RESUMEN
Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.
Asunto(s)
Enfermedad de Alexander , Ratones , Animales , Enfermedad de Alexander/metabolismo , Enfermedad de Alexander/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Astrocitos/metabolismo , Microglía/metabolismo , Clopidogrel/metabolismo , Calcio/metabolismo , Progresión de la Enfermedad , Adenosina Trifosfato/metabolismoRESUMEN
Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.
Asunto(s)
Lipopolisacáridos , Microglía , Ratones , Animales , Lipopolisacáridos/toxicidad , Macrófagos , Encéfalo , Infarto de la Arteria Cerebral Media , InflamaciónRESUMEN
Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Trasplante de Células Madre , Animales , Encéfalo/fisiología , Diferenciación Celular/fisiología , Humanos , Ratones , Microglía , Nariz , Trasplante de Células Madre/métodosRESUMEN
Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.
Asunto(s)
Astrocitos , Receptor de Adenosina A2B , Receptor del Glutamato Metabotropico 5 , Adenosina/metabolismo , Adenosina/farmacología , Animales , Astrocitos/metabolismo , Proteínas Portadoras/metabolismo , Ratones , Receptor de Adenosina A2B/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismoRESUMEN
In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca2+ signals in pathological brains and found that reactive astrocytes share abnormal Ca2+ signals, even in different types of diseases. In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca2+ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca2+ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca2+ signals. Because the abnormal astrocytic Ca2+ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP3RII), we reanalyzed these pathological events using IP3RII-deficient mice and found that all abnormal Ca2+ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca2+ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca2+ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.
Asunto(s)
Enfermedad de Alexander , Astrocitos , Señalización del Calcio , Calcio , Animales , Enfermedad de Alexander/metabolismo , Astrocitos/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , RatonesRESUMEN
Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+ signals. Astrocytes display numerous spontaneous microdomain Ca2+ signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+ signals. Instead, Ca2+ waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.SIGNIFICANCE STATEMENT Astrocytes are thought to enwrap synapses with their processes to receive neuronal information via Gq-protein coupled receptors (GqPCRs). Astrocyte processes display numerous microdomain Ca2+ signals that occur spontaneously. To determine whether GqPCRs play a role in microdomain Ca2+ signals and the timeframe in which astrocytes sense neuronal information, we engineered mice whose astrocytes specifically overexpress the P2Y1 receptor, a major GqPCR in astrocytes. We found that overexpression of P2Y1 receptors in astrocytes did not increase microdomain Ca2+ signals in astrocyte processes but caused Ca2+ wavelike signals. Our data indicate that spontaneous microdomain Ca2+ signals do not require activation of P2Y1 receptors.
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Astrocitos/fisiología , Señalización del Calcio/fisiología , Receptores Purinérgicos P2Y1/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Señalización del Calcio/efectos de los fármacos , Femenino , Hipocampo/fisiología , Masculino , Ratones , Ratones Transgénicos , Probenecid/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/genética , Sinapsis/fisiologíaRESUMEN
Astrocytes are abundant cells in the brain that regulate multiple aspects of neural tissue homeostasis by providing structural and metabolic support to neurons, maintaining synaptic environments and regulating blood flow. Recent evidence indicates that astrocytes also actively participate in brain functions and play a key role in brain disease by responding to neuronal activities and brain insults. Astrocytes become reactive in response to injury and inflammation, which is typically described as hypertrophy with increased expression of glial fibrillary acidic protein (GFAP). Reactive astrocytes are frequently found in many neurological disorders and are a hallmark of brain disease. Furthermore, reactive astrocytes may drive the initiation and progression of disease processes. Recent improvements in the methods to visualize the activity of reactive astrocytes in situ and in vivo have helped elucidate their functions. Ca2+ signals in reactive astrocytes are closely related to multiple aspects of disease and can be a good indicator of disease severity/state. In this review, we summarize recent findings concerning reactive astrocyte Ca2+ signals. We discuss the molecular mechanisms underlying aberrant Ca2+ signals in reactive astrocytes and the functional significance of aberrant Ca2+ signals in neurological disorders.
Asunto(s)
Astrocitos/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Animales , Humanos , Ratones , RatasRESUMEN
Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca2+ signals "AxCa signals", which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.
Asunto(s)
Enfermedad de Alexander/metabolismo , Astrocitos/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alexander/patología , Animales , Astrocitos/patología , Cationes Bivalentes/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
AIMS: The sensation of bladder fullness (SBF) is triggered by the release of ATP. Therefore, the aim of this study was to investigate whether time-dependent changes in the levels of stretch-released ATP in mouse primary-cultured urothelial cells (MPCUCs) is regulated by circadian rhythm via clock genes. METHODS: MPCUCs were derived from wild-type and Clock mutant mice (ClockΔ19/Δ19 ), presenting a nocturia phenotype. They were cultured in elastic silicone chambers. Stretch-released ATP was quantified every 4 h by ATP photon count. An experiment was also performed to determine whether ATP release correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Connexin26 (Cx26) in MPCUCs regulated by clock genes with circadian rhythms. MPCUCs were treated with carbenoxolone, an inhibitor of gap junction protein; were derived from VNUT-KO mice; or treated with Piezo1-siRNA, TRPV4-siRNA, and Cx26-siRNA. RESULTS: Stretch-released ATP showed time-dependent changes in wild-type mice and correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Cx26. However, these rhythms were disrupted in ClockΔ19/Δ19 mice. Carbenoxolone eliminated the rhythmicity of ATP release in wild-type mice. However, time-dependent ATP release changes were maintained when a single gene was deficient such as VNUT-KO, Piezo1-, TRPV4-, and Cx26-siRNA. CONCLUSIONS: ATP release in the bladder urothelium induces SBF and may have a circadian rhythm regulated by the clock genes. In the bladder urothelium, clock gene abnormalities may disrupt circadian ATP release by inducing Piezo1, TRPV4, VNUT, and Cx26. All these genes can trigger nocturia.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/fisiología , Urotelio/metabolismo , Animales , Carbenoxolona/farmacología , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/genética , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Nocturia/genética , Proteínas de Transporte de Nucleótidos/genética , Cultivo Primario de Células , Urotelio/citologíaRESUMEN
AIMS: To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm. METHODS: The gene expression rhythms of the clock genes, mechano-sensors such as Piezo1 and TRPV4, ATP release mediated molecules (ARMM) such as Cx26 and VNUT were investigated in mouse primary cultured urothelial cells (UCs) of wild-type (WT) and Clock mutant (ClockΔ19/Δ19 ) mice using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting analysis. The long-term oscillation of the clock genes in UC was investigated by measuring bioluminescence from UC isolated from Period2luciferase knock-in mice (Per2::luc) and Per2::luc with ClockΔ19/Δ19 using a luminometer. The mRNA expression rhythms after treatment with Clock short interfering RNA (siRNA) were also measured to compare differences between Clock point mutations and Clock deficiency. RESULTS: The UCs from WT mice showed the time-dependent gene expressions for clock genes, mechano-sensors, and ARMM. The abundances of the products of these genes also correlated with the mRNA expression rhythms in UCs. The bioluminescence of Per2::Luc in UCs showed a circadian rhythm. By contrast, all the gene expressions rhythms observed in WT mice were abrogated in the ClockΔ19/Δ19 mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation. CONCLUSIONS: We demonstrated that the time-dependent gene expressions, including clock genes, mechano-sensors, and ARMM, were reproducible in UCs. These findings demonstrated that UCs have the potential to progress research into the circadian functions of the lower urinary tract regulated by clock genes.
Asunto(s)
Proteínas CLOCK/metabolismo , Conexina 26/metabolismo , Canales Iónicos/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Canales Catiónicos TRPV/metabolismo , Urotelio/metabolismo , Animales , Proteínas CLOCK/genética , Células Cultivadas , Ritmo Circadiano/genética , Conexina 26/genética , Expresión Génica , Canales Iónicos/genética , Ratones , Proteínas de Transporte de Nucleótidos/genética , Canales Catiónicos TRPV/genética , Urotelio/citologíaRESUMEN
AIMS: The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (ClockΔ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP. METHODS: Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 ClockΔ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods. RESULTS: No significant differences were observed in behavior patterns between ClockΔ19/Δ19 and WT mice. ClockΔ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in ClockΔ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in ClockΔ19/Δ19 mice than in WT mice. CONCLUSIONS: We demonstrated that ClockΔ19/Δ19 mice showed the phenotype of NOC/NP. The ClockΔ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas CLOCK/genética , Ritmo Circadiano/genética , Nocturia/genética , Poliuria/genética , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Müller cells, the primary macroglia of the retina, support various functions of retinal ganglion cells (RGCs). Here, we demonstrate a nucleotide-mediated communication between these two types of cells, by which Müller cells control neurite outgrowth of RGCs by activation of P2 receptors such as P2Y6 . Cultured mouse RGCs had significantly enhanced neurite outgrowth when cultured with either cultured mouse Müller cells or conditioned medium derived from Müller cells, and this was completely inhibited by the nucleotide-degrading enzyme, apyrase. This increase in outgrowth was mimicked by exogenously applied nucleotides such as ATP, uridine triphosphate, and uridine diphosphate. Pharmacological and genetic analysis revealed that P2Y6 receptor in RGCs was responsible for the increased neurite outgrowth. P2Y6 receptor was expressed in the ganglion cell layer of the retina and in RGC primary cultures. High performance liquid chromatography has revealed that Müller cells constitutively release uridine triphosphate, which is immediately metabolized into uridine diphosphate, an endogenous agonist for P2Y6 receptor. In the in vitro ocular hypertension model (i.e., glaucoma model), neurite outgrowth in RGCs was significantly reduced, which was associated with a decrease in P2Y6 receptors. Taken together, Müller cells control neurite outgrowth of RGCs by activating P2 receptors such as P2Y6 receptor, and the receptor expression level might be down-regulated in glaucoma. Müller cells support various functions of retina including those of retinal ganglion cells (RGCs). Here, we report an importance of nucleotide-mediated communication between these two types of cells. Müller cells were found to release uridine diphosphate (UTD), uridine triphosphate (UTP), and activate P2Y6 receptors in RGCs, which was essential for neurite outgrowth of RGCs. In addition, P2Y6 receptors in RGCs were reduced in a glaucoma model in vitro, suggesting an involvement of their dysfunction in the pathogenesis of glaucoma.
RESUMEN
Astrocytes are found throughout the brain where they make extensive contacts with neurons and synapses. Astrocytes are known to display intracellular Ca(2+) signals and release signaling molecules such as D-serine into the extracellular space. However, the role(s) of astrocyte Ca(2+) signals in hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, remains unclear. Here, we explored a recently discovered novel TRPA1 channel-mediated transmembrane Ca(2+) flux pathway in astrocytes. Specifically, we determined whether block or genetic deletion of TRPA1 channels affected LTP of Schaffer collateral to CA1 pyramidal neuron synapses. Using pharmacology, TRPA1(-/-) mice, imaging, electrophysiology, and D-serine biosensors, our data indicate that astrocyte TRPA1 channels contribute to basal Ca(2+) levels and are required for constitutive D-serine release into the extracellular space, which contributes to NMDA receptor-dependent LTP. The findings have broad relevance for the study of astrocyte-neuron interactions by demonstrating how TRPA1 channel-mediated fluxes contribute to astrocyte basal Ca(2+) levels and neuronal function via constitutive D-serine release.
Asunto(s)
Astrocitos/metabolismo , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Potenciación a Largo Plazo/fisiología , Microdominios de Membrana/metabolismo , Serina/metabolismo , Acetanilidas/farmacología , Animales , Astrocitos/citología , Astrocitos/ultraestructura , Región CA3 Hipocampal/citología , Células Cultivadas , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas HSP90 de Choque Térmico , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/deficiencia , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Potenciación a Largo Plazo/genética , Microdominios de Membrana/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Técnicas de Placa-Clamp , Purinas/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , beta-Alanina/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Reactive astrocytes play a pivotal role in the pathogenesis of neurological diseases; however, their functional phenotype and the downstream molecules by which they modify disease pathogenesis remain unclear. Here, we genetically increase P2Y1 receptor (P2Y1R) expression, which is upregulated in reactive astrocytes in several neurological diseases, in astrocytes of male mice to explore its function and the downstream molecule. This astrocyte-specific P2Y1R overexpression causes neuronal hyperexcitability by increasing both astrocytic and neuronal Ca2+ signals. We identify insulin-like growth factor-binding protein 2 (IGFBP2) as a downstream molecule of P2Y1R in astrocytes; IGFBP2 acts as an excitatory signal to cause neuronal excitation. In neurological disease models of epilepsy and stroke, reactive astrocytes upregulate P2Y1R and increase IGFBP2. The present findings identify a mechanism underlying astrocyte-driven neuronal hyperexcitability, which is likely to be shared by several neurological disorders, providing insights that might be relevant for intervention in diverse neurological disorders.
Asunto(s)
Astrocitos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Neuronas , Receptores Purinérgicos P2Y1 , Regulación hacia Arriba , Animales , Astrocitos/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neuronas/metabolismo , Masculino , Ratones , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y1/genética , Ratones Transgénicos , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/fisiopatología , Ratones Endogámicos C57BL , Humanos , Señalización del Calcio , Modelos Animales de EnfermedadRESUMEN
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Asunto(s)
Señalización del Calcio , Colorantes Fluorescentes/química , Fluorometría/métodos , Proteínas Fluorescentes Verdes/química , Neuroimagen/métodos , Neuronas/química , Péptidos/química , Transmisión Sináptica , Animales , Astrocitos/química , Astrocitos/ultraestructura , Caenorhabditis elegans , Cristalografía por Rayos X , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Colorantes Fluorescentes/análisis , Genes Sintéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Células HEK293/química , Células HEK293/ultraestructura , Hipocampo/química , Hipocampo/citología , Humanos , Larva , Rayos Láser , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Neuromuscular/química , Unión Neuromuscular/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Neurópilo/química , Neurópilo/fisiología , Neurópilo/ultraestructura , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Neuronas Receptoras Olfatorias/ultraestructura , Péptidos/análisis , Péptidos/genética , Estimulación Luminosa , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Células Bipolares de la Retina/química , Células Bipolares de la Retina/fisiología , Células Bipolares de la Retina/ultraestructura , Pez Cebra/crecimiento & desarrolloRESUMEN
Astrocytes are present throughout the brain and intimately interact with neurons and blood vessels. Three decades of research have shown that astrocytes reciprocally communicate with neurons and other non-neuronal cells in the brain and dynamically regulate cell function. Astrocytes express numerous receptors for neurotransmitters, neuromodulators, and cytokines and receive information from neurons, other astrocytes, and other non-neuronal cells. Among those receptors, the main focus has been G-protein coupled receptors. Activation of G-protein coupled receptors leads to dramatic changes in intracellular signaling (Ca2+ and cAMP), which is considered a form of astrocyte activity. Methodological improvements in measurement and manipulation of astrocytes have advanced our understanding of the role of astrocytes in circuits and have begun to reveal unexpected functions of astrocytes in behavior. Recent studies have suggested that astrocytic activity regulates behavior flexibility, such as coping strategies for stress exposure, and plays an important role in behaviors related to emotion and motivation. Preclinical evidence suggests that impairment of astrocytic function contributes to psychiatric diseases, especially major depression. Here, we review recent progress on the role of astrocytes in behaviors related to emotion and motivation.
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Astrocitos , Motivación , Astrocitos/fisiología , Neuronas/fisiología , Receptores Acoplados a Proteínas G , EmocionesRESUMEN
Extracellular ATP and adenosine are neuromodulators that regulate numerous neuronal functions in the brain. Neuronal activity and brain insults such as ischemic and traumatic injury upregulate these neuromodulators, which exert their effects by activating purinergic receptors. In addition, extracellular ATP/adenosine signaling plays a pivotal role in the pathogenesis of neurological diseases. Virtually every cell type in the brain contributes to the elevation of ATP/adenosine, and various mechanisms underlying this increase have been proposed. Extracellular adenosine is thought to be mainly produced via the degradation of extracellular ATP. However, adenosine is also released from neurons and glia in the brain. Therefore, the regulation of extracellular ATP/adenosine in physiological and pathophysiological conditions is likely far more complex than previously thought. To elucidate the complex mechanisms that regulate extracellular ATP/adenosine levels, accurate methods of assessing their spatiotemporal dynamics are needed. Several novel techniques for acquiring spatiotemporal information on extracellular ATP/adenosine, including fluorescent sensors, have been developed and have started to reveal the mechanisms underlying the release, uptake and degradation of ATP/adenosine. Here, we review methods for analyzing extracellular ATP/adenosine dynamics as well as the current state of knowledge on the spatiotemporal dynamics of ATP/adenosine in the brain. We focus on the mechanisms used by neurons and glia to cooperatively produce the activity-dependent increase in ATP/adenosine and its physiological and pathophysiological significance in the brain.
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AIMS: Bladder function is regulated by clock genes and dysregulation of circadian bladder function can cause nocturia. The blood concentration of palmitoylethanolamide (PEA), a fatty acid metabolite, changes with circadian rhythm. Clock gene abnormalities demonstrate the highest PEA levels during the sleep phase. PEA is a GPR55 agonist that influences urination; therefore, increased PEA during the sleep phase may cause nocturia. Herein, we investigated the function of GPR55 to evaluate the relationship between GPR55 and nocturia that evoked higher PEA during the sleep phase in patients with circadian rhythm disorders. MAIN METHODS: Male C57BL/6 mice were used. GPR55 localization was evaluated by immunofluorescence staining, qRT-PCR, and western blotting. Variations in PEA-induced intracellular Ca2+ concentrations were measured in primary cultured mouse urothelial cells (UCs) using Ca2+ imaging. PEA-induced NGF and PGI2 release in UCs was measured by ELISA. The micturition reflex pathway after PEA administration was evaluated using immunofluorescence staining. KEY FINDINGS: GPR55 was predominant in the UC layer. PEA induced release of Ca2+ from the endoplasmic reticulum into the UC cytoplasm. ELISA and immunofluorescence staining revealed that NGF and PGI2 were released from bladder UCs, stimulated the pontine micturition center in mice, and induced nocturia. SIGNIFICANCE: The loss of regular circadian metabolizing rhythm in fatty acids causes higher blood PEA levels during the sleep phase. Binding of PEA to GPR55 in UC may activate the downstream processes of the micturition reflex, leading to nocturia. These findings suggest a new mechanism for nocturia and its potential as a therapeutic target.
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Vascular cognitive impairment (VCI) refers to cognitive alterations caused by vascular disease, which is associated with various types of dementia. Because chronic cerebral hypoperfusion (CCH) induces VCI, we used bilateral common carotid artery stenosis (BCAS) mice as a CCH-induced VCI model. Transient receptor potential ankyrin 1 (TRPA1), the most redox-sensitive TRP channel, is functionally expressed in the brain. Here, we investigated the pathophysiological role of TRPA1 in CCH-induced VCI. During early-stage CCH, cognitive impairment and white matter injury were induced by BCAS in TRPA1-knockout but not wild-type mice. TRPA1 stimulation with cinnamaldehyde ameliorated BCAS-induced outcomes. RNA sequencing analysis revealed that BCAS increased leukemia inhibitory factor (LIF) in astrocytes. Moreover, hydrogen peroxide-treated TRPA1-stimulated primary astrocyte cultures expressed LIF, and culture medium derived from these cells promoted oligodendrocyte precursor cell myelination. Overall, TRPA1 in astrocytes prevents CCH-induced VCI through LIF production. Therefore, TRPA1 stimulation may be a promising therapeutic approach for VCI.
Asunto(s)
Isquemia Encefálica , Disfunción Cognitiva , Canales de Potencial de Receptor Transitorio , Sustancia Blanca , Ratones , Animales , Astrocitos , Canal Catiónico TRPA1/genética , Factor Inhibidor de Leucemia/farmacología , Disfunción Cognitiva/complicaciones , Isquemia Encefálica/complicaciones , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving â¼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.