RESUMEN
The temporal effect of discharge and limnology on fish composition and species diversity in a floodplain lake at the confluence of the Amazon and Negro Rivers was evaluated. Species richness, abundance and assemblage composition were strongly influenced by seasonal discharge of the Amazon and Negro Rivers, which affects lateral connectivity, water conductivity and temperature. As a consequence, temporal ß-diversity was high in the lake and the assemblage was dominated by seasonally transient species. Relatively large species known to feed on resources within the floodplain were captured almost exclusively during the flood period. During the dry season, the assemblage was dominated by fishes adapted to harsh conditions of high temperature and low dissolved oxygen concentrations. An open system with high spatial and temporal heterogeneity created by the meeting of two large rivers with different water chemistry, Lago Catalão has a dynamic fish assemblage. Given its high temporal ß-diversity and abundance of fishes, many of great importance in local fisheries, Lago Catalão and other floodplain lakes in this region merit special attention for conservation.
Asunto(s)
Ecosistema , Peces , Estaciones del Año , Animales , Brasil , Inundaciones , Lagos , RíosRESUMEN
Botulinum toxin type-A is currently thought to be effective and safe for hemifacial spasm (HFS). The pre-synaptic block of acetylcholine release at the neuromuscular junction induces depression of orbicularis oculi muscle compound motor action potential (CMAP). The aim of our study was to evaluate at what extent end-plate functional recovery is possible even in botulinum toxin treatments lasting up to 15 years. We examined 81 outpatients with primary HFS (mean treatment duration = 7.2 ± 4.2 years) who underwent neurophysiologic study, once clinical effect of the previous treatment had vanished. The mean CMAP amplitude, mean rectified amplitude of response 1 (R1) of the blink reflex and area of response 2 (R2) of treated orbicularis oculi muscle were measured in comparison to the controlateral side. Mean amplitude of the above mentioned parameters was slightly lower (about 20%; p < 0.001) in the treated side at the end of the follow-up period (4.7 ± 1.7 months). The CMAP amplitude reduction weakly correlated with the interval from last treatment, while other neurophysiologic parameters did not change due to treatment duration or total toxin amount. Our study demonstrates that botulinum toxin affects compound motor action potential and blink-reflex responses for at least 4-5 months in HFS patients. The residual block is slight and does not increase with repeated injections after several years of treatment. Our study, beside confirming the long-term efficacy of botulinum toxin treatment for HFS, provides neurophysiologic evidence that therapeutic effect may be obtained without hindering the regenerative potential of the nerve-muscle complex.
Asunto(s)
Toxinas Botulínicas Tipo A/uso terapéutico , Espasmo Hemifacial/tratamiento farmacológico , Placa Motora/fisiología , Conducción Nerviosa/fisiología , Fármacos Neuromusculares/uso terapéutico , Adulto , Anciano , Toxinas Botulínicas Tipo A/farmacología , Electromiografía , Femenino , Espasmo Hemifacial/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Placa Motora/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiopatología , Conducción Nerviosa/efectos de los fármacos , Fármacos Neuromusculares/farmacología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Resultado del TratamientoRESUMEN
Enzymatically derived NO and extracellular ATP are receiving greater attention due to their role as messengers in the CNS during different physiological and pathological processes. Ionotropic (P2XR) and metabotropic (P2YR) purinergic receptors mediate ATP effects and are present throughout the body. Particularly P2XR are crucial for brain plasticity mechanisms, and are involved in the pathogenesis of different CNS illnesses. NO does not have a specific receptor and its actions are directly dependent on the production on demand by different nitric oxide synthase isoforms. NO synthesizing enzymes are present virtually in all tissues, and NO influences multifarious physiological and pathological functions. Interestingly, various are the tissue and organs modulated by both ATP and NO, such as the immune, brain and vascular systems. Moreover, direct interactions between purinergic and nitrergic mechanisms outside the CNS are well documented, with several studies also indicating that ATP and NO do participate to the same CNS functions. In the past few years, further experimental evidence supported the physiological and pathological relevance of ATP and NO direct interactions in the CNS. The aim of the present review is to provide an account of the available information on the interplay between purinergic and nitrergic systems, focussing on the CNS. The already established relevance of ATP and NO in different pathological processes would predict that the knowledge of ATP/NO cross-talk mechanisms would support pharmacological approaches toward the development of novel ATP/NO combined pharmacological agents.
Asunto(s)
Adenosina Trifosfato/metabolismo , Sistema Nervioso Central/metabolismo , Óxido Nítrico/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiología , Animales , Comunicación Celular/fisiología , Humanos , Neuroglía/metabolismo , Neuronas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Receptores Purinérgicos P2XRESUMEN
Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.
Asunto(s)
Sistema Nervioso Central/inmunología , Terapia Genética/métodos , Interleucina-4/genética , Esclerosis Múltiple/terapia , Linfocitos T Reguladores/inmunología , Adenoviridae/genética , Animales , Sistema Nervioso Central/patología , Quimiocinas/inmunología , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Virus Helper/genética , Humanos , Interleucina-4/análisis , Interleucina-4/inmunología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética/métodosRESUMEN
Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disorder resulting from the inherited deficiency of the arylsulfatase A (ARSA) enzyme. Currently, no valid therapeutic options are available for affected patients. A thorough knowledge of disease progression in its diverse clinical variants, together with the identification of reliable prognostic factors, could be instrumental in accurate patient selection for new upcoming therapeutic opportunities, such as enzyme replacement and gene therapy. The described correlation between genotype and clinical presentation proved helpful in predicting patient's prognosis, only in the minority of MLD patients harboring common mutations. Molecular characterization of a cohort of 26 MLD patients allowed us to identify 18 mutations, excluding the common 0 and R alleles, 10 of which are rare and 8 are novel. By categorizing the rare mutations, we were able to confirm a correlation between ARSA gene mutations, age at onset and patterns of disease progression, not only in those patients bearing common mutations, but also in those carrying rare mutant alleles. Moreover, in the case of absent or delayed molecular diagnosis, or of newly identified mutations, the involvement of peripheral nervous system from disease onset proved to be a sensitive prognostic marker predicting a severe progression.
Asunto(s)
Genotipo , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Mutación/genética , Alelos , Encéfalo/patología , Cerebrósido Sulfatasa/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Familia , Femenino , Humanos , Leucodistrofia Metacromática/enzimología , Masculino , FenotipoRESUMEN
In the CNS, nucleotide receptors termed P2 receptors are identified on neurons and glial cells, mediating neuron-neuron, glia-glia and glia-neuron communication. In the present work, we qualify in vivo in the adult rat CNS the cellular/subcellular distribution of P2Y12 receptor protein in cerebral cortex, white matter and subcortical nuclei (striatum and substantia nigra), by means of immunofluorescence-confocal, electron microscopy and Western blot analysis. P2Y12 receptor immunoreactivity colocalizes neither with markers such as neuronal nuclei, neurofilament light chain, calbindin and tyrosine hydroxylase, nor with glial fibrillary acidic protein and isolectin B4, but with myelin basic protein and the oligodendrocyte marker RIP, in both cell bodies and processes, indicating therefore oligodendrocyte localization. Electron microscopy identifies P2Y12 receptors in both the perikaryon and under the plasmalemma of oligodendrocyte cell bodies and radiating processes, until the paranodal region of fibers. By Western blot analysis, P2Y12 receptor shows a specific band of 42-44 kDa, matching the molecular mass predicted from amino acid sequencing. Since in platelets P2Y12 receptor is known to regulate adhesion/activation and thrombus growth/stability, from our results we could speculate by analogy that, in oligodendrocytes, P2Y12 receptor signaling might contribute to the migration and adhesion of the glial processes to axons to be myelinated.
Asunto(s)
Encéfalo/citología , Proteínas de la Membrana/metabolismo , Oligodendroglía/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Western Blotting/métodos , Inmunohistoquímica/métodos , Lectinas/metabolismo , Microscopía Confocal/métodos , Microscopía Electrónica de Transmisión/métodos , Proteína Básica de Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/ultraestructura , Ratas , Ratas Wistar , Receptores Purinérgicos P2Y12RESUMEN
BACKGROUND: Evoked potentials are used in the functional assessment of sensory and motor pathways. Their usefulness in monitoring the evolution of multiple sclerosis has not been fully clarified. OBJECTIVE: The aim of this longitudinal study was to examine the usefulness of multimodal evoked potential in predicting paraclinical outcomes of disease severity and as a prognostic marker in multiple sclerosis. METHODS: Eighty four patients with clinically definite multiple sclerosis underwent Expanded Disability Status Scale (EDSS) and functional system scoring at study entry and after a mean (standard deviation) follow-up of 30.5 (11.7) months. Sensory and motor evoked potentials were obtained in all patients at study entry and at follow-up in 64 of them, and quantified according to a conventional score. RESULTS: Cross-sectionally, the severity of each evoked potential score significantly correlated with the corresponding functional system (0.32 < R < 0.60, p < 0.01, for all but follow-up visual evoked potential) and with EDSS (0.34 < R < 0.61; p < 0.001 for all but brain stem evoked potential). EDSS significantly correlated with global evoked potential score severity (baseline R = 0.60, follow-up R = 0.46, p < 0.001). Using longitudinal analysis, only changes in somatosensory evoked potential scores were significantly correlated with changes of sensory functional system (R = 0.34, p = 0.006). However, patients with multiple sclerosis with disability progression at follow-up had more severe baseline evoked potential scores than patients who remained stable. Patients with severe baseline global evoked potential score (higher than the median value) had a risk of 72.5% to progress on disability at follow-up, whereas patients with multiple sclerosis with lower scores had a risk of only 36.3%. CONCLUSIONS: These results suggest that evoked potential is a good marker of the severity of nervous damage in multiple sclerosis and may have a predictive value regarding the evolution of disability.
Asunto(s)
Potenciales Evocados , Esclerosis Múltiple/fisiopatología , Adulto , Biomarcadores , Personas con Discapacidad/clasificación , Progresión de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Understanding the means by which microglia self-regulate the neuroinflammatory response helps modulating their reaction during neurodegeneration. In amyotrophic lateral sclerosis (ALS), classical NF-κB pathway is related to persistent microglia activation and motor neuron injury; however, mechanisms of negative control of NF-κB activity remain unexplored. One of the major players in the termination of classical NF-κB pathway is the ubiquitin-editing enzyme A20, which has recognized anti-inflammatory functions. Lately, microRNAs are emerging as potent fine-tuners of neuroinflammation and reported to be regulated in ALS, for instance, by purinergic P2X7 receptor activation. In this work, we uncover an interplay between miR-125b and A20 protein in the modulation of classical NF-κB signaling in microglia. In particular, we establish the existence of a pathological circuit in which termination of A20 function by miR-125b strengthens and prolongs the noxious P2X7 receptor-dependent activation of NF-κB in microglia, with deleterious consequences on motor neurons. We prove that, by restoring A20 levels, miR-125b inhibition then sustains motor neuron survival. These results introduce miR-125b as a key mediator of microglia dynamics in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , MicroARNs/fisiología , Microglía/metabolismo , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Muerte Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Microglía/inmunología , Neuronas Motoras/fisiología , Mutación Missense , Cultivo Primario de Células , Interferencia de ARN , Superóxido Dismutasa-1 , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Spinocerebellar ataxia type 2 (SCA2) is caused by the expansion of a polyglutamine tract in ataxin-2, the SCA2 gene product. In spite of the identification of the genetic defect and the coded protein, the function of wild-type ataxin-2 has not been clarified. In order to identify the possible resistance of ataxin-2-containing neurons to degeneration, we investigated in this study the distribution and the characteristics of cell reaction to axotomy in ataxin-2-positive olivary and pontine neurons in a model of cerebellar damage represented by hemicerebellectomy. We also performed double immunofluorescence studies of ataxin-2 and purinergic receptors to characterize ataxin-2-positive surviving neurons. The present data demonstrated that after axotomy olivary and pontine ataxin-2-expressing neurons survived longer than the ataxin-2-negative cell population. Cell counting performed in the different olivary subdivisions failed to reveal any topographical prevalence in the distribution of ataxin-2-positive neurons. Therefore, the relative resistance to axotomy appears to be an intrinsic property of the ataxin-2 cell population. In addition, the capacity to modify the pattern of purinergic receptor expression in response to damage was present in only one subset of ataxin-2-positive surviving neurons. These data suggest that ataxin-2 is involved in resistance to degeneration phenomena which may be lost after mutation.
Asunto(s)
Axotomía , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Núcleo Olivar/citología , Núcleo Olivar/fisiología , Puente/citología , Puente/fisiología , Animales , Especificidad de Anticuerpos , Ataxinas , Western Blotting , Núcleos Cerebelosos/citología , Núcleos Cerebelosos/fisiología , Cerebelo/citología , Cerebelo/fisiología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Microscopía Confocal , Ratas , Ratas WistarRESUMEN
In this study we investigate the presence, modulation and biological function of P2 receptors and extracellular ATP in cultured cerebellar granule neurons. As we demonstrate by RT-PCR and western blotting, both P2X and P2Y receptor subtypes are expressed and furthermore regulated as a function of neuronal maturation. In early primary cultures, mRNA for most of the P2 receptor subtypes, except P2X(6), are found, while in older cultures only P2X(3), P2Y(1) and P2Y(6) mRNA persist. In contrast, P2 receptor proteins are more prominent in mature neurons, with the exception of P2Y(1). We also report that extracellular ATP acts as a cell death mediator for fully differentiated and mature granule neurons, for dissociated striatal primary cells and hippocampal organotypic cultures, inducing both apoptotic and necrotic features of degeneration. ATP causes cell death with EC(50) in the 20-50 microM range within few minutes of exposure and with a time lapse of at most two hours. Additional agonists for P2 receptors induce toxic effects, whereas selected antagonists are protective. Cellular swelling, lactic dehydrogenase release and nuclei fragmentation are among the features of ATP-evoked cell death, which also include direct P2 receptor modulation. Comparably to P2 receptor antagonists previously shown preventing glutamate-toxicity, here we report that competitive and non-competitive NMDA receptor antagonists inhibit the detrimental consequences of extracellular ATP. Due to the massive extracellular release of purine nucleotides and nucleosides often occurring during a toxic insult, our data indicate that extracellular ATP can now be included among the potential causes of CNS neurodegenerative events.
Asunto(s)
Adenosina Trifosfato/toxicidad , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/fisiología , Animales , Células Cultivadas , Sistema Nervioso Central/citología , Cerebelo/citología , Cerebelo/fisiología , Relación Dosis-Respuesta a Droga , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/fisiología , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Neuronas/clasificación , Neuronas/citología , Técnicas de Placa-Clamp , Agonistas del Receptor Purinérgico P2 , Ratas , Ratas WistarRESUMEN
In a previous study we used P2 receptor antagonists to inhibit diverse responses that nerve growth factor (NGF) promotes and coordinates in PC12 cells and we suggested that P2 receptors partake in the NGF signalling cascade. In this paper, we examine the direct role of extracellular P2 receptor agonists as neurotrophic factors. ATP and 2-Cl-ATP promote neurite regeneration after priming PC12 cells with NGF and the effect is dose-dependent, with an EC(50) of about 5 and 3 microM, respectively. The number of cell clumps bearing neurites was maximally induced in day 1 and it was maintained up to about one week by ATP, or up to at least 2 weeks by 2-Cl-ATP. The involvement of P1 receptors or intracellular inosine in these actions was excluded, whereas various antagonists of P2 receptors were inhibitory. Moreover, NGF and ATP caused a direct up-regulation of P2X(2), P2X(3), P2X(4) and P2Y(2), but not P2Y(4) receptor proteins under neurite-regenerating conditions, as well as extracellular signal-regulated kinase (Erk)1-2 tyrosine/threonine phosphorylation and activation. Finally, ATP, 2-Cl-ATP and ATPgammaS enhanced neurite initiation evoked by sub-optimal NGF concentrations and ATP and 2-Cl-ATP fully sustained survival of PC12 cells after serum deprivation. Our results establish that P2 receptor agonists can behave as neurotrophic factors for neuronal cells and suggest a potential interplay between ATP and NGF in the signalling pathways triggered on their target cells.
Asunto(s)
Adenosina Trifosfato/fisiología , Factor de Crecimiento Nervioso/fisiología , Neuritas/fisiología , Neuronas/fisiología , Transducción de Señal/fisiología , Adenosina Trifosfato/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero/farmacología , Activación Enzimática , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Regeneración Nerviosa/efectos de los fármacos , Neuritas/efectos de los fármacos , Células PC12 , Isoformas de Proteínas/metabolismo , Agonistas del Receptor Purinérgico P2 , Ratas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y2 , Regulación hacia ArribaRESUMEN
In the present work we examined the involvement of selected P2X receptors for extracellular ATP in the onset of neuronal cell death caused by glucose/oxygen deprivation. The in vitro studies of organotypic cultures from hippocampus evidenced that P2X2 and P2X4 were up-regulated by glucose/oxygen deprivation. Moreover, we showed that ischemic conditions induced specific neuronal loss not only in hippocampal, but also in cortical and striatal organotypic cultures and the P2 receptor antagonists basilen blue and suramin prevented these detrimental effects. In the in vivo experiments we confirmed the induction of P2X receptors in the hippocampus of gerbils subjected to bilateral common carotid occlusion. In particular, P2X2 and P2X4 proteins became significantly up-regulated, although to different extent and in different cellular phenotypes. The induction was confined to the pyramidal cell layer of the CA1 subfield and to the transition zone of the CA2 subfield and it was coincident with the area of neuronal damage. P2X2 was expressed in neuronal cell bodies and fibers in the CA1 pyramidal cell layer and in the strata oriens and radiatum. Intense P2X4 immunofluorescence was localized to microglia cells. Our results indicate a direct involvement of P2X receptors in the mechanisms sustaining cell death evoked by metabolism impairment and suggest the use of selected P2 antagonists as effective neuroprotecting agents.
Asunto(s)
Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Gerbillinae , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X4 , Regulación hacia Arriba/fisiologíaRESUMEN
Exercise-induced asthma is a well-known phenomenon, particularly affecting children, with an important social impact. In order to assess the usefulness of nedocromil sodium in the prevention of exercise-induced asthma, we studied 49 (15 females, 34 males) children who suffered from asthma; their mean +/- SD age was 9.2 +/- 3.0 (range: 3.3-19.1) years. On the first day respiratory function was evaluated by spirometry, basally and after 6 minutes. The inhalation of nedocromil sodium had a great influence on post-exercise lung function measurements; in fact on the day of nedocromil sodium pretreatment, our patients showed an increase of respiratory function, significantly different from the parameters recorded during the first day. Our findings suggest that nedocromil sodium is effective in the prevention of exercise-induced asthma, in pediatric age.
Asunto(s)
Antiasmáticos/uso terapéutico , Asma Inducida por Ejercicio/prevención & control , Nedocromil/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Resultado del TratamientoAsunto(s)
Alucinógenos/efectos adversos , Heroína/efectos adversos , N-Metil-3,4-metilenodioxianfetamina/efectos adversos , Narcóticos/efectos adversos , Enfermedades de la Médula Espinal/inducido químicamente , Enfermedad Aguda , Administración por Inhalación , Adolescente , Sobredosis de Droga/complicaciones , Femenino , Alucinógenos/administración & dosificación , Heroína/administración & dosificación , Humanos , Región Lumbosacra , Imagen por Resonancia Magnética , N-Metil-3,4-metilenodioxianfetamina/administración & dosificación , Narcóticos/administración & dosificación , Paraplejía/inducido químicamente , Enfermedades de la Médula Espinal/patologíaRESUMEN
OBJECTIVES: The time course of mu and beta sensorimotor rhythms, with event-related desynchronisation (ERD) to preparation and execution of voluntary movement followed by synchronisation (ERS) after movement, is considered to indicate cortical activation and idling, respectively. We investigated ERD and ERS in amyotrophic lateral sclerosis (ALS) patients and the relationship with anatomical and neurophysiological measures of corticospinal tract damage. METHODS: Pre-movement mu and beta ERD, and post-movement beta ERS were analysed in 16 ALS patients and 15 healthy controls performing self-paced brisk right thumb extensions. Apparent diffusion coefficient (ADC) of corticospinal tract was measured with magnetic resonance imaging (MRI). Motor-evoked potentials (MEPs) to the right abductor pollicis brevis were obtained using transcranial magnetic stimulation (TMS). RESULTS: Movement-related electromyographic activity was similar in the two groups. Post-movement ERS was significantly reduced in ALS group and negatively correlated with the amount of corticospinal damage as from MRI and TMS measures. ERD did not significantly differ between groups. CONCLUSIONS: Alterations of cortical activity in ALS patients were limited to the post-movement phase, as indicated by reduced ERS, and could be linked to reduced cortical inhibition rather than to generalised hyperexcitability. SIGNIFICANCE: The correlation between ERS and corticospinal damage severity might be interpreted as a functional compensation or dysfunction of inhibitory systems paralleling corticospinal damage.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Corteza Cerebral/fisiopatología , Potenciales Evocados Motores/fisiología , Movimiento/fisiología , Tractos Piramidales/fisiopatología , Anciano , Mapeo Encefálico , Electroencefalografía , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fuerza Muscular/fisiologíaRESUMEN
BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.
Asunto(s)
Encéfalo/metabolismo , Galactosilceramidasa/genética , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Lentivirus/genética , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Bioensayo , Encéfalo/citología , Encéfalo/fisiología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , ADN Complementario , Modelos Animales de Enfermedad , Galactosilceramidasa/análisis , Genética , Células HeLa , Hemaglutininas/química , Homocigoto , Humanos , Inmunohistoquímica , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patología , Leucodistrofia de Células Globoides/terapia , Lisosomas/enzimología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas/metabolismo , Oligodendroglía/metabolismoRESUMEN
Extracellular nucleotides act as potent signaling molecules in the neuron-glia and glia-glia communication, via the activation of specific ligand-gated P2X and G-protein-coupled metabotropic P2Y receptors. Most of the data available about the effects of P2 receptor activation in the CNS concern astrocytes, microglia, and neurons. To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in rat oligodendrocyte progenitors (OPs) and investigated the effects of ATP and its breakdown products on their functions. We describe here that rat OPs express different types of P2 receptors and that nucleotide-induced Ca(2+) raises in these progenitor cells are mainly due to the activation of P2X(7) ionotropic and ADP-sensitive P2Y(1) metabotropic receptors. We also show that ATP and ADP stimulate OP migration, inhibit the mitogenic response of OPs to PDGF and promote oligodendrocyte differentiation. The pharmacological profile of the nucleotide-induced effects demonstrates the important regulatory role of P2Y(1) receptor signaling in OP functions. These findings suggest that ATP, which is released in high amounts under inflammatory conditions and following cell death, might regulate remyelination processes in inflammatory demyelinating diseases of the CNS, like multiple sclerosis.
Asunto(s)
Adenosina Trifosfato/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Células Madre/efectos de los fármacos , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Modelos Biológicos , Oligodendroglía/citologíaRESUMEN
To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X(1,2,3,4,7)) and P2Y (P2Y(1,2,4)) receptors. Intracellular Ca(2+) recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPbetaS = ADP = Benzoyl ATP > ATP > ATPgammaS > UTP, alpha,beta-meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of alpha,betameATP-induced inward current in whole-cell recording, P2X(7) and P2Y(1) were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y(1)-specific agonist ADPbetaS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet-derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y(1) antagonist MRS2179. By confocal laser scanning microscopy, P2Y(1) receptors were localized in NG2-labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y(1) receptors.
Asunto(s)
Oligodendroglía/metabolismo , Agonistas del Receptor Purinérgico P2 , Células Madre/metabolismo , Adenosina Difosfato/fisiología , Adenosina Trifosfato/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Movimiento Celular , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Quimiotaxis de Leucocito/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Electrofisiología , Inmunohistoquímica , Microscopía Confocal , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2Y1RESUMEN
We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.