RESUMEN
Ubiquitin (Ub) extension proteins (UEPs) are fusion proteins of a Ub at the N terminus to a ribosomal protein. They are the main source of Ub and the only source of extension ribosomal protein. Although important roles of the Ub-26S proteasome system in various biological processes have been well established, direct evidence for the role of UEP genes in plant defense is rarely reported. In this study, we cloned a Ub-S27a-type UEP gene (NbUEP1) from Nicotiana benthamiana and demonstrated its function in cell death and disease resistance. Virus-induced gene silencing of NbUEP1 led to intensive cell death, culminating in whole-seedling withering. Transient RNA interference (RNAi) of NbUEP1 caused strong cell death in infiltrated areas, while stable NbUEP1-RNAi tobacco plants constitutively formed necrotic lesions in leaves. NbUEP1-RNAi plants exhibited increased resistance to the oomycete Pythium aphanidermatum and viruses Tobacco mosaic virus and Cucumber mosaic virus while displaying decreased resistance to the nematode Meloidogyne incognita compared with non-RNAi control plants. Transcription profiling analysis indicated that jasmonate and ethylene pathways, lipid metabolism, copper amine oxidase-mediated active species generation, glycine-rich proteins, vacuolar processing enzyme- and RD21-mediated cell death and defense regulation, and autophagy might be associated with NbUEP1-mediated cell death and resistance. Our results provided evidence for the important roles of plant UEPs in modulating plant cell death and disease resistance.
Asunto(s)
Nicotiana , Enfermedades de las Plantas/microbiología , Proteínas de Plantas , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Animales , Muerte Celular , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/efectos de los fármacos , Nicotiana/efectos de los fármacos , Nicotiana/crecimiento & desarrollo , UbiquitinasRESUMEN
[Introduction] Seven smoke constituents, including hydrogen cyanide (HCN), ammonia (NH3), phenol, benzo[α]pyrene (B[a]P), carbon monoxide (CO)¸ crotonaldehyde, and 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK), are proposed be the most relevant constituents for smoking-related diseases. [Methods] Different combinations of leaf stalk positions, varieties and locations were used to create variable chemistry of cigarette filler and smoke. Experimental cigarettes were measured for emission level of seven smoke toxicants and content of seventy-three filler components. [Results] The ranges of coefficient of variation (CV) for seven smoke toxicants were 15.43%-43.15%. The emission pattern of NNK and crotonaldehyde were different from that of other five smoke toxicants. Most of the seven smoke toxicants were influenced in following order: stalk positionâ¯>â¯locationâ¯>â¯variety. The leaf constitutes closely correlated with seven smoke toxicants were analyzed. [Conclusions] The results showed that seven toxicants were significantly influenced by leaf position and location, and closely correlated with leaf components, such as potassium, malate and alkaloid contents. The results provide useful and comprehensive information on the affecting factors and correlating leaf constituents for the variations of seven smoke toxicants.
Asunto(s)
Sustancias Peligrosas/análisis , Nicotiana/química , Hojas de la Planta/química , Humo/análisis , Productos de Tabaco/análisis , Sustancias Peligrosas/químicaRESUMEN
BACKGROUND: Solid tumors are becoming prevalent affecting both old and young populations. Numerous solid tumors are associated with high cMET expression. The complexity of solid tumors combined with the highly interconnected nature of the cMET/HGF pathway with other cellular pathways make the pursuit of finding an effective treatment extremely challenging. The current standard of care for these malignancies is mostly small molecule-based chemotherapy. Antibody-based therapeutics as well as antibody drug conjugates are promising emerging classes against cMET-overexpressing solid tumors. RESEARCH DESIGN AND METHODS: In this study, we described the design, synthesis, in vitro and in vivo characterization of cMET-targeting Fab drug conjugates (FDCs) as an alternative therapeutic strategy. The format is comprised of a Fab conjugated to a potent cytotoxic drug via a cleavable linker employing lysine-based and cysteine-based conjugation chemistries. RESULTS: We found that the FDCs have potent anti-tumor efficacies in cancer cells with elevated overexpression of cMET. Moreover, they demonstrated a remarkable anti-tumor effect in a human gastric xenograft mouse model. CONCLUSIONS: The FDC format has the potential to overcome some of the challenges presented by the other classes of therapeutics. This study highlights the promise of antibody fragment-based drug conjugate formats for the treatment of solid tumors.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias , Humanos , Animales , Ratones , Inmunoconjugados/uso terapéutico , Proteínas Proto-Oncogénicas c-met/metabolismo , Neoplasias/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Anticuerpos , Línea Celular TumoralRESUMEN
Activation of interferon (IFN) signaling in the central nervous system (CNS) is usually associated with inflammation. However, a robust activation of type I IFN-stimulated genes (ISGs) at pre-symptomatic stages occurs in the spinal cord of SOD1(G93A) mice, an amyotrophic lateral sclerosis (ALS) animal model, without obvious signs of inflammation. To determine if the same signaling pathway is elevated in other types of neuronal injuries, we examined the protein expression levels of an IFN-stimulated gene, ISG15, in mouse models of acute and chronic neuronal injuries. We found that ISG15 protein was dramatically increased in the brains of mice subjected to global ischemia and traumatic brain injury, and in transgenic mice overexpressing HIV gp120 protein. These results suggest that activation of ISGs is a shared feature of neuronal injuries and that ISG15 may be a suitable biomarker for detecting neuronal injuries in the CNS.
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Sistema Nervioso Central/fisiopatología , Citocinas/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Lesiones Encefálicas/fisiopatología , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Ubiquitinas/metabolismoRESUMEN
We investigated potassium (K) accumulation characteristics and expression of K metabolism related genes in one high-K variety (ND202) and a common variety (NC89) of tobacco (Nicotiana tabacum L.). Results showed that K accumulation and leaf K content in ND202 were higher than those in NC89. The distribution rate and K accumulation in the leaves of ND202 increased significantly, while the distribution rate in the roots and stems had lower values. In addition, the maximum K accumulation rate and high-speed K accumulation duration in ND202 were found to be better than those in NC89. The expression of NKT1 in the upper and middle leaves of ND202 had an advantage, and the relative expression of NtKC1 and NtTPK1 in both the upper and middle leaves, as well as the roots, was also significantly upregulated. Conversely, the expression of NTRK1 in the lower leaves and roots of ND202 was weaker. ND202 had significantly greater expression levels of NtHAK1 than NC89 in the upper and middle leaves and roots; moreover, the expression of NtKT12 in the upper leaves and roots of ND202 was also higher. In comparison with common varieties, high-K varieties had a stronger ability to absorb and accumulate K. They also possessed higher expression of K+ channel- and transporter-related genes and showed a superior K accumulation rate and longer duration of high-speed K accumulation. Furthermore, K accumulation rate at 40-60days can be suggested as an important reference for the selection of high-K tobacco varieties.
Asunto(s)
Nicotiana , Potasio , Hojas de la Planta/genética , Raíces de Plantas/genética , Potasio/metabolismo , Nicotiana/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting predominantly motor neurons. Recent studies suggest that the disease progression of ALS is non-cell-autonomous, although the interaction between neurons and glial cells in different disease stages is not entirely clear. Here, we demonstrate that the interferon (IFN) signaling pathway is activated in human SOD1(G93A) transgenic mice, a rodent model of ALS. IFN-stimulated genes (ISGs) increased in the spinal cord of SOD1(G93A) mice at a presymptomatic age. In addition, the up-regulated ISGs, and most likely their transcriptional activators, were found specifically in astrocytes surrounding motor neurons, suggesting that IFN signaling in astrocytes was triggered by specific pathologic changes in motor neurons. Furthermore, induction of ISGs in cultured astrocytes was highly sensitive to IFN, especially Type I IFN. ISGs in astrocytes were activated specifically by endoplasmic reticulum stress-induced neurodegeneration in vitro, implicating a similar process in the presymptomatic stage of SOD1 mutant mice. Finally, reduction or deletion of IFNα receptor 1 inhibited IFN signaling and increased the life-span of SOD1(G93A) mice. Thus, the activation of IFN signaling pathways represents an early "dialogue" between motor neurons and astrocytes in response to pathological changes in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/metabolismo , Interferón Tipo I/fisiología , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Médula Espinal/patologíaRESUMEN
Organic acids secreted from the roots of plants play important roles in nutrient acquisition and metal detoxification; however, the precise underlying mechanisms of these processes remain poorly understood. In the present study we examined the content of organic acids exuded from roots and the effects of these organic acids on the activation of slowly available potassium (K) at different K levels, including normal K supply and K-deficient conditions. In addition, the study system also comprised a high-K tobacco variety (ND202) and two common ones (K326 and NC89). Our results showed that high-K varieties exhibited significantly higher contents of organic acids in its root exudates and available K in both rhizosphere and non-rhizosphere soils than the other varieties. This research also suggested that a cyclic process in which soil was acidified after being complexed by organic acids was involved in the release of slowly available K, and that this process primarily depended on the soil pH at high organic acids concentrations, but the complexation of organic ligands became dominant at low concentrations. In conclusion, tobacco roots secrete organic acids to increase available K content and improve the utilisation rate of soil K. High-K varieties probably enhance slowly available K activation by secreting relatively high amounts of organic acids, thus leading to more available K in soil for absorption by plants.
Asunto(s)
Nicotiana , Suelo , Raíces de Plantas , Potasio , RizosferaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes.
Asunto(s)
Epilepsia/genética , Mutación , Valina-ARNt Ligasa/genética , Alelos , Anticodón , Niño , Preescolar , Progresión de la Enfermedad , Epilepsia/enzimología , Epilepsia/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Estudios Longitudinales , Mutación con Pérdida de Función , Masculino , Microcefalia/enzimología , Microcefalia/genética , Modelos Moleculares , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Linaje , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , ARN de Transferencia/genética , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
ENHANCED DISEASE RESISTANCE1 (EDR1) encodes a Raf-like mitogen-activated protein kinase, and it acts as a negative regulator of disease resistance and ethylene-induced senescence. Mutations in the EDR1 gene can enhance resistance to powdery mildew both in monocotyledonous and dicotyledonous plants. However, little is known about EDR1-like gene members from a genome-wide perspective in plants. In this study, the tobacco (Nicotiana tabacum)EDR1-like gene family was first systematically analyzed. We identified 19 EDR1-like genes in tobacco, and compared them to those from Arabidopsis, tomato and rice. Phylogenetic analyses divided the EDR1-like gene family into six clades, among them monocot and dicot plants were respectively divided into two sub-clades. NtEDR1-1A and NtEDR1-1B were classified into clade I in which the other members have been reported to negatively regulate plant resistance to powdery mildew. The expression patterns of tobacco EDR1-like genes were analyzed after plants were challenged by Golovinomyces orontii, and showed that several other EDR1-like genes were induced after infection, as well as NtEDR1-1A and NtEDR1-1B. Expression analysis showed that NtEDR1-13 and NtEDR1-16 had exclusively abundant expression patterns in roots and leaves, respectively, and the remaining NtEDR1-like members were actively expressed in most of the tissue/organ samples investigated. Our findings will contribute to further study of the physiological functions of EDR1-like genes in tobacco.
RESUMEN
Neuronal migration defects, including pachygyria, are among the most severe developmental brain defects in humans. Here, we identify biallelic truncating mutations in CTNNA2, encoding αN-catenin, in patients with a distinct recessive form of pachygyria. CTNNA2 was expressed in human cerebral cortex, and its loss in neurons led to defects in neurite stability and migration. The αN-catenin paralog, αE-catenin, acts as a switch regulating the balance between ß-catenin and Arp2/3 actin filament activities1. Loss of αN-catenin did not affect ß-catenin signaling, but recombinant αN-catenin interacted with purified actin and repressed ARP2/3 actin-branching activity. The actin-binding domain of αN-catenin or ARP2/3 inhibitors rescued the neuronal phenotype associated with CTNNA2 loss, suggesting ARP2/3 de-repression as a potential disease mechanism. Our findings identify CTNNA2 as the first catenin family member with biallelic mutations in humans, causing a new pachygyria syndrome linked to actin regulation, and uncover a key factor involved in ARP2/3 repression in neurons.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/genética , Movimiento Celular/genética , Corteza Cerebral/fisiología , Neuronas/patología , alfa Catenina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Embrión de Mamíferos , Genoma Humano , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Linaje , alfa Catenina/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity. The molecules exhibit excellent selectivity and display efficacy in a SOD1 mutant mouse model of ALS. Interestingly, the molecules appear to act as agonists for the receptor in certain surrogate cellular assays. While the exact mechanisms responsible for the therapeutic effect of the new agonists remain to be elucidated, we believe that the described agent represents both an invaluable pharmacological tool to further decipher the role of the EphA4 in ALS and potentially other human diseases, and a significant stepping stone for the development of novel treatments.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Receptor EphA4/agonistas , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Sitios de Unión , Células Cultivadas , Modelos Animales de Enfermedad , Diseño de Fármacos , Semivida , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptor EphA4/química , Receptor EphA4/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Hitherto, membralin has been a protein of unknown function. Here, we show that membralin mutant mice manifest a severe and early-onset motor neuron disease in an autosomal recessive manner, dying by postnatal day 5-6. Selective death of lower motor neurons, including those innervating the limbs, intercostal muscles, and diaphragm, is predominantly responsible for this fatal phenotype. Neural expression of a membralin transgene completely rescues membralin mutant mice. Mechanistically, we show that membralin interacts with Erlin2, an endoplasmic reticulum (ER) membrane protein that is located in lipid rafts and known to be important in ER-associated protein degradation (ERAD). Accordingly, the degradation rate of ERAD substrates is attenuated in cells lacking membralin. Membralin mutations or deficiency in mouse models induces ER stress, rendering neurons more vulnerable to cell death. Our study reveals a critical role of membralin in motor neuron survival and suggests a novel mechanism for early-onset motor neuron disease.
Asunto(s)
Supervivencia Celular/fisiología , Enfermedad de la Neurona Motora/genética , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/metabolismo , Animales , Northern Blotting , Cartilla de ADN/genética , Estrés del Retículo Endoplásmico/fisiología , Degradación Asociada con el Retículo Endoplásmico/fisiología , Genes Recesivos , Vectores Genéticos/genética , Células HEK293 , Técnicas Histológicas , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Enfermedad de la Neurona Motora/fisiopatología , Mutación/genética , Proteínas del Tejido Nervioso/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.
Asunto(s)
1-Metil-4-fenilpiridinio/farmacología , Manganeso/administración & dosificación , Neurotoxinas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Concentración Osmolar , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.
Asunto(s)
Metilación de ADN , Nicotiana/genética , Nicotiana/virología , Virus del Mosaico del Tabaco/fisiología , Análisis por Conglomerados , ADN de Plantas/química , ADN de Plantas/genética , Epigénesis Genética , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Nicotiana/clasificaciónRESUMEN
All botulinum toxins (BoNTs, types A-G) inhibit synaptic transmitter release from motoneurons, and thus result in respiratory arrest and death. Rapid treatment with anti-BoNT antibodies can prevent progression, but recovery still requires weeks on a ventilator. Even after recovery, there is a potential for persistent fatigue in some cases of botulism even years after the insult, possibly because of motoneuron dropout for previously unknown reasons. Unique among BoNTs, the C-type (BoNT/C) cleaves two proteins involved in neurotransmitter release, syntaxin and SNAP-25, and induces apoptotic cell death in cultured cerebellar neurons. It is not clear, however, whether BoNT/C also affects neurons that encounter toxin in vivo, namely motoneurons. Here, we provide experimental evidence that BoNT/C causes a slow degeneration of motoneurons both in vitro and in vivo. This novel form of BoNT/C-induced cell death may require new treatment strategies.
Asunto(s)
Toxinas Botulínicas/toxicidad , Neuronas Motoras/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Venenos/toxicidad , Animales , Recuento de Células/métodos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Lateralidad Funcional , Fosfolipasas A2 Grupo II , Indoles , Ratones , Ratones Endogámicos C57BL , Proteínas de Neurofilamentos/metabolismo , Médula Espinal/citologíaRESUMEN
The subunit composition of N-methyl-D-aspartate (NMDA) receptors affects their function under normal and pathological conditions. Functional NMDA receptors are expressed in lower motor neurons, but their subunit composition has not been defined. Here, we employed electrophysiology, quantitative PCR, and immunohistochemistry to investigate the subunit composition of NMDA receptors in postnatal motor neurons of the Wistar rat facial nucleus (FN). Whole-cell patch clamp recordings of acutely dissociated motor neurons from postnatal days 3 and 4 (P3-P4) showed that ifenprodil, a specific antagonist of the NMDA receptor 2B (NR2B) subunit, inhibited 91.62%+/-2.02% of NMDA-induced current, whereas NVP-AAM007, a specific antagonist of the NMDA receptor 2A (NR2A) subunit, inhibited much less of the current (16.69%+/-3.28%). Starting from P5, the inhibitory effects of ifenprodil and NVP-AAM007 gradually decreased and increased, respectively, such that the effect of NVP-AAM007 exceeded that of ifenprodil by P10. At P14, most of the NMDA-induced current was inhibited by NVP-AAM007 (84.59%+/-3.35%). Consistent with this, NR2B mRNA and protein were expressed highly at P3 and then gradually decreased by more than 75% by P14 in FN motor neurons, while NR1 was expressed stably over the same ages. However, NR2A mRNA and protein showed relatively constant levels between P3-P10 and decreased to 45% and 75% of the P3 level, respectively, by P14. Thus, analysis of functional NMDA receptors is critical to revealing subunit switching, which may be an important step in postnatal development of FN motor neurons.
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Nervio Facial/crecimiento & desarrollo , Nervio Facial/metabolismo , Neuronas Motoras/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Rombencéfalo/crecimiento & desarrollo , Rombencéfalo/metabolismo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Nervio Facial/citología , Ácido Glutámico/metabolismo , Inmunohistoquímica , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Motoras/efectos de los fármacos , Técnicas de Placa-Clamp , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/genética , Rombencéfalo/citología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which results from selective loss of upper and lower motor neurons. Mouse models of ALS, such as one carrying the G93A mutant of the human Cu-Zn superoxide dismutase gene[SOD1(G93A)], develop motor neuron pathology and clinical symptoms similar to those observed in ALS patients. There is compelling evidence that both direct and indirect glutamate toxicity contribute to the pathogenesis of motor neuron degeneration. However, the therapeutic effect of various glutamate receptor antagonists has not been clearly demonstrated. Memantine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. It has been shown to protect neurons against NMDA- or glutamate-induced toxicity in vitro and in animal models of neurodegenerative diseases. In the current study, we have examined the therapeutic efficacy of memantine in an ALS mouse model carrying a high copy number of SOD1(G93A). Memantine treatment significantly delayed the disease progression and increased the life span of SOD1(G93A) mice, from 121.4 +/- 5.5 to 129.7 +/- 4.5 days (P = 0.032). Furthermore, NMDA receptor subunits were reliably detected in the spinal cord of SOD1(G93A) mice and their expression levels were similar to those in the wild-type littermate control. Therefore, the neuroprotective effect of memantine in SOD1(G93A) mice is most probably due to the inhibition of spinal cord NMDA receptors. In view of the long-term usage of memantine for dementia patients, with excellent tolerance and safety, these data suggest that memantine may be used in ALS patients alone or in combination with other therapies to prolong survival.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Memantina/uso terapéutico , Esclerosis Amiotrófica Lateral/mortalidad , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Receptores de N-Metil-D-Aspartato/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Superóxido Dismutasa/genética , Análisis de Supervivencia , Factores de Tiempo , Resultado del TratamientoRESUMEN
AIM: To study the expressions of vascular endothelial growth factor (VEG F), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1, and Tie-2 in C57BL/6 mouse brain after permanent focal cerebral ischemia. METHODS: The mRNA levels of VEGF, Ang-1, Ang-2, Tie-1, and Tie-2 were measured by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The protein express ions of VEGF and Ang-2 were determined by immunohistochemistry. RESULTS: Low mRNA levels of VEGF, Ang-1, Ang-2, Tie-1, and Tie-2 were constitutively expressed in the normal cortex of mouse. After middle cerebral artery occlusion (MCAO), the expressions of VEGF, Ang-2, and Tie-2 mRNA were dramatically increased in the infarcted cortex and the elevation was remained through 7 d of ischemia. However, the levels of Ang-1 and Tie-1 mRNA were unchanged in the infarcted cortex. Immunoreactivities of Ang-2 or VEGF were hardly observed in the normal cortex. Ang-2 protein was evidently detected in the infarct core 8 h after MCAO and in t he perifocal area 1 d after MCAO. Expression of VEGF protein was elevated in the infarct core 2 h after MCAO and in the perifocal area 1 d after MCAO. Immunoreaction was restricted to endothelial cells and glial-like cells within the infarct core and perifocal area. CONCLUSION: The expressions of An g-2 and VEGF are induced after focal cerebral ischemia, which may contribute to the angiogenic response in the cortex of ischemic brain.