RESUMEN
Huntington's disease (HD) is a fatal neurodegenerative disorder with no effective cure currently available. Over the past few years our research has shown that alterations in sphingolipid metabolism represent a critical determinant in HD pathogenesis. In particular, aberrant metabolism of sphingosine-1-phosphate (S1P) has been reported in multiple disease settings, including human postmortem brains from HD patients. In this study, we investigate the potential therapeutic effect of the inhibition of S1P degradative enzyme SGPL1, by the chronic administration of the 2-acetyl-5-tetrahydroxybutyl imidazole (THI) inhibitor. We show that THI mitigated motor dysfunctions in both mouse and fly models of HD. The compound evoked the activation of pro-survival pathways, normalized levels of brain-derived neurotrophic factor, preserved white matter integrity, and stimulated synaptic functions in HD mice. Metabolically, THI restored normal levels of hexosylceramides and stimulated the autophagic and lysosomal machinery, facilitating the reduction of nuclear inclusions of both wild-type and mutant huntingtin proteins.
Asunto(s)
Enfermedad de Huntington , Ratones , Humanos , Animales , Enfermedad de Huntington/tratamiento farmacológico , Modelos Teóricos , Imidazoles/farmacología , Glicoesfingolípidos , Modelos Animales de Enfermedad , Proteína Huntingtina/genéticaRESUMEN
Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.
Asunto(s)
Dolor Crónico , Corteza Somatosensorial , Animales , Dolor Crónico/inducido químicamente , Dolor Crónico/metabolismo , Matriz Extracelular/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Parvalbúminas/metabolismo , Corteza Somatosensorial/metabolismoRESUMEN
Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.
Asunto(s)
Compuestos de Amonio/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Estilbenos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ligandos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
BACKGROUND: Intracellular Ca2+ modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca2+ concentration ([Ca2+]i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca2+-mediated response triggered by purinergic signaling. METHODS: Fura-2 based fluorescence microscopy was used to measure [Ca2+]i in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca2+-dependent whole-cell currents. RESULTS: In human microglia extracellular ATP evoked [Ca2+]i increases depend on Ca2+ entry from the extracellular space and on Ca2+ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca2+]i increases were prevented by removing external Ca2+ and using an intracellular Ca2+ chelator. TRAM-34, a selective KCa3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl- in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca2+ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca2+ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca2+-evoked K+ current led to a less sustained ATP-evoked Ca2+ entry, as shown by the faster Ca2+ transient kinetics observed in LPS-treated microglia. CONCLUSIONS: Our study confirms a functional role for KCa3.1 channels in human microglia, linking ATP-evoked Ca2+ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the KCa3.1-mediated microglial response to purinergic signaling may be reduced.
Asunto(s)
Adenosina Trifosfato/farmacología , Calcio/metabolismo , Epilepsia Refractaria/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Microglía/metabolismo , Lóbulo Temporal/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Epilepsia Refractaria/patología , Humanos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Lipopolisacáridos/toxicidad , Microglía/efectos de los fármacos , Lóbulo Temporal/efectos de los fármacos , Lóbulo Temporal/patologíaRESUMEN
Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.
Asunto(s)
Corteza Cerebral/anomalías , Embrión de Mamíferos/anomalías , Neuronas GABAérgicas/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Biomarcadores , Corteza Cerebral/metabolismo , Femenino , Regulación de la Expresión Génica , Genes Homeobox , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , ARN Mensajero , Receptores de Glutamato Metabotrópico/genéticaRESUMEN
The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.
Asunto(s)
Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Sitios de Unión/fisiología , Membrana Celular/metabolismo , Células Cultivadas , HumanosRESUMEN
BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.
Asunto(s)
Cromonas/farmacología , Glioma/tratamiento farmacológico , Glioma/metabolismo , Metabolómica , Animales , Proliferación Celular/efectos de los fármacos , Glioma/diagnóstico , Glucólisis/efectos de los fármacos , Humanos , Masculino , Ratones , Neoplasias Experimentales/diagnóstico , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
OBJECTIVE: γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in adult central nervous system, and profound alterations of GABA receptor functions are linked to temporal lobe epilepsy (TLE). Here we describe the functional relationships between GABA receptors type B (GABAB R) and type A (GABAA R) in human temporal cortex and how TLE affects this aspect of GABAergic signaling. METHODS: Miniature inhibitory postsynaptic currents (mIPSCs) were recorded by patch-clamp techniques from human L5 pyramidal neurons in slices from temporal cortex tissue obtained from surgery. RESULTS: We describe a constitutive functional crosstalk between GABAB Rs and GABAA Rs in human temporal layer 5 pyramidal neurons, which is lost in epileptic tissues. The activation of GABAB Rs by baclofen, in addition to the expected reduction of mIPSC frequency, produced, in cortex of nonepileptic patients, the prolongation of mIPSC rise and decay times, thus increasing the inhibitory net charge associated with a single synaptic event. Block of K+ channels did not prevent the increase of decay time and charge. Protein kinase A (PKA) blocker KT5720 and pertussis toxin inhibited the action of baclofen, whereas 8Br-cAMP mimicked the GABAB R action. The same GABAB R-mediated modulation of GABAA Rs was observed in pyramidal neurons of rat temporal cortex, with both PKA and PKC involved in the process. In cortices from TLE patients and epileptic rats, baclofen lost its ability to modulate mIPSCs. SIGNIFICANCE: Our results highlight the association of TLE with functional changes of GABAergic signaling that may be related to seizure propagation, and suggest that the selective activation of a definite subset of nonpresynaptic GABAB Rs may be therapeutically useful in TLE.
Asunto(s)
Epilepsia del Lóbulo Temporal/metabolismo , Neocórtex/metabolismo , Células Piramidales/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Lóbulo Temporal/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adolescente , Adulto , Animales , Baclofeno/farmacología , Carbazoles/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Modelos Animales de Enfermedad , Epilepsia Refractaria/metabolismo , Epilepsia Refractaria/fisiopatología , Epilepsia Refractaria/cirugía , Inhibidores Enzimáticos/farmacología , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Epilepsia/fisiopatología , Epilepsia del Lóbulo Temporal/fisiopatología , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Agonistas de Receptores GABA-B/farmacología , Humanos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Persona de Mediana Edad , Agonistas Muscarínicos/toxicidad , Neocórtex/efectos de los fármacos , Neocórtex/fisiopatología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Pilocarpina/toxicidad , Proteína Quinasa C/metabolismo , Células Piramidales/efectos de los fármacos , Pirroles/farmacología , Ratas , Lóbulo Temporal/efectos de los fármacos , Lóbulo Temporal/fisiopatologíaRESUMEN
RATIONALE: Long living individuals show delay of aging, which is characterized by the progressive loss of cardiovascular homeostasis, along with reduced endothelial nitric oxide synthase activity, endothelial dysfunction, and impairment of tissue repair after ischemic injury. OBJECTIVE: Exploit genetic analysis of long living individuals to reveal master molecular regulators of physiological aging and new targets for treatment of cardiovascular disease. METHODS AND RESULTS: We show that the polymorphic variant rs2070325 (Ile229Val) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) associates with exceptional longevity, under a recessive genetic model, in 3 independent populations. Moreover, the expression of BPIFB4 is instrumental to maintenance of cellular and vascular homeostasis through regulation of protein synthesis. BPIFB4 phosphorylation/activation by protein-kinase-R-like endoplasmic reticulum kinase induces its complexing with 14-3-3 and heat shock protein 90, which is facilitated by the longevity-associated variant. In isolated vessels, BPIFB4 is upregulated by mechanical stress, and its knock-down inhibits endothelium-dependent vasorelaxation. In hypertensive rats and old mice, gene transfer of longevity-associated variant-BPIFB4 restores endothelial nitric oxide synthase signaling, rescues endothelial dysfunction, and reduces blood pressure levels. Furthermore, BPIFB4 is implicated in vascular repair. BPIFB4 is abundantly expressed in circulating CD34(+) cells of long living individuals, and its knock-down in endothelial progenitor cells precludes their capacity to migrate toward the chemoattractant SDF-1. In a murine model of peripheral ischemia, systemic gene therapy with longevity-associated variant-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization, and reperfusion of the ischemic muscle. CONCLUSIONS: Longevity-associated variant-BPIFB4 may represent a novel therapeutic tool to fight endothelial dysfunction and promote vascular reparative processes.
Asunto(s)
Células Progenitoras Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Longevidad/genética , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas 14-3-3/metabolismo , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Presión Sanguínea , Movimiento Celular , Modelos Animales de Enfermedad , Europa (Continente) , Femenino , Estudios de Asociación Genética , Terapia Genética , Genotipo , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Miembro Posterior , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipertensión/terapia , Péptidos y Proteínas de Señalización Intercelular , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatología , Isquemia/terapia , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo , Fosforilación , Interferencia de ARN , Ratas Endogámicas SHR , Transducción de Señal , Estrés Mecánico , Transfección , Estados Unidos , Vasodilatación , eIF-2 Quinasa/metabolismoRESUMEN
Huntington disease (HD) is a genetic neurodegenerative disorder for which there is currently no cure and no way to stop or even slow the brain changes it causes. In the present study, we aimed to investigate whether FTY720, the first approved oral therapy for multiple sclerosis, may be effective in HD models and eventually constitute an alternative therapeutic approach for the treatment of the disease. Here, we utilized preclinical target validation paradigms and examined the in vivo efficacy of chronic administration of FTY720 in R6/2 HD mouse model. Our findings indicate that FTY720 improved motor function, prolonged survival and reduced brain atrophy in R6/2 mice. The beneficial effect of FTY720 administration was associated with a significant strengthening of neuronal activity and connectivity and, with reduction of mutant huntingtin aggregates, and it was also paralleled by increased phosphorylation of mutant huntingtin at serine 13/16 residues that are predicted to attenuate protein toxicity.
Asunto(s)
Enfermedad de Huntington/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Glicoles de Propileno/uso terapéutico , Esfingosina/análogos & derivados , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod , Enfermedad de Huntington/metabolismo , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Esfingosina/uso terapéuticoRESUMEN
Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (α5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the α5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca(2+) concentration elevations and that in α5(-/-) mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the α5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how α5DN impairs heteromeric nAChR functions, we coexpressed α4, α5, or α5DN subunits with a dimeric concatemer (ß2α4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both α5(ß2α4)2 and α5DN(ß2α4)2 nAChRs yielded similar levels of functional expression and Ca(2+) permeability, measured as fractional Ca(2+) currents (8.2 ± 0.7% and 8.0 ± 1.9%, respectively), 2-fold higher than α4(ß2α4)2. Our results indicate that the loss of function of nicotinic responses observed in α5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca(2+) permeability but likely to intracellular modulation.
Asunto(s)
Calcio/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Nicotina/metabolismoRESUMEN
Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.
Asunto(s)
Adenosina Quinasa/fisiología , Adenosina/fisiología , Homeostasis/fisiología , Sinapsis/fisiología , Adenosina Quinasa/genética , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/fisiología , Región CA1 Hipocampal/fisiología , Región CA3 Hipocampal/fisiología , Fenómenos Electrofisiológicos/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Espacio Extracelular/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fibras Musgosas del Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Técnicas de Placa-Clamp , Purinas/metabolismo , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/fisiología , Receptores de GABA-A/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
INTRODUCTION: Fluoxetine is an open channel blocker of fetal muscle acetylcholine (ACh) receptor (AChR) and slow-channel mutant AChRs. It is used commonly to treat patients with slow-channel congenital myasthenic syndromes. Fluoxetine effects on adult wild-type endplate AChR are less characterized, although muscle AChR isoforms are differentially modulated by some drugs. METHODS: Excitotoxicity assays and patch clamp recordings were performed in human embryonic kidney 293 (HEK) cells expressing wild-type or slow-channel mutant human AChRs. RESULTS: Fluoxetine (2-10 µM) abolished ACh-induced death and decreased ACh-activated whole-cell currents in cells expressing all AChR types. In outside-out patches, fluoxetine rapidly curtailed ACh evoked unitary activity and macroscopic currents. The effect was increased if fluoxetine was applied before ACh. CONCLUSIONS: Fluoxetine is an open channel blocker, but it also affects AChR in the closed state. AChR blockade likely underlies the rescue of HEK cells from ACh-induced death.
Asunto(s)
Acetilcolina/efectos adversos , Fluoxetina/farmacología , Placa Motora/efectos de los fármacos , Neurotoxinas/efectos adversos , Receptores Colinérgicos/efectos de los fármacos , Acetilcolina/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células HEK293 , Humanos , Técnicas In Vitro , Placa Motora/fisiología , Síndromes Miasténicos Congénitos/fisiopatología , Neurotoxinas/farmacología , Técnicas de Placa-Clamp , Receptores Colinérgicos/fisiología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
Excessive Ca2+ influx through N-methyl-D-aspartate type glutamate receptors (NMDAR) is associated with excitotoxicity and neuronal death, but the inhibition of this receptor-channel causes severe adverse effects. Thus, a selective reduction of NMDA-mediated Ca2+ entry, leaving unaltered the Na+ current, could represent a valid neuroprotective strategy. We developed a new two-fluorophore approach to efficiently assess the Ca2+ permeability of ligand-gated ion channels, including NMDARs, in different conditions. This technique was able to discriminate differential Ca2+/Na+ permeation ratio through different receptor channels, and through the same channel in different conditions. With this method, we confirmed that EU1794-4, a negative allosteric modulator of NMDARs, decreased their Ca2+ permeability. Furthermore, we measured for the first time the fractional Ca2+ current (Pf, i.e. the percentage of the total current carried by Ca2+ ions) of human NMDARs in the presence of EU1794-4, exhibiting a 40% reduction in comparison to control conditions. EU1794-4 was also able to reduce NMDA-mediated Ca2+ entry in human neurons derived from induced pluripotent stem cells. This last effect was stronger in the absence of extracellular Mg2+, but still significant in its presence, supporting the hypothesis to use NMDA-selective allosteric modulators to lower Ca2+ influx in human neurons, to prevent Ca2+-dependent excitotoxicity and consequent neurodegeneration.
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Calcio , Células Madre Pluripotentes Inducidas , Receptores de N-Metil-D-Aspartato , Sodio , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Calcio/metabolismo , Sodio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células HEK293 , Neuronas/metabolismo , Neuronas/efectos de los fármacos , N-Metilaspartato/farmacologíaRESUMEN
Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients' cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.
RESUMEN
PURPOSE: The chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ-aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABAA receptor (GABAA R) mediated transmission, with a particular focus on the action of CX3CL1 on the use-dependent decrease (rundown) of the GABA-evoked currents (IGABA ), a feature underlying the reduction of GABAergic function in epileptic tissue. METHODS: Patch-clamp recordings were obtained from cortical pyramidal neurons in slices from six MTLE patients after surgery. Alternatively, the cell membranes from epileptic brain tissues of 17 MTLE patients or from surgical samples and autopsies of nonepileptic patients were microtransplanted into Xenopus oocytes, and IGABA were recorded using the standard two-microelectrode voltage-clamp technique. Immunohistochemical staining and double-labeling studies were carried out on the same brain tissues to analyze CX3CR1 expression. KEY FINDINGS: In native pyramidal neurons from cortical slices of patients with MTLE, CX3CL1 reduced IGABA rundown and affected the recovery of IGABA amplitude from rundown. These same effects were confirmed in oocytes injected with cortical and hippocampal MTLE membranes, whereas CX3CL1 did not influence IGABA in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with MTLE, CX3CR1 immunoreactivity was higher in MTLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells. SIGNIFICANCE: These findings indicate a role for CX3CL1 in MTLE, supporting recent evidence on the relevance of brain inflammation in human epilepsies. Our data demonstrate that in MTLE tissues the reduced GABAergic function can be modulated by CX3CL1. The increased CX3CR1 expression in microglia and the modulation by CX3CL1 of GABAergic currents in human epileptic brain suggests new therapeutic approaches for drug-resistant epilepsies based on the evidence that the propagation of seizures can be influenced by inflammatory processes.
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Quimiocina CX3CL1/fisiología , Epilepsia del Lóbulo Temporal/fisiopatología , Receptores de GABA-A/fisiología , Potenciales de Acción/fisiología , Adulto , Animales , Western Blotting , Encéfalo/fisiopatología , Membrana Celular/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oocitos/fisiología , Células Piramidales/fisiología , Xenopus laevis , Adulto JovenRESUMEN
The binding mode of nicotinic agonists has been thoroughly investigated in the last decades. It is now accepted that the charged amino group is bound by a cation-π interaction to a conserved tryptophan residue, and that the aromatic moiety is projected into a hydrophobic pocket deeply located inside the binding cleft. A hydrogen bond donor/acceptor, maybe a water molecule solvating this receptor subsite, contributes to further stabilize the nicotinic ligands. The position of this water molecule has been established by several X-ray structures of the acetylcholine-binding protein. In this study, we computationally analyzed the role of this water molecule as a putative hydrogen bond donor/acceptor moiety in the agonist binding site of the three most relevant heteromeric (α4ß2, α3ß4) and homomeric (α7) neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Our theoretical investigation made use of epibatidine 1 and deschloroepibatidine 2 as molecular probes, and was then extended to their analogues 3 and 4, which were subsequently synthesized and tested at the three target receptor subtypes. Although the pharmacological data for the new ligands 3 and 4 indicated a reduction of the affinity at the studied nAChRs with respect to reference agonists, a variation of the selectivity profile was clearly evidenced.
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Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacología , Piridinas/química , Piridinas/farmacología , Receptores Colinérgicos/metabolismo , Agua/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Sondas Moleculares/química , Datos de Secuencia Molecular , Receptores Colinérgicos/químicaRESUMEN
Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABA(A) receptors. In particular, the use-dependent decrease (run-down) of the currents (I(GABA)) evoked by the repetitive activation of GABA(A) receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of I(GABA) run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in I(GABA) run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in alpha4-, relative to alpha1-containing GABA(A) receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic.
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Epilepsia del Lóbulo Temporal/fisiopatología , Hipocampo/fisiología , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Electrofisiología , Fluoresceínas , Técnica del Anticuerpo Fluorescente , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Oocitos/metabolismo , Compuestos Orgánicos , Pilocarpina , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , XenopusRESUMEN
BACKGROUND: Schwann cells (SCs) are glial cells involved in peripheral axon myelination. SCs also play a strategic role after peripheral nerve injury, regulating local inflammation and axon regeneration. Our previous studies demonstrated the presence of cholinergic receptors in SCs. In particular, the α7 nicotinic acetylcholine receptors (nAChRs) are expressed in SCs after peripheral axotomy, suggesting their involvement in the regulation of SC-regenerating properties. To clarify the role that α7 nAChRs may play after peripheral axon damage, in this study we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. METHODS: Both ionotropic and metabotropic cholinergic signaling were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation. In addition, the expression of c-Jun and α7 nAChRs was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. RESULTS: Activation of α7 nAChRs, activated by the selective partial agonist ICH3, did not induce calcium mobilization but positively modulated the PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was also supported by the up-regulated expression of its specific p-p70 S6KThr389 target. Moreover, up-regulation of p-AMPKThr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses proved that α7 nAChR activation also promotes SC migration. CONCLUSIONS: Our data demonstrate that α7 nAChRs, expressed by SCs only after peripheral axon damage and/or in an inflammatory microenvironment, contribute to improve the SCs regenerating properties. Indeed, α7 nAChR stimulation leads to an upregulation of c-Jun expression and promotes Schwann cell migration by non-canonical pathways involving the mTORC1 activity.
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Axones , Receptor Nicotínico de Acetilcolina alfa 7 , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Axones/metabolismo , Calcio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regeneración Nerviosa , Transducción de Señal/fisiología , Células de Schwann/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no effective therapy, causing progressive loss of motor neurons in the spinal cord, brainstem, and motor cortex. Regardless of its genetic or sporadic origin, there is currently no cure for ALS or therapy that can reverse or control its progression. In the present study, taking advantage of a human superoxide dismutase-1 mutant (hSOD1-G93A) mouse that recapitulates key pathological features of human ALS, we investigated the possible role of voltage-gated potassium channel Kv1.3 in disease progression. We found that chronic administration of the brain-penetrant Kv1.3 inhibitor, PAP-1 (40 mg/Kg), in early symptomatic mice (i) improves motor deficits and prolongs survival of diseased mice (ii) reduces astrocyte reactivity, microglial Kv1.3 expression, and serum pro-inflammatory soluble factors (iii) improves structural mitochondrial deficits in motor neuron mitochondria (iv) restores mitochondrial respiratory dysfunction. Taken together, these findings underscore the potential significance of Kv1.3 activity as a contributing factor to the metabolic disturbances observed in ALS. Consequently, targeting Kv1.3 presents a promising avenue for modulating disease progression, shedding new light on potential therapeutic strategies for ALS.