RESUMEN
Calcium ions (Ca2+) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.
Asunto(s)
Canales de Calcio , Dependovirus , Ratones , Animales , Dependovirus/metabolismo , Optogenética , Región CA1 Hipocampal/metabolismo , Aprendizaje , Calcio/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
The incidence of pulmonary arterial hypertension (PAH) is a significant co-morbidity observed in HIV (+) individuals. Pulmonary artery smooth muscle cells (PASMCs)-key components of the arterial vessel wall that regulate vessel diameter, demonstrate increased proliferation and hypertrophy in the lungs of HIV infected individuals, underscoring the role of these cells in the pathogenesis of HIV-associated PAH. While several pathways have been implicated in enhanced proliferation of PASMCs, detailed molecular mechanism(s) underlying HIV-associated PASMC proliferation still remain elusive. In the current study, we sought to investigate the effects HIV protein transactivator of transcription (TAT)-mediated proliferation on PASMCs. In agreement with earlier findings, our results also demonstrated TAT-mediated proliferation of human PASMCs. We identified activation of a novel Notch3 signaling pathway in TAT-mediated proliferation of PASMCs. Further validation of the Notch 3 pathway was demonstrated using both pharmacological (γ-secretase inhibitor, DAPT), as well as genetic approaches (Notch3 siRNA). Vascular endothelial growth factor A (VEGF-A) was identified as a novel downstream molecule that was induced following Notch activation. Findings from in vitro studies were further validated in archived simian immunodeficiency virus (SIV)-infected monkey lung tissues. There was increased activation of Notch3 signaling as well as enhanced expression of VEGF-A in the lungs of SIV-infected macaques compared with the lungs of SIV(-) controls. Taken together, we demonstrated that HIV-TAT increased the proliferation of PASMCs via the Notch3/VEGF-A axis. Targeting the Notch3/VEGF-A axis could thus be considered a potential therapeutic approach for the treatment of HIV-associated PAH.
RESUMEN
Cocaine is known to activate microglia both in vitro and in vivo. High expression of microglial Toll-like receptors (TLRs) and their downstream signal transducers play critical roles in determining microglial activation status. Emerging reports have also demonstrated that cocaine can enhance the strength of TLR signaling. Detailed molecular mechanisms underlying this phenomenon, however, remain elusive. In this study, we investigated the role(s) of miR-124 in regulating microglial TLR4 signaling in the context of cocaine. Herein, we found a dose- and time-dependent upregulation of KLF4 in cocaine-exposed BV-2 cells and rat primary microglial cells (rPMs). KLF4 also identified as a novel 3'-UTR target directly regulated by miR-124. In parallel, miR-124 regulated multiple TLR4 signaling molecules including TLR4, MyD88, TRAF6, and IRAK1. Repeated doses of cocaine (20 mg/kg; i.p.) administration in mice for 7 days further validated the in vitro key findings. Also, miR-124 overexpression significantly blocked the cocaine-mediated upregulation of pro-inflammatory cytokines. In contrast, miR-124 overexpression notably increased the expression of anti-inflammatory mediators in cocaine-exposed microglial cells. Intriguingly, stereotactic administration of lentivirus-miR-124 in the striatum significantly inhibited cocaine-mediated microglial activation and locomotor hyperactivity in vivo. In summary, these findings implicate the role of miR-124 in regulating TLR4 signaling, thereby indicating a new pathway responsible for cocaine-mediated microglial activation.
Asunto(s)
Cocaína/farmacología , Regulación hacia Abajo , Factores de Transcripción de Tipo Kruppel/metabolismo , MicroARNs/metabolismo , Microglía/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/metabolismo , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Microglía/efectos de los fármacos , Modelos Biológicos , Factor 88 de Diferenciación Mieloide/metabolismo , Ratas Sprague-Dawley , Factor 6 Asociado a Receptor de TNF/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Neuroinflammation plays a critical role in the development of reward-related behavior in cocaine self-administration rodents. Cocaine, one of most commonly abused drugs, has been shown to activate microglia both in vitro and in vivo. Detailed molecular mechanisms underlying cocaine-mediated microglial activation remain poorly understood. microRNAs (miRs) belonging to a class of small noncoding RNA superfamily have been shown to modulate the activation status of microglia. miR-124, one of the microglia-enriched miRs, functions as an anti-inflammatory regulator that maintains microglia in a quiescent state. To date, the possible effects of cocaine on microglial miR-124 levels and the associated underlying mechanisms have not been explored. In the current study, we demonstrated that cocaine exposure decreased miR-124 levels in both BV-2 cells and rat primary microglia. These findings were further validated in vivo, wherein we demonstrated decreased abundance of miR-124 in purified microglia isolated from cocaine-administered mice brains compared with cells from saline administered animals. Molecular mechanisms underlying these effects involved cocaine-mediated increased mRNA and protein expression of DNMTs in microglia. Consistently, cocaine substantially increased promoter DNA methylation levels of miR-124 precursors (pri-miR-124-1 and -2), but not that of pri-miR-124-3, both in vitro and in vivo. In summary, our findings demonstrated that cocaine exposure increased DNA methylation of miR-124 promoter resulting into its downregulation, which, in turn, led to microglial activation. Our results thus implicate that epigenetic modulation of miR-124 could be considered as a potential therapeutic approach to ameliorate microglial activation and, possibly, the development of cocaine addiction.
Asunto(s)
Cocaína/toxicidad , Metilación de ADN/genética , MicroARNs/genética , Animales , Células Cultivadas , Cocaína/metabolismo , Metilación de ADN/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , MicroARNs/biosíntesis , Microglía/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , RatasRESUMEN
Cocaine is a highly addictive narcotic associated with dendritic spine plasticity in the striatum. However, it remains elusive whether cocaine modifies spines in a cell type-specific or region-specific manner or whether it alters different types of synapses in the brain. In addition, there is a paucity of data on the regulatory mechanism(s) involved in cocaine-induced modification of spine density. In the current study, we report that cocaine exposure differentially alters spine density, spine morphology, and the types of synapses in hippocampal and cortical neurons. Cocaine exposure in the hippocampus resulted in increased spine density, but had no significant effect on cortical neurons. Although cocaine exposure altered spine morphology in both cell types, the patterns of spine morphology were distinct for each cell type. Furthermore, we observed that cocaine selectively affects the density of excitatory synapses. Intriguingly, in hippocampal neurons cocaine-mediated effects on spine density and morphology involved sigma-1 receptor (Sig-1 R) and its downstream TrkB signaling, which were not the case in cortical neurons. Furthermore, pharmacological inhibition of Sig-1 R prevented cocaine-induced TrkB activation in hippocampal neurons. Our findings reveal a novel mechanism by which cocaine induces selective changes in spine morphology, spine density, and synapse formation, and could provide insights into the cellular basis for the cognitive impairment observed in cocaine addicts.
Asunto(s)
Cocaína/farmacología , Espinas Dendríticas/metabolismo , Hipocampo/citología , Receptor trkB/genética , Receptores sigma/metabolismo , Activación Transcripcional/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Cocaína/administración & dosificación , Espinas Dendríticas/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Biológicos , Morfogénesis/efectos de los fármacos , Ratas Sprague-Dawley , Receptor trkB/metabolismo , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Receptor Sigma-1RESUMEN
Changes in routine mouse home-cage behavioral activities have been used recently to study alterations of neural circuits caused by genetic and environmental modifications and by drug administration. Nevertheless, automatic assessment of mouse home-cage behaviors remains challenging due to the cost of proprietary systems and to the difficulty in adjusting systems to different monitoring conditions. Here we present software for the automatic quantification of multiple facets of mouse home-cage behaviors, suitable for continuous 24 h video monitoring. We used this program to assess behavioral changes in male and female R6/2 transgenic mouse models of Huntington's disease over a 10-week period. Consistent with the well-known progressive motor coordination deficits of R6/2 mice, their hanging, rearing, and climbing activity declined as the disease progressed. R6/2 mice also exhibited frequent disturbances in their resting activity compared to wild-type mice, suggesting that R6/2 mice are more restless and wakeful. Behavioral differences were seen earlier for male R6/2 mice than female R6/2 mice, and "behavioral signatures" based on multiple behaviors enabled us to distinguish male R6/2 mice from sex- and age-matched wild-type controls as early as 5 weeks of age. These results demonstrate that the automated behavioral classification software that we developed ("OpenCage") provides a powerful tool for analyzing natural home-cage mouse behaviors, and for constructing behavioral signatures that will be useful for assessing therapeutic strategies. The OpenCage software is available under an open-source GNU General Public License, allowing other users to freely modify and extend it to suit their purposes.
Asunto(s)
Conducta Animal/fisiología , Procesamiento Automatizado de Datos , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/fisiopatología , Caracteres Sexuales , Factores de Edad , Animales , Peso Corporal/genética , Computadores , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Conducta de Ingestión de Líquido/fisiología , Conducta Alimentaria/fisiología , Femenino , Lateralidad Funcional/genética , Proteína Huntingtina , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Proteínas del Tejido Nervioso , Proteínas Nucleares , Programas Informáticos , Expansión de Repetición de Trinucleótido , Grabación en VideoRESUMEN
The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson's disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP(+))-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP(+) treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP(+)-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP(+)-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Eritropoyetina/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson Secundaria/metabolismo , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Citocinas/farmacología , Modelos Biológicos , Células PC12 , Enfermedad de Parkinson Secundaria/patología , Ratas , Proteína p53 Supresora de TumorRESUMEN
PURPOSE: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization. METHOD: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied. RESULTS: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts. CONCLUSIONS: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Cafeína/administración & dosificación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Fármacos Sensibilizantes a Radiaciones/administración & dosificaciónRESUMEN
PURPOSE: The purpose of the present study was to evaluate the efficacy of mild hyperthermia to potentiate the anticancer effects of beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells. EXPERIMENTAL DESIGN: Effects of beta-lapachone alone or in combination with mild heating on the clonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro was determined. Effects of heating on the NQO1 level in the cancer cells in vitro were assessed using Western blot analysis for NQO1 expression, biochemical determination of NQO1 activity, and immunofluorescence microscopy for NQO1 expression. Growth of FSaII tumors in the hind legs of C3H mice was determined after treating the host mice with i.p. injection of 45 mg/kg beta-lapachone followed by heating the tumors at 42 degrees C for 1 hour every other day for four times. RESULTS: Incubation of FSaII tumor cells and A549 tumor cells with beta-lapachone at 37 degrees C reduced clonogenic survival of the cells in dose-dependent and incubation time-dependent manner. NQO1 level in the cancer cells in vitro increased within 1 hour after heating at 42 degrees C for 1 hour and remained elevated for >72 hours. The clonogenic cell death caused by beta-lapachone increased in parallel with the increase in NQO1 levels in heated cells. Heating FSaII tumors in the legs of C3H mice enhanced the effect of i.p.-injected beta-lapachone in suppressing tumor growth. CONCLUSION: We observed for the first time that mild heat shock up-regulates NQO1 in tumor cells. The heat-induced up-regulation of NQO1 enhanced the anticancer effects of beta-lapachone in vitro and in vivo.