RESUMEN
Rett Syndrome is a devastating neurodevelopmental disorder resulting from mutations in the gene MECP2. Mutations of Mecp2 that are restricted to GABAergic cell types largely replicate the behavioral phenotypes associated with mouse models of Rett Syndrome, suggesting a pathophysiological role for inhibitory interneurons. Recent work has suggested that vasoactive intestinal peptide-expressing (VIP) interneurons may play a critical role in the proper development and function of cortical circuits, making them a potential key point of vulnerability in neurodevelopmental disorders. However, little is known about the role of VIP interneurons in Rett Syndrome. Here we find that loss of MeCP2 specifically from VIP interneurons replicates key neural and behavioral phenotypes observed following global Mecp2 loss of function.
Asunto(s)
Interneuronas/fisiología , Proteína 2 de Unión a Metil-CpG/metabolismo , Síndrome de Rett/genética , Péptido Intestinal Vasoactivo/metabolismo , Animales , Modelos Animales de Enfermedad , Neuronas GABAérgicas/fisiología , Proteína 2 de Unión a Metil-CpG/genética , Ratones Transgénicos , Síndrome de Rett/fisiopatologíaRESUMEN
Although failure of GABAergic inhibition is a commonly hypothesized mechanism underlying seizure disorders, the series of events that precipitate a rapid shift from healthy to ictal activity remain unclear. Furthermore, the diversity of inhibitory interneuron populations poses a challenge for understanding local circuit interactions during seizure initiation. Using a combined optogenetic and electrophysiological approach, we examined the activity of identified mouse hippocampal interneuron classes during chemoconvulsant seizure induction in vivo. Surprisingly, synaptic inhibition from parvalbumin- (PV) and somatostatin-expressing (SST) interneurons remained intact throughout the preictal period and early ictal phase. However, these two sources of inhibition exhibited cell-type-specific differences in their preictal firing patterns and sensitivity to input. Our findings suggest that the onset of ictal activity is not associated with loss of firing by these interneurons or a failure of synaptic inhibition but is instead linked with disruptions of the respective roles these interneurons play in the hippocampal circuit.
Asunto(s)
Hipocampo/patología , Interneuronas/fisiología , Convulsiones/fisiopatología , Potenciales de Acción , Animales , Electroencefalografía , Ratones , Optogenética , Convulsiones/inducido químicamenteRESUMEN
KCNQ2 and KCNQ3 potassium channels have emerged as central regulators of pyramidal neuron excitability and spiking behavior. However, despite an abundance of evidence demonstrating that KCNQ2/3 heteromers underlie critical potassium conductances, it is unknown whether KCNQ2, KCNQ3, or both are obligatory for maintaining normal pyramidal neuron excitability. Here, we demonstrate that conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons in mice results in abnormal electrocorticogram activity and early death, whereas similar deletion of Kcnq3 does not. At the cellular level, Kcnq2-null, but not Kcnq3-null, CA1 pyramidal neurons show increased excitability manifested as a decreased medium afterhyperpolarization and a longer-lasting afterdepolarization. As a result, these Kcnq2-deficient neurons are hyperexcitable, responding to current injections with an increased number and frequency of action potentials. Biochemically, the Kcnq2 deficiency secondarily results in a substantial loss of KCNQ3 and KCNQ5 protein levels, whereas loss of Kcnq3 only leads to a modest reduction of other KCNQ channels. Consistent with this finding, KCNQ allosteric activators can still markedly dampen neuronal excitability in Kcnq3-null pyramidal neurons, but have only weak effects in Kcnq2-null pyramidal neurons. Together, our data reveal the indispensable function of KCNQ2 channels at both the cellular and systems levels, and demonstrate that pyramidal neurons have near normal excitability in the absence of KCNQ3 channels.
Asunto(s)
Potenciales de Acción , Epilepsia/genética , Eliminación de Gen , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/fisiología , Animales , Epilepsia/metabolismo , Epilepsia/fisiopatología , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/genética , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Células Piramidales/metabolismoRESUMEN
The secosteroidal hormone 1,25-dihyroxyvitamin D [1,25(OH)(2)D(3)] and its receptor, the vitamin D receptor (VDR), are crucial regulators of epidermal proliferation and differentiation. However, the effects of 1,25(OH)(2)D(3)-directed signaling on oral keratinocyte pathophysiology have not been well studied. We examined the role of 1,25(OH)(2)D(3) in regulating proliferation and differentiation in cultured oral keratinocytes and on the oral epithelium in vivo. Using lentiviral-mediated shRNA to silence VDR, we generated an oral keratinocyte cell line with stable knockdown of VDR expression. VDR knockdown significantly enhanced proliferation and disrupted calcium- and 1,25(OH)(2)D(3)-induced oral keratinocyte differentiation, emphasizing the anti-proliferative and pro-differentiation effects of 1,25(OH)(2)D(3) in oral keratinocytes. Using vitamin D(3)-deficient diets, we induced chronic vitamin D deficiency in mice as evidenced by decreased serum 25-hydroxyvitamin D (25OHD) concentrations. The vitamin D-deficient mice manifested increased proliferation of the tongue epithelium, but did not develop any morphological or histological abnormalities in the oral epithelium, suggesting that vitamin D deficiency alone is insufficient to alter oral epithelial homeostasis and provoke carcinogenesis. Immunohistochemical analyses of human and murine oral squamous cell carcinomas showed increased VDR expression. Overall, our results provide strong support for a crucial role for vitamin D signaling in oral keratinocyte pathophysiology.
Asunto(s)
Calcitriol/farmacología , Queratinocitos/metabolismo , Boca/citología , Receptores de Calcitriol/genética , Deficiencia de Vitamina D/patología , Vitaminas/farmacología , Animales , Calcitriol/deficiencia , Carcinoma de Células Escamosas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Epidermis/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neoplasias de la Boca/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Maintenance of oral epithelial homeostasis requires a fine balance between cell proliferation and differentiation. However, the molecular mechanisms that couple these processes, and its deregulation in tumorigenesis are not fully understood. Cyclin D1 and its kinase partners CDK4 and CDK6 play an important role in regulating the G1-S phase of the cell cycle. Deregulation of cyclin D1 is a frequent event in oral squamous cell carcinoma. Here, we examined whether overexpression of cyclin D1, CDK4 and CDK6 can deregulate the link between oral keratinocyte proliferation and differentiation. Our results show that cyclin D1 and its kinase partners CDK4 and CDK6 enhance keratinocyte proliferation, but are not sufficient to block calcium-induced keratinocyte differentiation and suggest that deregulation of these G1-regulatory kinases alone is insufficient to uncouple the link between proliferation and differentiation.
Asunto(s)
Diferenciación Celular/genética , Proliferación Celular , Ciclina D1/fisiología , Quinasa 4 Dependiente de la Ciclina/fisiología , Quinasa 6 Dependiente de la Ciclina/fisiología , Queratinocitos/fisiología , Mucosa Bucal/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Queratinocitos/metabolismo , Mucosa Bucal/fisiología , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Transfección , Regulación hacia Arriba/genéticaRESUMEN
Bromelain (Br), a proteolytic enzyme extracted from the stem of the pineapple, is known to possess anti-inflammatory activity and has been shown to reduce blood viscosity, prevent the aggregation of blood platelets, and improve ischemia-reperfusion (I/R) injury in a skeletal muscle model. We investigated the capacity of Br to limit myocardial injury in a global I/R model. Adult male Sprague-Dawley rats were divided into two groups: control (PBS) and Br at 10 mg/kg in PBS administered via intraperitoneal injection (twice/day) for 15 consecutive days. On day 16, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Br treatment showed higher left ventricular functional recovery throughout reperfusion compared with the controls [maximum rate of rise in intraventricular pressure (dP/dt max), 2,225 vs. 1,578 mmHg/s at 2 h reperfusion]. Aortic flow was also found to be increased in Br treatment when compared with that in untreated rats (11 vs. 1 ml). Furthermore, Br treatment reduced both the infarct size (34% vs. 43%) and the degree of apoptosis (28% vs. 37%) compared with the control animals. Western blot analysis showed an increased phosphorylation of both Akt and FOXO3A in the treatment group compared with the control. These results demonstrated for the first time that Br triggers an Akt-dependent survival pathway in the heart, revealing a novel mechanism of cardioprotective action and a potential therapeutic target against I/R injury.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Bromelaínas/uso terapéutico , Cardiotónicos , Factores de Transcripción Forkhead/fisiología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Citosol/metabolismo , Pruebas de Función Cardíaca , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/fisiopatología , Proteínas Nucleares/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Función Ventricular Izquierda/fisiologíaRESUMEN
Hypercholesterolemia (HC) is a common health problem that significantly increases risk of cardiovascular disease. Both statin (S) and resveratrol (R) demonstrated cardioprotection through nitric oxide-dependent mechanism. Therefore, the present study was undertaken to determine whether combination therapy with statin and resveratrol is more cardioprotective than individual treatment groups in ischemic rat heart model. The rats were fed with 2% high cholesterol diet and after 8 weeks of high cholesterol diet the animals were treated with statin (1 mg/kg bw/day) and resveratrol (20 mg/kg bw/day) for 2 weeks. The rats were assigned to: (1) Control (C), (2) HC, (3) HCR, (4) HCS and (5) HCRS. The hearts, subjected to 30-min global ischemia followed by 120-min reperfusion were used as experimental model. The left ventricular functional recovery (+dp/dt(max)) was found to be significantly better in the HCRS (1926+/-43), HCR (1556+/-65) and HCS (1635+/-40) compared to HC group (1127+/-16). The infarct sizes in the HCRS, HCS and HCR groups were 37+/-3.6, 43+/-3.3 and 44+/-4.2 respectively compared to 53+/-4.6 in HC. The lipid level was found to be decreased in all the treatment groups when compared to HC more significantly in HCS and HCRS groups when compared to HCR. Increased phosphorylation of Akt and eNOS was also observed in all the treatment groups resulting in decreased extent of cardiomyocyte apoptosis but the extent of reduction in apoptosis was more significant in HCRS group compared to all other groups. In vivo rat myocardial infarction (MI) model subjected to 1 week of permanent left descending coronary artery (LAD) occlusion documented increased capillary density in HCR and HCRS treated group when compared to HCS treatment group. We also documented increased beta-catenin translocation and increased VEGF mRNA expression in all treatment groups. Thus, we conclude that the acute as well as chronic protection afforded by combination treatment with statin and resveratrol may be due to pro-angiogenic, anti-hyperlipidemic and anti-apoptotic effects and long-term effects may be caused by increased neo-vascularization of the MI zone leading to less ventricular remodeling.