RESUMEN
Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.
Asunto(s)
Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Canales Iónicos/biosíntesis , Mecanotransducción Celular/fisiología , Arteria Pulmonar/metabolismo , Regulación hacia Arriba/fisiología , Adulto , Anciano , Animales , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Hipertensión Pulmonar/patología , Indoles/farmacología , Masculino , Mecanotransducción Celular/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Slow type anion channels (SLAC/SLAHs) play important roles during anion transport, growth and development, abiotic stress responses and hormone responses in plants. However, there is few report on SLAC/SLAHs in rapeseed (Brassica napus). Genome-wide identification and expression analysis of SLAC/SLAH gene family members were performed in B. napus. A total of 23 SLAC/SLAH genes were identified in B. napus. Based on the structural characteristics and phylogenetic analysis of these members, the SLAC/SLAHs could be classified into three main groups. Transcriptome data demonstrated that BnSLAH3 genes were detected in various tissues of the rapeseed and could be up-regulated by low nitrate treatment in roots. BnSLAC/SLAHs were exclusively localized on the plasma membrane in transient expression of tobacco leaves. These results will increase our understanding of the evolution and expression of the SLAC/SLAHs and provide evidence for further research of biological functions of candidates in B. napus.
Asunto(s)
Brassica napus/genética , Brassica napus/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Canales Iónicos/metabolismo , Nitratos/metabolismo , Filogenia , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Estrés Fisiológico , Transcriptoma , Canales Aniónicos Dependientes del Voltaje/biosíntesisRESUMEN
Thalamocortical neurons (TCNs) play a critical role in the maintenance of thalamocortical oscillations, dysregulation of which can result in certain types of seizures. Precise control over firing rates of TCNs is foundational to these oscillations, yet the transcriptional mechanisms that constrain these firing rates remain elusive. We hypothesized that Shox2 is a transcriptional regulator of ion channels important for TCN function and that loss of Shox2 alters firing frequency and activity, ultimately perturbing thalamocortical oscillations into an epilepsy-prone state. In this study, we used RNA sequencing and quantitative PCR of control and Shox2 knockout mice to determine Shox2-affected genes and revealed a network of ion channel genes important for neuronal firing properties. Protein regulation was confirmed by Western blotting, and electrophysiological recordings showed that Shox2 KO impacted the firing properties of a subpopulation of TCNs. Computational modeling showed that disruption of these conductances in a manner similar to Shox2's effects modulated frequency of oscillations and could convert sleep spindles to near spike and wave activity, which are a hallmark for absence epilepsy. Finally, Shox2 KO mice were more susceptible to pilocarpine-induced seizures. Overall, these results reveal Shox2 as a transcription factor important for TCN function in adult mouse thalamus.
Asunto(s)
Potenciales de Acción/fisiología , Corteza Cerebral/metabolismo , Proteínas de Homeodominio/biosíntesis , Neuronas/metabolismo , Convulsiones/metabolismo , Tálamo/metabolismo , Animales , Proteínas de Homeodominio/genética , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Red Nerviosa/metabolismo , Convulsiones/genética , Convulsiones/prevención & control , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The effects of piezo-type mechanosensitive ion channel component 1 (Piezo1) in sensing extracellular mechanical stress have been well investigated. Recently, Piezo1's vital role in cancerogenesis has been demonstrated by many studies. Nonetheless, the prognostic value of Piezo1 in cancer still remains unexplored and unclear. This article aims to investigate the prognostic value of Piezo1 in breast cancer. Human Protein Atlas and the Cancer Genome Atlas (TCGA) databases were used to examine Piezo1 expression in different human tissues and human cell lines. The discrepancies of Piezo1 mRNA expression in breast cancer patients with different clinicopathological features were assessed using bc-GenExMiner. The prognostic value of Piezo1 in breast cancer patients was evaluated using Kaplan-Meier plotter. Piezo1 mRNA was extensively expressed in human tissues and cell lines, particularly in breast and cancerous breast cancer cell line MCF7. High Piezo1 expression was found correlated with poor prognosis of breast cancer. Survival analysis further confirmed unfavorable prognosis of high Piezo1 expression in breast cancer patients with lymph node positive, estrogen receptor positive, Grade 2 (Scarff-Bloom-Richardson grading system), luminal A, and human epidermal growth factor receptor 2 overexpression, respectively. This study suggested that Piezo1 can serve as a prognostic indicator of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Canales Iónicos/biosíntesis , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Canales Iónicos/genética , Células MCF-7 , Persona de Mediana Edad , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Ventricular tachyarrhythmias and sudden cardiac death show a circadian pattern of occurrence in patients with heart failure. In the rodent ventricle, a significant portion of genes, including some ion channels, shows a circadian pattern of expression. However, genes that define electrophysiological properties in failing human heart ventricles have not been examined for a circadian expression pattern. METHODS: Ventricular tissue samples were collected from patients at the time of cardiac transplantation. Two sets of samples (n=37 and 46, one set with a greater arrhythmic history) were selected to generate pseudo-time series according to their collection time. A third set (n=27) of samples was acquired from the nonfailing ventricles of brain-dead donors. The expression of 5 known circadian clock genes and 19 additional ion channel genes plausibly important to electrophysiological properties were analyzed by real-time polymerase chain reaction and then analyzed for the percentage of expression variation attributed to a 24-hour circadian pattern. RESULTS: The 5 known circadian clock gene transcripts showed a strong circadian expression pattern. Compared with rodent hearts, the human circadian clock gene transcripts showed a similar temporal order of acrophases but with a ≈7.6 hours phase shift. Five of the ion channel genes also showed strong circadian expression. Comparable studies of circadian clock gene expression in samples recovered from nonheart failure brain-dead donors showed acrophase shifts, or weak or complete loss of circadian rhythmicity, suggesting alterations in circadian gene expression. CONCLUSIONS: Ventricular tissue from failing human hearts display a circadian pattern of circadian clock gene expression but phase-shifted relative to rodent hearts. At least 5 ion channels show a circadian expression pattern in the ventricles of failing human hearts, which may underlie a circadian pattern of ventricular tachyarrhythmia/sudden cardiac death. Nonfailing hearts from brain-dead donors show marked differences in circadian clock gene expression patterns, suggesting fundamental deviations from circadian expression.
Asunto(s)
Ritmo Circadiano , Expresión Génica , Insuficiencia Cardíaca/genética , Miocardio/metabolismo , Adulto , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUD: The TBX3(T-box 3)transcription factor is considered as an essential factor in sinoatrial node formation. While the effect of TBX3 in the differentiation of sinoatrial node cells from embryonic stem cells(ESCs) has been recognized, its role in human induced pluripotent stem cell derived cardiomyocytes(hiPSCMs) has not been addressed. Therefore, the purpose of the present study was to investigate whether overexpression of TBX3 in hiPSCs could increase their differentiation into pacemaker-like cells. METHODS: The hiPSCs were transfected with TBX3 gene during differentiation into cardiomyocytes(CMs). The hiPSCMs were analyzed using immunofluorescence, RT-qPCR, flow cytometry, whole-cell patch clamp recording to identify the differentiation effect exerted by TBX3. We discovered that hiPSCs transfected with TBX3 showed more proportions of NKX2.5-cTNT + sinoatrial node cells and faster contracting rates. RESULTS: The results showed increment in transcription factor TBX18, SHOX2; hyperpolarization-activated cyclic nucleotide (HCN) channel: HCN1, HCN2, HCN4, connexin 45(CX45), Na + Ca2+ exchanger(NCX) in TBX3 transfected hiPSCMs. Sinoatrial node cell specific If current and action potential were also confirmed by patch clamp in TBX3 transfected hiPSCMs and the pacemaker-like cells were able to pace hiPSCMs ex vivo. CONCLUSION: In conclusion, the present study demonstrated that overexpression of TBX3 could increase the differentiation of hiPSCs into pacemaker-like cells. Our study provide new strategy to construct a biological pacemaker, however, further study is still needed to identify the efficacy and safety of using the pacemaker-like cells to produce biological pacemaker in vivo.
Asunto(s)
Relojes Biológicos/genética , Diferenciación Celular/genética , Corazón/inervación , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Potenciales de Acción , Señalización del Calcio/genética , Conexinas/metabolismo , Humanos , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Nodo Sinoatrial/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Previous studies have suggested that increased mortality and disability in patients with brain tumor are associated with peritumoral brain edema. However, the mechanism of peritumoral brain edema in brain tumors is unknown. This study aimed to investigate the effect of Piezo1 overexpression on peritumoral brain edema in glioblastomas. MATERIALS AND METHODS: The Piezo1 expression in cell lines and paired samples was detected by quantitative reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry. Sixty-four patients with glioblastomas were analyzed retrospectively. The Piezo1 expression of tumor tissue was detected by immunohistochemistry. The diameters of tumor and edema were measured by preoperative MR imaging, and the edema index value was calculated. RESULTS: Western blot and quantitative reverse transcription polymerase chain reaction showed that Piezo1 expression was higher in 6 glioma cell lines than in the normal astrocyte cell line. Compared with peritumoral tissues, Piezo1 was up-regulated in tumor tissues. Sixty-four patients with glioblastomas were enrolled in further study. Piezo1 was higher in the moderate edema group than in the mild edema group (P < .001), higher in the severe edema group than in the moderate edema group (P < .001), and correlated with the edema index (r = 0.73; P < .001). Receiver operating characteristic curve analysis showed that the edema index yielded an area under the curve of 0.867 (95% CI, 0.76-0.97; P < .001), with a sensitivity of 100% and a specificity of 70%. CONCLUSIONS: Piezo1 overexpression is positively correlated with the degree of peritumoral brain edema in glioblastomas. Predicting high Piezo1 expression in tumor tissues based on the edema extent shows good sensitivity and specificity.
Asunto(s)
Edema Encefálico/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/patología , Canales Iónicos/biosíntesis , Adulto , Anciano , Edema Encefálico/etiología , Neoplasias Encefálicas/complicaciones , Femenino , Glioblastoma/complicaciones , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Curva ROC , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Purpose: To clarify the expression profiles of ion channels and transporters of metabolic substrates among heterogeneous cultured human corneal endothelial cells (cHCECs) distinct in their effectiveness in reconstituting the corneal endothelium. Methods: Integrated proteomics for cell lysates by liquid chromatography-tandem mass spectrometry was carried out from three aliquots of cHCECs enriched in either cluster of definition (CD)44-/+ (mature) cHCECs or CD44++/+++ cell-state transition (CST) cHCECs. The expression profiles of cations/anions, monocarboxylic acid transporters (MCTs), and solute carrier (SLC) family proteins, as well as carbonic anhydrases (CAs), were investigated. Results: The polarized expression of cations/anions, MCTs, and SLC family proteins, as well as CAs, was clarified for mature and CST cHCECs. Most SLC4 family members, including SLC4A11 and SLC4A4 (NBCe1), were upregulated in the CST cHCECs, whereas SLC9A1 (Na+/H+ exchanger isoform one [NHE1]) and CA5B were detected only in the mature cHCECs. In addition, SLC25A42, catalyzing the entry of coenzyme A into the mitochondria, and SLC25A18, functioning as a mitochondrial glutamate carrier 2 (both relevant for providing the substrates for mitochondrial bioenergetics), were selectively expressed in the mature cHCECs. Conclusions: Our findings may suggest the relevance of qualifying the polarized expression of these ion channels and transporter-like proteins to ensure not only the suitability but also the in vivo biological functionality of cHCECs selected for use in a cell-injection therapy.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Corneal/citología , Canales Iónicos/biosíntesis , Proteínas Transportadoras de Solutos/biosíntesis , Células Cultivadas , HumanosRESUMEN
AIMS: Atrial remodeling, including structural and electrical remodeling, is considered as the substrate in the development of atrial fibrillation (AF). Structural remodeling mainly involves atrial fibrosis, and electrical remodeling is closely related to the changes of ion channels in atrial myocytes. In this study, we aimed to investigate the changes of ion channels in atrial remodeling induced by CIH in rats, which provide the explication for the mechanisms of AF. MATERIALS AND METHODS: 80 male Sprague-Dawley rats were randomized into two groups: Control and CIH group (n = 40). CIH rats were subjected to CIH 8 h/d for 30 days. Atrial epicardial conduction velocity, conduction inhomogeneity and AF inducibility were examined. Masson's trichrome staining was used to evaluate the extent of atrial fibrosis, and the expression levels of ion channel subunits were measured by RT-qPCR, Western blot, and IHC, respectively. The remaining 40 rats were used for whole-cell patch clamp experiments. Action potential, INa, ICa-L, Ito were recorded and compared between two groups. KEY FINDINGS: CIH rats showed increased AF inducibility, atrial interstitial collagen deposition, APD, expression levels of RyR2, p-RyR2, CaMKII, p-CaMKII, and decreased atrial epicardial conduction velocity, expression levels of Nav1.5, Cav1.2, Kv1.5, Kv4.2, Kv4.3 compared to the Control rats, and the current density of INa, ICa-L, Ito were significantly decreased in CIH group. SIGNIFICANCE: We observed significant atrial remodeling induced by CIH in our rat model, which was characterized by changes in ion channels. These changes may be the mechanisms of CIH promoting AF.
Asunto(s)
Remodelación Atrial/fisiología , Hipoxia/fisiopatología , Canales Iónicos/fisiología , Potenciales de Acción/fisiología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/biosíntesis , Enfermedad Crónica , Fibrosis/complicaciones , Fibrosis/patología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Hipoxia/complicaciones , Hipoxia/metabolismo , Canales Iónicos/biosíntesis , Masculino , Potenciales de la Membrana/fisiología , Ratas , Canal Liberador de Calcio Receptor de Rianodina/biosíntesisRESUMEN
The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H+extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.
Asunto(s)
Corion/metabolismo , Regulación de la Expresión Génica , Canales Iónicos/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Corion/citología , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
OBJECTIVE: Increasing evidence has revealed that mechanical stress and elevated mechanical signals promote malignant tumor transformation and metastasis. This study aimed to explore the function of the mechanically activated ion-channel Piezo1 in the colon cancer metastasis and its potential regulatory mechanism. METHODS: First, we examined the expression levels of Piezo1 and mitochondrial calcium uniporter (MCU) both in colon cancer tissues and assessed the prognostic value of Piezo1 and MCU in a colon cancer cohort (n = 110). Second, functional assays were performed to investigate the effects of Piezo1 and MCU on colon cancer cell migration, invasion, and mitochondrial membrane potential. Third, we analyzed the expression of Piezo1, MCU, and HIF-1α by overexpressing/silencing each other's expression. RESULTS: We found that Piezo1 was up-regulated and MCU was down-regulated in colon cancer tissues. Piezo1 and MCU were both correlated with poor prognosis of patients with colon cancer. Overexpressing Piezo1 and silencing MCU could promote colon cancer cell migration and metastasis, reduce mitochondrial membrane potential, and promote each other's expression. We also found that HIF-1α was up-regulated in colon cancer tissues. Additionally, silencing Piezo1 inhibited the expression of HIF-1α and VEGF, which was contrary to MCU silencing. Intriguingly, Piezo1-overexpressing cells did not regain their migration behaviors when HIF-1α expression was inhibited, which was accompanied with the re-expression of MCU and VEGF. CONCLUSION: In our study, Piezo1 is involved in colon cancer cell metastasis. Furthermore, our findings indicated a possible Piezo1-MCU-HIF-1α-VEGF axis, which still need further exploration.
Asunto(s)
Neoplasias del Colon/metabolismo , Canales Iónicos/metabolismo , Canales de Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Estudios de Cohortes , Neoplasias del Colon/patología , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Canales Iónicos/biosíntesis , Potencial de la Membrana Mitocondrial , Metástasis de la Neoplasia , Pronóstico , Transducción de Señal , Regulación hacia ArribaRESUMEN
During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na+) leak channel, nonselective (NALCN), was shown to contribute to a Na+ leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.
Asunto(s)
Estradiol/farmacología , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Miocitos del Músculo Liso/metabolismo , Miometrio/metabolismo , Progesterona/farmacología , Adulto , Línea Celular , Femenino , Humanos , Mutación/genética , Miocitos del Músculo Liso/efectos de los fármacos , Miometrio/efectos de los fármacos , Embarazo , ARN Mensajero/biosíntesis , Elementos de Respuesta/efectos de los fármacosRESUMEN
The sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, lrrc8a, was investigated in the zebrafish model. RT-PCR and whole-mount in situ hybridization indicated that both genes were ubiquitously expressed through to 12â hpf, and around the ventricular layer of neural tubes and the cardiogenic region at 24â hpf. Knocking down either one lrrc8a paralog with morpholino oligos resulted in abnormalities in circulation at 32â hpf. Morpholino oligos or CRISPR interference against either paralog led to smaller brain ventricles at 24â hpf. Either lrrc8aa or lrrc8ab mRNA rescued the phenotypic penetrance in both lrrc8aa and lrrc8ab morphants. Supplementation of taurine in the E3 medium and overexpression csad mRNA also rescued lrrc8aa and lrrc8ab morphants. Our results indicate that the two zebrafish lrrc8a paralogs are maternal message genes and are ubiquitously expressed in early embryos. The two genes play redundant roles in the expansion of brain ventricles and the circulatory system and taurine contributes to brain ventricle expansion via the volume-regulated anion and organic osmolyte channels.
Asunto(s)
Encéfalo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Canales Iónicos , Osmorregulación/fisiología , Proteínas de Pez Cebra , Pez Cebra , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genéticaRESUMEN
Computations that require speed and temporal precision are implemented throughout the nervous system by neurons capable of firing at very high rates, rapidly encoding and transmitting a rich amount of information, but with substantial metabolic and physical costs. For economical fast spiking and high throughput information processing, neurons need to optimize multiple biophysical properties in parallel, but the mechanisms of this coordination remain unknown. We hypothesized that coordinated gene expression may underlie the coordinated tuning of the biophysical properties required for rapid firing and signal transmission. Taking advantage of the diversity of fast-spiking cell types in the medial vestibular nucleus of mice of both sexes, we examined the relationship between gene expression, ionic currents, and neuronal firing capacity. Across excitatory and inhibitory cell types, genes encoding voltage-gated ion channels responsible for depolarizing and repolarizing the action potential were tightly coexpressed, and their absolute expression levels increased with maximal firing rate. Remarkably, this coordinated gene expression extended to neurofilaments and specific presynaptic molecules, providing a mechanism for coregulating axon caliber and transmitter release to match firing capacity. These findings suggest the presence of a module of genes, which is coexpressed in a graded manner and jointly tunes multiple biophysical properties for economical differentiation of firing capacity. The graded tuning of fast-spiking capacity by the absolute expression levels of specific ion channels provides a counterexample to the widely held assumption that cell-type-specific firing patterns can be achieved via a vast combination of different ion channels.SIGNIFICANCE STATEMENT Although essential roles of fast-spiking neurons in various neural circuits have been widely recognized, it remains unclear how neurons efficiently coordinate the multiple biophysical properties required to maintain high rates of action potential firing and transmitter release. Taking advantage of diverse fast-firing capacities among medial vestibular nucleus neurons of mice, we identify a group of ion channel, synaptic, and structural genes that exhibit mutually correlated expression levels, which covary with firing capacity. Coexpression of this fast-spiking gene module may be a basic strategy for neurons to efficiently and coordinately tune the speed of action potential generation and propagation and transmitter release at presynaptic terminals.
Asunto(s)
Canales Iónicos/biosíntesis , Proteínas de Neurofilamentos/biosíntesis , Neuronas/metabolismo , Sinapsis/genética , Núcleos Vestibulares/metabolismo , Potenciales de Acción , Animales , Axones/metabolismo , Axones/fisiología , Fenómenos Electrofisiológicos/genética , Femenino , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Núcleos Vestibulares/citologíaRESUMEN
Perioperative neurocognitive disorders have been widely recognized as common adverse events after surgical intervention. Aging is one of the most important independent risk factors for worsened cognitive outcome, and this deterioration is linked to exacerbated microglia-mediated neuroinflammation in the aged brain. Under pathological stimulation, microglia are capable of polarizing toward proinflammatory M1 and anti-inflammatory M2 phenotypes. In the present study, we examined how aging affects microglial responses and neuroinflammation following peripheral surgery. Adult (2-3 months) and aged (18 months old) male C57/BL6 mice were subjected to tibial fracture or sham surgery. Aged mice exhibited higher level of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the hippocampus. The expression of synaptic protein synaptophysin (SYP) was also markedly reduced in the aged brain after the surgery. Both adult and aged mice showed significant increases in M1 microglial polarization (CD16/32). In contrast, tibial fracture surgery induced a decreased M2 microglial polarization (CD206, Ym1/2, Arg1) in aged brain but enhanced M2 microglial polarization in adult brain. Aged mice have upregulated voltage-gated proton channel (Hv1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit expression compared with adult mice. The percentage of CD16/32-positive M1 microglia colabeling with Hv1 was higher in aged mice after tibial fracture surgery. Thus, Hv1/NADPH oxidase upregulation in the aged brain may shift the dynamic equilibrium of microglial activation toward M1 polarization and exaggerate postoperative neuroinflammatory responses after peripheral surgical intervention.
Asunto(s)
Envejecimiento/metabolismo , Polaridad Celular/fisiología , Fijación de Fractura/efectos adversos , Mediadores de Inflamación/metabolismo , Canales Iónicos/biosíntesis , Microglía/metabolismo , Envejecimiento/inmunología , Animales , Inmunidad Innata/fisiología , Inflamación/etiología , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/inmunología , Canales Iónicos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Fracturas de la Tibia/inmunología , Fracturas de la Tibia/metabolismo , Fracturas de la Tibia/cirugíaRESUMEN
Excitation-contraction coupling in normal cardiac function is performed with well balanced and coordinated functioning but with complex dynamic interactions between functionally connected membrane ionic currents. However, their genomic investigations provide essential information on the regulation of diseases by their transcripts. Therefore, we examined the gene expression levels of the most important voltage-gated ionic channels such as Na+-channels (SCN5A), Ca2+-channels (CACNA1C and CACNA1H), and K+-channels, including transient outward (KCND2, KCNA2, KCNA5, KCNA8), inward rectifier (KCNJ2, KCNJ12, KCNJ4), and delayed rectifier (KCNB1) in left ventricular tissues from either ischemic or dilated cardiomyopathy (ICM or DCM). We also examined the mRNA levels of ATP-dependent K+-channels (KCNJ11, ABCC9) and ERG-family channels (KCNH2). We further determined the mRNA levels of ryanodine receptors (RyR2; ARVC2), phospholamban (PLB or PLN), SR Ca2+-pump (SERCA2; ATP2A1), an accessory protein FKBP12 (PPIASE), protein kinase A (PPNAD4), and Ca2+/calmodulin-dependent protein kinase II (CAMK2G). The mRNA levels of SCN5A, CACNA1C, and CACNA1H in both groups decreased markedly in the heart samples with similar significance, while KvLQT1 genes were high with depressed Kv4.2. The KCNJ11 and KCNJ12 in both groups were depressed, while the KCNJ4 level was significantly high. More importantly, the KCNA5 gene was downregulated only in the ICM, while the KCNJ2 was upregulated only in the DCM. Besides, mRNA levels of ARVC2 and PLB were significantly high compared to the controls, whereas others (ATP2A1, PPIASE, PPNAD4, and CAMK2G) were decreased. Importantly, the increases of KCNB1 and KCNJ11 were more prominent in the ICM than DCM, while the decreases in ATP2A1 and FKBP1A were more prominent in DCM compared to ICM. Overall, this study was the first to demonstrate that the different levels of changes in gene profiles via different types of cardiomyopathy are prominent particularly in some K+-channels, which provide further information about our knowledge of how remodeling processes can be differentiated in HF originated from different pathological conditions.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Regulación de la Expresión Génica , Insuficiencia Cardíaca/metabolismo , Canales Iónicos/biosíntesis , Miocitos Cardíacos/metabolismo , Anciano , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Femenino , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/patologíaRESUMEN
Astrocytes are important for information processing in the brain and they achieve this by fine-tuning neuronal communication via continuous uptake and release of biochemical modulators of neurotransmission and synaptic plasticity. Often overlooked are their important functions in mechanosensation. Indeed, astrocytes can detect pathophysiological changes in the mechanical properties of injured, ageing, or degenerating brain tissue. We have recently shown that astrocytes surrounding mechanically-stiff amyloid plaques upregulate the mechanosensitive ion channel, Piezo1. Moreover, ageing transgenic Alzheimer's rats harboring a chronic peripheral bacterial infection displayed enhanced Piezo1 expression in amyloid plaque-reactive astrocytes of the hippocampus and cerebral cortex. Here, we have shown that the bacterial endotoxin, lipopolysaccharide (LPS), also upregulates Piezo1 in primary mouse cortical astrocyte cultures in vitro. Activation of Piezo1, via the small molecule agonist Yoda1, enhanced Ca2+ influx in both control and LPS-stimulated astrocytes. Moreover, Yoda1 augmented intracellular Ca2+ oscillations but decreased subsequent Ca2+ influx in response to adenosine triphosphate (ATP) stimulation. Neither blocking nor activating Piezo1 affected cell viability. However, LPS-stimulated astrocyte cultures exposed to the Piezo1 activator, Yoda1, migrated significantly slower than reactive astrocytes treated with the mechanosensitive channel-blocking peptide, GsMTx4. Furthermore, our data show that activating Piezo1 channels inhibits the release of cytokines and chemokines, such as IL-1ß, TNFα, and fractalkine (CX3 CL1), from LPS-stimulated astrocyte cultures. Taken together, our results suggest that astrocytic Piezo1 upregulation may act to dampen neuroinflammation and could be a useful drug target for neuroinflammatory disorders of the brain.
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Astrocitos/metabolismo , Señalización del Calcio/fisiología , Citocinas/metabolismo , Canales Iónicos/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Femenino , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.
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Dióxido de Carbono/metabolismo , Carbonatos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Canales Iónicos/biosíntesis , Proteínas de Plantas/biosíntesis , Tilacoides/metabolismo , Chlamydomonas reinhardtii/genética , Canales Iónicos/genética , Proteínas de Plantas/genética , Tilacoides/genéticaRESUMEN
BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously found that neurons respond to prolonged BDNF exposure (both in vivo (in models of epilepsy and TBI) and in vitro (in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory cytokines in non-neuronal cells. RESULTS: Here, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is specifically regulated by agents that inhibit JAK/STAT signaling. Surprisingly, the BDNF-induced JAK/STAT transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this dataset has revealed a unique non-canonical mechanism of JAK/STATs in neurons as differential gene expression mediated by STAT3 is not solely dependent upon phosphorylation at residue 705 and may involve a BDNF-induced interaction of STAT3 with Heterochromatin Protein 1 alpha (HP1α). CONCLUSIONS: These findings suggest that the neuronal BDNF-induced JAK/STAT pathway involves more than STAT3 phosphorylation at 705, providing the first evidence for a non-canonical mechanism that may involve HP1α. Our analysis reveals that JAK/STAT signaling regulates many of the genes associated with epilepsy syndromes where BDNF levels are markedly elevated. Uncovering the mechanism of this novel form of BDNF signaling in the brain may provide a new direction for epilepsy therapeutics and open a window into the complex mechanisms of STAT3 transcriptional regulation in neurological disease.
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Factor Neurotrófico Derivado del Encéfalo/farmacología , Encéfalo/metabolismo , Quinasas Janus/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Encéfalo/enzimología , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Inhibidores de las Cinasas Janus/farmacología , Quinasas Janus/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , RNA-Seq , Ratas , Ratas Sprague-Dawley , Receptores de Neurotransmisores/biosíntesis , Receptores de Neurotransmisores/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal , TranscriptomaRESUMEN
Bladder cystopathy and autonomic dysfunction are common complications of diabetes, and have been associated with changes in ganglionic transmission and some measures of neuronal excitability in male mice. To determine whether type II diabetes also impacts excitability of ganglionic neurons in females, we investigated neuronal excitability and firing properties, as well as underlying ion channel expression, in major pelvic ganglion (MPG) neurons in control, 10-week, and 21-week Leprdb/db mice. Type II diabetes in Leprdb/db animals caused a non-linear change in excitability and firing properties of MPG neurons. At 10â¯weeks, cells exhibited increased excitability as demonstrated by an increased likelihood of firing multiple spikes upon depolarization, decreased rebound spike latency, and overall narrower action potential half-widths as a result of increased depolarization and repolarization slopes. Conversely, at 21â¯weeks MPG neurons of Leprdb/db mice reversed these changes, with spiking patterns and action-potential properties largely returning to control levels. These changes are associated with numerous time-specific changes in calcium, sodium, and potassium channel subunit mRNA levels. However, Principal Components Analysis of channel expression patterns revealed that rectification of excitability is not simply a return to control levels, but rather a distinct ion channel expression profile in 21-week Leprdb/db neurons. These data indicate that type II diabetes can impact the excitability of post-ganglionic, autonomic neurons of female mice, and suggest that the non-linear progression of these properties with diabetes may be the result of compensatory changes in channel expression that act to rectify disrupted firing patterns of Leprdb/db MPG neurons.