RESUMEN
Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.
Asunto(s)
Conducta Adictiva/metabolismo , Receptores de Neurotensina/metabolismo , beta-Arrestinas/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Conducta Adictiva/tratamiento farmacológico , Línea Celular , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Brain-wide fluctuations in local field potential oscillations reflect emergent network-level signals that mediate behavior. Cracking the code whereby these oscillations coordinate in time and space (spatiotemporal dynamics) to represent complex behaviors would provide fundamental insights into how the brain signals emotional pathology. Using machine learning, we discover a spatiotemporal dynamic network that predicts the emergence of major depressive disorder (MDD)-related behavioral dysfunction in mice subjected to chronic social defeat stress. Activity patterns in this network originate in prefrontal cortex and ventral striatum, relay through amygdala and ventral tegmental area, and converge in ventral hippocampus. This network is increased by acute threat, and it is also enhanced in three independent models of MDD vulnerability. Finally, we demonstrate that this vulnerability network is biologically distinct from the networks that encode dysfunction after stress. Thus, these findings reveal a convergent mechanism through which MDD vulnerability is mediated in the brain.
Asunto(s)
Encéfalo/fisiología , Depresión/patología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Depresión/fisiopatología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Electrodos Implantados , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ketamina/farmacología , Aprendizaje Automático , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenómenos Fisiológicos/efectos de los fármacos , Corteza Prefrontal/fisiología , Estrés PsicológicoRESUMEN
The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.
Asunto(s)
Neurotensina , Receptores de Neurotensina , Receptores de Neurotensina/química , Receptores de Neurotensina/metabolismo , Neurotensina/metabolismo , Transducción de Señal , Péptidos/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The United States is experiencing a dramatic increase in maternal opioid misuse and, consequently, the number of individuals exposed to opioids in utero. Prenatal opioid exposure has both acute and long-lasting effects on health and wellbeing. Effects on the brain, often identified at school age, manifest as cognitive impairment, attention deficit, and reduced scholastic achievement. The neurobiological basis for these effects is poorly understood. Here, we examine how in utero exposure to heroin affects brain development into early adolescence in a mouse model. Pregnant C57BL/6J mice received escalating doses of heroin twice daily on gestational days 4-18. The brains of offspring were assessed on postnatal day 28 using 9.4 T diffusion MRI of postmortem specimens at 36 µm resolution. Whole-brain volumes and the volumes of 166 bilateral regions were compared between heroin-exposed and control offspring. We identified a reduction in whole-brain volume in heroin-exposed offspring and heroin-associated volume changes in 29 regions after standardizing for whole-brain volume. Regions with bilaterally reduced standardized volumes in heroin-exposed offspring relative to controls include the ectorhinal and insular cortices. Regions with bilaterally increased standardized volumes in heroin-exposed offspring relative to controls include the periaqueductal gray, septal region, striatum, and hypothalamus. Leveraging microscopic resolution diffusion tensor imaging and precise regional parcellation, we generated whole-brain structural MRI diffusion connectomes. Using a dimension reduction approach with multivariate analysis of variance to assess group differences in the connectome, we found that in utero heroin exposure altered structure-based connectivity of the left septal region and the region that acts as a hub for limbic regulatory actions. Consistent with clinical evidence, our findings suggest that prenatal opioid exposure may have effects on brain morphology, connectivity, and, consequently, function that persist into adolescence. This work expands our understanding of the risks associated with opioid misuse during pregnancy and identifies biomarkers that may facilitate diagnosis and treatment.
Asunto(s)
Trastornos Relacionados con Opioides , Efectos Tardíos de la Exposición Prenatal , Humanos , Embarazo , Femenino , Animales , Ratones , Heroína/efectos adversos , Imagen de Difusión Tensora/métodos , Analgésicos Opioides/farmacología , Ratones Endogámicos C57BL , EncéfaloRESUMEN
BACKGROUND & AIMS: Mood disorders and constipation are often comorbid, yet their shared etiologies have rarely been explored. The neurotransmitter serotonin (5-HT) regulates central nervous system and enteric nervous system (ENS) development and long-term functions, including gastrointestinal (GI) motility and mood. Therefore, defects in neuron production of 5-HT might result in brain and intestinal dysfunction. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis. A variant of TPH2 that encodes the R441H substitution (TPH2-R441H) was identified in individuals with severe depression. We studied mice with an analogous mutation (TPH2-R439H), which results in a 60%-80% decrease in levels of 5-HT in the central nervous system and behaviors associated with depression in humans. Feeding chow that contains 5-HTP slow release (5-HTP SR) to TPH2-R439H mice restores levels of 5-HT in the central nervous system and reduces depressive-like behaviors. METHODS: We compared the effects of feeding chow, with or without 5-HTP SR, to mice with the TPH2-R439H mutation and without this mutation (control mice). Myenteric and submucosal plexuses were isolated from all 4 groups of mice, and immunocytochemistry was used to quantify total enteric neurons, serotonergic neurons, and 5-HT-dependent subsets of neurons. We performed calcium imaging experiments to evaluate responses of enteric neurons to tryptamine-evoked release of endogenous 5-HT. In live mice, we measured total GI transit, gastric emptying, small intestinal transit, and propulsive colorectal motility. To measure colonic migrating motor complexes (CMMCs), we isolated colons and constructed spatiotemporal maps along the proximodistal length to quantify the frequency, velocity, and length of CMMCs. We measured villus height, crypt perimeter, and relative densities of enterochromaffin and enteroendocrine cells in small intestinal tissue. RESULTS: Levels of 5-HT were significantly lower in enteric neurons from TPH2-R439H mice than from control mice. TPH2-R439H mice had abnormalities in ENS development and ENS-mediated GI functions, including reduced motility and intestinal epithelial growth. Total GI transit and propulsive colorectal motility were slower in TPH2-R439H mice than controls, and CMMCs were slower and less frequent. Villus height and crypt perimeter were significantly decreased in colon tissues from TPH2-R439H mice compared with controls. Administration of 5-HTP SR to adult TPH2-R439H mice restored 5-HT to enteric neurons and reversed these abnormalities. Adult TPH2-R439H mice given oral 5-HTP SR had normalized numbers of enteric neurons, total GI transit, and colonic motility. Intestinal tissue from these mice had normal measures of CMMCs and enteric epithelial growth CONCLUSIONS: In studies of TPH2-R439H mice, we found evidence for reduced release of 5-HT from enteric neurons that results in defects in ENS development and GI motility. Our findings indicate that neuron production of 5-HT links constipation with mood dysfunction. Administration of 5-HTP SR to mice restored 5-HT to the ENS and normalized GI motility and growth of the enteric epithelium. 5-HTP SR might be used to treat patients with intestinal dysfunction associated with low levels of 5-HT.
Asunto(s)
5-Hidroxitriptófano/administración & dosificación , Estreñimiento/tratamiento farmacológico , Depresión/tratamiento farmacológico , Tracto Gastrointestinal/fisiopatología , Serotonina/metabolismo , Animales , Estreñimiento/etiología , Estreñimiento/fisiopatología , Preparaciones de Acción Retardada/administración & dosificación , Depresión/complicaciones , Depresión/genética , Depresión/fisiopatología , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/fisiopatología , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/inervación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Resultado del Tratamiento , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
Psychostimulants and opioids increase dopamine (DA) neurotransmission, activating D1 and D2 G protein-coupled receptors. ß-arrestin2 (ßarr2) desensitizes and internalizes these receptors and initiates G protein-independent signaling. Previous work revealed that mice with a global or cell-specific knockout of ßarr2 have altered responses to certain drugs; however, the effects of ßarr2 on the excitability of medium spiny neurons (MSNs), and its role in mediating the rewarding effects of drugs of abuse are unknown. D1-Cre and D2-Cre transgenic mice were crossed with floxed ßarr2 mice to eliminate ßarr2 specifically in cells containing either D1 (D1ßarr2-KO ) or D2 (D2ßarr2-KO ) receptors. We used slice electrophysiology to characterize the role of ßarr2 in modulating D1 and D2 nucleus accumbens MSN intrinsic excitability in response to DA and tested the locomotor-activating and rewarding effects of cocaine and morphine in these mice. Eliminating ßarr2 attenuated the ability of DA to inhibit D2-MSNs and altered the DA-induced maximum firing rate in D1-MSNs. While D1ßarr2-KO mice had mostly normal drug responses, D2ßarr2-KO mice showed dose-dependent reductions in acute locomotor responses to cocaine and morphine, attenuated locomotor sensitization to cocaine, and blunted cocaine reward measured with conditioned place preference. Both D2ßarr2-KO and D1ßarr2-KO mice displayed an enhanced conditioned place preference for the highest dose of morphine. These results indicate that D1- and D2-derived ßarr2 functionally contribute to DA-induced changes in MSN intrinsic excitability and behavioral responses to psychostimulants and opioids dose-dependently.
Asunto(s)
Analgésicos Opioides/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Núcleo Accumbens/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Recompensa , Arrestina beta 2/metabolismo , Analgésicos Opioides/administración & dosificación , Animales , Estimulantes del Sistema Nervioso Central/administración & dosificación , Cocaína/administración & dosificación , Cocaína/farmacología , Femenino , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfina/administración & dosificación , Morfina/farmacología , Núcleo Accumbens/fisiopatología , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genéticaRESUMEN
The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and ß-arrestins (ßarrs). Selectively engaging either the G protein or ßarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to ßarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates ßarr recruitment, bridge these pathways. Therefore, ßarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on ßarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or ßarrs as well as a ßarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the ßarr-preferring D2R achieves ßarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the ßarr-preferring D2R and also contributes to ßarr recruitment at the WT D2R. Additionally, we found an additive interaction between the ßarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving ßarr-biased signaling.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Agonistas de Dopamina/farmacología , Transferencia de Energía , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 3 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Células HEK293 , Humanos , Modelos Teóricos , Mutación , Toxina del Pertussis/metabolismo , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Dopamina D2/genética , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.
Asunto(s)
Antipsicóticos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Arrestina beta 2/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Antagonistas de los Receptores de Dopamina D2/farmacología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Interneuronas/metabolismo , Ligandos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Fenciclidina/toxicidad , Transducción de Señal/efectos de los fármacosRESUMEN
The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/ß-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.
Asunto(s)
Clatrina/química , Endocitosis , Leucina/química , Receptores Acoplados a Proteínas G/química , Adenosina Trifosfatasas/química , Animales , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Dioxolanos/química , Epitelio/metabolismo , Femenino , Homeostasis , Humanos , Lignanos/química , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Ratas , Células Madre/citología , Procesos Estocásticos , Vía de Señalización WntRESUMEN
Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.
Asunto(s)
Isquemia Encefálica/metabolismo , Proteínas de Choque Térmico/metabolismo , Neuroprotección/fisiología , Proteínas Priónicas/metabolismo , Receptores de Activinas Tipo I/metabolismo , Animales , Apoptosis/fisiología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Transducción de Señal/fisiología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and ß-arrestin-dependent intracellular signaling. Ex vivo and in vivo, protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.
Asunto(s)
Heparina/farmacología , Protaminas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Receptores de Apelina , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through ßarrestin-2 to regulate actin/stress fiber rearrangement, suggesting ßarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific ßarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The ßarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for ßarrestin-2 and show that pharmacological inhibition of ßarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of ßarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate ßarrestin-2 activity differentially from G protein activity may be required.
Asunto(s)
Trastornos Relacionados con Cocaína/metabolismo , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Actividad Motora/efectos de los fármacos , Receptores de Ghrelina/antagonistas & inhibidores , Arrestina beta 2/metabolismo , Animales , Línea Celular Tumoral , Fármacos del Sistema Nervioso Central , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Femenino , Ghrelina/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/fisiología , Piperidinas/farmacología , Quinazolinonas/farmacología , Receptores de Ghrelina/metabolismo , Arrestina beta 2/genéticaRESUMEN
Brain serotonin (5-HT) deficiency and exposure to psychosocial stress have both been implicated in the etiology of depression and anxiety disorders, but whether 5-HT deficiency influences susceptibility to depression- and anxiety-like phenotypes induced by psychosocial stress has not been formally established. Most clinically effective antidepressants increase the extracellular levels of 5-HT, and thus it has been hypothesized that antidepressant responses result from the reversal of endogenous 5-HT deficiency, but this hypothesis remains highly controversial. Here we evaluated the impact of brain 5-HT deficiency on stress susceptibility and antidepressant-like responses using tryptophan hydroxylase 2 knockin (Tph2KI) mice, which display 60-80% reductions in brain 5-HT. Our results demonstrate that 5-HT deficiency leads to increased susceptibility to social defeat stress (SDS), a model of psychosocial stress, and prevents the fluoxetine (FLX)-induced reversal of SDS-induced social avoidance, suggesting that 5-HT deficiency may impair antidepressant responses. In light of recent clinical and preclinical studies highlighting the potential of inhibiting the lateral habenula (LHb) to achieve antidepressant and antidepressant-like responses, we also examined whether LHb inhibition could achieve antidepressant-like responses in FLX-insensitive Tph2KI mice subjected to SDS. Our data reveal that using designer receptors exclusively activated by designer drugs (DREADDs) to inhibit LHb activity leads to reduced SDS-induced social avoidance behavior in both WT and Tph2KI mice. This observation provides additional preclinical evidence that inhibiting the LHb might represent a promising alternative therapeutic approach under conditions in which selective 5-HT reuptake inhibitors are ineffective.
Asunto(s)
Antidepresivos/uso terapéutico , Encéfalo/metabolismo , Serotonina/deficiencia , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismo , Animales , Antidepresivos/farmacología , Encéfalo/efectos de los fármacos , Susceptibilidad a Enfermedades , Fluoxetina/farmacología , Fluoxetina/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Serotonina/metabolismo , Triptófano Hidroxilasa/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (L-DOPA), but its prolonged use causes dyskinesias referred to as L-DOPA-induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. ß-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting ß-arrestins in PD L-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson's symptoms that genetic deletion of ß-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic L-DOPA treatment. Viral rescue or overexpression of ß-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine-treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated nonhuman primates, ß-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of L-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance ß-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.
Asunto(s)
Arrestinas/metabolismo , Discinesias/metabolismo , Levodopa/química , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/química , Animales , Arrestinas/genética , Conducta Animal , Modelos Animales de Enfermedad , Dopamina/metabolismo , Discinesia Inducida por Medicamentos/metabolismo , Eliminación de Gen , Macaca , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Oxidopamina/química , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Regulación hacia Arriba , beta-ArrestinasRESUMEN
The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G-protein and ß-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased signal transduction for either the G-protein or ß-arrestin interactions. These mutants were used to resolve the role of G proteins and ß-arrestins in D2R signaling assays. The results show that D2R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D2R canonical MAP kinase signaling cascade activation, whereas ß-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the ß-arrestin-biased D2R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D2R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and ß-arrestin signaling pathways in D2R pharmacology, neurobiology, and associated pathologies.
Asunto(s)
Arrestinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Modelos Moleculares , Receptores de Dopamina D2/metabolismo , Animales , Arrestinas/química , Cuerpo Estriado/citología , Cristalografía , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Mutagénesis , Neuronas/metabolismo , Conformación Proteica , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Análisis de Regresión , beta-ArrestinasRESUMEN
Embryonic development and adult tissue homeostasis require precise information exchange between cells and their microenvironment to coordinate cell behavior. A specialized class of ultra-long actin-rich filopodia, termed cytonemes, provides one mechanism for this spatiotemporal regulation of extracellular cues. We provide here a mechanism whereby the stem-cell marker Lgr5, and its family member Lgr4, promote the formation of cytonemes. Lgr4- and Lgr5-induced cytonemes exceed lengths of 80â µm, are generated through stabilization of nascent filopodia from an underlying lamellipodial-like network and functionally provide a pipeline for the transit of signaling effectors. As proof-of-principle, we demonstrate that Lgr5-induced cytonemes act as conduits for cell signaling by demonstrating that the actin motor and filopodial cargo carrier protein myosin X (Myo10) and the G-protein-coupled receptor (GPCR) signaling effector ß-arrestin-2 (Arrb2) transit into cytonemes. This work delineates a biological function for Lgr4 and Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 function and cytonemes in mammalian stem cell and cancer stem cell behavior.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Adulto , Arrestinas/metabolismo , Transporte Biológico , Western Blotting , Células HEK293 , Humanos , Inmunoprecipitación , Seudópodos/fisiología , Transducción de Señal , Células Madre/citología , Arrestina beta 2 , beta-ArrestinasRESUMEN
GPR55, a G protein-coupled receptor, is an attractive target to alleviate inflammatory and neuropathic pain and treat osteoporosis and cancer. Identifying a potent and selective ligand will aid to further establish the specific physiological roles and pharmacology of the receptor. Towards this goal, a targeted library of 22 compounds was synthesized in a modular fashion to obtain structure-activity relationship information. The general route consisted of coupling a variety of p-aminophenyl sulfonamides to isothiocyanates to form acylthioureas. For the synthesis of a known naphthyl ethyl alcohol motif, route modification led to a shorter and more efficient process. The 22 analogues were analyzed for their ability to serve as agonists at GPR55 and valuable information for both ends of the molecule was ascertained.
Asunto(s)
Diseño de Fármacos , Receptores Acoplados a Proteínas G/agonistas , Tiourea/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Receptores de Cannabinoides , Relación Estructura-Actividad , Tiourea/análogos & derivados , Tiourea/síntesis químicaRESUMEN
A series of 1,3,4-oxadiazol-2-ones was synthesized and tested for activity as antagonists at GPR55 in cellular beta-arrestin redistribution assays. The synthesis was designed to be modular in nature so that a sufficient number of analogues could be rapidly accessed to explore initial structure-activity relationships. The design of analogues was guided by the docking of potential compounds into a model of the inactive form of GPR55. The results of the assays were used to learn more about the binding pocket of GPR55. With this oxadiazolone scaffold, it was determined that modification of the aryl group adjacent to the oxadiazolone ring was often detrimental and that the distal cyclopropane was beneficial for activity. These results will guide further exploration of this receptor.
Asunto(s)
Diseño de Fármacos , Oxadiazoles/química , Oxadiazoles/farmacología , Piperidinas/química , Piperidinas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Arrestinas/metabolismo , Células CHO , Cricetulus , Humanos , Simulación del Acoplamiento Molecular , Oxadiazoles/síntesis química , Piperidinas/síntesis química , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-Actividad , beta-ArrestinasRESUMEN
BACKGROUND: Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed. RESULTS: This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane. CONCLUSION: The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.