RESUMEN
The mechanism underlying the antiepileptic actions of norepinephrine (NE) is unclear with conflicting results. Our objectives are to conclusively delineate the specific adrenergic receptor (AR) involved in attenuating hippocampal CA3 epileptiform activity and assess compounds for lead drug development. We utilized the picrotoxin model of seizure generation in rat brain slices using electrophysiological recordings. Epinephrine (EPI) reduced epileptiform burst frequency in a concentration-dependent manner. To identify the specific receptor involved in this response, the equilibrium dissociation constants were determined for a panel of ligands and compared with established binding values for α1, α2, and other receptor subtypes. Correlation and slope of unity were found for the α2A-AR, but not other receptors. Effects of different chemical classes of α-AR agonists at inhibiting epileptiform activity by potency (pEC50) and relative efficacy (RE) were determined. Compared with NE (pEC50, 6.20; RE, 100%), dexmedetomidine, an imidazoline (pEC50, 8.59; RE, 67.1%), and guanabenz, a guanidine (pEC50, 7.94; RE, 37.9%), exhibited the highest potency (pEC50). In contrast, the catecholamines, EPI (pEC50, 6.95; RE, 120%) and α-methyl-NE (pEC50, 6.38; RE, 116%) were the most efficacious. These findings confirm that CA3 epileptiform activity is mediated solely by α2A-ARs without activation of other receptor systems. These findings suggest a pharmacotherapeutic target for treating epilepsy and highlight the need for selective and efficacious α2A-AR agonists that can cross the blood-brain barrier.
Asunto(s)
Agonistas alfa-Adrenérgicos , Región CA3 Hipocampal , Norepinefrina , Convulsiones , Animales , Ratas , Agonistas alfa-Adrenérgicos/farmacología , Epinefrina/farmacología , Ligandos , Norepinefrina/farmacología , Receptores Adrenérgicos , Región CA3 Hipocampal/fisiopatología , Convulsiones/tratamiento farmacológico , Técnicas In VitroRESUMEN
UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.
Asunto(s)
Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Microglía/metabolismo , Adaptación Ocular/genética , Adaptación Ocular/fisiología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/farmacología , Animales , Astrocitos/metabolismo , Proliferación Celular/genética , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinos/farmacología , Mutación/genética , Fenotipo , Presenilina-1/genética , Presenilina-1/metabolismoRESUMEN
Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of Gαq/11 and TASK-3 channels suggesting a direct coupling of Gαq/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory.
Asunto(s)
Giro Dentado/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Neuronas/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio/metabolismo , Receptores de Neurotensina/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Giro Dentado/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Células HEK293 , Humanos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neurotensina/metabolismo , Canales de Potasio/genética , Ratas Sprague-Dawley , Receptores de Neurotensina/genética , Técnicas de Cultivo de TejidosRESUMEN
Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimer's disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K(+) channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Corteza Entorrinal/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotensina/farmacología , Receptores de Neurotensina/agonistas , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Enfermedad de Alzheimer/psicología , Animales , Modelos Animales de Enfermedad , Corteza Entorrinal/fisiopatología , Aprendizaje por Laberinto/fisiología , Ratones , Neuronas/fisiologíaRESUMEN
Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson's disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity. Therefore, we hypothesized that the MPP(+) toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP(+) increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP(+) can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Reguladoras de la Apoptosis , Beclina-1 , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de la Membrana , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Although cholecystokinin (CCK) has long been known to exert anxiogenic effects in both animal anxiety models and humans, the underlying cellular and molecular mechanisms are ill-defined. CCK interacts with CCK-1 and CCK-2 receptors resulting in up-regulation of phospholipase C (PLC) and protein kinase C (PKC). However, the roles of PLC and PKC in CCK-mediated anxiogenic effects have not been determined. We have shown previously that CCK facilitates glutamate release in the hippocampus especially at the synapses formed by the perforant path and dentate gyrus granule cells via activations of PLC and PKC. Here we further demonstrated that CCK enhanced NMDA receptor function in dentate gyrus granule cells via activation of PLC and PKC pathway. At the single-channel level, CCK increased NMDA single-channel open probability and mean open time, reduced the mean close time, and had no effects on the conductance of NMDA channels. Because elevation of glutamatergic functions results in anxiety, we explored the roles of PLC and PKC in CCK-induced anxiogenic actions using the Vogel Conflict Test (VCT). Our results from both pharmacological approach and knockout mice demonstrated that microinjection of CCK into the dentate gyrus concentration-dependently increased anxiety-like behavior via activation of PLC and PKC. Our results provide a novel unidentified signaling mechanism whereby CCK increases anxiety.
Asunto(s)
Ansiedad/inducido químicamente , Ansiedad/enzimología , Colecistoquinina/administración & dosificación , Proteína Quinasa C/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Fosfolipasas de Tipo C/fisiología , Animales , Colecistoquinina/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Femenino , Activación del Canal Iónico/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microinyecciones , Ratas , Ratas Sprague-DawleyRESUMEN
Prostanoids are potent inflammatory mediators that play a regulatory role in the innate immune activation of the adaptive immune response to determine the duration of protection against infection. We aim to quantify the modulation of prostanoids profiles in lipopolysaccharide (LPS)-stimulated THP-1 cells treated with the novel pertussis antigen BscF. We compared the effect with pertussis antigens present in the current Tdap vaccine to understand the immunomodulatory effect that might contribute to the diminished Tdap vaccine effectiveness. The inflammatory challenge with LPS induced a robust elevation of most prostanoid family members compared to the control treatment. Treatment with BscF and Tdap significantly reduced the LPS-stimulated elevation of prostaglandins (PGs) D2, E2, and F2α, as well as thromboxane (TX) A2 levels. An opposite trend was observed for PGI2, as both antigens accelerated the LPS-stimulated upregulation. Further, we quantified cyclooxygenases (COXs) that catalyze the biosynthesis of prostanoids and found that both antigens significantly reduced LPS-stimulated COX-1 and COX-2, demonstrating that the waning of acellular pertussis vaccines' protective immunity may be due to other downstream enzymes not related to COXs. Our present study validates the potential role of BscF as an adjuvant, resulting in the next-generation pertussis vaccine discovery.
Asunto(s)
Vacunas contra Difteria, Tétanos y Tos Ferina Acelular , Tos Ferina , Anticuerpos Antibacterianos , Antígenos Bacterianos , Bordetella pertussis , Humanos , Lipopolisacáridos/farmacología , Monocitos , Prostaglandinas , Tos Ferina/prevención & controlRESUMEN
BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.
RESUMEN
Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.
Asunto(s)
Colecistoquinina/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Animales , Calcio/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/crecimiento & desarrollo , Técnicas In Vitro , Espacio Intracelular/fisiología , Canales de Potasio/metabolismo , Terminales Presinápticos/fisiología , Probabilidad , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Colecistoquinina B/metabolismo , Receptores AMPA/metabolismo , Sinapsis/fisiología , Fosfolipasas de Tipo C/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Catecholamines released from the sympathetic nervous system in response to stress or injury affect expression of inflammatory cytokines generated by immune cells. α(1)-Adrenergic receptors (ARs) are expressed on innate immune cell populations, but their subtype expression patterns and signaling characteristics are not well characterized. Primary human monocytes, a human monocytic cell line, and monocyte-derived macrophage cells were used to measure expression of the proinflammatory mediator interleukin (IL)-1ß responding to lipopolysaccharide (LPS) in the presence or absence of α(1)-AR activation. Based on our previous findings, we hypothesized that α(1)-AR stimulation on innate immune cells positively regulates LPS-initiated IL-1ß production. IL-1ß production in response to LPS was synergistically higher for both monocytes and macrophages in the presence of the selective α(1)-AR agonist (R)-(-)-phenylephrine hydrochloride (PE). This synergistic IL-1ß response could be blocked with a selective α(1)-AR antagonist as well as inhibitors of protein kinase C (PKC). Radioligand binding studies characterized a homogenous α(1B)-AR subtype population on monocytes, which changed to a heterogeneous receptor subtype expression pattern when differentiated to macrophages. Furthermore, increased p38 mitogen-activated protein kinase (MAPK) activation was observed only with concurrent PE and LPS stimulation, peaking after 120 and 30 min in monocytes and macrophages, respectively. Blocking the PKC/p38 MAPK signaling pathway in both innate immune cell types inhibited the synergistic IL-1ß increase observed with concurrent PE and LPS treatments. This study characterizes α(1)-AR subtype expression on both human monocyte and macrophage cells and illustrates a mechanism by which increased IL-1ß production can be modulated by α(1)-AR input.
Asunto(s)
Macrófagos/metabolismo , Monocitos/metabolismo , Subunidades de Proteína/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Receptor Toll-Like 4/fisiología , Regulación hacia Arriba/inmunología , Adulto , Diferenciación Celular/inmunología , Línea Celular Transformada , Células Cultivadas , Humanos , Inmunidad Innata , Mediadores de Inflamación/fisiología , Interleucina-1beta/biosíntesis , Interleucina-1beta/sangre , Lipopolisacáridos/fisiología , Macrófagos/inmunología , Macrófagos/patología , Monocitos/inmunología , Monocitos/patología , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/sangre , Receptores Adrenérgicos alfa 1/biosíntesis , Receptores Adrenérgicos alfa 1/sangre , Transducción de Señal/inmunología , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/sangreRESUMEN
Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP-PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27-FAK interaction. In human Caco-2 cells, Zn27-stimulated FAK-Tyr-397 phosphorylation despite PTP-PEST inhibition and did not affect PTP1B-FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full-length 125 kDa FAK and its 35 kDa kinase domain. The ATP-competitive FAK inhibitor PF573228 reduced basal and ZN27-stimulated FAK phosphorylation in Caco-2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose-dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax ) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.
Asunto(s)
Activadores de Enzimas/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Regulación Alostérica , Células CACO-2 , Pruebas de Enzimas , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/enzimología , Mucosa Gástrica/lesiones , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/lesiones , Fosforilación/efectos de los fármacos , Unión Proteica , Quinolonas/farmacología , Sulfonas/farmacologíaRESUMEN
Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. Here, we determined the gene expression and DNA methylation changes in microglia induced by aggregate α-syn. Transgenic murine Thy-1 promoter (mThy1)-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-regulated changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data. Microglial DNA isolated from our 3-month cohort had 5315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3742 DMGs and 3766 DEGs in 13-month mThy1-Asyn mice. These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months. We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.
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Microglía , alfa-Sinucleína , Animales , Metilación de ADN , Modelos Animales de Enfermedad , Expresión Génica , Inflamación , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.
RESUMEN
Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson's disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug-induced neurotoxicity. Therefore, we hypothesized that METH-generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell line as our model system, we found that METH-induced autophagy by inhibiting dissociation of Bcl-2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti-apoptotic protein Bcl-2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl-2 was activated by c-Jun N-terminal kinase 1 (JNK 1), which is upstream of Bcl-2 phosphorylation, to induce Bcl-2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl-2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl-2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.
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Autofagia/efectos de los fármacos , Melatonina/farmacología , Metanfetamina/toxicidad , Fármacos Neuroprotectores/farmacología , Análisis de Varianza , Antracenos/farmacología , Western Blotting , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/metabolismo , Arrestinas/fisiología , Citoprotección/fisiología , Proteínas de Choque Térmico HSP27/fisiología , Receptores Adrenérgicos beta 2/fisiología , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis/agonistas , Proteínas Reguladoras de la Apoptosis/fisiología , Arrestinas/genética , Línea Celular Transformada , Citoprotección/efectos de los fármacos , Proteínas de Choque Térmico HSP27/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/fisiología , Interferencia de ARN , Urotelio/citología , Urotelio/efectos de los fármacos , Urotelio/metabolismo , beta-ArrestinasRESUMEN
Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes. In this study, we employed a systems biology approach to identify DPN-related transcriptional pathways conserved across human and various murine models. Eight microarray datasets on peripheral nerve samples from murine models of type 1 (streptozotocin-treated) and type 2 (db/db and ob/ob) diabetes of various ages and human subjects with non-progressive and progressive DPN were collected. Differentially expressed genes (DEGs) were identified between non-diabetic and diabetic samples in murine models, and non-progressive and progressive human samples using a unified analysis pipeline. A transcriptional network for each DEG set was constructed based on literature-derived gene-gene interaction information. Seven pairwise human-vs-murine comparisons using a network-comparison program resulted in shared sub-networks including 46 to 396 genes, which were further merged into a single network of 688 genes. Pathway and centrality analyses revealed highly connected genes and pathways including LXR/RXR activation, adipogenesis, glucocorticoid receptor signalling, and multiple cytokine and chemokine pathways. Our systems biology approach identified highly conserved pathways across human and murine models that are likely to play a role in DPN pathogenesis and provide new possible mechanism-based targets for DPN therapy.
Asunto(s)
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Neuropatías Diabéticas/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Neuropatías Diabéticas/complicaciones , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Biología de SistemasRESUMEN
Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG)-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of "bullets" carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.
RESUMEN
BACKGROUND: Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. RESULTS: Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant but similar loss of binding for all leucine mutant and wild type CLR because the important receptor contact on the neuropeptide was missing in all experimental situations. CONCLUSION: These results are consistent with previous structure-function investigations of the neuropeptide and are the first to propose specific CLR binding contacts for the amidated F37 of CGRP that are important for docking but not activation of the mature CGRP receptor.