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Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.
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AIMS: Cytoplasmic dynein heavy chain (DYNC1H1) is a multi-subunit protein complex that provides motor force for movement of cargo on microtubules and traffics them back to the soma. In humans, mutations along the DYNC1H1 gene result in intellectual disabilities, cognitive delays, and neurologic and motor deficits. The aim of the study was to generate a mouse model to a newly identified de novo heterozygous DYNC1H1 mutation, within a functional ATPase domain (c9052C > T(P3018S)), identified in a child with motor deficits, and intellectual disabilities. RESULTS: P3018S heterozygous (HET) knockin mice are viable; homozygotes are lethal. Metabolic and EchoMRI™ testing show that HET mice have a higher metabolic rate, are more active, and have less body fat compared to wildtype mice. Neurobehavioral studies show that HET mice perform worse when traversing elevated balance beams, and on the negative geotaxis test. Immunofluorescent staining shows neuronal migration abnormalities in the dorsal and lateral neocortex with heterotopia in layer I. Neuron-subtype specific transcription factors CUX1 and CTGF identified neurons from layers II/III and VI respectively in cortical layer I, and abnormal pyramidal neurons with MAP2+ dendrites projecting downward from the pial surface. CONCLUSION: The HET mice are a good model for the motor deficits seen in the child, and highlights the importance of cytoplasmic dynein in the maintenance of cortical function and dendritic orientation relative to the pial surface. Our results are discussed in the context of other dynein mutant mice and in relation to clinical presentation in humans with DYNC1H1 mutations.
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Dineínas Citoplasmáticas , Mutación , Animales , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Ratones , Mutación/genética , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ratones Transgénicos , Masculino , Discapacidad Intelectual/genética , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
ABSTRACT: Binge drinking is a risk factor for cardiac arrhythmias, known as the holiday heart syndrome. Atrial fibrillation (AF) is the most frequently diagnosed arrhythmia in this condition. Recent reports indicated that cardiac ryanodine receptor (RyR2) dysfunction and Ca 2+ leak contribute to alcohol-enhanced AF. In this study, we investigated whether stabilizing RyR2 with dantrolene treatment can prevent alcohol-enhanced AF in rats. A binge drinking rat model was established with alcohol (2 g /kg, IP) delivered once every other day for 4 times. The study consisted of following 3 groups: control group (n = 9), binge alcohol group (n = 10), and binge alcohol + dantrolene (A+D) group (dantrolene, 10 mg/kg, IP before each alcohol injection, n = 9). Echocardiography, left ventricular hemodynamics, in vivo atrial electrophysiology and AF inducibility test, RyR2 phosphorylation level, and blood norepinephrine level were studied 24 hours after the last injection. Ca 2+ leak in isolated atrial myocytes from control and binge alcohol rats was examined. Binge alcohol significantly increased AF inducibility (1/9 in control vs. 8/9 in binge alcohol group, P < 0.05) and AF duration. Dantrolene treatment significantly reduced both AF inducibility (2/9 in dantrolene group, P < 0.05) and AF duration. Binge alcohol significantly increased Ca 2+ leak in isolated atrial myocytes, which was reduced by dantrolene treatment. Blood norepinephrine,7 RyR2 phosphorylation level, cardiac echocardiography, and left ventricular hemodynamics were not significantly affected 24 hours after binge drinking. In conclusion, stabilizing RyR2 with dantrolene treatment significantly attenuated binge drinking-enhanced AF, suggesting that therapeutic strategies stabilizing RyR2 could be a preventive measure to blunt binge drinking-enhanced AF arrhythmogenesis.
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Fibrilación Atrial , Consumo Excesivo de Bebidas Alcohólicas , Ratas , Animales , Dantroleno/farmacología , Canal Liberador de Calcio Receptor de Rianodina , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/etiología , Fibrilación Atrial/prevención & control , Consumo Excesivo de Bebidas Alcohólicas/complicaciones , Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Etanol , Norepinefrina , Calcio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 µg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.
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Señalización del Calcio/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Hipotiroidismo/tratamiento farmacológico , Triyodotironina/administración & dosificación , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Hipotiroidismo/sangre , Hipotiroidismo/inducido químicamente , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Sarcómeros/metabolismo , Resultado del Tratamiento , Triyodotironina/sangre , Función Ventricular/efectos de los fármacosRESUMEN
There is both anecdotal and quantitative evidence that undergraduate neuroscience education has grown substantially in the US. Therefore, efforts to continue to track changes in undergraduate neuroscience education are important. Here we provide quantitative data that both public and private institutions are creating new undergraduate neuroscience programs. In addition, we demonstrate that the number of graduates from undergraduate neuroscience programs continues to increase compared to graduates from other life sciences programs. These data are important to faculty and administrators at institutions that currently have or seek to establish new undergraduate neuroscience programs.
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Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.
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Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Proteínas Luminiscentes/metabolismo , Conexina 26 , Conexina 30 , Uniones Comunicantes/química , Células HeLa , Humanos , Conformación ProteicaRESUMEN
Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanisms for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrated that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we showed that targeting dCas9 to telomeres using sgTelo impeded DNA replication at telomeres and induced a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigated the genome-wide features of telomeres in the dCas9/sgTelo cells and observed a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-.Consistently, we also observed an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncovered many novel molecular and structural features of the ITCB and demonstrated that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.
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Background: Alcohol and caffeine are the 2 frequently consumed substances in the general population, and the 2 substances are frequently co-consumed. Both substances may increase cardiac arrhythmia risk. However, it is unknown whether alcohol and caffeine co-consumption can synergistically enhance cardiac arrhythmogenesis. Objective: The study sought to investigate whether caffeine and binge drinking synergistically affect cardiac arrhythmogenesis. Methods: A binge drinking rat model (alcohol 2 g/kg, intraperitoneal, every other day for 3 times) was used. Rats (4 months old, both sexes) were randomized into the following 4 groups: binge alcohol-only group (A) (n = 8), nonalcohol, caffeine-only (60 mg/kg, intraperitoneal) group (C) (n = 8), binge alcohol plus caffeine group (A+C) (n = 8), and binge alcohol + caffeine + dantrolene group (A+D) (n = 7, treated with dantrolene 10 mg/kg before each alcohol injection). We also investigated whether alcohol induces Ca2+ sparks and dantrolene treatment attenuates alcohol-induced Ca2+ leak in ventricular myocytes. Results: No arrhythmia was induced with caffeine alone (group C, n = 0 of 8) or alcohol alone (group A, n = 0 of 8). However, alcohol + caffeine induced spontaneous ventricular tachyarrhythmias in all rats (group A+C, n = 8 of 8; P < .001 vs group C or A). Dantrolene prevented ventricular tachyarrhythmia induction in all 7 rats (group A+D, n = 0 of 7; P < .001 vs group A+C). In isolated ventricular myocytes, alcohol significantly increased Ca2+ sparks and dantrolene treatment reduced alcohol-induced Ca2+ sparks. Conclusion: Co-consumption of caffeine and binge drinking synergistically promote spontaneous ventricular tachyarrhythmias in rats. Dantrolene treatment can decrease alcohol-enhanced Ca2+ sparks in vitro and prevented alcohol and caffeine induced ventricular tachyarrhythmias in vivo.
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Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca(2+) signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca(2+) -dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer's disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Neuroglía/metabolismo , Neuroglía/patología , Animales , Humanos , Neuronas/metabolismo , Neuronas/patología , Transmisión Sináptica/fisiologíaRESUMEN
Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood-brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impairment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.
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Conexina 43 , Retinopatía Diabética , Animales , Conexina 43/genética , Conexina 43/metabolismo , Células Endoteliales , Uniones Comunicantes/metabolismo , Ratones , RetinaRESUMEN
The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if BBB integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.
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Acuaporina 4/metabolismo , Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Permeabilidad de la Membrana Celular , Conexina 43/fisiología , Conexinas/metabolismo , Endotelio Vascular/metabolismo , Animales , Acuaporina 4/química , Acuaporina 4/genética , Conexinas/química , Conexinas/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína alfa-5 de Unión ComunicanteRESUMEN
We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.
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Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions, providing exchange of metabolites, signaling molecules, and ionic currents. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.
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Conexinas/metabolismo , Citoplasma/metabolismo , Uniones Comunicantes/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Línea Celular Tumoral , Humanos , Canales Iónicos/metabolismo , Ratones , Transporte de Proteínas/fisiología , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
We profiled the transcriptomes of primary mouse cortical astrocytes cultured alone or co-cultured with immortalized precursor oligodendrocytes (Oli-neu cells). Filters between the cell types prevented formation of hetero-cellular gap junction channels but allowed for free exchange of the two culture media. We previously reported that major functional pathways in the Oli-neu cells are remodeled by the proximity of non-touching astrocytes and that astrocytes and oligodendrocytes form a panglial transcriptomic syncytium in the brain. Here, we present evidence that the astrocyte transcriptome likewise changes significantly in the proximity of non-touching Oli-neu cells. Our results indicate that the cellular environment strongly modulates the transcriptome of each cell type and that integration in a heterocellular tissue changes not only the expression profile but also the expression control and networking of the genes in each cell phenotype. The significant decrease of the overall transcription control suggests that in the co-culture astrocytes are closer to their normal conditions from the brain. The Oli-neu secretome regulates astrocyte genes known to modulate neuronal synaptic transmission and remodels calcium, chemokine, NOD-like receptor, PI3K-Akt, and thyroid hormone signaling, as well as actin-cytoskeleton, autophagy, cell cycle, and circadian rhythm pathways. Moreover, the co-culture significantly changes the gene hierarchy in the astrocytes.
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Astrocitos/fisiología , Transcriptoma , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Astrocitos/ultraestructura , Comunicación Celular , Ciclo Celular/genética , Forma de la Célula , Células Cultivadas , Corteza Cerebral/citología , Ritmo Circadiano/genética , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica/genética , Ontología de Genes , Redes Reguladoras de Genes , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Oligodendrocitos/citología , Transducción de Señal/genéticaRESUMEN
Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.
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Astrocitos/metabolismo , Metabolismo Energético/fisiología , Potenciación a Largo Plazo/fisiología , Neuronas/fisiología , Estrés Psicológico/metabolismo , Adaptación Psicológica/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Glucosa/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Redes y Vías Metabólicas/fisiología , Ratones , Neocórtex/citología , Neocórtex/metabolismo , Técnicas de Placa-ClampRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αV ß3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αV ß3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to ß3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2 × 7R and the low voltage transiently opened T-type calcium channel CaV3.2-1 all reside in close proximity to ß3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:642-652, 2018.
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Canales de Calcio Tipo T/metabolismo , Conexinas/metabolismo , Integrina beta3/metabolismo , Mecanotransducción Celular , Proteínas del Tejido Nervioso/metabolismo , Osteocitos/fisiología , Animales , Línea Celular , Masculino , Ratones Endogámicos C57BL , Receptores Purinérgicos/metabolismoRESUMEN
Gap junctions are cellular contact sites composed of clustered connexin transmembrane proteins that act in dual capacities as channels for direct intercellular exchange of small molecules and as structural adhesion complexes known as gap junction nexuses. Depending on the connexin isoform, the cluster of channels (the gap junction plaque) can be stably or fluidly arranged. Here we used confocal microscopy and mutational analysis to identify the residues within the connexin proteins that determine gap junction plaque stability. We found that stability is altered by changing redox balance using a reducing agent-indicating gap junction nexus stability is modifiable. Stability of the arrangement of connexins is thought to regulate intercellular communication by establishing an ordered supramolecular platform. By identifying the residues that establish plaque stability, these studies lay the groundwork for exploration of mechanisms by which gap junction nexus stability modulates intercellular communication.
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Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Animales , Comunicación Celular/fisiología , Línea Celular , Conexinas/fisiología , Cisteína/metabolismo , Citoplasma , Citosol/metabolismo , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Uniones Comunicantes/fisiología , Células HeLa , Humanos , Proteínas de la Membrana , Dominios Proteicos , Isoformas de Proteínas , RatasRESUMEN
In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P < 0.05), the frequency of ATP-evoked transient (P < 0.001) and full-fusion exocytotic (P < 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca2+ activity in astrocytes by ~70 % (P < 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca2+ excitability.
Asunto(s)
Adenosina Trifosfato/farmacología , Astrocitos/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Vesículas Citoplasmáticas/metabolismo , Exocitosis/efectos de los fármacos , Ketamina/farmacología , Animales , Astrocitos/efectos de los fármacos , Biomarcadores/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Concentración de Iones de Hidrógeno , Fusión de Membrana/efectos de los fármacos , Purinas/metabolismo , Ratas Wistar , Proteínas SNARE/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and associated with legal and ethical constrains. Various biochemical, optical and mechanical methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin's enzymatic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 derivative. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concentration was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophysical mechanisms for biopolymer transportation.