RESUMEN
Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle-derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm-SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , Músculo Esquelético/citología , Trasplante de Células Madre , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Línea Celular , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Neuronas Motoras/patología , Médula Espinal/patologíaRESUMEN
Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.
Asunto(s)
Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Fármacos Neuroprotectores/metabolismo , Médula Espinal/patología , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Técnicas de Cultivo de Célula , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Ligandos , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neuronas Motoras/efectos de los fármacos , Músculos/efectos de los fármacos , Músculos/patología , Neurotoxinas/toxicidad , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Neuronal ceroid lipofuscinoses (NCLs) are a group of hereditary childhood diseases characterized mainly by lipopigment accumulation and a multisystemic pattern of symptoms including mental retardation, seizures, motor impairment, and blindness. The mnd mouse, carrying a mutation in the Cln8 gene, has been proposed as a model of epilepsy with mental retardation (EPMR, ornorthern epilepsy). We recently showed neuronal hyperexcitability and seizure hypersusceptibility in mnd mice. To elucidate the cellular mechanisms related to hippocampal hyperexcitability, the glutamatergic transmission and the expression of postsynaptic glutamate receptors were investigated in hippocampus. A significant increase in either spontaneous or KCl-stimulated overflow of [³H]D-aspartate was found in mnd mice compared with controls. This increase was maintained after DL-threo-ß-benzyloxyaspartic acid (TBOA) treatment, suggesting a nonrelevant role for transporter-mediated release and supporting the involvement of exocytotic [³H]D-aspartate release. Accordingly, Ca²âº-dependent overflow induced by ionomycin was also increased in mnd mice. Levels of glutamate 1-3 AMPA receptor subunits were increased, and levels of the NR2A NMDA receptor subunit were decreased in the hippocampus of mnd mice, suggesting an adaptive response to glutamate overstimulation.
Asunto(s)
Ácido D-Aspártico/metabolismo , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Receptores de Glutamato/metabolismo , Análisis de Varianza , Animales , Ácido Aspártico/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ionóforos de Calcio/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ionomicina/farmacología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Cloruro de Potasio/farmacología , Receptores de Glutamato/clasificación , Receptores de Glutamato/genética , Sinaptosomas/metabolismo , Tritio/metabolismoRESUMEN
A group of spirocyclic tropanyl-Δ(2)-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC(50) >> 10 µM), while some had an IC(50) value in the range 5-10 µM (8a-c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3'-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5'(4'H)-isoxazole 7a, was able to enhance at a concentration of 10 µM both [(3)H]citalopram and [(3)H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of B(max)) and [(3)H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.
Asunto(s)
Citalopram/farmacología , Isoxazoles/farmacología , Paroxetina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Serotonina/farmacocinética , Compuestos de Espiro/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Citalopram/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Relación Dosis-Respuesta a Droga , Isoxazoles/síntesis química , Isoxazoles/química , Estructura Molecular , Paroxetina/química , Ratas , Serotonina/química , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-Actividad , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Experimentally induced autoimmune encephalomyelitis (EAE) in mice provides an animal model that shares many features with human demyelinating diseases such as multiple sclerosis (MS). To what extent the cerebral cortex is affected by the process of demyelination and how the corollary response of the oligodendrocyte lineage is explicated are still not completely known aspects of EAE. By performing a detailed in situ analysis of expression of myelin and oligodendrocyte markers we have identified areas of subpial demyelination in the cerebral cortex of animals with conventionally induced EAE conditions. On EAE-affected cerebral cortices, the distribution and relative abundance of cells of the oligodendrocyte lineage were assessed and compared with control mouse brains. The analysis demonstrated that A2B5(+) glial restricted progenitors (GRPs) and NG2(+)/PDGFR-α(+) oligodendrocyte precursor cells (OPCs) were increased in number during "early" disease, 20 days post MOG immunization, whereas in the "late" disease, 39 days post-immunization, they were strongly diminished, and there was an accompanying reduction in NG2(+)/O4(+) pre-oligodendrocytes and GST-π mature oligodendrocytes. These results, together with the observed steady-state amount of NG2(-)/O4(+) pre-myelinating oligodendrocytes, suggested that oligodendroglial precursors attempted to compensate for the progressive loss of myelin, although these cells appeared to fail to complete the last step of their differentiation program. Our findings confirm that this chronic model of EAE reproduces the features of neocortex pathology in progressive MS and suggest that, despite the proliferative response of the oligodendroglial precursors, the failure to accomplish final differentiation may be a key contributing factor to the impaired remyelination that characterizes these demyelinating conditions.
Asunto(s)
Células Madre Adultas/patología , Corteza Cerebral/patología , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/patología , Oligodendroglía/patología , Células Madre Adultas/metabolismo , Animales , Linaje de la Célula/fisiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas Mielínicas/patología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismoRESUMEN
Three different series of 1H-pyrrolopyrimidine-2,4-dione derivatives were designed and synthesized as ligands for the α(1)-adrenergic receptors (α(1)-ARs). A microwave-assisted protocol was developed in order to improve purity and yields of some final products. The majority of the synthesized compounds, tested in binding assays, displayed α(1)-AR affinities in the nanomolar range. Highest affinity values were found in derivatives 10b and 10c (K(i)=1.4 nM for both) whereas compound 10e was endowed with the best profile in term of α(1)-AR affinity (K(i)=2.71 nM) coupled with high selectivity towards 5-HT(1A) receptors (K(i) >10,000). Molecular docking studies were performed on human α(1)-ARs and human 5-HT(1A) receptors in order to rationalize the observed experimental affinity and selectivity; these computational studies helped to clarify molecular requirements for the design of high-selective α(1)-adrenergic ligands.
Asunto(s)
Ligandos , Modelos Moleculares , Pirimidinas/química , Pirroles/química , Receptores Adrenérgicos alfa 1/química , Animales , Sitios de Unión , Simulación por Computador , Humanos , Microondas , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirroles/síntesis química , Pirroles/farmacología , Ratas , Receptor de Serotonina 5-HT1A/química , Receptor de Serotonina 5-HT1A/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Relación Estructura-ActividadRESUMEN
Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the beta common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58-82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia-reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.
Asunto(s)
Eritropoyetina/uso terapéutico , Papiledema/tratamiento farmacológico , Reconocimiento Visual de Modelos/fisiología , Péptidos/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Cicatrización de Heridas/genética , Animales , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Eritropoyetina/genética , Riñón/lesiones , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Hippocampal excitability and the metabolic glial-neuronal interactions were investigated in 22-week-old mice with motor neuron degeneration (mnd), a model of progressive epilepsy with mental retardation. Mnd mice developed spontaneous spikes in the hippocampus and were more susceptible to kainate-induced seizures compared with control mice. Neuronal hyperexcitability in their hippocampus was confirmed by the selective increase of c-Fos positive nuclei. Glial activation and pro-inflammatory cytokines over-expression were observed in the hippocampus of mnd mice, even in the absence of marked hippocampal neurodegeneration, as suggested by unchanged amounts of neuroactive amino acids and N-acetyl aspartate. Concentration of other amino acids, including GABA and glutamate, was not changed as well. However, ex vivo(13) C magnetic resonance spectroscopy, after simultaneous injection of [1-(13) C]glucose and [1,2-(13) C]acetate, followed by decapitation, showed decreased [1,2-(13) C]GABA formation from hippocampal astrocytic precursors and a marked reduction in [4,5-(13) C]glutamate derived from glutamine. We suggest that astrocyte dysfunction plays a primary role in the pathology and that mnd mice are of value to investigate early pathogenetic mechanism of progressive epilepsy with mental retardation.
Asunto(s)
Comunicación Celular/fisiología , Modelos Animales de Enfermedad , Epilepsia/patología , Hipocampo/patología , Discapacidad Intelectual/patología , Neuroglía/patología , Neuronas/patología , Convulsiones/patología , Animales , Epilepsia/complicaciones , Epilepsia/metabolismo , Femenino , Hipocampo/metabolismo , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Neuronas/metabolismo , Convulsiones/complicaciones , Convulsiones/metabolismoRESUMEN
Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.
Asunto(s)
Apoptosis/fisiología , Isquemia Encefálica/inmunología , Citocinas/biosíntesis , Eritropoyetina/fisiología , Inflamación/metabolismo , Neuronas/metabolismo , Animales , Apoptosis/inmunología , Isquemia Encefálica/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Eritropoyetina/farmacología , Humanos , Infarto de la Arteria Cerebral Media , Inflamación/inmunología , Lipopolisacáridos/farmacología , Masculino , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/citología , Fármacos Neuroprotectores/metabolismo , Ratas , Receptores de Eritropoyetina/metabolismo , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The wobbler mouse is a model of selective motor neuron degeneration in the cervical spinal cord. Comparing cervical and lumbar tracts of control and diseased mice at the early stage of pathology by proteomic analysis, we identified 31 proteins by peptide mass fingerprint after tryptic digestion and MALDI-TOF analysis, that were differently represented among the four experimental groups. In healthy mice, patterns of protein expression differed between cervical and lumbar tract: proteins of cellular energetic metabolism pathway showed lower expression in the cervical tract, while cellular trafficking proteins were overrepresented. In wobbler mice, these differences disappeared and the expression pattern was similar between cervical and lumbar spinal cord. We found that most of the proteins differentially regulated in wobbler with respect to control cervical tract were related to astrogliosis or involved in glutamate-glutamine cycle, energy transduction and redox functions. Proteins overrepresented in the wobbler lumbar spinal cord were cytoskeleton proteins and cellular transport proteins, in particular the vesicle fusing ATPase and the isoform 2 of syntaxin-binding protein 1, involved in vesicle trafficking. We suggest that overexpression of proteins involved in vesicle trafficking, together with proteins counteracting mitochondrial dysfunction can have neuroprotective effects, preserving lumbar spinal cord motor neurons in wobbler mice.
Asunto(s)
Vértebras Cervicales , Vértebras Lumbares , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso , Proteoma/análisis , Médula Espinal , Animales , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Consumo de Oxígeno , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
We studied two non-neurotoxic amphetamine derivatives (methyl-thioamphetamine, MTA and N,N-dimethylMTA, DMMTA) interacting with serotonin (5-HT) transporters (SERTs) with affinities comparable to that of p-Cl-amphetamine (pCA). The rank order for their maximal effects in inducing both [(3)H]5-HT release from rat brain synaptosomes or hSERT-expressing HEK-293 cells, and currents in hSERT-expressing oocytes, was pCA >> MTA > or = DMMTA. A correlation between drug-induced release and currents is also strengthened by the similar bell shape of the dose-response curves. Release experiments indicated that MTA and DMMTA are SERT substrates although MTA is taken up by HEK-293 cells with a V(max) 40% lower than pCA. The weak effects of MTA and DMMTA in vitro might therefore be due to their properties as 'partial substrates' on the mechanisms, other than translocation, responsible for currents and/or release. After either local or systemic in vivo administration, MTA and DMMTA release 5-HT in a manner comparable to pCA. These findings confirm that the neurotoxic properties of some amphetamine derivatives are independent of their 5-HT-releasing activity in vivo. It is worth noting that only those amphetamine derivatives with high efficiency in inducing 5-HT release and currents in vitro have neurotoxic properties.
Asunto(s)
Anfetamina/farmacología , Anfetaminas/farmacología , Metanfetamina/análogos & derivados , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Serotonina/metabolismo , Transmisión Sináptica/fisiología , Anfetamina/química , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Metanfetamina/farmacología , Ratas , Serotonina/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/fisiología , Transmisión Sináptica/efectos de los fármacos , Xenopus laevisRESUMEN
TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/prevención & control , Neuronas Motoras/patología , Receptores Tipo I de Factores de Necrosis Tumoral/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Receptores Señuelo del Factor de Necrosis Tumoral/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Recuento de Células/métodos , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes Neurológicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Receptores Señuelo del Factor de Necrosis Tumoral/administración & dosificación , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS.
Asunto(s)
Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Eritropoyetina/metabolismo , Eritropoyetina/fisiología , Animales , Línea Celular Tumoral , Eritropoyetina/genética , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Interferón gamma/farmacología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
(+/-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (+/-)-3-hydroxy-4,5,6, 6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-beta-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (-)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1-3-expressing cells. Comparable IC(50) values were found on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (-)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 microM (-)-HIP-A, but not with 10 to 30 microM TBOA or 100 microM (-)-HIP-A. The effect of (-)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (-)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.
Asunto(s)
Ácidos Carboxílicos/farmacología , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Ácido Glutámico/metabolismo , Fármacos Neuroprotectores/farmacología , Oxazoles/farmacología , Sinaptosomas/efectos de los fármacos , Animales , Transporte Biológico , Ácidos Carboxílicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Transportador 1 de Aminoácidos Excitadores/biosíntesis , Humanos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Estructura Molecular , Fármacos Neuroprotectores/química , Oxazoles/química , Ratas , Ratas Endogámicas , Estereoisomerismo , Sinaptosomas/metabolismoRESUMEN
Primary motor neuron cultures are widely used as in vitro model to study the early mechanisms involved in the aetiology of amyotrophic lateral sclerosis. In this study, we directly compared the morphological features and the responses to AMPA receptor (AMPAR) activation of mouse spinal cord motor neurons under different culture conditions (OptiPrep-purified, mixed anterior horn or motor neuron/glia cocultures). Motor neurons cocultured with a confluent glial layer had significant improvements in axonal length and in somata perimeter and area, compared both to mixed anterior horn cultures and to purified cultures, suggesting that the presence of more "mature" glial cells was determinant to obtain healthier motor neurons. By immuno-cytochemical assays we found that both in mixed anterior horn cultures and in cocultures, lower AMPA (0.3 microM) or kainate (5 microM) concentrations, but not the higher (1 or 15 microM, respectively), induced classical apoptotic events such as the nuclear fragmentation, the membrane externalization of phosphatidylserine residues and the activation of caspases-9 and -3. The morphological features and the different degenerative pathways induced by AMPAR agonist concentrations suggest that the experimental conditions used for in vitro studies are key factors that should be deeply considered to obtain more valid and reproducible results.
Asunto(s)
Células del Asta Anterior/fisiología , Aminoácidos Excitadores/toxicidad , Neuronas Motoras/patología , Neuroglía/fisiología , Receptores AMPA/efectos de los fármacos , Animales , Anexina A5 , Células del Asta Anterior/efectos de los fármacos , Células del Asta Anterior/ultraestructura , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Muerte Celular/fisiología , Separación Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Agonistas de Aminoácidos Excitadores/farmacología , Colorantes Fluorescentes , Inmunohistoquímica , Ácido Kaínico/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Neuroglía/efectos de los fármacos , Neuroglía/ultraestructura , Receptores AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Riluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigated whether this drug affects glutamate transporter-mediated uptake, using clonal cell lines stably expressing the rat glutamate transporters GLAST, GLT1 or EAAC1. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner; kinetic analysis indicated that the apparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate the buffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partly mediated by its activating effect on glutamate uptake.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/agonistas , Corteza Cerebral/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores/agonistas , Transportador 3 de Aminoácidos Excitadores/agonistas , Ácido Glutámico/metabolismo , Fármacos Neuroprotectores/farmacología , Riluzol/farmacología , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Línea Celular , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Humanos , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Cinética , Masculino , Ratas , Serina/análogos & derivados , Serina/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , TransfecciónRESUMEN
In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompetitive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones possess marked anticonvulsant properties and high affinity for the noncompetitive binding site of the AMPAR complex. In this paper, we report the synthesis and pharmacological characterization of a full set of 2,3-benzodiazepin-4-ones in order to better define the structure-activity relationship (SAR) of this class of compounds. Binding assays and functional tests were performed to evaluate the antagonistic activity at the AMPARs. Through these results we have identified a potent AMPAR antagonist, 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (5c). This compound noncompetitively inhibited AMPAR-mediated toxicity in primary mouse hippocampal cultures with an IC(50) of 1.6muM and blocked kainate-induced calcium influx in rat cerebellar granule cells with an IC(50) of 6.4muM. Thus, 5c has the in vitro potential as therapeutic drug in the treatment of various neurological disorders.
Asunto(s)
Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacología , Fármacos Neuroprotectores/farmacología , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Animales , Benzodiazepinas/síntesis química , Benzodiazepinonas/química , Calcio/metabolismo , Células Cultivadas , Hipocampo/citología , Concentración 50 Inhibidora , Ácido Kaínico/toxicidad , Ligandos , Ratones , Estructura Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
The 21-amino acid peptide endothelin-1 (ET-1) is the predominant isoform of the endothelin peptide family, which includes ET-2, and ET-3. These peptides display a variety of physiological activities including vasoconstriction and the stimulation of cell proliferation in tissues both within and outside of the cardiovascular system. They exert their actions via activation of two distinct receptor subtypes, ET(A) and ET(B), belonging to the G protein-coupled receptor (GPCR) superfamily. Ligands of these receptors have received numerous citations in the recent pharmaceutical literature. In particular receptor antagonists, both ET(A)- and ET(B)-selective, as well as non-selective, have been described due to their wide therapeutic potential. As a part of our program toward the development of selective ET(A) ligands we have designed and we now report new molecules based on 2-substituted-4-aryl-3-quinolinecarboxylic acid moiety. Binding profile for some compounds (40, 44, 46, and 47) of this class showed a reasonable affinity and selectivity for ET(A) receptors.
Asunto(s)
Quinolinas/química , Receptores de Endotelina/metabolismo , Ácidos Carboxílicos/química , Humanos , Ligandos , Unión Proteica , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Relación Estructura-ActividadRESUMEN
Amyotrophic lateral sclerosis (ALS) is a severe clinical condition characterized by upper and lower motor neuron degeneration for which there is no truly effective treatment. The absence of an effective treatment can be explained in part by the complex and heterogeneous genetic, biochemical, and clinical features of ALS. While ALS accounts for the majority of the motor neuron diseases, the recognition of disease variants and mimic syndromes may lead to further insights into possible causes for the generality of ALS. From a biochemical perspective, the process of motor neuron degeneration is complex and the multifactorial influences and potential biomarkers of ALS have never been assessed in the light of the clinical heterogeneity of ALS. Several genes and environmental influences have been suggested as possible risk factors of ALS. A better understanding of interactions between these risk factors, potential biomarkers and heterogeneous clinical features may lead to more clearly defined pathological profiles among individuals or groups of ALS patients and in turn lead to more focused therapeutic trials.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/epidemiología , Animales , Biomarcadores , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Humanos , Resultado del TratamientoRESUMEN
The aim of the present study was to investigate the biological profile of new substituted 1-phenyl-2-cyclopropylmethylamines. High affinity for both sigma subtypes was achieved when 4-phenylpiperidin-4-ol (4a-e) and 4-benzylpiperidine moieties were present (5a-e). (1R,2S/1S,2R)-2-[4-Hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate (4b) showed high affinity for the sigma1 sites (Ki = 1.5 nM) and the most favorable sigma1/sigma2 selectivity (Ki(sigma2)/Ki(sigma1) = 33.9). Binding affinity studies showed that 4b binding on N-methyl-d-aspartate (NMDA), dopaminergic (D1, D2, D3), muscarinic, histaminergic H1, adrenergic (alpha1, alpha2), serotoninergic (5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT6), DA (DAT), and 5-HT (SERT) transporters was not significant. Interestingly, sigma ligands differently induced the expression of tissue transglutaminase (TG-2) in primary astroglial cell cultures. We suggest that 4b may act as a sigma1/sigma2 agonist and that the sigma ligands may modulate TG-2 differently.