Fisiología de los receptores de glutamato en transmisión y plasticidad sinápticas en el hipocampo y el cerebelo de ratón

Programa de Doctorado en NeurocienciasLínea de Investigación: Bases moleculares y celulares de la plasticidad neuronal. Mecanismos moleculares y celulares relacionados con la plasticidad neuronal a largo plazoClave Programa: DNFCódigo Línea: 94Glutamato es el neurotransmisor excitador más importante del sistema nervioso central de los mamíferos. Las acciones fisiológicas de glutamato están mediadas por la activación de sus receptores (ionotrópicos y metabotrópicos) y sistemas de señalización. Los receptores ionotrópicos de glutamato se clasifican en tres tipos, en función del agonista que los activa con mayor afinidad en receptores de tipo NMDA (NMDAR), de tipo AMPA (AMPAR) y receptores de tipo kainato (KAR). El glutamato participa en la transmisión sináptica normal, así como en los procesos de potenciación y depresión de larga duración que son considerados los correlatos celulares de los procesos de aprendizaje, memoria, neurodesarrollo, entrenamiento sensorial y otras funciones. En el presente trabajo se ha estudiado la fisiología de los receptores presinápticos de glutamato de tipo kainato en el hipocampo y el cerebelo de ratón y los mecanismos celulares que subyacen a dichas acciones. Así se observó la facilitación de la liberación de glutamato mediada por la activación de estos receptores en la sinapsis establecida entre las fibras paralelas y las células de Purkinje (PF-PuC) de cerebelo. En las sinapsis descritas, la facilitación observada se debe a la activación de KARs presinápticos permeables a calcio, es dependiente de los niveles de calcio citoplasmático e involucra a la cascada de señalización intracelular Ca2+-calmodulina/adenilato ciclasa (AC)/ adenosín monofosfato cíclico (cAMP)/Proteína quinasa A (PKA). Asimismo, se ha estudiado el efecto depresor de la transmisión glutamatérgica mediado por la activación de KARs presinápticos en la sinapsis PF-PuC de cerebelo y las sinapsis de las colaterales de Schaffer y Cornus Ammonis 2 (SC-CA2) de hipocampo de ratón. En el cerebelo esta depresión está mediada por KAR e involucra a la PKA. En el hipocampo necesita el correcto funcionamiento de la PKA y proteínas G. En este trabajo también se han estudiados los mecanismos moleculares y celulares de la Spike timing depend plasticity (STDP). Tanto de depresión de larga duración (t-LTD) como potenciación de larga duración (t-LTP). Así se observó una t-LTD en las sinapsis SC-CA1 de hipocampo, que es evidente hasta la tercera semana posnatal en ratones, desapareciendo durante la cuarta semana. En etapas más maduras, se encontró que el protocolo que inducía t-LTD indujo t-LTP. Se ha caracterizó esta forma de t-LTP y los mecanismos involucrados en su inducción, así como también los que impulsan este cambio de t-LTD a t-LTP. Se observó que esta t-LTP se expresa presinápticamente en las sinapsis CA3-CA1, como lo demuestra el análisis del coeficiente de variación, del número de fallos, la relación de pulsos pareados y el análisis de respuestas miniatura. Además, esta forma de t-LTP presináptica no requiere NMDAR, sino la activación de receptores metabotrópicos de glutamato (mGluR), la entrada de Ca2+ en la neurona postsináptica a través de canales de Ca2+ dependientes de voltaje de tipo L y la liberación de Ca2+ de organulos intracelulares. El óxido nítrico (NO) también se requiere como mensajero de la neurona postsináptica. De manera crucial, se requiere la liberación de adenosina y glutamato por parte de los astrocitos para la inducción de esta t-LTP y para el cambio de t-LTD a t-LTP. Por lo tanto, se ha descubierto un cambio durante el desarrollo de la transmisión sináptica de t-LTD a t-LTP en las sinapsis CA3-CA1 del hipocampo en las que los astrocitos juegan un papel central revelando una forma de LTP presináptica y los mecanismos para su inducción.Universidad Pablo de Olavide de Sevilla. Departamento de Fisiología, Anatomía y Biología Celula

Kainate Receptors Modulating Glutamate Release in the Cerebellum

Glutamate receptors of the kainate type (Kainate receptors, KARs), are mediators of ionotropic postsynaptic synaptic transmission, as well as presynaptic modulators of neurotransmitter release where they show both ionotropic and metabotropic actions regulating glutamate and γ-aminobutiric acid (GABA) release. The mechanisms underlying these modulatory roles are starting to be understood at some brain regions. Here we review the KARs roles and mechanisms involved in the modulation of glutamate release in the cerebellum at parallel fibers (PF)-Purkinje Cells (PuC) synapses. KARs activation mediate a biphasic effect on glutamate release at this synapse, with low kainate (KA) concentrations mediating a facilitation of glutamate release and higher KA concentrations mediating a depression of glutamate release. KA-mediated facilitation is prevented by antagonizing KARs, by inhibition of PKA or stimulation of adenylyl cyclase (AC), by blocking Ca2+ permeant KARs, by depleting intracellular Ca2+ stores and by blocking calmodulin. Thus, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling. KAR-mediated depression of glutamate release involves the AC/cAMP/PKA pathway as for facilitation but not Ca2+-calmodulin, being in this case AC activated by a Gi/o protein to mediate a depression of glutamate release

Metabotropic actions of kainate receptors modulating glutamate release

Presynaptic kainate (KA) receptors (KARs) modulate GABA and glutamate release in the central nervous system of mammals. While some of the actions of KARs are ionotropic, metabotropic actions for these receptors have also been seen to modulate both GABA and glutamate release. In general, presynaptic KARs modulate glutamate release through their metabotropic actions in a biphasic manner, with low KA concentrations producing an increase in glutamate release and higher concentrations of KA driving weaker release of this neurotransmitter. Different molecular mechanisms are involved in this modulation of glutamate release, with a G-protein independent, Ca2+-calmodulin adenylate cyclase (AC) and protein kinase A (PKA) dependent mechanism facilitating glutamate release, and a G-protein, AC and PKA dependent mechanism mediating the decrease in neurotransmitter release. Here, we describe the events underlying the KAR modulation of glutamatergic transmission in different brain regions, addressing the possible functions of this modulation and proposing future research lines in this field.Universidad Pablo de Olavid

Kainate receptors: from synaptic activity to disease

Kainate receptors (KARs) are glutamate receptors that participate in the postsynaptic transmission of information and in the control of neuronal excitability, as well as presynaptically modulating the release of the neurotransmitters GABA and glutamate. These modulatory effects, general follow a biphasic pattern, with low KA concentrations provoking an increase in GABA and glutamate release, and higher concentrations mediating a decrease in the release of these neurotransmitters. In addition, KARs are involved in different forms of long- and short-term plasticity. Importantly, altered activity of these receptors has been implicated in different central nervous system diseases and disturbances. Here, we describe the pre- and postsynaptic actions of KARs, and the possible role of these receptors in disease, a field that has seen significant progress in recent years.Universidad Pablo de Olavid

Vitamin E prevents lipid peroxidation and iron accumulation in PLA2G6-Associated Neurodegeneration

23 Páginas.-- 17 FigurasPLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease.This work was supported by FIS PI16/00786 and PI19/00377 grants, Instituto de Salud Carlos III, Spain and Fondo Europeo de Desarrollo Regional (FEDR-Unión Europea), Proyectos de Investigación de Excelencia de la Junta de Andalucía CTS-5725 and PY18-850 and by AIDNAI (Association Internationale de Dystrophie Neuro Axonale Infantile), ENACH (Asociación de Enfermos de Neurodegeneración con Acumulación Cerebral de Hierro), AEPMI (Asociación de Enfermos de Patología Mitocondrial), FEDER (Federación Española de Enfermedades Raras) and Fundación MERK Salud. S. Povea-Cabello is a recipient of Ayudas para la Formación del Profesorado Universitario (FPU) from Ministerio de Universidades de España.Peer reviewe

Kainate Receptor-Mediated Depression of Glutamate Release Involves Protein Kinase A in the Cerebellum

Kainate (KA) receptors (KAR) have important modulatory roles of synaptic transmission. In the cerebellum, the action mechanisms of KAR-mediated glutamatergic depression are unknown. We studied these mechanisms by recording evoked excitatory postsynaptic currents (eEPSCs) from cerebellar slices using the whole-cell configuration of the patch-clamp technique. We observed that 3 μM KA decreased the amplitude of eEPSCs and increased the number of failures at the synapses established between parallel fibers (PF) and Purkinje neurons, and the effect was antagonized by NBQX under the condition where AMPA receptors were previously blocked. The inhibition of protein kinase A (PKA) suppressed the effect of KAR activation on eEPSC, and effect was not prevented by protein kinase C inhibitors. Furthermore, in the presence of Pertussis toxin, the depression of glutamate release mediated by KAR activation was prevented, invoking the participation of a Gi/o protein in this modulation. Finally, the KAR-mediated depression of glutamate release was not prevented by blocking calcium-permeable KARs or by treatments that affect calcium release from intracellular stores. We conclude that KARs present at these synapses mediate an inhibition of glutamate release through a mechanism that involves the activation of G-protein and protein kinase A

Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

We elucidated the mechanisms underlying the kainate receptor (KAR)-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM) increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs) at synapses between axon terminals of parallel fibers (PF) and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA) suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC). KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling

Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.This work was supported by FIS PI16/00786 grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), Spanish Ministry of Education, Culture and Sports, “Ayudas para la Formación de Profesorado Universitario” (FPU) and AEPMI (Asociación de Enfermos de Patología Mitocondrial) and ENACH (Asociación de enfermos de Neurodegeneración con Acumulación Cerebral de Hierro).Peer reviewe

Astrocyte-mediated switch in spike timing-dependent plasticity during hippocampal development

Presynaptic spike timing-dependent long-term depression at hippocampal CA3-CA1 synapses is evident until the third postnatal week in mice. The authors show that maturation beyond four weeks is associated with a switch to long-term potentiation in which astrocytes play a central role

Cannabinoid type-1 receptor blockade restores neurological phenotypes in two models for Down syndrome

Intellectual disability is the most limiting hallmark of Down syndrome, for which there is no gold-standard clinical treatment yet. The endocannabinoid system is a widespread neuromodulatory system involved in multiple functions including learning and memory processes. Alterations of this system contribute to the pathogenesis of several neurological and neurodevelopmental disorders. However, the involvement of the endocannabinoid system in the pathogenesis of Down syndrome has not been explored before. We used the best-characterized preclinical model of Down syndrome, the segmentally trisomic Ts65Dn model. In male Ts65Dn mice, cannabinoid type-1 receptor (CB1R) expression was enhanced and its function increased in hippocampal excitatory terminals. Knockdown of CB1R in the hippocampus of male Ts65Dn mice restored hippocampal-dependent memory. Concomitant with this result, pharmacological inhibition of CB1R restored memory deficits, hippocampal synaptic plasticity and adult neurogenesis in the subgranular zone of the dentate gyrus. Notably, the blockade of CB1R also normalized hippocampal-dependent memory in female Ts65Dn mice. To further investigate the mechanisms involved, we used a second transgenic mouse model overexpressing a single gene candidate for Down syndrome cognitive phenotypes, the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). CB1R pharmacological blockade similarly improved cognitive performance, synaptic plasticity and neurogenesis in transgenic male Dyrk1A mice. Our results identify CB1R as a novel druggable target potentially relevant for the improvement of cognitive deficits associated with Down syndrome.A.N.-R. is the recipient of a predoctoral fellowship (Ministerio de Educación y Cultura, Spain). A.V.-O. is the recipient of a predoctoral fellowship from Jérôme Lejeune Foundation, France (Spanish Delegation). M.G.-G. was partially supported by FRAXA Research Foundation, United States. A.B-G. was the recipient of a predoctoral fellowship (Ministerio de Educación y Cultura). This study was supported by Jérôme Lejeune Foundation (French Delegation) to A.O.; Instituto de Salud Carlos III, Spain (#RD16/0017/0020) to R.M.; Ministerio de Economía, Innovación y Competitividad (MINECO), Spain (#BFU2015-68568-P to A.O., #SAF2014-59648-P and #SAF2017- 84060-R to R.M.; SAF2016-79956-R to M.D.); Catalan Foundation ‘La Marató de TV3’, Spain (#2016/20-30) to E.M.-G.; Junta de Andalucía, Spain (CVI-7290 to A.R.-M.); Generalitat de Catalunya, Spain (2014SGR-1547 and 2017SGR-669 to R.M.; 2014SGR-1125 to M.D.) and ICREA (Institució Catalana de Recerca i Estudis Avançats, Spain) Academia to A.O. and R.M. Grant “Unidad de Excelencia María de Maeztu”, funded by the MINECO (#MDM-2014-0370); PLAN E, Spain (Plan Español para el Estímulo de la Economía y el Empleo); FEDER, European Commission funding is also acknowledge