RESUMEN
Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.
Asunto(s)
Epilepsia del Lóbulo Temporal , Estado Epiléptico , Ratones , Animales , Humanos , Epilepsia del Lóbulo Temporal/patología , Astrocitos/patología , Factor de Necrosis Tumoral alfa , Microglía/patología , Hipocampo/patología , Convulsiones/patología , Estado Epiléptico/patología , Ácido Kaínico/toxicidad , Modelos Animales de Enfermedad , Ratones NoqueadosRESUMEN
NG2 glia represents a distinct type of macroglial cells in the CNS and is unique among glia because they receive synaptic input from neurons. They are abundantly present in white and gray matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of gray matter NG2 glia and their synaptic input are still ill defined. Here, we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Kir4.1 was deleted at postnatal day 23-26 (recombination efficiency about 75%) and mice were investigated 3-8 weeks later. Notably, these mice with dysfunctional NG2 glia demonstrated improved spatial memory as revealed by testing new object location recognition while working and social memory remained unaffected. Focussing on the hippocampus, we found that loss of Kir4.1 potentiated synaptic depolarizations of NG2 glia and stimulated the expression of myelin basic protein while proliferation and differentiation of hippocampal NG2 glia remained largely unaffected. Mice with targeted deletion of the K+ channel in NG2 glia showed impaired long-term potentiation at CA3-CA1 synapses, which could be fully rescued by extracellular application of a TrkB receptor agonist. Our data demonstrate that proper NG2 glia function is important for normal brain function and behavior.
Asunto(s)
Neuroglía , Proteoglicanos , Ratones , Animales , Proteoglicanos/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Plasticidad Neuronal , Antígenos/metabolismoRESUMEN
NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron-glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABAA receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron-glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABAA receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity.
Asunto(s)
Receptores de GABA-A , Animales , Ratones , Ácido gamma-Aminobutírico , Hipocampo , Interneuronas , NeuroglíaRESUMEN
OBJECTIVE: Growing evidence suggests that dysfunctional astrocytes are crucial players in the development of mesial temporal lobe epilepsy (MTLE). Using a mouse model closely recapitulating key alterations of chronic human MTLE with hippocampal sclerosis, here we asked whether death of astrocytes contributes to the initiation of the disease and investigated potential underlying molecular mechanisms. METHODS: Antibody staining was combined with confocal imaging and semiquantitative real-time polymerase chain reaction analysis to identify markers of different cellular death mechanisms between 4 h and 3 days after epilepsy induction. RESULTS: Four hours after kainate-mediated induction of status epilepticus (SE), we found a significant reduction in the density of astrocytes in the CA1 stratum radiatum (SR) of the ipsilateral hippocampus. This reduction was transient, as within the next 3 days, astrocyte cell numbers recovered to the initial values, which was accompanied by enhanced proliferation. Four hours after SE induction, a small proportion of astrocytes in the ipsilateral CA1 SR expressed autophagy-related genes and proteins, whereas we did not find astrocytes positive for cleaved caspase 3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling, ruling out apoptosis-related astrocytic death. Importantly, at the same early time point post-SE, many astrocytes in the ipsilateral CA1 SR showed strong expression of genes encoding pro-necroptosis factors, including receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Phosphorylation of MLKL (pMLKL), formation of necrosome complexes composed of RIPK3 and pMLKL, and translocation of pMLKL to the nucleus and to the plasma membrane were often observed in astrocytes of the ipsilateral hippocampus 4 h post-SE. SIGNIFICANCE: The present study revealed that astrocytes die shortly after induction of SE. Our expression data and immunohistochemistry suggest that necroptosis and autophagy contribute to astrocytic death. These findings help to better understand how dysfunctional and pathological remodeling of astrocytes contributes to the initiation of temporal lobe epilepsy.
Asunto(s)
Astrocitos/patología , Región CA1 Hipocampal/patología , Muerte Celular , Epilepsia/patología , Animales , Autofagia/genética , Caspasa 3/genética , Recuento de Células , Proliferación Celular , Convulsivantes , Epilepsia/inducido químicamente , Ácido Kaínico , Masculino , Ratones , Microglía/patología , Proteínas Quinasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Estado Epiléptico/inducido químicamente , Estado Epiléptico/patologíaRESUMEN
The astroglial gap junctional network formed by connexin (Cx) channels plays a central role in regulating neuronal activity and network synchronization. However, its involvement in the development and progression of epilepsy is not yet understood. Loss of interastrocytic gap junction (GJ) coupling has been observed in the sclerotic hippocampus of patients with mesial temporal lobe epilepsy (MTLE) and in mouse models of MTLE, leading to the suggestion that it plays a causative role in the pathogenesis. To further elucidate this clinically relevant question, we investigated consequences of astrocyte disconnection on the time course and severity of kainate-induced MTLE with hippocampal sclerosis (HS) by comparing mice deficient for astrocytic Cx proteins with wild-type mice (WT). Continuous telemetric EEG recordings and video monitoring performed over a period of 4 weeks after epilepsy induction revealed substantially higher seizure and interictal spike activity during the chronic phase in Cx deficient versus WT mice, while the severity of status epilepticus was not different. Immunohistochemical analysis showed that, despite the elevated chronic seizure activity, astrocyte disconnection did not aggravate the severity of HS. Indeed, the extent of CA1 pyramidal cell loss was similar between the experimental groups, while astrogliosis, granule cell dispersion, angiogenesis, and microglia activation were even reduced in Cx deficient as compared to WT mice. Interestingly, seizure-induced neurogenesis in the adult dentate gyrus was also independent of astrocytic Cxs. Together, our data indicate that constitutive loss of GJ coupling between astrocytes promotes neuronal hyperexcitability and attenuates seizure-induced histopathological outcomes.
Asunto(s)
Astrocitos/metabolismo , Conexinas/deficiencia , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Eliminación de Gen , Ácido Kaínico/toxicidad , Animales , Astrocitos/efectos de los fármacos , Conexinas/genética , Epilepsia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Microglia, the central nervous system resident innate immune cells, cluster around Aß plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aß plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aß plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes. In addition, patch-clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K+ currents in plaque-associated but not plaque-distant microglia. Within the subgroup of plaque-associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single-cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in- and outward K+ conductance in plaque-associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Microglía/patología , Microglía/fisiología , Placa Amiloide/patología , Placa Amiloide/fisiopatología , Animales , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Cationes Monovalentes/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Potenciales de la Membrana/fisiología , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Potasio/metabolismo , Canales de Potasio/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Neurogenesis is sustained throughout life in the mammalian brain, supporting hippocampus-dependent learning and memory. Its permanent alteration by status epilepticus (SE) is associated with learning and cognitive impairments. The mechanisms underlying the initiation of altered neurogenesis after SE are not understood. Glial fibrillary acidic protein-positive radial glia (RG)-like cells proliferate early after SE, but their proliferation dynamics and signaling are largely unclear. We have previously reported a polarized distribution of AMPA receptors (AMPARs) on RG-like cells in vivo and postulated that these may signal their proliferation. Here, we examined the acute effects of kainate on hippocampal precursor cells in vitro and in kainate-induced SE on proliferating and quiescent clones of 5-bromo-2-deoxyuridine prelabeled hippocampal precursors in vivo. In vitro, we found that 5 µM kainate shortened the cell cycle time of RG-like cells via AMPAR activation and accelerated cell cycle re-entry of their progeny. It also shifted their fate choice expanding the population of RG-like cells and reducing the population of downstream amplifying neural progenitors. Kainate enhanced the survival of all precursor cell subtypes. Pharmacologically, kainate's proliferative and survival effects were abolished by AMPAR blockade. Functional AMPAR expression was confirmed on RG-like cells in vitro. In agreement with these observations, kainate/seizures enhanced the proliferation and expansion predominantly of constitutively cycling RG-like cell clones in vivo. Our results identify AMPARs as key potential players in initiating the proliferation of dentate RG-like cells and unravel a possible receptor target for modifying the radial glia-like cell response to SE.
Asunto(s)
Proliferación Celular/fisiología , Hipocampo/citología , Neuroglía/patología , Receptores AMPA/metabolismo , Convulsiones/patología , Células Madre/patología , Animales , Animales Recién Nacidos , Benzodiazepinas/farmacología , Muerte Celular/genética , Células Cultivadas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Kaínico/farmacología , Antígeno Ki-67/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Proteínas del Tejido Nervioso/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Receptores AMPA/genéticaRESUMEN
The contribution of glial cells to normal and impaired hippocampal function is increasingly being recognized, although important questions as to the mechanisms that these cells use for their crosstalk with neurons and capillaries are still unanswered or lead to controversy. Astrocytes in the hippocampus are morphologically variable and a single cell contacts with its processes more than 100,000 synapses. They predominantly express inward rectifier K+ channels and transporters serving homeostatic function but may also release gliotransmitters to modify neuronal signaling and brain circulation. Intracellular Ca2+ transients are key events in the interaction of astrocytes with neurons and the vasculature. Hippocampal NG2 glia represent a population of cells with proliferative capacity throughout adulthood. Intriguingly, they receive direct synaptic input from pyramidal neurons and interneurons and express a multitude of ion channels and receptors. Despite in-depth knowledge about the features of these transmembrane proteins, the physiological impact of NG2 glial cells and their synaptic input remain nebulous. Because of the low abundance of oligodendrocytes in the hippocampus, limited information is available about their specific properties. Given the multitude of signaling molecules expressed by the various types of hippocampal glial cells (and because of space constraints), we focus, in this review, on those properties that are considered key for the interaction of the respective cell type with its neighborhood.
Asunto(s)
Hipocampo/fisiología , Neuroglía/citología , Neuroglía/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Señalización del Calcio , Comunicación Celular , Interneuronas/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Neurológicos , Oligodendroglía/metabolismo , Células Piramidales/metabolismo , Sinapsis/metabolismoRESUMEN
Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of â¼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.
Asunto(s)
Adenosina Trifosfato/metabolismo , Astrocitos/citología , Uniones Comunicantes/metabolismo , Ácido Glutámico/metabolismo , Transducción de Señal/fisiología , Sodio/metabolismo , Sistema de Transporte de Aminoácidos X-AG/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Astrocitos/efectos de los fármacos , Conexina 30/deficiencia , Conexina 30/genética , Conexina 43/deficiencia , Conexina 43/genética , Ácido D-Aspártico/farmacología , Femenino , Uniones Comunicantes/efectos de los fármacos , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons.
Asunto(s)
Antígenos/genética , Hipocampo/citología , Neuroglía/citología , Neuronas/citología , Proteoglicanos/genética , Sinapsis/metabolismo , Transmisión Sináptica/genética , Animales , Ácido Glutámico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Receptores AMPA/metabolismoRESUMEN
NG2 cells, a main pool of glial progenitors, express γ-aminobutyric acid A (GABA(A)) receptors (GABA(A)Rs), the functional and molecular properties of which are largely unknown. We recently reported that transmission between GABAergic interneurons and NG2 cells drastically changes during development of the somatosensory cortex, switching from synaptic to extrasynaptic communication. Since synaptic and extrasynaptic GABA(A)Rs of neurons differ in their subunit composition, we hypothesize that GABA(A)Rs of NG2 cells undergo molecular changes during cortical development accompanying the switch of transmission modes. Single-cell RT-PCR and the effects of zolpidem and α5IA on evoked GABAergic currents reveal the predominance of functional α1- and α5-containing GABA(A)Rs at interneuron-NG2 cell synapses in the second postnatal week, while the α5 expression declines later in development when responses are exclusively extrasynaptic. Importantly, pharmacological and molecular analyses demonstrate that γ2, a subunit contributing to the clustering of GABA(A)Rs at postsynaptic sites in neurons, is down-regulated in NG2 cells in a cell type-specific manner in concomitance with the decline of synaptic activity and the switch of transmission mode. In keeping with the synaptic nature of γ2 in neurons, the down-regulation of this subunit is an important molecular hallmark of the change of transmission modes between interneurons and NG2 cells during development.
Asunto(s)
Neocórtex/crecimiento & desarrollo , Células-Madre Neurales/fisiología , Oligodendroglía/fisiología , Receptores de GABA-A/metabolismo , Sinapsis/fisiología , Animales , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Regulación hacia Abajo , Estimulación Eléctrica , Agonistas de Receptores de GABA-A/farmacología , Interneuronas/efectos de los fármacos , Interneuronas/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones Transgénicos , Neocórtex/efectos de los fármacos , Neocórtex/fisiología , Células-Madre Neurales/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Piridinas/farmacología , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/fisiología , Sinapsis/efectos de los fármacos , Zolpidem , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.
Asunto(s)
Astrocitos/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Hipocampo/metabolismo , Neocórtex/metabolismo , Oligodendroglía/metabolismo , Tálamo/metabolismo , Animales , Conexina 30 , Femenino , Hipocampo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Neocórtex/citología , Red Nerviosa/citología , Red Nerviosa/metabolismo , Tálamo/citologíaRESUMEN
Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/crecimiento & desarrollo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células HeLa , Humanos , Ratones , Neuronas/metabolismo , Isoformas de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Receptor EphA4/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , beta Catenina/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Panglial networks are essential for normal physiology in the CNS, and the function of distinct connexins participating in these networks is not well understood. We generated Connexin32 (Cx32)-deficient mice with additional deletion of astrocytic Cx43 to explore the role of both connexins in panglial networks. Cx43/Cx32 double knock-out (dKO) mice revealed strong microglial activation in corpus callosum and cingulum along with severe astrogliosis and scar formation. In addition, most of the fine myelinated fibers projecting from the corpus callosum into the cortex were lost. Myelin loss was caused by a strong decrease of oligodendrocytes in the cingulum of Cx43/Cx32dKO mice. Immunoblot analyses using newly generated specific Cx47 antibodies revealed that oligodendrocytic Cx47 is phosphorylated in vivo depending on astrocytic Cx43 expression. In Cx43-deficient mice, Cx47 protein levels were strongly decreased, whereas Cx47 mRNA levels were not altered. Using Cx43G138R/Cx30KO mice, we show that Cx47 expression depends on the presence of astrocytic Cx43 protein and that its gap junctional channel function is not necessary for Cx47 stabilization. In consequence, Cx43/Cx32dKO mice additionally lack Cx47 expression and therefore cannot form oligodendrocytic gap junctions, which explains the phenotypic similarities to Cx32/Cx47dKO mice. Our findings provide strong evidence that phosphorylation and stability of oligodendrocytic Cx47 proteins is dependent on astrocytic Cx43 expression. These results further unravel the complexity of panglial networks and show that results of previous studies using astrocytic Cx43-deficient mice have to be reconsidered.
Asunto(s)
Astrocitos/fisiología , Conexina 43/metabolismo , Conexinas/metabolismo , Oligodendroglía/fisiología , Factores de Edad , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Nervioso Central/citología , Conexina 43/genética , Conexinas/genética , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vaina de Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Factor de Transcripción 2 de los Oligodendrocitos , Fosforilación , ARN Mensajero/metabolismo , Proteína beta1 de Unión ComunicanteRESUMEN
NG2 cells are equipped with transmitter receptors and receive direct synaptic input from glutamatergic and GABAergic neurons. The functional impact of these neuron-glia synapses is still unclear. Here, we combined functional and molecular techniques to analyze properties of GABA(A) receptors in NG2 cells of the juvenile mouse hippocampus. GABA activated slowly desensitizing responses in NG2 cells, which were mimicked by muscimol and inhibited by bicuculline. To elucidate the subunit composition of the receptors we tested its pharmacological properties. Coapplication of pentobarbital, benzodiazepines, and zolpidem all significantly increased the GABA-evoked responses. The presence of small tonic currents indicated the presence of extrasynaptic GABA(A) receptors. To further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of NG2 cells. The subunits α1, α2, ß3, γ1, and γ2 were most abundantly expressed, matching properties resulting from pharmacological characterization. Importantly, lack of the γ2-subunit conferred a high Zn²âº sensitivity to the GABA(A) receptors of NG2 cells. Judging from the zolpidem sensitivity, postsynaptic GABA(A) receptors in NG2 cells contain the γ2-subunit, in contrast to extrasynaptic receptors, which were not modulated by zolpidem. To determine the effect of GABA(A) receptor activation on membrane potential, perforated patch recordings were obtained from NG2 cells. In the current-clamp mode, GABA depolarized the cells to approximately -30 mV, indicating a higher intracellular Clâ» concentration (â¼50 mM) than previously reported. GABA-induced depolarization in NG2 cells might trigger Ca²âº influx through voltage-activated Ca²âº channels.
Asunto(s)
Hipocampo/metabolismo , Neuroglía/metabolismo , Subunidades de Proteína/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Benzodiazepinas/farmacología , Bicuculina/farmacología , Diazepam/farmacología , Agonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Muscimol/farmacología , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Pentobarbital/farmacología , Subunidades de Proteína/genética , Receptores de GABA-A/genética , Sinapsis/efectos de los fármacos , Sinapsis/genética , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
RATIONALE: The gap junctional protein connexin (Cx) 45 is strongly expressed in the early embryonic myocardium. In the adult hearts of mice and humans, the expression mainly is restricted to the cardiac conduction system. Cx45 plays an essential role for development and function of the embryonic heart because general and cardiomyocyte-directed deficiencies of Cx45 in mice lead to embryonic lethality attributable to morphological and functional cardiovascular defects. The function of Cx45 in the adult mouse has not yet been cleared. OBJECTIVE: To clarify the function of Cx45 in the adult mouse heart. METHODS AND RESULTS: To circumvent the embryonic lethality resulting from Cx45 deficiency, mice were generated in which deletion of Cx45 specifically was induced in cardiomyocytes of adult mice. These Cx45-deficient mice were viable but showed a decrease in atrioventricular nodal conductivity. In addition, the Cx30.2 protein that is coexpressed with Cx45 in the cardiac conduction system was posttranscriptionally reduced by 70% in mutant hearts. Furthermore, deletion of both Cx45 and Cx30.2 resulted in viable mice that, however, showed stronger impairment of atrioventricular nodal conduction than the single Cx45-deficient mice. CONCLUSIONS: Cx45 is required for optimal impulse propagation in the atrioventricular node and stabilizes the level of the coexpressed Cx30.2 protein in the adult mouse heart. In contrast to the embryo, Cx45 is not essential for the viability of adult mice.
Asunto(s)
Nodo Atrioventricular/embriología , Nodo Atrioventricular/metabolismo , Conexinas/fisiología , Corazón/embriología , Corazón/fisiología , Animales , Conexinas/deficiencia , Conexinas/genética , Sistema de Conducción Cardíaco/embriología , Sistema de Conducción Cardíaco/metabolismo , Ratones , Ratones NoqueadosRESUMEN
In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and â¼40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.
Asunto(s)
Sistema Nervioso Central/citología , Uniones Comunicantes/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Vaina de Mielina/fisiología , Neuroglía/citología , Oligodendroglía/citología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Actinas/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biofisica , Sistema Nervioso Central/crecimiento & desarrollo , Conexina 30 , Conexinas/deficiencia , Conexinas/metabolismo , Estimulación Eléctrica , Conducta Exploratoria/fisiología , Uniones Comunicantes/ultraestructura , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Habituación Psicofisiológica/genética , Técnicas In Vitro , Estimación de Kaplan-Meier , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Actividad Motora/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/fisiología , Neuroglía/ultraestructura , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/fisiología , Oligodendroglía/ultraestructura , Técnicas de Placa-Clamp , Desempeño Psicomotor/fisiología , ARN Mensajero/metabolismo , Reconocimiento en Psicología/fisiología , Tinción con Nitrato de Plata , Estadísticas no Paramétricas , Proteína beta1 de Unión ComunicanteRESUMEN
Glial fibrillary acidic protein (GFAP)-positive astrocytes with radial processes [radial glia (RG)-like cells] in the postnatal dentate gyrus share many of the characteristics of embryonic radial glia and appear to act as precursor cells for adult dentate neurogenesis, a process important for pattern separation and hippocampus-dependent learning. Although much work has delineated the mechanisms underlying activity-neurogenesis coupling via gamma-amino butyric acid (GABA)ergic neurotransmission on GFAP-negative transient-amplifying cells and neuroblasts, little is known regarding the effects of neurotransmitters on RG-like cells. Conflicting evidence exists for both GABA and glutamate receptors on these cells. Here, using GFAP reporter mice, we show that the somatic membrane of RG-like cells carries GABAA receptors and glutamate transporters but not ionotropic glutamate receptors, whereas 2-amino-3-(hydroxyl-5-methylisoxazole-4-yl) propionic acid (AMPA) and GABAA receptors are expressed on the processes of these cells. Almost all RG-like cells expressed the GluA2 subunit, which restricts the Ca(2+) permeability of AMPA receptors. The glial GABAA receptors mainly comprised α2/α4, ß1, and γ1/γ3. The selective presence of AMPA receptors on the radial processes may be important for sensing and responding to local activity-driven glutamate release and supports the concept that RG-like astrocytes are composed of functional and structural domains.
Asunto(s)
Giro Dentado/citología , Neuroglía/fisiología , Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Bicuculina/farmacología , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , GABAérgicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas In Vitro , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Muscimol/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Tetraetilamonio/farmacologíaRESUMEN
In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.
Asunto(s)
Neoplasias Encefálicas/genética , Adhesiones Focales/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Glioblastoma/genética , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Hibridación Genómica Comparativa , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Modelos Animales de Enfermedad , Femenino , Adhesiones Focales/inmunología , Adhesiones Focales/metabolismo , Gadolinio , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Técnicas In Vitro , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neuroglía/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Transfección , Ensayo de Tumor de Célula Madre/métodos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Vinculina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Astrocytes are endowed with the machinery to sense and respond to neuronal activity. Recent work has demonstrated that astrocytes play important physiological roles in the CNS, e.g., they synchronize action potential firing, ensure ion homeostasis, transmitter clearance and glucose metabolism, and regulate the vascular tone. Astrocytes are abundantly coupled through gap junctions, which is a prerequisite to redistribute elevated K(+) from sites of excessive neuronal activity to sites of lower extracellular K(+) concentration. Recent studies identified dysfunctional astrocytes as crucial players in epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial inwardly rectifying K(+) (Kir) channels, particularly Kir4.1, which is suspected to entail impaired K(+) buffering. Gap junctions in astrocytes appear to play a dual role: on the one hand they counteract the generation of hyperactivity by facilitating clearance of elevated extracellular K(+) levels while in contrast, they constitute a pathway for energetic substrate delivery to fuel neuronal (hyper)activity. Recent work suggests that astrocyte dysfunction is causative of the generation or spread of seizure activity. Thus, astrocytes should be considered as promising targets for alternative antiepileptic therapies.