RESUMEN
G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners. For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1.
Asunto(s)
Proteínas Portadoras , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , Membrana Celular , Inmunoprecipitación , Espectrometría de MasasRESUMEN
BACKGROUND: Neuroinflammation is a widely studied phenomenon underlying various neurodegenerative diseases. Earlier study demonstrated that pharmacological activation of GPR110 in both central and peripheral immune cells cooperatively ameliorates neuroinflammation caused by systemic lipopolysaccharide (LPS) administration. Ethanol consumption has been associated with exacerbation of neurodegenerative and systemic inflammatory conditions. The goal of this study is to determine the effects of single-dose acute ethanol exposure and GPR110 activation on the neuro-inflammation mechanisms. METHODS: For in vivo studies, GPR110 wild type (WT) and knockout (KO) mice at 10-12 weeks of age were given an oral gavage of ethanol (3 g/kg) or maltose (5.4 g/kg) at 1-4 h prior to the injection of LPS (1 mg/kg, i.p.) followed by the GPR110 ligand, synaptamide (5 mg/kg). After 2-24 h, brains were collected for the analysis of gene expression by RT-PCR or protein expression by western blotting and enzyme-linked immunosorbent assay (ELISA). Microglial activation was assessed by western blotting and immunohistochemistry. For in vitro studies, microglia and peritoneal macrophages were isolated from adult WT mice and treated with 25 mM ethanol for 4 h and then with LPS (100 ng/ml) followed by 10 nM synaptamide for 2 h for gene expression and 12 h for protein analysis. RESULTS: Single-dose exposure to ethanol by gavage before LPS injection upregulated pro-inflammatory cytokine expression in the brain and plasma. The LPS-induced Iba-1 expression in the brain was significantly higher after ethanol pretreatment in both WT and GPR110KO mice. GPR110 ligand decreased the mRNA and/or protein expression of these cytokines and Iba-1 in the WT but not in GPR110KO mice. In the isolated microglia and peritoneal macrophages, ethanol also exacerbated the LPS-induced expression of pro-inflammatory cytokines which was mitigated at least partially by synaptamide. The expression of an inflammasome marker NLRP3 upregulated by LPS was further elevated with prior exposure to ethanol, especially in the brains of GPR110KO mice. Both ethanol and LPS reduced adenylate cyclase 8 mRNA expression which was reversed by the activation of GPR110. PDE4B expression at both mRNA and protein level in the brain increased after ethanol and LPS treatment while synaptamide suppressed its expression in a GPR110-dependent manner. CONCLUSION: Single-dose ethanol exposure exacerbated LPS-induced inflammatory responses. The GPR110 ligand synaptamide ameliorated this effect of ethanol by counteracting on the cAMP system, the common target for synaptamide and ethanol, and by regulating NLRP3 inflammasome.
Asunto(s)
Etanol , Enfermedades Neuroinflamatorias , Receptores Acoplados a Proteínas G , Animales , Ratones , Citocinas/metabolismo , Etanol/toxicidad , Inflamasomas/metabolismo , Ligandos , Lipopolisacáridos/toxicidad , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , ARN Mensajero/metabolismoRESUMEN
Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin. By removing non-specific partners by comparing the binding proteins of GPR110 WT and an inactive mutant exhibiting impaired surface expression, occludin was distinguished as a true binding partner which was further confirmed by reciprocal co-immunoprecipitation assay. Deletion of GPR110 in mice led to the disruption of blood-brain barrier (BBB) and reduced occludin phosphorylation at Y285 in the brain. The Y285 phosphorylation increased upon the ligand-induced activation of GPR110. These data suggest an important role of GPR110-occludin interaction in BBB function and association of previously unknown GPR110-dependent occludin phosphorylation at Y285 with BBB integrity.
RESUMEN
It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.
Asunto(s)
Traumatismos del Nervio Óptico , Animales , Ratones , Axones , Ligandos , Ratones Noqueados , Compresión Nerviosa , Regeneración Nerviosa/fisiología , Receptores Acoplados a Proteínas G/genética , Retina , Células Ganglionares de la Retina/fisiologíaRESUMEN
The neurodevelopmental and neuroprotective actions of docosahexaenoic acid (DHA) are mediated by mechanisms involving membrane- and metabolite-related signal transduction. A key characteristic in the membrane-mediated action of DHA results from the stimulated synthesis of neuronal phosphatidylserine (PS). The resulting DHA-PS-rich membrane domains facilitate the translocation and activation of kinases such as Raf-1, protein kinase C (PKC), and Akt. The activation of these signaling pathways promotes neuronal development and survival. DHA is also metabolized in neural tissues to bioactive mediators. Neuroprotectin D1, a docosatriene synthesized by the lipoxygenase activity, has an anti-inflammatory property, and elovanoids formed from DHA elongation products exhibit antioxidant effects in the retina. Synaptamide, an endocannabinoid-like lipid mediator synthesized from DHA in the brain, promotes neurogenesis and synaptogenesis and exerts anti-inflammatory effects. It binds to the GAIN domain of the GPR110 (ADGRF1) receptor, triggers the cAMP/protein kinase A (PKA) signaling pathway, and activates the cAMP-response element binding protein (CREB). The DHA status in the brain influences not only the PS-dependent signal transduction but also the metabolite formation and expression of pre- and post-synaptic proteins that are downstream of the CREB and affect neurotransmission. The combined actions of these processes contribute to the neurodevelopmental and neuroprotective effects of DHA.
Asunto(s)
Ácidos Docosahexaenoicos , Neuroprotección , Antiinflamatorios/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Endocannabinoides/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450 mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. METHODS AND RESULTS: Young adult male rats sustaining upper-moderate BACs (~150 mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1 week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. CONCLUSIONS: The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.
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Antiinflamatorios/farmacología , Etanol/farmacología , Etanolaminas/metabolismo , Fosfolipasas A2 Calcio-Independiente/farmacología , Fosfolipasas A2 Citosólicas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Fosfolipasas A2/metabolismo , RatasRESUMEN
BACKGROUND: Repetitive mild traumatic brain injury (mTBI) can result in chronic visual dysfunction. G-protein receptor 110 (GPR110, ADGRF1) is the target receptor of N-docosahexaenoylethanolamine (synaptamide) mediating the anti-neuroinflammatory function of synaptamide. In this study, we evaluated the effect of an endogenous and a synthetic ligand of GPR110, synaptamide and (4Z,7Z,10Z,13Z,16Z,19Z)-N-(2-hydroxy-2-methylpropyl) docosa-4,7,10,13,16,19-hexaenamide (dimethylsynaptamide, A8), on the mTBI-induced long-term optic tract histopathology and visual dysfunction using Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA), a clinically relevant model of mTBI. METHODS: The brain injury in wild-type (WT) and GPR110 knockout (KO) mice was induced by CHIMERA applied daily for 3 days, and GPR110 ligands were intraperitoneally injected immediately following each impact. The expression of GPR110 and proinflammatory mediator tumor necrosis factor (TNF) in the brain was measured by using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in an acute phase. Chronic inflammatory responses in the optic tract and visual dysfunction were assessed by immunostaining for Iba-1 and GFAP and visual evoked potential (VEP), respectively. The effect of GPR110 ligands in vitro was evaluated by the cyclic adenosine monophosphate (cAMP) production in primary microglia isolated from adult WT or KO mouse brains. RESULTS: CHIMERA injury acutely upregulated the GPR110 and TNF gene level in mouse brain. Repetitive CHIMERA (rCHIMERA) increased the GFAP and Iba-1 immunostaining of glia cells and silver staining of degenerating axons in the optic tract with significant reduction of N1 amplitude of visual evoked potential at up to 3.5 months after injury. Both GPR110 ligands dose- and GPR110-dependently increased cAMP in cultured primary microglia with A8, a ligand with improved stability, being more effective than synaptamide. Intraperitoneal injection of A8 at 1 mg/kg or synaptamide at 5 mg/kg significantly reduced the acute expression of TNF mRNA in the brain and ameliorated chronic optic tract microgliosis, astrogliosis, and axonal degeneration as well as visual deficit caused by injury in WT but not in GPR110 KO mice. CONCLUSION: Our data demonstrate that ligand-induced activation of the GPR110/cAMP system upregulated after injury ameliorates the long-term optic tract histopathology and visual impairment caused by rCHIMERA. Based on the anti-inflammatory nature of GPR110 activation, we suggest that GPR110 ligands may have therapeutic potential for chronic visual dysfunction associated with mTBI.
Asunto(s)
Conmoción Encefálica/complicaciones , Etanolaminas/metabolismo , Etanolaminas/farmacología , Gliosis/tratamiento farmacológico , Gliosis/metabolismo , Tracto Óptico/efectos de los fármacos , Tracto Óptico/patología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Conmoción Encefálica/patología , Técnicas de Cultivo de Célula , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Potenciales Evocados Visuales , Gliosis/complicaciones , Inflamación , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Tracto Óptico/lesiones , Factor de Necrosis Tumoral alfa/metabolismo , Visión OcularRESUMEN
Repeated mild traumatic brain injury (TBI) can cause persistent neuropathological effects and is a major risk factor for chronic traumatic encephalopathy. PUFAs (n-3 polyunsaturated fatty acids) were shown to improve acute TBI outcomes in single-injury models in most cases. In this study, we demonstrate positive effects of dietary n-3 PUFA on long-term neuropathological and functional outcome in a clinically relevant model of repeated mild TBI using the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA). Adult mice, reared on n-3 PUFA adequate (higher n-3 PUFA) or deficient (lower n-3 PUFA) diets, were given a mild CHIMERA daily for 3 consecutive days. At 2 months after injury, visual function and spatial memory were evaluated. Glia cell activation was assessed by immunostaining using antibodies of ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein, and axonal damage was examined using silver staining. Repeated CHIMERA (rCHIMERA)-induced gliosis was significantly suppressed in the optic tract, corpus callosum, and hippocampus of mice fed the n-3 PUFA adequate diet compared to the deficient diet group. Considerable axonal damage was detected in the optic tract after rCHIMERA, but the adequate diet group displayed less axonal damage compared to the deficient diet group. rCHIMERA induced a drastic reduction in N1 amplitude of the visual evoked potential in both diet groups and the a-wave amplitude of the electroretinogram in the deficient diet group. However, reduction of N1 and a-wave amplitude were less severe in the adequate diet group. The Morris water maze probe test indicated a significant decrease in the number of platform crossings in the deficient diet group compared to the adequate group. In summary, dietary n-3 PUFA can attenuate persistent glial cell activation and axonal damage and improve deficits in visual function and spatial memory after repeated mild TBI. These data support the neuroprotective potential of a higher n-3 PUFA diet in ameliorating the adverse outcome of repeated mild TBI.
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Conmoción Encefálica/tratamiento farmacológico , Conmoción Encefálica/psicología , Dieta , Ácidos Grasos Omega-3/uso terapéutico , Enfermedades del Sistema Nervioso/etiología , Animales , Axones/patología , Ácidos Grasos Omega-3/metabolismo , Femenino , Inmunohistoquímica , Activación de Macrófagos , Masculino , Ratones Endogámicos C57BL , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/psicología , Neuroglía/efectos de los fármacos , Tracto Óptico/patología , Embarazo , Recurrencia , Memoria Espacial , Resultado del Tratamiento , Visión OcularRESUMEN
Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.
Asunto(s)
Axones/metabolismo , Etanolaminas/farmacología , Regeneración Nerviosa , Receptores Acoplados a Proteínas G/metabolismo , Animales , Femenino , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microfluídica , Simulación del Acoplamiento Molecular , Compresión Nerviosa , Neurogénesis , Neuronas/metabolismo , Nervio Óptico/metabolismo , Retina/metabolismoRESUMEN
We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.
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Ácidos Araquidónicos/biosíntesis , Ácidos Docosahexaenoicos/metabolismo , Endocannabinoides/biosíntesis , Etanolaminas/metabolismo , Lisofosfatidilcolinas/metabolismo , Neuronas/metabolismo , Animales , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/aislamiento & purificación , Bitionol/farmacología , Isótopos de Carbono , Línea Celular Tumoral , Cromatografía Liquida , Endocannabinoides/antagonistas & inhibidores , Endocannabinoides/aislamiento & purificación , Etanolaminas/antagonistas & inhibidores , Etanolaminas/aislamiento & purificación , Hexaclorofeno/farmacología , Cinética , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Plasmalógenos/antagonistas & inhibidores , Plasmalógenos/biosíntesis , Plasmalógenos/aislamiento & purificación , Alcamidas Poliinsaturadas/antagonistas & inhibidores , Alcamidas Poliinsaturadas/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
Previous studies suggest that long-term supplementation and dietary intake of omega-3 polyunsaturated fatty acids (PUFAs) may have neuroprotective effects following brain injury. The objective of this study was to investigate potential neuroprotective effects of omega-3 PUFAs on white matter following closed-head trauma. The closed-head injury model of engineered rotational acceleration (CHIMERA) produces a reproducible injury in the optic tract and brachium of the superior colliculus in mice. Damage is detectable using diffusion tensor imaging (DTI) metrics, particularly fractional anisotropy (FA), with sensitivity comparable to histology. We acquired in vivo (n = 38) and ex vivo (n = 41) DTI data in mice divided into sham and CHIMERA groups with two dietary groups: one deficient in omega-3 PUFAs and one adequate in omega-3 PUFAs. We examined injury effects (reduction in FA) and neuroprotection (FA reduction modulated by diet) in the optic tract and brachium. We verified that diet did not affect FA in sham animals. In injured animals, we found significantly reduced FA in the optic tract and brachium (~10% reduction, p < 0.001), and Bayes factor analysis showed strong evidence to reject the null hypothesis. However, Bayes factor analysis showed substantial evidence to accept the null hypothesis of no diet-related FA differences in injured animals in the in vivo and ex vivo samples. Our results indicate no neuroprotective effect from adequate dietary omega-3 PUFA intake on white matter damage following traumatic brain injury. Since damage from CHIMERA mainly affects white matter, our results do not necessarily contradict previous findings showing omega-3 PUFA-mediated neuroprotection in gray matter.
Asunto(s)
Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Dieta , Ácidos Grasos Omega-3/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/lesiones , Animales , Teorema de Bayes , Imagen de Difusión Tensora , Sustancia Gris/patología , Traumatismos Cerrados de la Cabeza/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Tracto Óptico/diagnóstico por imagen , Tracto Óptico/lesiones , Colículos Superiores/diagnóstico por imagen , Colículos Superiores/lesionesRESUMEN
Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.
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Etanolaminas/farmacología , Proteínas Oncogénicas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Sitios de Unión , Etanolaminas/metabolismo , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Visual dysfunction is a common occurrence after traumatic brain injury (TBI). We investigated in this study effects of single or multiple mild TBI on visual function in mice using a closed head injury model that permits unconstrained head movement after impact. Adult mice were briefly anesthetized with isoflurane and given one or three mild TBI with the closed head injury by mechanically engineered rotational acceleration (CHIMERA) device with an interinjury interval of 24 h. Mice were then tested in the Morris water maze, visual cliff, and open field tests from day 19 to day 32 and for visual evoked potential at 5 weeks after the last injury and euthanized. Mice with multiple TBI showed impaired performance in the visible platform water maze test and had increased errors in the visual cliff test. Further, there was a graded difference in visual evoked potential, with the single injury mice showing modest reduction in N1 amplitude whereas the multiple injuries produced significant reduction compared to sham and single injury groups. The optic tract of the injured mice showed increases in glial cell immunostaining. The increase in glial fibrillary acid protein immunostaining reached statistical significance for both injured groups whereas the ionized calcium binding adaptor molecule 1 immunostaining was only significantly increased in the optic tract of repeatedly injured mice. These results indicate that multiple injuries using CHIMERA may result in visual deficits, which can affect certain behavioral performances. The change in vision may be a useful marker when monitoring repeated TBI outcome and screening for protective agents from TBI.
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Lesiones Traumáticas del Encéfalo/patología , Potenciales Evocados Visuales/fisiología , Traumatismos Cerrados de la Cabeza/patología , Tracto Óptico/patología , Animales , Lesiones Traumáticas del Encéfalo/etiología , Modelos Animales de Enfermedad , Traumatismos Cerrados de la Cabeza/complicaciones , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Neuroinflammation is a widely accepted underlying condition for various pathological processes in the brain. In a recent study, synaptamide, an endogenous metabolite derived from docosahexaenoic acid (DHA, 22:6n-3), was identified as a specific ligand to orphan adhesion G-protein-coupled receptor 110 (GPR110, ADGRF1). Synaptamide has been shown to suppress lipopolysaccharide (LPS)-induced neuroinflammation in mice, but involvement of GPR110 in this process has not been established. In this study, we investigated the possible immune regulatory role of GPR110 in mediating the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. METHODS: For in vitro studies, we assessed the role of GPR110 in synaptamide effects on LPS-induced inflammatory responses in adult primary mouse microglia, immortalized murine microglial cells (BV2), primary neutrophil, and peritoneal macrophage by using quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) as well as neutrophil migration and ROS production assays. To evaluate in vivo effects, wild-type (WT) and GPR110 knock-out (KO) mice were injected with LPS intraperitoneally (i.p.) or TNF intravenously (i.v.) followed by synaptamide (i.p.), and expression of proinflammatory mediators was measured by qPCR, ELISA, and western blot analysis. Activated microglia in the brain and NF-kB activation in cells were examined microscopically after immunostaining for Iba-1 and RelA, respectively. RESULTS: Intraperitoneal (i.p.) administration of LPS increased TNF and IL-1ß in the blood and induced pro-inflammatory cytokine expression in the brain. Subsequent i.p. injection of the GPR110 ligand synaptamide significantly reduced LPS-induced inflammatory responses in wild-type (WT) but not in GPR110 knock-out (KO) mice. In cultured microglia, synaptamide increased cAMP and inhibited LPS-induced proinflammatory cytokine expression by inhibiting the translocation of NF-κB subunit RelA into the nucleus. These effects were abolished by blocking synaptamide binding to GPR110 using an N-terminal targeting antibody. GPR110 expression was found to be high in neutrophils and macrophages where synaptamide also caused a GPR110-dependent increase in cAMP and inhibition of LPS-induced pro-inflammatory mediator expression. Intravenous injection of TNF, a pro-inflammatory cytokine that increases in the circulation after LPS treatment, elicited inflammatory responses in the brain which were dampened by the subsequent injection (i.p.) of synaptamide in a GPR110-dependent manner. CONCLUSION: Our study demonstrates the immune-regulatory function of GPR110 in both brain and periphery, collectively contributing to the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. We suggest GPR110 activation as a novel therapeutic strategy to ameliorate inflammation in the brain as well as periphery.
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Antiinflamatorios/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Etanolaminas/farmacología , Inflamación/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1⯱â¯0.3â¯g/kg/day), or control (dextrose)⯱â¯veliparib (25â¯mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.
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Trastornos del Sistema Nervioso Inducidos por Alcohol/prevención & control , Bencimidazoles/uso terapéutico , Proteínas del Tejido Nervioso/biosíntesis , Fosfolipasas A2 Citosólicas/biosíntesis , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Trastornos del Sistema Nervioso Inducidos por Alcohol/tratamiento farmacológico , Trastornos del Sistema Nervioso Inducidos por Alcohol/enzimología , Animales , Bencimidazoles/farmacología , Consumo Excesivo de Bebidas Alcohólicas , Giro Dentado/efectos de los fármacos , Giro Dentado/enzimología , Giro Dentado/patología , Modelos Animales de Enfermedad , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/enzimología , Corteza Entorrinal/patología , Inducción Enzimática/efectos de los fármacos , Masculino , Proteínas del Tejido Nervioso/genética , Fosfolipasas A2 Citosólicas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Humanos , Receptores Acoplados a Proteínas G/químicaRESUMEN
Shortly after the discovery that linoleic acid was an essential fatty acid in 1930, α-linolenic acid also was reported to prevent the fatty acid deficiency syndrome in animals. However, several prominent laboratories could not confirm the findings with α-linolenic acid, and as a result there was a loss of interest in omega-3 fatty acids in lipid research. Even the findings that a prostaglandin can be synthesized from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is necessary for optimum retinal function generated only limited interest in omega-3 fatty acids. The breakthrough came in the 1970s when Dyerberg and Bang reported that the low incidence of atherosclerotic coronary disease in Greenland Eskimos was due to the high marine lipid content of their diet. They subsequently found that EPA, which was increased in Eskimo plasma, inhibited platelet aggregation, and they concluded that the low incidence of coronary artery disease was due to the anti-thrombotic effect of EPA. This stimulated widespread interest and research in EPA and DHA, leading to the present view that, like their omega-6 counterparts, omega-3 fatty acids have important physiological functions and are essential fatty acids.
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Investigación Biomédica , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-3/fisiología , Fibrinolíticos/farmacología , Animales , Ácido Araquidónico/fisiología , Enfermedad de la Arteria Coronaria/sangre , Trombosis Coronaria/sangre , Dieta , Humanos , Inuk , Ácido Linoleico/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , RatasRESUMEN
PURPOSE: Measure concentrations of the neurogenic, pro-neurogenic, pro-synaptogenic and anti-inflammatory mediator N-docosahexaenoylethanolamine (synaptamide) in relation to its precursor docosahexaenoic acid (DHA) in breast milk. DESIGN AND METHODS: Postpartum women were recruited prior to discharge. We supplemented half the subjects with omega-3 fatty acids. Breast milk samples were collected at 1, 4 and 8 weeks. Synaptamide and DHA concentrations were determined by liquidchromatography/tandem mass spectrometry (LC-MS/MS) and gas chromatography, respectively. RESULTS: Synaptamide was detected in all breast milk samples. The concentration ranged from 44 to 257 fmol/mL. Omega-3 fatty acid supplementation did not affect DHA or synaptamide concentration in breast milk due to a high-DHA-containing diet self-selected by control mothers. Nevertheless, synaptamide levels significantly correlated with DHA concentration in breast milk (râ¯=â¯0.624, Pâ¯<â¯0.001). CONCLUSION: This is the first demonstration of detectable concentrations of synaptamide in human breast milk. Although the attempt to raise the milk DHA content by omega-3 fatty acid supplementation was not successful in the current study, the positive correlation observed between synaptamide and DHA concentration suggests that synaptamide levels in human milk can be raised by proper omega-3 fatty acid supplementation that is known to increase DHA.
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Ácidos Docosahexaenoicos/química , Etanolaminas/química , Ácidos Grasos Omega-3/administración & dosificación , Leche Humana/química , Adulto , Ácidos Docosahexaenoicos/aislamiento & purificación , Etanolaminas/aislamiento & purificación , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Leche Humana/efectos de los fármacos , Periodo Posparto/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
N-Docosahexaenoylethanolamine (synaptamide) is an endocannabinoid-like metabolite endogenously synthesized from docosahexaenoic acid (DHA, 22:6n-3), the major omega-3 polyunsaturated fatty acid present in the brain. Although its biosynthetic mechanism has yet to be established, there is a closely linked relationship between the levels of synaptamide and its precursor DHA in the brain. Synaptamide at nanomolar concentrations promotes neurogenesis, neurite outgrowth and synaptogenesis in developing neurons. Synaptamide also attenuates the lipopolysaccharide-induced neuroinflammatory response and reduces the deleterious effects of ethanol on neurogenic differentiation of neural stem cells (NSCs). These actions are mediated by a specific target receptor of synaptamide GPR110 (ADGRF1), a G-protein coupled receptor that is highly expressed in NSCs and the brain during development. Synaptamide binding to GPR110 induces cAMP production and phosphorylation of protein kinase A (PKA) and the cAMP response element binding protein (CREB). This signaling pathway leads to the expression of neurogenic and synaptogenic genes and suppresses the expression of proinflammatory genes. The GPR110-dependent cellular effects of synaptamide are recapitulated in animal models, suggesting that synaptamide-derived mechanisms may have translational implications. The synaptamide bioactivity transmitted by newly deorphanized GPR110 provides a novel target for neurodevelopmental and neuroprotective control as well as new insight into mechanisms for DHA's beneficial effects on the central nervous system.
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Encéfalo/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Etanolaminas/metabolismo , Proteínas Oncogénicas/genética , Receptores Acoplados a Proteínas G/genética , Animales , Encéfalo/patología , Diferenciación Celular/genética , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Endocannabinoides/genética , Endocannabinoides/metabolismo , Etanolaminas/uso terapéutico , Ácidos Grasos Omega-3/genética , Ácidos Grasos Omega-3/metabolismo , Humanos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuronas/metabolismoRESUMEN
Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol's brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day-binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors-olaparib, veliparib, and 4-aminobenzamide-provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol's amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective-thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2âphospholipidâARA and HMGB1âTLR4âproinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration.