RESUMO
Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes , Rodopsina , Camundongos , Animais , Rodopsina/genética , Retina , Mutação/genética , Adaptação à Escuridão/genéticaRESUMO
Light-dependent activation of chloroplast enzymes is required for the rapid induction of photosynthesis after a shift from dark to light. The thioredoxin (Trx) system plays a central role in this process. In chloroplasts, the Trx system consists of two pathways: the ferredoxin (Fd)/Trx pathway and the nicotinamide adenine dinucleotide phosphate (NADPH)-Trx reductase C (NTRC) pathway. In Arabidopsis (Arabidopsis thaliana) mutants defective in either pathway, the photoreduction of thiol enzymes was impaired, resulting in decreased carbon fixation. The close relationship between the Fd/Trx pathway and proton gradient regulation 5 (PGR5)-dependent photosystem I cyclic electron transport (PSI CET) in the induction of photosynthesis was recently elucidated. However, how the PGR5-dependent pathway is involved in the NTRC pathway is unclear, although NTRC has been suggested to physically interact with PGR5. In this study, we analyzed Arabidopsis mutants lacking either the PGR5 or the chloroplast NADH dehydrogenase-like complex (NDH)-dependent PSI CET pathway in the ntrc mutant background. The ntrc pgr5 double mutant suppressed both the growth defects and the high non-photochemical quenching phenotype of the ntrc mutant when grown under long-day conditions. By contrast, the inactivation of NDH activity with the chlororespiratory reduction 2-2 mutant failed to suppress either phenotype. We discovered that the phenotypic rescue of ntrc by pgr5 is caused by the partial restoration of Trx-dependent reduction of thiol enzymes. These results suggest that electron partitioning to the PGR5-dependent pathway and the Trx system needs to be properly regulated for the activation of the Calvin-Benson-Bassham cycle enzymes during the induction of photosynthesis.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Redes e Vias Metabólicas/efeitos da radiação , Oxirredução/efeitos da radiação , Tiorredoxina Dissulfeto Redutase/metabolismo , Adaptação Ocular/genética , Adaptação Ocular/fisiologia , Adaptação à Escuridão/genética , Adaptação à Escuridão/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Redes e Vias Metabólicas/genética , Mutação , Fotossíntese/fisiologia , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
Jasmonate (JA)-induced plant senescence has been mainly studied with a dark/starvation-promoted system using detached leaves; yet, the induction of whole-plant senescence by JA remains largely unclear. This work reports the finding of a JA-induced whole-plant senescence of tobacco under light/non-starvation conditions and the investigation of underlying regulations. Methyl jasmonate (MeJA) treatment induces the whole-plant senescence of tobacco in a light-intensity-dependent manner, which is suppressed by silencing of NtCOI1 that encodes the receptor protein of JA-Ile (the bioactive derivative of JA). MeJA treatment could induce the senescence-specific cysteine protease gene SAG12 and another cysteine protease gene SAG-L1 to high expression levels in the detached leaf patches under dark conditions but failed to induce their expression in tobacco whole plants under light conditions. Furthermore, MeJA attenuates the RuBisCo activase (RCA) level in the detached leaves but has no effect on this protein in the whole plant under light conditions. A genome-wide transcriptional assay also supports the presence of a differential regulatory pattern of senescence-related genes during MeJA-induced whole-plant senescence under non-starvation conditions and results in the finding of a chlorophylase activity increase in this process. We also observed that the MeJA-induced senescence of tobacco whole plants is reversible, which is accompanied by a structural change of chloroplasts. This work provides novel insights into JA-induced plant senescence under non-starvation conditions and is helpful to dissect the JA-synchronized process of whole-plant senescence.
Assuntos
Ciclopentanos/efeitos adversos , Nicotiana/genética , Nicotiana/fisiologia , Oxilipinas/efeitos adversos , Senescência Vegetal/efeitos dos fármacos , Senescência Vegetal/genética , Adaptação Ocular/genética , Adaptação Ocular/fisiologia , Adaptação à Escuridão/genética , Adaptação à Escuridão/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de PlantasRESUMO
Impaired dark adaptation (DA), a defect in the ability to adjust to dimly lit settings, is a universal hallmark of aging. However, the mechanisms responsible for impaired DA are poorly understood. Vitamin A byproducts, such as vitamin A dimers, are small molecules that form in the retina during the vitamin A cycle. We show that later in life, in the human eye, these byproducts reach levels commensurate with those of vitamin A. In mice, selectively inhibiting the formation of these byproducts, with the investigational drug C20D3-vitamin A, results in faster DA. In contrast, acutely increasing these ocular byproducts through exogenous delivery leads to slower DA, with otherwise preserved retinal function and morphology. Our findings reveal that vitamin A cycle byproducts alone are sufficient to cause delays in DA and suggest that they may contribute to universal age-related DA impairment. Our data further indicate that the age-related decline in DA may be tractable to pharmacological intervention by C20D3-vitamin A.
Assuntos
Adaptação à Escuridão/fisiologia , Retina/metabolismo , Vitamina A/metabolismo , Envelhecimento , Animais , Adaptação à Escuridão/genética , Olho/efeitos dos fármacos , Olho/metabolismo , Humanos , Degeneração Macular/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Retina/efeitos dos fármacos , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Acuidade Visual/efeitos dos fármacos , Acuidade Visual/fisiologia , Vitamina A/antagonistas & inibidores , Vitamina A/fisiologiaRESUMO
The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. We used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was over 100 times slower in the ∆petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P800 (the primary donor of the photochemical reaction center) were much higher in the ∆petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The ∆petCBDA mutant was unable to grow phototrophically on acetate plus CO2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.
Assuntos
Sobrevivência Celular/fisiologia , Clostridiales/genética , Clostridiales/metabolismo , Citocromos/genética , Citocromos/metabolismo , Deleção de Genes , Fotossíntese/fisiologia , Adaptação Ocular/genética , Adaptação Ocular/fisiologia , Adaptação à Escuridão/genética , Adaptação à Escuridão/fisiologia , Mutação , Fotossíntese/genéticaRESUMO
The Chlamydomonas reinhardtii Compromised Hydrolysis of Triacylglycerols7 (CHT7) protein has been previously implicated in the regulation of DNA metabolism and cell-cycle-related gene expression during nitrogen (N) deprivation, and its predicted protein interaction domains are necessary for function. Here, we examined impacts of the cht7 mutation during the cell division cycle under nutrient deficiency in light-dark synchronized cultures. We explored the potential mechanisms affecting CHT7 complex activities during the cell cycle and N starvation, with a focus on the possible interaction between CHT7 and the C. reinhardtii retinoblastoma tumor suppressor (RB) protein homolog MAT3. Notably, the absence of CHT7 did not negatively impact the synchrony of cell division and cell cycle progression during diel growth. Although the majority of CHT7 and MAT3/RB proteins were observed in separate complexes by blue native-PAGE, the two proteins coimmunoprecipitated both during synchronized growth and following N deprivation, suggesting the presence of low abundance subcomplexes containing CHT7 and MAT3/RB. Furthermore, we observed several phosphorylated isoforms of CHT7 under these conditions. To test the potential role of phosphorylation on the structure and function of CHT7, we performed site-directed mutagenesis of previously identified phosphorylated amino acids within CHT7. These phosphorylated residues were dispensable for CHT7 function, but phosphorylated variants of CHT7 persisted, indicating that yet-unidentified residues within CHT7 are also likely phosphorylated. Based on the interaction of CHT7 and MAT3/RB, we postulate the presence of a low-abundance or transient regulatory complex in C. reinhardtii that may be similar to DREAM-like complexes in other organisms.
Assuntos
Adaptação Ocular/fisiologia , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiologia , Adaptação à Escuridão/fisiologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologia , Nitrogênio/fisiologia , Adaptação Ocular/genética , Adaptação à Escuridão/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , MutaçãoRESUMO
In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K+ Efflux Antiporter3 (KEA3) is a putative thylakoid H+/K+ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m-2 s-1 after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m-2 s-1, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO2 fixation was delayed after overnight dark adaptation.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Adaptação à Escuridão/fisiologia , NADH Desidrogenase/fisiologia , Fotossíntese/fisiologia , Antiportadores de Potássio-Hidrogênio/fisiologia , Adaptação à Escuridão/genética , Variação Genética , Genótipo , Mutação , NADH Desidrogenase/genética , Fenótipo , Fotossíntese/genética , Plantas Geneticamente Modificadas , Antiportadores de Potássio-Hidrogênio/genéticaRESUMO
While reducing the investment in the visual system of nocturnal/cave-dwelling species appears to be an evolutionarily stable strategy in response to the difficulty of locating food in the dark, relying on visual information for diurnal species is crucial for their survival and reproduction. However, the manner in which species evolve and adapt to the energetic demands placed upon them by environmental changes is not perfectly understood. In particular, if life in the dark is associated with a reduction in energetic demand, would relocation to a well-lit environment increase energetic demand? This question has a bearing upon our understanding of factors that influence the ability of species to adapt to new habitats. After observing that a sub-population of "Dark-flies" (i.e., fruit flies bred in the dark for more than 60 years) has been selected with a larger visual system (optic lobes) and brain over the course of being maintained in normal lighting conditions for 3 years (DFLight), we used the CAFÉ assay method to investigate the differences in the two strains' energetic demands in the present study. We therefore measured brain size, body size, and food consumption in Dark-flies, DFLight, and Oregon flies (i.e., the fly species most genetically similar to Dark-flies). We found that the DFLight consumed more food solution than the Dark-flies, which correlates with that strain's larger brain size and improved visual capability compared to the Dark-flies. In addition, and although the -Oregon flies initially consumed less food solution than the DFLight, the amount consumed by these two strains by the end of the CAFÉ assay was approximately the same. This suggests that the Dark-flies have adapted their metabolism or feeding strategies in response to a dark environment. Our investigation therefore provides empirical evidence elucidating the manner in which energetic demands change in response to environmental changes and the cross-generational effect upon sensory-system investment.
Assuntos
Adaptação à Escuridão/genética , Comportamento Alimentar/fisiologia , Adaptação Fisiológica/genética , Animais , Tamanho Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Luz , Masculino , Lobo Óptico de Animais não Mamíferos/metabolismo , Tamanho do Órgão/genéticaRESUMO
For night blindness, a detailed structural exploration of the interactions among G-protein receptor rhodopsin, transducin and arrestin was performed. Rhodopsin is responsible for dim light vision while a point mutation (G90âD90) results in an adverse change in its photo-transduction. The validated 3D models of the three proteins were utilized, and upon mutation and interactions, rhodopsin attained higher stability (evaluated through thermodynamic energy calculations, electrostatic surface potential and solvent accessible area), thereby participating strongly with transducin. Conformational switches in mutated rhodopsin also depicted a firm conformation with few 310 helices accompanied by increased percentage of pure α-helices and sheets. All evaluations were corroborated through paired T-tests. Glu33 (glycosylated unit in the N-terminal zone) of rhodopsin plays a chief role in the overall interaction pattern. Arg69 and Glu33 from wild-type rhodopsin participated in ionic interactions, while the latter set of ionic interaction remained preserved even after mutation. Cys323 (C-terminal residue) and Arg69 formed H-bonds from the wild-type rhodopsin. Cys323 exceptionally supports cellular signaling pattern in the non-mutated situation and for the non-sufferers of night-blindness. Ser297 and Tyr43 from mutated rhodopsin reside in helices and interact with Thr32 of transducin, preserving the steady conformation in activated interacted state, even in the dark. Ser297 lies adjoined to Lys296 (retinal attachment site), which resides in NPXXY motif (an "activation switch" for signal transduction). Thus, the molecular facet for involvement of photo-transduction, which holds a paramount zone in ophthalmology, was dealt with. This might instigate the future prospect for drug discovery to prevent such mutations.
Assuntos
Adaptação à Escuridão/genética , Cegueira Noturna/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas/genética , Rodopsina/genética , Substituição de Aminoácidos , Ácido Aspártico/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Glicina/genética , Humanos , Modelos Moleculares , Cegueira Noturna/metabolismo , Ligação Proteica/genética , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína/genética , Rodopsina/química , Rodopsina/metabolismo , Transdução de Sinais/genética , Relação Estrutura-AtividadeRESUMO
Little is known about the exact genes that confer vulnerability or resilience to environmental stressors during early neurodevelopment. Partial genetic deletion of neuregulin 1 (Nrg1) moderates the neurobehavioural effects of stressors applied in adolescence and adulthood, however, no study has yet examined its impact on prenatal stress. Here we examined whether Nrg1 deficiency in mice modulated the impact of prenatal stress on various behaviours in adulthood. Male heterozygous Nrg1 mice were mated with wild-type female mice who then underwent daily restraint stress from days 13 to 19 of gestation. Surprisingly, prenatal stress had overall beneficial effects by facilitating sensorimotor gating, increasing sociability, decreasing depressive-like behaviour, and improving spatial memory in adulthood. Such benefits were not due to any increase in maternal care, as prenatal stress decreased nurturing of the offspring. Nrg1 deficiency negated the beneficial behavioural effects of prenatal stress on all measures except sociability. However, Nrg1 deficiency interacted with prenatal stress to trigger locomotor hyperactivity. Nrg1 deficiency, prenatal stress or their combination failed to alter acute stress-induced plasma corticosterone concentrations. Collectively these results demonstrate that Nrg1 deficiency moderates the effects of prenatal stress on adult behaviour, but it does so in a complex, domain-specific fashion.
Assuntos
Sintomas Comportamentais/etiologia , Neuregulina-1/deficiência , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Estresse Psicológico/fisiopatologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Sintomas Comportamentais/genética , Corticosterona/sangue , Adaptação à Escuridão/genética , Comportamento Exploratório/fisiologia , Feminino , Relações Interpessoais , Masculino , Comportamento Materno/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuregulina-1/genética , Gravidez , Reconhecimento Psicológico/fisiologia , Filtro Sensorial/genética , Filtro Sensorial/fisiologia , Estresse Psicológico/genética , Natação/psicologiaRESUMO
The (pro)renin receptor [(P)RR], also known as ATP6AP2 [ATPase 6 accessory protein 2], is highly expressed in the brain. ATP6AP2 plays a role in early brain development, adult hippocampal neurogenesis and in cognitive functions. Lack of ATP6AP2 has deleterious effects, and mutations of ATP6AP2 in humans are associated with, e.g. X-linked intellectual disability. However, little is known about the effects of over-expression of ATP6AP2 in the adult brain. We hypothesized that mice over-expressing ATP6AP2 in the brain might exhibit altered neuroanatomical features and behavioural responses. To this end, we investigated heterozygous transgenic female mice and confirmed increased levels of ATP6AP2 in the brain. Our data show that over-expression of ATP6AP2 does not affect adult hippocampal neurogenesis, exercise-induced cell proliferation, or dendritic spine densities in the hippocampus. Only a reduced ventricular volume on the gross morphological level was found. However, ATP6AP2 over-expressing mice displayed altered exploratory behaviour with respect to the hole-board and novel object recognition tests. Moreover, primary adult hippocampal neural stem cells over-expressing ATP6AP2 exhibit a faster cell cycle progression and increased cell proliferation. Together, in contrast to the known deleterious effects of ATP6AP2 depletion, a moderate over-expression results in moderate behavioural changes and affects cell proliferation rate in vitro.
Assuntos
Comportamento Animal/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Neurônios/citologia , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Ventrículos Cerebrais/anatomia & histologia , Adaptação à Escuridão/genética , Espinhas Dendríticas/metabolismo , Proteínas do Domínio Duplacortina , Epêndima/metabolismo , Comportamento Exploratório , Hipocampo/diagnóstico por imagem , Histonas/metabolismo , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Neuropeptídeos/metabolismo , ATPases Translocadoras de Prótons/genética , Receptores de Superfície Celular/genética , Reconhecimento Psicológico/fisiologia , Caracteres SexuaisRESUMO
The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.
Assuntos
Ansiedade/genética , Ansiedade/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Neurônios/fisiologia , Núcleos Septais/patologia , Transdução de Sinais/fisiologia , Animais , Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Mapeamento Encefálico , Antagonistas de Receptores de Canabinoides/farmacologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Adaptação à Escuridão/efeitos dos fármacos , Adaptação à Escuridão/genética , Modelos Animais de Doenças , Estrenos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Comportamento Exploratório/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Pirrolidinonas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/fisiologia , Rimonabanto/farmacologia , Núcleos Septais/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/uso terapêutico , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein invitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats.
Assuntos
Ciclídeos/genética , Adaptação à Escuridão/genética , Rodopsina/genética , Animais , Evolução Biológica , América Central , Ecossistema , Evolução Molecular , Proteínas do Olho/genética , Variação Genética/genética , Lagos , Luz , Mutagênese Sítio-Dirigida , FilogeniaRESUMO
Complex behaviors are often observed at a spectrum in the population, and psychiatric disorders represent extremes of such behavioral spectra. While grasping the underlying cellular and molecular basis of these disorders represents a major challenge, it is believed that studies of complex behaviors in model organisms, where genotyping and phenotyping can be more conveniently carried out and cause-effect relationships can be further discerned, will help address this challenge. Here we report the characterization of a natural dark aversion behavior in larval zebrafish, which is previously shown to be fear or anxiety-associated. Phenotyping â¼200 individuals using a light/dark choice assay uncovered that, while a majority of individuals displayed medium level of dark aversion (mda), a small number of individuals exhibited strong dark aversion (sda), and a third small cohort showed variable dark aversion (vda). Through selective breeding and phenotyping of the next generation, we demonstrated that both the sda and vda traits are heritable, with sda being invariable while vda being highly variable across multiple trials. Additionally, sda appears to be recessive and vda appears to be dominant over the common allele(s) in the population. Moreover, compared to vda, sda showed increased thigmotaxis (preference for the walls in an open field), another measure of anxiety. Together, these findings reveal a naturally heritable variation of anxiety-like behavior in a tractable model organism, thereby laying foundation for future dissection of the underlying molecular and cellular mechanisms.
Assuntos
Ansiedade/genética , Aprendizagem da Esquiva/fisiologia , Modelos Animais de Doenças , Larva/fisiologia , Proteínas de Peixe-Zebra/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Comportamento de Escolha/fisiologia , Adaptação à Escuridão/genética , Embrião não Mamífero , Feminino , Genótipo , Masculino , Fatores Sexuais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.
Assuntos
Adaptação à Escuridão/genética , Técnicas de Introdução de Genes , Genes Dominantes , Mutação/genética , Vias Visuais/metabolismo , cis-trans-Isomerases/genética , Animais , Cromatografia Líquida de Alta Pressão , Eletrorretinografia , Heterozigoto , Isomerases/metabolismo , Camundongos Mutantes , Modelos Animais , Opsinas/metabolismo , Fotodegradação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Retina/metabolismo , Retina/patologia , Retinoides/metabolismo , cis-trans-Isomerases/metabolismoRESUMO
Developmental dyslexia is a common disorder with a strong genetic component, but the underlying molecular mechanisms are still unknown. Several candidate dyslexia-susceptibility genes, including KIAA0319, DYX1C1, and DCDC2, have been identified in humans. RNA interference experiments targeting these genes in rat embryos have shown impairments in neuronal migration, suggesting that defects in radial cortical migration could be involved in the disease mechanism of dyslexia. Here we present the first characterisation of a Kiaa0319 knockout mouse line. Animals lacking KIAA0319 protein do not show anatomical abnormalities in any of the layered structures of the brain. Neurogenesis and radial migration of cortical projection neurons are not altered, and the intrinsic electrophysiological properties of Kiaa0319-deficient neurons do not differ from those of wild-type neurons. Kiaa0319 overexpression in cortex delays radial migration, but does not affect final neuronal position. However, knockout animals show subtle differences suggesting possible alterations in anxiety-related behaviour and in sensorimotor gating. Our results do not reveal a migration disorder in the mouse model, adding to the body of evidence available for Dcdc2 and Dyx1c1 that, unlike in the rat in utero knockdown models, the dyslexia-susceptibility candidate mouse homolog genes do not play an evident role in neuronal migration. However, KIAA0319 protein expression seems to be restricted to the brain, not only in early developmental stages but also in adult mice, indicative of a role of this protein in brain function. The constitutive and conditional knockout lines reported here will be useful tools for further functional analyses of Kiaa0319.
Assuntos
Movimento Celular/genética , Dislexia/genética , Dislexia/patologia , Neocórtex/patologia , Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Ansiedade/etiologia , Ansiedade/genética , Encéfalo/metabolismo , Adaptação à Escuridão/genética , Modelos Animais de Doenças , Dislexia/complicações , Eletroporação , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Técnicas In Vitro , Antígeno Ki-67/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Fator de Transcrição PAX6/metabolismo , Técnicas de Patch-Clamp , Gravidez , Inibição Pré-Pulso/genética , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Filtro Sensorial/genética , Proteínas com Domínio T/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Neurotrimin (Ntm) belongs to the IgLON family of cell adhesion molecules with Lsamp, Obcam and kilon that regulate the outgrowth of neurites mostly by forming heterodimers. IgLONs have been associated with psychiatric disorders, intelligence, body weight, heart disease and tumours. This study provides an initial behavioural and pharmacological characterization of the phenotype of Ntm-deficient mice. We expected to see at least some overlap with the phenotype of Lsamp-deficient mice as Ntm and Lsamp are the main interaction partners in the IgLON family and are colocalized in some brain regions. However, Ntm-deficient mice displayed none of the deviations in behaviour that we have previously shown in Lsamp-deficient mice, but differently from Lsamp-deficient mice, had a deficit in emotional learning in the active avoidance task. The only overlap was decreased sensitivity to the locomotor stimulating effect of amphetamine in both knockout models. Thus, despite being interaction partners, on the behavioural level Lsamp seems to play a much more central role than Ntm and the roles of these two proteins seem to be complementary rather than overlapping.
Assuntos
Emoções/fisiologia , Deficiências da Aprendizagem/genética , Moléculas de Adesão de Célula Nervosa/deficiência , Anfetamina/farmacologia , Animais , Aprendizagem da Esquiva/fisiologia , Peso Corporal/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Adaptação à Escuridão/efeitos dos fármacos , Adaptação à Escuridão/genética , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Jejum/fisiologia , Medo/efeitos dos fármacos , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Locomoção/efeitos dos fármacos , Locomoção/genética , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/genética , Reflexo/genéticaRESUMO
UNLABELLED: Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that is activated when its 11-cis-retinal moiety is photoisomerized to all-trans retinal. This step initiates a cascade of reactions by which rods signal changes in light intensity. Like other GPCRs, rhodopsin is deactivated through receptor phosphorylation and arrestin binding. Full recovery of receptor sensitivity is then achieved when rhodopsin is regenerated through a series of steps that return the receptor to its ground state. Here, we show that dephosphorylation of the opsin moiety of rhodopsin is an extremely slow but requisite step in the restoration of the visual pigment to its ground state. We make use of a novel observation: isolated mouse retinae kept in standard media for routine physiologic recordings display blunted dephosphorylation of rhodopsin. Isoelectric focusing followed by Western blot analysis of bleached isolated retinae showed little dephosphorylation of rhodopsin for up to 4 h in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometeric determinations of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single-cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. We propose a model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires >3 h in complete darkness. SIGNIFICANCE STATEMENT: G-protein-coupled receptors (GPCRs) constitute the largest superfamily of proteins that compose â¼4% of the mammalian genome whose members share a common membrane topology. Signaling by GPCRs regulate a wide variety of physiological processes, including taste, smell, hearing, vision, and cardiovascular, endocrine, and reproductive homeostasis. An important feature of GPCR signaling is its timely termination. This normally occurs when, after their activation, GPCRs are rapidly phosphorylated by specific receptor kinases and subsequently bound by cognate arrestins. Recovery of receptor sensitivity to the ground state then requires dephosphorylation of the receptor and unbinding of arrestin, processes that are poorly understood. Here we investigate in mouse rod photoreceptors the relationship between rhodopsin dephosphorylation and recovery of visual sensitivity.
Assuntos
Adaptação à Escuridão/genética , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/metabolismo , Animais , Biofísica , Adaptação à Escuridão/efeitos dos fármacos , Eletrorretinografia , Receptor Quinase 1 Acoplada a Proteína G/genética , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Focalização Isoelétrica , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microespectrofotometria , Mutação/genética , Opsinas/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Retina/citologia , Retina/efeitos dos fármacos , Retinaldeído/farmacologiaRESUMO
Benzodiazepines have been widely used for their anxiolytic actions. However, the contribution of GABAA receptor subtypes to anxiolysis is still controversial. Studies with mutant mice harboring diazepam-insensitive α-subunits α1, α2, α3, or α5 have revealed that α2-containing GABAA receptors (α2-GABAARs) are required for diazepam-induced anxiolysis, with no evidence for an involvement of any other α-subunit, whereas TP003, described as a selective modulator of α3-containing GABAA receptors, was shown to be anxiolytic. Here, we describe a novel, systematic approach to evaluate the role of positive allosteric modulation of each of the four diazepam-sensitive α-subtypes in anxiety-related behavioral paradigms. By combining H to R point mutations in three out of the four diazepam-sensitive α-subunits in mice with a 129X1/SvJ background, diazepam becomes a subtype-specific modulator of the remaining non-mutated α-subtype. Modulation of α5-GABAARs, but not of α2-GABAARs, increased the time in the light side of the light-dark box as well as open-arm exploration in the elevated plus maze. In contrast, modulation of α3-GABAARs decreased open-arm exploration, whereas modulation of α2-GABAARs increased time in the center in the open-field test. Modulation of any single α-subtype had no effect on stress-induced hyperthermia. Our results provide evidence that modulation of α5-GABAARs elicits anxiolytic-like actions, whereas our data do not provide evidence for an anxiolytic-like action of α3-GABAARs. Thus, α5-GABAARs may be suitable targets for novel anxiolytic drugs.