function of plasmolipin during myelination in the central as well as in the peripheral nervous system.

Asunto(s)

Química Encefálica , Proteínas de la Membrana , Proteínas del Tejido Nervioso/genética , Proteolípidos/genética , ARN Mensajero/genética , Nervio Ciático/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Biblioteca de Genes , Hibridación in Situ , Modelos Moleculares , Vaina de Mielina/metabolismo , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Compresión Nerviosa , Regeneración Nerviosa , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Neuroglía/metabolismo , Conformación Proteica , Proteolípidos/análisis , Proteolípidos/biosíntesis , Proteolípidos/química , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Células de Schwann/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/fisiología , Homología de Secuencia de Ácido Nucleico