RESUMEN
G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.
Asunto(s)
Calcio , Complemento C3a , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismo , Calcio/metabolismo , Complemento C3a/metabolismo , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Animales , Ratones , Línea CelularRESUMEN
OBJECTIVE: Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. METHODS: We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21âA). RESULTS: We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue. CONCLUSIONS: The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.
Asunto(s)
Metabolismo Energético , Neuropéptidos , Hormonas Peptídicas , Animales , Ratones , Dieta , Homeostasis , Neuropéptidos/genética , Neuropéptidos/química , Fragmentos de Péptidos/farmacología , Metabolismo Energético/genética , Metabolismo Energético/fisiologíaRESUMEN
Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. Here, we developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R 21 âA). We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by a temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.
RESUMEN
The TLQP-21 neuropeptide has been implicated in functions as diverse as lipolysis, neurodegeneration and metabolism, thus suggesting an important role in several human diseases. Three binding targets have been proposed for TLQP-21: C3aR1, gC1qR and HSPA8. The aim of this review is to critically evaluate the molecular identity of the TLQP-21 receptor and the proposed multi-receptor mechanism of action. Several studies confirm a critical role for C3aR1 in TLQP-21 biological activity and a largely conserved mode of binding, receptor activation and signaling with C3a, its first-identified endogenous ligand. Conversely, data supporting a role of gC1qR and HSPA8 in TLQP-21 activity remain limited, with no signal transduction pathways being described. Overall, C3aR1 is the only receptor for which a necessary and sufficient role in TLQP-21 activity has been confirmed thus far. This conclusion calls into question the validity of a multi-receptor mechanism of action for TLQP-21 and should inform future studies.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas Mitocondriales/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Complemento/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Fragmentos de Péptidos/genética , Transducción de Señal/fisiologíaRESUMEN
Aside from the classical motor symptoms, Parkinson's disease also has various non-classical symptoms. Interestingly, orexin neurons, involved in the regulation of exploratory locomotion, spontaneous physical activity, and energy expenditure, are affected in Parkinson's. In this study, we hypothesized that Parkinson's-disease-associated pathology affects orexin neurons and therefore impairs functions they regulate. To test this, we used a transgenic animal model of Parkinson's, the A53T mouse. We measured body composition, exploratory locomotion, spontaneous physical activity, and energy expenditure. Further, we assessed alpha-synuclein accumulation, inflammation, and astrogliosis. Finally, we hypothesized that chemogenetic inhibition of orexin neurons would ameliorate observed impairments in the A53T mice. We showed that aging in A53T mice was accompanied by reductions in fat mass and increases in exploratory locomotion, spontaneous physical activity, and energy expenditure. We detected the presence of alpha-synuclein accumulations in orexin neurons, increased astrogliosis, and microglial activation. Moreover, loss of inhibitory pre-synaptic terminals and a reduced number of orexin cells were observed in A53T mice. As hypothesized, this chemogenetic intervention mitigated the behavioral disturbances induced by Parkinson's disease pathology. This study implicates the involvement of orexin in early Parkinson's-disease-associated impairment of hypothalamic-regulated physiological functions and highlights the importance of orexin neurons in Parkinson's disease symptomology.
Asunto(s)
Modelos Animales de Enfermedad , Metabolismo Energético/genética , Actividad Motora/genética , Neuronas/metabolismo , Orexinas/genética , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Animales , Composición Corporal/genética , Gliosis/genética , Gliosis/fisiopatología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Orexinas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , alfa-Sinucleína/metabolismoRESUMEN
Parkinson's disease (PD), classically defined as a progressive motor disorder accompanied with dopaminergic neuron loss and presence of Lewy bodies, is the second most common neurodegenerative disease. PD also has various non-classical symptoms, including cognitive impairments. In addition, inflammation and astrogliosis are recognized as an integral part of PD pathology. The hippocampus (Hipp) is a brain region involved in cognition and memory, and the neuropeptide orexin has been shown to enhance learning and memory. Previous studies show impairments in Hipp-dependent memory in a transgenic mouse model of Parkinson's disease (A53T mice), and we hypothesized that increasing orexin tone will reverse this. To test this, we subjected 3, 5, and 7-month old A53T mice to a Barnes maze and a contextual object recognition test to determine Hipp dependent memory. Inflammation and astrogliosis markers in the Hipp were assessed by immuno-fluorescence densitometry. The data show that early cognitive impairment is coupled with an increase in expression of inflammatory and astrogliosis markers. Next, in two separate experiments, mice were given intra-hippocampal injections of orexin or chemogenetic viral injections of an orexin neuron specific Designer Receptor Exclusively Activated by Designer Drug (DREADD). For the pharmacological approach mice were intracranially treated with orexin A, whereas the chemogenetic approach utilized clozapine N-oxide (CNO). Both pharmacological orexin A intervention as well as chemogenetic activation of orexin neurons ameliorated Hipp-dependent early memory impairment observed in A53T mice. This study implicates orexin in PD-associated cognitive impairment and suggests that exogenous orexin treatment and/or manipulation of endogenous orexin levels may be a potential strategy for addressing early cognitive loss in PD.
Asunto(s)
Hipocampo/metabolismo , Hipocampo/patología , Trastornos de la Memoria/genética , Orexinas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Animales , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Clozapina/análogos & derivados , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/complicaciones , Gliosis/patología , Inflamación/complicaciones , Inflamación/patología , Inyecciones , Masculino , Aprendizaje por Laberinto , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Enfermedad de Parkinson/complicaciones , Reproducibilidad de los ResultadosRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease. PD symptomology is recognized as heterogeneous and in addition to motor function decline includes cognitive, mood, sleep, and metabolic disorders. Previous studies showed early reductions in anxiety and locomotion in the A53T mice model of PD. Since inflammation and astrogliosis are an integral part of PD pathology and impair proper neuronal function, we were keen to investigate if behavioral changes in A53T mice are accompanied by increased inflammation and astrogliosis in the hippocampus (Hipp) and motor cortex (mCtx) brain regions involved in the regulation of anxiety and locomotion, respectively. To test this, we used 3-, 5-, and 7-month-old A53T mice to examine anxiety-like behavior, locomotion, and expression of inflammation and astrogliosis markers in the Hipp and mCtx. Further, we examined the presence of alpha-synuclein accumulation in orexin neurons and orexin neuronal loss. The data show early reductions in anxiety-like behavior as well as increased locomotor activity, which was accompanied by inflammation and astrogliosis in the Hipp and mCtx. Due to the persistence of the orexin neuron population in A53T mice and the involvement of orexin in anxiety and locomotor regulation, we hypothesized that chemogenetic modulation of orexin neurons would reverse the observed reductions in anxiety-like behavior and the increases in locomotor activity in these animals. We showed that chemogenetic activation of orexin neurons in A53T mice restores anxiety-like behavior back to control levels without affecting locomotor activity, whereas the inhibition of orexin neurons reverses the elevated locomotor activity without any effects on anxiety-like behavior. This study exemplifies the complex role of orexin neurons in this model of PD and demonstrates the novel finding that changes in locomotor and anxiety-like behavior are accompanied by inflammation and astrogliosis. Together, these data suggest that the orexin system may play a significant role in early and late stages of PD.
RESUMEN
Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) play a critical role in immune-mediated demyelinating diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), by regulating the viability of oligodendrocytes. Our previous studies show that activation of the PERK branch of the UPR protects myelinating oligodendrocytes against ER stress in young, developing mice that express IFN-γ, a key pro-inflammatory cytokine in MS and EAE, in the CNS. Several studies also demonstrate that PERK activation preserves oligodendrocyte viability and function, protecting mice against EAE. While evidence suggests activation of the ATF6α branch of the UPR in oligodendrocytes under normal and disease conditions, the effects of ATF6α activation on oligodendrocytes in immune-mediated demyelinating diseases remain unknown. Herein, we showed that ATF6α deficiency had no effect on oligodendrocytes under normal conditions. Interestingly, we showed that ATF6α deficiency exacerbated ER stressed-induced myelinating oligodendrocyte death and subsequent myelin loss in the developing CNS of IFN-γ-expressing mice. Moreover, we found that ATF6α deficiency increased EAE severity and aggravated EAE-induced oligodendrocyte loss and demyelination, without affecting inflammation. Thus, these data suggest the protective effects of ATF6α activation on oligodendrocytes in immune-mediated demyelinating diseases.