RESUMEN
Bioluminescence resonance energy transfer (BRET) is widely employed for real-time monitoring of G protein-coupled receptor activity, interactions, and trafficking in heterologous cell lines, yet its use in neuronal systems remains limited. Here, we present a protocol to apply BRET assays to primary neuronal cultures from mouse embryos. We describe steps and key concepts for generating plasmid constructs and lentivirus preparations, plating and lentiviral transduction of primary cultured neurons in 96-well plates, and BRET data collection and analysis. For complete details on the use and execution of this protocol, please refer to George et al.1.
RESUMEN
Oxytocin plays critical roles in the brain as a neuromodulator, regulating social and other affective behavior. However, the regulatory mechanisms controlling oxytocin receptor (OXTR) signaling in neurons remain unexplored. In this study, we have identified robust and rapid-onset desensitization of OXTR response in multiple regions of the mouse brain. Both cell autonomous spiking response and presynaptic activation undergo similar agonist-induced desensitization. G-protein-coupled receptor kinases (GRK) GRK2, GRK3, and GRK6 are recruited to the activated OXTR in neurons, followed by recruitment of ß-arrestin-1 and -2. Neuronal OXTR desensitization was impaired by suppression of GRK2/3/6 kinase activity but remained unaltered with double knockout of ß-arrestin-1 and -2. Additionally, we observed robust agonist-induced internalization of neuronal OXTR and its Rab5-dependent recruitment to early endosomes, which was impaired by GRK2/3/6 inhibition. This work defines distinctive aspects of the mechanisms governing OXTR desensitization and internalization in neurons compared to prior studies in heterologous cells.
RESUMEN
AMPA receptors (AMPAR) are organized into supramolecular complexes in association with other membrane proteins that provide exquisite regulation of their biophysical properties and subcellular trafficking. Proline-rich transmembrane protein 1 (PRRT1), also named as SynDIG4, is a component of native AMPAR complexes in multiple brain regions. Deletion of PRRT1 leads to altered surface levels and phosphorylation status of AMPARs, as well as impaired forms of synaptic plasticity. Here, we have investigated the mechanisms underlying the observed regulation of AMPARs by investigating the interaction properties and subcellular localization of PRRT1. Our results show that PRRT1 can interact physically with all AMPAR subunits GluA1-GluA4. We decipher the membrane topology of PRRT1 to find that contrary to the predicted dual membrane pass, only the second hydrophobic segment spans the membrane completely, and is involved in mediating the interaction with AMPARs. We also report a physical interaction of PRRT1 with phosphatase PP2B that dephosphorylates AMPARs during synaptic plasticity. Our co-localization analysis in primary neuronal cultures identifies that PRRT1 associates with AMPARs extrasynaptically where it localizes to early and recycling endosomes as well as to the plasma membrane. These findings advance the understanding of the mechanisms by which PRRT1 regulates AMPARs under basal conditions and during synaptic plasticity.
RESUMEN
Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized. LC8 dimers could bind in either a paired "in-register" or a heterogeneous off-register manner to any of the available sites on a multivalent partner. Here, using NMR chemical shift perturbation, analytical ultracentrifugation, and native electrospray ionization MS, we show that LC8 forms a predominantly in-register complex when bound to an IDP domain of the multivalent regulatory protein ASCIZ. Using saturation transfer difference NMR, we demonstrate that at substoichiometric LC8 concentrations, the IDP domain preferentially binds to one of the three LC8 recognition motifs. Further, the differential dynamic behavior for the three sites and the size of the fully bound complex confirmed an in-register complex. Dynamics measurements also revealed that coupling between sites depends on the linker length separating these sites. These results identify linker length and motif specificity as drivers of in-register binding in the multivalent LC8-IDP complex assembly and the degree of compositional and conformational heterogeneity as a promising emerging mechanism for tuning of binding and regulation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Dineínas/química , Dineínas/genética , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Modelos Moleculares , Conformación Proteica , Homología de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: This study aims to ascertain and compare the prevalence and correlates of alcohol-related harms to children cross-nationally. METHOD: National and regional sample surveys of randomly selected households included 7,848 carers (4,223 women) from eight countries (Australia, Chile, Ireland, Lao People's Democratic Republic [PDR], Nigeria, Sri Lanka, Thailand, and Vietnam). Country response rates ranged from 35% to 99%. Face-to-face or telephone surveys asking about harm from others' drinking to children ages 0-17 years were conducted, including four specific harms: that because of others' drinking in the past year children had been (a) physically hurt, (b) verbally abused, (c) exposed to domestic violence, or (d) left unsupervised. RESULTS: The prevalence of alcohol-related harms to children varied from a low of 4% in Lao PDR to 14% in Vietnam. Alcohol-related harms to children were reported by a substantial minority of families in most countries, with only Lao PDR and Nigeria reporting significantly lower levels of harm. Alcohol-related harms to children were dispersed sociodemographically and were concentrated in families with heavy drinkers. CONCLUSIONS: Family-level drinking patterns were consistently identified as correlates of harm to children because of others' drinking, whereas sociodemographic factors showed few obvious correlations.
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Consumo de Bebidas Alcohólicas/epidemiología , Maltrato a los Niños/estadística & datos numéricos , Adolescente , Adulto , Intoxicación Alcohólica/epidemiología , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Prevalencia , Encuestas y Cuestionarios , Adulto JovenRESUMEN
With motorcycle ownership high and rising in Viet Nam, and motorcycle riders vulnerable to both fatal and non-fatal injury, the re-introduction of mandatory helmet legislation in 2007 has been a priority for the Vietnamese government. The paper uses a qualitative analysis of web-based versions of the eight most popular newspapers in Viet Nam to track reporting over four phases of the implementation of the legislation, identifying codes and constructing the dominant themes of the media coverage. The study documents the justification and promotion of the legislation, and the mechanisms for preparing for its implementation at a national and local level, developing solutions and encouraging the replication of successful strategies. It records opposition and obstacles to helmet use, and concerns raised around the quality of helmets purchased. In return, the press notes the response of the market in innovative solutions to these problems. With the successful implementation of the legislation, the functions of the print media in promulgating and promoting the legislation, together with the reporting of ongoing resistance to the process, serve to enable a dialogue between the State and population around expressed concerns. In highlighting quality control of helmets as a key issue, the media have identified a potential ongoing role in monitoring the state's initiative in reducing the road toll from traumatic brain injury in motorcyclists.
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Accidentes de Tránsito/estadística & datos numéricos , Lesiones Encefálicas/prevención & control , Dispositivos de Protección de la Cabeza/estadística & datos numéricos , Medios de Comunicación de Masas/estadística & datos numéricos , Motivación , Periódicos como Asunto/estadística & datos numéricos , Política Pública , Lesiones Encefálicas/epidemiología , Promoción de la Salud , Humanos , Mercadeo Social , Vietnam/epidemiología , Heridas y Lesiones/epidemiología , Heridas y Lesiones/prevención & controlRESUMEN
BACKGROUND: Road traffic accidents are the leading cause of fatal and non-fatal injuries in Vietnam. The purpose of this study is to estimate the costs, in the first year post-injury, of non-fatal traumatic brain injury (TBI) in motorcycle users not wearing helmets in Hanoi, Vietnam. The costs are calculated from the perspective of the injured patients and their families, and include quantification of direct, indirect and intangible costs, using years lost due to disability as a proxy. METHODS: The study was a retrospective cross-sectional study. Data on treatment and rehabilitation costs, employment and support were obtained from patients and their families using a structured questionnaire and The European Quality of Life instrument (EQ6D). RESULTS: Thirty-five patients and their families were interviewed. On average, patients with severe, moderate and minor TBI incurred direct costs at USD 2,365, USD 1,390 and USD 849, with time lost for normal activities averaging 54 weeks, 26 weeks and 17 weeks and years lived with disability (YLD) of 0.46, 0.25 and 0.15 year, respectively. CONCLUSION: All three component costs of TBI were high; the direct cost accounted for the largest proportion, with costs rising with the severity of TBI. The results suggest that the burden of TBI can be catastrophic for families because of high direct costs, significant time off work for patients and caregivers, and impact on health-related quality of life. Further research is warranted to explore the actual social and economic benefits of mandatory helmet use.