RESUMO
The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating. How acidity is sensed and translated into an appropriate behavioural response is poorly understood. Here we describe a functionally segregated population of olfactory sensory neurons in the fruitfly, Drosophila melanogaster, that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors. In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery.
Assuntos
Ácidos/análise , Drosophila melanogaster/fisiologia , Odorantes/análise , Condutos Olfatórios/fisiologia , Ácido Acético/análise , Ácido Acético/farmacologia , Ácidos/farmacologia , Animais , Antenas de Artrópodes/anatomia & histologia , Antenas de Artrópodes/citologia , Antenas de Artrópodes/efeitos dos fármacos , Antenas de Artrópodes/inervação , Sinalização do Cálcio/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Ácido Carbônico/metabolismo , Ácido Carbônico/farmacologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos dos fármacos , Aprendizagem em Labirinto , Condutos Olfatórios/citologia , Condutos Olfatórios/efeitos dos fármacos , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores Odorantes/metabolismo , Sensilas/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , TemperaturaRESUMO
Animals across various phyla exhibit odor-evoked innate attraction behavior that is developmentally programmed. The mechanism underlying such behavior remains unclear because the odorants that elicit robust attraction responses and the neuronal circuits that mediate this behavior have not been identified. Here, we describe a functionally segregated population of olfactory sensory neurons (OSNs) and projection neurons (PNs) in Drosophila melanogaster that are highly specific to ammonia and amines, which act as potent attractants. The OSNs express IR92a, a member of the chemosensory ionotropic receptor (IR) family and project to a pair of glomeruli in the antennal lobe, termed VM1. In vivo calcium-imaging experiments showed that the OSNs and PNs innervating VM1 were activated by ammonia and amines but not by nonamine odorants. Flies in which the IR92a(+) neurons or IR92a gene was inactivated had impaired amine-evoked physiological and behavioral responses. Tracing neuronal pathways to higher brain centers showed that VM1-PN axonal projections within the lateral horn are topographically segregated from those of V-PN and DC4-PN, which mediate innate avoidance behavior to carbon dioxide and acidity, respectively, suggesting that these sensory stimuli of opposing valence are represented in spatially distinct neuroanatomic loci within the lateral horn. These experiments identified the neurons and their cognate receptor for amine detection, and mapped amine attractive olfactory inputs to higher brain centers. This labeled-line mode of amine coding appears to be hardwired to attraction behavior.
Assuntos
Aminas/metabolismo , Amônia/metabolismo , Anopheles/fisiologia , Quimiotaxia/fisiologia , Drosophila/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Vias Aferentes , Análise de Variância , Animais , Animais Geneticamente Modificados , Imuno-Histoquímica , Receptores Ionotrópicos de Glutamato/metabolismo , Especificidade da EspécieRESUMO
Drosophila olfactory sensory neurons express either odorant receptors or ionotropic glutamate receptors (IRs). The sensory neurons that express IR64a, a member of the IR family, send axonal projections to either the DC4 or DP1m glomeruli in the antennal lobe. DC4 neurons respond specifically to acids/protons, whereas DP1m neurons respond to a broad spectrum of odorants. The molecular composition of IR64a-containing receptor complexes in either DC4 or DP1m neurons is not known, however. Here, we immunoprecipitated the IR64a protein from lysates of fly antennal tissue and identified IR8a as a receptor subunit physically associated with IR64a by mass spectrometry. IR8a mutants and flies in which IR8a was knocked down by RNAi in IR64a+ neurons exhibited defects in acid-evoked physiological and behavioral responses. Furthermore, we found that the loss of IR8a caused a significant reduction in IR64a protein levels. When expressed in Xenopus oocytes, IR64a and IR8a formed a functional ion channel that allowed ligand-evoked cation currents. These findings provide direct evidence that IR8a is a subunit that forms a functional olfactory receptor with IR64a in vivo to mediate odor detection.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Receptores Ionotrópicos de Glutamato/fisiologia , Animais , Animais Geneticamente Modificados , Comportamento Animal/fisiologia , Western Blotting , Cálcio/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Proteínas de Drosophila/genética , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Imunoprecipitação , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Oócitos/metabolismo , Receptores Ionotrópicos de Glutamato/genética , Olfato/fisiologia , XenopusRESUMO
OBJECTIVE: Nausea and vomiting remain life-threatening obstacles to successful treatment of chronic diseases, despite a cadre of available antiemetic medications. Our inability to effectively control chemotherapy-induced nausea and vomiting (CINV) highlights the need to anatomically, molecularly, and functionally characterize novel neural substrates that block CINV. METHODS: Behavioral pharmacology assays of nausea and emesis in 3 different mammalian species were combined with histological and unbiased transcriptomic analyses to investigate the beneficial effects of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism on CINV. RESULTS: Single-nuclei transcriptomics and histological approaches in rats revealed a topographical, molecularly distinct, GABA-ergic neuronal population in the dorsal vagal complex (DVC) that is modulated by chemotherapy but rescued by GIPR agonism. Activation of DVCGIPR neurons substantially decreased behaviors indicative of malaise in cisplatin-treated rats. Strikingly, GIPR agonism blocks cisplatin-induced emesis in both ferrets and shrews. CONCLUSION: Our multispecies study defines a peptidergic system that represents a novel therapeutic target for the management of CINV, and potentially other drivers of nausea/emesis.
Assuntos
Antineoplásicos , Cisplatino , Animais , Ratos , Cisplatino/efeitos adversos , Furões , Náusea/induzido quimicamente , Náusea/tratamento farmacológico , Náusea/epidemiologia , Vômito/induzido quimicamente , Vômito/tratamento farmacológico , Antineoplásicos/efeitos adversosRESUMO
OBJECTIVE: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective medications to reduce appetite and body weight. These actions are centrally mediated; however, the neuronal substrates involved are poorly understood. METHODS: We employed a combination of neuroanatomical, genetic, and behavioral approaches in the mouse to investigate the involvement of caudal brainstem cholecystokinin-expressing neurons in the effect of the GLP-1RA exendin-4. We further confirmed key neuroanatomical findings in the non-human primate brain. RESULTS: We found that cholecystokinin-expressing neurons in the caudal brainstem are required for the anorectic and body weight-lowering effects of GLP-1RAs and for the induction of GLP-1RA-induced conditioned taste avoidance. We further show that, while cholecystokinin-expressing neurons are not a direct target for glucose-dependent insulinotropic peptide (GIP), GIP receptor activation results in a reduced recruitment of these GLP-1RA-responsive neurons and a selective reduction of conditioned taste avoidance. CONCLUSIONS: In addition to disclosing a neuronal population required for the full appetite- and body weight-lowering effect of GLP-1RAs, our data also provide a novel framework for understanding and ameliorating GLP-1RA-induced nausea - a major factor for withdrawal from treatment.
Assuntos
Colecistocinina/farmacologia , Polipeptídeo Inibidor Gástrico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Animais , Apetite/efeitos dos fármacos , Depressores do Apetite/farmacologia , Glicemia/efeitos dos fármacos , Exenatida/farmacologia , Feminino , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/fisiologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Liraglutida/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismoRESUMO
The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist reduced conditioned taste avoidance (CTA) without affecting hypophagia mediated by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were found to be expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in detecting aversive stimuli. Peripheral administration of a GIPR agonist induced neuronal activation (cFos) in the AP. Further, whole-brain cFos analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), a brainstem nucleus that relays aversive/emetic signals to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPR agonist treatment reduces PYY-induced nausea-like behavior. Together, the results of our study indicate a novel mechanism by which GIP-based therapeutics may have benefit in improving the tolerability of weight loss agents.
Assuntos
Fármacos Antiobesidade , Peptídeo YY , Receptores dos Hormônios Gastrointestinais , Animais , Fármacos Antiobesidade/efeitos adversos , Camundongos , Náusea/induzido quimicamente , Náusea/tratamento farmacológico , Obesidade/tratamento farmacológico , Peptídeo YY/efeitos adversos , Receptores dos Hormônios Gastrointestinais/agonistasRESUMO
Glucagon-like peptide 1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by adverse side effects, including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Using single-nuclei RNA sequencing, we identified the cellular phenotypes of AP/NTS cells expressing GIPR and GLP-1R on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in γ-aminobutyric acid-ergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Náusea/induzido quimicamente , Náusea/tratamento farmacológico , Receptores dos Hormônios Gastrointestinais/agonistas , Animais , Peso Corporal/efeitos dos fármacos , Comportamento Alimentar , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Musaranhos , VômitoRESUMO
We extend the spectrum of phenotypes caused by mutations in the Wnt/Norrin coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) by identifying two novel types of mutation in related individuals whose presenting features were profound muscle hypotonia, mild mental retardation, blindness, and growth retardation. One mutation removes 6 out of 9 consecutive leucine residues in the LRP5 signal peptide (c.43_60del or p.Leu15_Leu20del), which impairs polypeptide entry into the endoplasmic reticulum (ER), trafficking to the cell membrane, and signal transduction. The second mutation resulted from nonhomologous recombination between Alu repeat sequences, which deleted exons 14-16 and would produce a nonfunctional, truncated, and frameshifted polypeptide, if expressed [chr11:g.(13871447_1387511)_(13879636_13879700)del (NW_925106.1) or p.Pro1010GlnfsX38]. We confirmed that the length of the LRP5 signal peptide poly-leucine repeat is polymorphic in the general population, and, importantly, we were able to demonstrate in independent in vitro assays that different allele sizes affect receptor processing and signal transduction. Consequently, this polymorphism may have physiologic effects in vivo. This latter finding is relevant since through a genomewide search we identified nearly 400 human proteins that contain poly-leucine repeats within their signal peptide. We chose 18 of these proteins and genotyped the underlying trinucleotide repeat in healthy Caucasian individuals. More than one length allele was observed in one-half of the proteins. We therefore propose that natural variation in poly-leucine-stretches within signal peptides constitutes a currently unrecognized source of variability in protein translation and expression.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Relacionadas a Receptor de LDL/genética , Mutação , Osteoporose/patologia , Sinais Direcionadores de Proteínas/genética , Repetições de Trinucleotídeos/genética , Anormalidades Múltiplas/patologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Oftalmopatias/patologia , Saúde da Família , Feminino , Frequência do Gene , Genótipo , Humanos , Leucina/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Luciferases/genética , Luciferases/metabolismo , Masculino , Linhagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Síndrome , TurquiaRESUMO
Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT's short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.
RESUMO
The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.
Assuntos
Doenças Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Mutação de Sentido Incorreto , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Doenças Ósseas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Relacionadas a Receptor de LDL/química , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Wnt , Proteína Wnt1RESUMO
Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Mutação/genética , Junção Neuromuscular/citologia , Terminações Pré-Sinápticas/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Potenciais de Ação/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Microscopia Confocal , Junção Neuromuscular/genética , Neurônios/fisiologia , Técnicas de Patch-Clamp , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
UNLABELLED: We studied whether the LRP5 gene contributes to the clinical phenotype of IO in men. Mutation analysis in 66 IO men revealed a range of sequence variants, of which two missense variants were shown to be of functional relevance. INTRODUCTION: Mutations in the LDL receptor-related protein 5 (LRP5) gene have been associated with extreme bone phenotypes, which makes LRP5 a plausible candidate gene for idiopathic osteoporosis (IO). MATERIALS AND METHODS: In 66 men with IO, all 23 exons and exon-intron boundaries of the LRP5 gene were screened for mutations, and functional analyses were performed for those that were putatively involved in the phenotype. RESULTS: Mutation analysis in the IO probands revealed five missense mutations, of which 1067C>T (S356L), 1364C>T (S455L), and 4609G>A (A1537T) were of potential functional significance because they were located in highly conserved regions of LRP5 and not found in a control panel. Segregation analysis in the respective families could not exclude their possible causality for IO. Furthermore, functional analyses clearly showed an inhibitory effect of mutations 1067C>T and 1364C>T on Wnt signal transduction. These effects are most likely caused by impaired LRP5 synthesis in the case of 1067C>T and failure of protein trafficking to the cell surface for 1364C>T. CONCLUSIONS: For 2 of 66 IO probands, a mutation in the LRP5 gene with proven functionality was found. The findings indicate that carrying an LRP5 mutation is a risk factor for IO, but that overall, IO in men is infrequently underlied by such a mutation.
Assuntos
Proteínas Relacionadas a Receptor de LDL/genética , Mutação de Sentido Incorreto/genética , Osteoporose/genética , Adulto , Idoso , Sequência de Aminoácidos , Densidade Óssea/genética , Doenças Ósseas Metabólicas/genética , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Éxons/genética , Frequência do Gene , Humanos , Íntrons/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Osteoporose/etiologia , Linhagem , Polimorfismo Genético/genética , Transporte Proteico/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transfecção , Proteína Wnt1/genéticaRESUMO
Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb's binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 µl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.
Assuntos
Anticorpos Monoclonais/imunologia , Artropatia Neurogênica/sangue , Coxa Vara/sangue , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/deficiência , Glicoproteínas/imunologia , Deformidades Congênitas da Mão/sangue , Articulações/metabolismo , Sinovite/sangue , Adulto , Idoso , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Estudos de Casos e Controles , Epitopos/química , Epitopos/imunologia , Feminino , Glicoproteínas/sangue , Glicoproteínas/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oligopeptídeos/química , Oligossacarídeos/química , Líquido Sinovial/metabolismoRESUMO
Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs--including GCaMP3, GCaMP5 and GCaMP6--can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.
Assuntos
Calmodulina/química , Proteínas de Fluorescência Verde/química , Quinase de Cadeia Leve de Miosina/química , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/química , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cor , Drosophila melanogaster , Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
UNLABELLED: Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.
Assuntos
Densidade Óssea , Deformidades Congênitas dos Membros/genética , Mutação , Receptores de LDL/genética , Animais , Relação Dose-Resposta a Droga , Extremidades/embriologia , Feminino , Genótipo , Heterozigoto , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microcirculação , Fenótipo , Ligação Proteica , Fatores Sexuais , Fatores de Tempo , Tomografia Computadorizada por Raios X , Proteínas WntRESUMO
Animals can determine the nutritional value of sugar without the influence of taste. We examined a Drosophila mutant that is insensitive to the nutritional value of sugars, responding only to the concentration (that is, sweetness). The affected gene encodes a sodium/solute co-transporter-like protein, designated SLC5A11 (or cupcake), which is structurally similar to mammalian sodium/glucose co-transporters that transport sugar across the intestinal and renal lumen. However, SLC5A11 was prominently expressed in 10-13 pairs of R4 neurons of the ellipsoid body in the brain and functioned in these neurons for selecting appropriate foods.
Assuntos
Encéfalo/metabolismo , Comportamento de Escolha/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Preferências Alimentares/fisiologia , Proteínas de Transporte de Sódio-Glucose/metabolismo , Paladar/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Sequência de Bases , Encéfalo/citologia , Antígenos CD8/genética , Relação Dose-Resposta a Droga , Proteínas de Drosophila/genética , Privação de Alimentos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glucose/administração & dosagem , Glicogênio/metabolismo , Proteínas de Fluorescência Verde/genética , Hemolinfa/metabolismo , Humanos , Mutação/genética , Neurônios/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Proteínas de Transporte de Sódio-Glucose/genética , Fatores de Transcrição/genéticaRESUMO
Low-density lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor, plays an important role in bone metabolism as loss-of-function and gain-of-function mutations in LRP5 result in the autosomal recessive osteoporosis-pseudoglioma syndrome and autosomal dominant high-bone mass (HBM) phenotypes, respectively. Prior studies suggested that the presence of HBM-associated LRP5 mutations results in decreased antagonism of LRP5-mediated Wnt signaling. In the present study, we investigated six different HBM-LRP5 mutations and confirm that neither Dickkopf1 (DKK1) nor sclerostin efficiently inhibits HBM-LRP5 signaling. In addition, when coexpressed, DKK1 and sclerostin do not inhibit HBM-LRP5 mutants better than either inhibitor by itself. Also, DKK1 and sclerostin do not simultaneously bind to wild-type LRP5, and DKK1 is able to displace sclerostin from previously formed sclerostin-LRP5 complexes. In conclusion, our results indicate that DKK1 and sclerostin are independent, and not synergistic, regulators of LRP5 signaling and that the function of each is impaired by HBM-LRP5 mutations.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Meios de Cultivo Condicionados/química , Marcadores Genéticos , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Ligação Proteica , Transdução de SinaisRESUMO
The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass. Loss-of-function mutations in LRP5 cause the human skeletal disease osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by severely reduced bone mass and strength. We investigated the role of LRP5 on bone strength using mice engineered with a loss-of-function mutation in the gene. We then tested whether the osteogenic response to mechanical loading was affected by the loss of Lrp5 signaling. Lrp5-null (Lrp5-/-) mice exhibited significantly lower bone mineral density and decreased strength. The osteogenic response to mechanical loading of the ulna was reduced by 88 to 99% in Lrp5-/- mice, yet osteoblast recruitment and/or activation at mechanically strained surfaces was normal. Subsequent experiments demonstrated an inability of Lrp5-/- osteoblasts to synthesize the bone matrix protein osteopontin after a mechanical stimulus. We then tested whether Lrp5-/- mice increased bone formation in response to intermittent parathyroid hormone (PTH), a known anabolic treatment. A 4-week course of intermittent PTH (40 microg/kg/day; 5 days/week) enhanced skeletal mass equally in Lrp5-/- and Lrp5+/+ mice, suggesting that the anabolic effects of PTH do not require Lrp5 signaling. We conclude that Lrp5 is critical for mechanotransduction in osteoblasts. Lrp5 is a mediator of mature osteoblast function following loading. Our data suggest an important component of the skeletal fragility phenotype in individuals affected with osteoporosis-pseudoglioma is inadequate processing of signals derived from mechanical stimulation and that PTH might be an effective treatment for improving bone mass in these patients.
Assuntos
Anabolizantes/administração & dosagem , Osso e Ossos/fisiologia , Proteínas Relacionadas a Receptor de LDL/fisiologia , Mecanotransdução Celular/fisiologia , Osteoblastos/metabolismo , Osteogênese , Hormônio Paratireóideo/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Proteínas Wnt/metabolismo , Animais , Peso Corporal/genética , Densidade Óssea/genética , Remodelação Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Movimento Celular/genética , Células Cultivadas , Feminino , Humanos , Proteínas Relacionadas a Receptor de LDL/deficiência , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/fisiologia , Osteogênese/genéticaRESUMO
Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Relacionadas a Receptor de LDL/genética , Mutação , Osteoporose/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Densidade Óssea/genética , Anormalidades do Olho/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Mutação de Sentido Incorreto , Osteoporose/fisiopatologia , Síndrome , Proteínas WntRESUMO
One of the well characterized cell biologic actions of lithium is the inhibition of glycogen synthase kinase-3beta and the consequent activation of canonical Wnt signaling. Because deficient Wnt signaling has been implicated in disorders of reduced bone mass, we tested whether lithium could improve bone mass in mice. We gavage-fed lithium chloride to 8-week-old mice from three different strains (Lrp5(-/-), SAMP6, and C57BL/6) and assessed the effect on bone metabolism after 4 weeks of therapy. Lrp5(-/-) mice lack the Wnt coreceptor low-density lipoprotein receptor-related protein 5 and have markedly reduced bone mass. Lithium, which is predicted to act downstream of this receptor, restored bone metabolism and bone mass to near wild-type levels in these mice. SAMP6 mice have accelerated osteoporosis due to inadequate osteoblast renewal. Lithium significantly improved bone mass in these mice and in wild-type C57BL/6 mice. We found that lithium activated canonical Wnt signaling in cultured calvarial osteoblasts from Lrp5(-/-) mice ex vivo and that lithium-treated mice had increased expression of Wnt-responsive genes in their bone marrow cells in vivo. These data lead us to conclude that lithium enhances bone formation and improves bone mass in mice and that it may do so via activation of the canonical Wnt pathway. Lithium has been used safely and effectively for over half a century in the treatment of bipolar illness. Prospective studies in patients receiving lithium should determine whether it also improves bone mass in humans.