RESUMO
Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.
Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Meios de Cultivo Condicionados , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Humanos , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Epitélio Pigmentado da Retina/citologia , Retinoides/metabolismo , Retinoides/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismoRESUMO
In the mouse 55°C warm-water tail-withdrawal assay, a single administration of nor-binaltorphimine (nor-BNI; 10 mg/kg i.p.) antagonized κ-opioid receptor (KOR) agonist-induced antinociception up to 14 days, whereas naloxone (10 mg/kg i.p.)-mediated antagonism lasted less than 1 day. In saturation binding experiments, mouse brain membranes isolated and washed 1 or 7 (but not 14) days after nor-BNI administration demonstrated a significant time-dependent decrease in maximal KOR agonist [(3)H]U69,593 binding. To determine whether brain concentrations of nor-BNI were sufficient to explain the antagonism of KOR-mediated antinociception, mouse blood and perfused brain were harvested at time points ranging from 30 minutes to 21 days after a single administration and analyzed for the presence of nor-BNI using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Nor-BNI was detected in the perfused brain homogenate up to 21 days after administration (30 nmol i.c.v. or 10 mg/kg i.p.). Subsequent experiments in which nor-BNI was administered at doses estimated from the amounts detected in the brain homogenates isolated from pretreated mice over time demonstrated significant antagonism of U50,488 antinociception in a manner consistent with the magnitude of observed KOR antagonism. The dose (1.4 nmol) approximating the lowest amount of nor-BNI detected in brain on day 14 did not antagonize U50,488-induced antinociception, consistent with the absence of U50,488 antagonism observed in vivo at this time point after pretreatment. Overall, the physical presence of nor-BNI in the mouse brain paralleled its in vivo pharmacological profile, suggesting physicochemical and pharmacokinetic properties of nor-BNI may contribute to the prolonged KOR antagonism.
Assuntos
Encéfalo/metabolismo , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides kappa/antagonistas & inibidores , Analgésicos/metabolismo , Analgésicos/farmacologia , Animais , Benzenoacetamidas/metabolismo , Benzenoacetamidas/farmacologia , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Injeções Intraventriculares , Membranas/efeitos dos fármacos , Membranas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Naloxona/farmacocinética , Naloxona/farmacologia , Naltrexona/farmacocinética , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacocinética , Medição da Dor/efeitos dos fármacos , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Padrões de Referência , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin beta-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20-25% of control animals survived after six to eight F.IX doses, 90-93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2-5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5-80 microg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (approximately 40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.
Assuntos
Administração Oral , Anafilaxia/prevenção & controle , Fator IX/administração & dosagem , Hemofilia B/sangue , Anafilaxia/mortalidade , Animais , Cloroplastos/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Fator IX/antagonistas & inibidores , Fator IX/química , Vetores Genéticos , Hemofilia B/complicações , Imunoglobulina E/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Modelos Genéticos , Nicotiana/genéticaRESUMO
Human immunodeficiency virus (HIV) infection is associated with mood disorders and behavioral disinhibition. Impairments in sensorimotor gating and associated neurocognitive disorders are reported, but the HIV-proteins and mechanisms involved are not known. The regulatory HIV-1 protein, Tat, is neurotoxic and its expression in animal models increases anxiety-like behavior concurrent with neuroinflammation and structural changes in limbic and extra-limbic brain regions. We hypothesized that conditional expression of HIV-1 Tat1-86 in the GT-tg bigenic mouse model would impair sensorimotor gating and increase microglial reactivity in limbic and extralimbic brain regions. Conditional Tat induction via doxycycline (Dox) treatment (0-125 mg/kg, i.p., for 1-14 days) significantly potentiated the acoustic startle reflex (ASR) of GT-tg mice and impaired prepulse inhibition (PPI) of this response in a dose-dependent manner when Dox (100mg/kg) was administered for brief (1 day) or prolonged (daily for 7 days) intervals. A greater proportion of active/reactive Iba1-labeled microglia was seen in the anterior cingulate cortex (ACC), dentate gyrus, and nucleus accumbens core when Tat protein was induced under either brief or prolonged expression conditions. Other subregions of the medial prefrontal cortex, amygdala, hippocampal formation, ventral tegmental area, and ventral pallidum also displayed Tat-induced microglial activation, but only the activation observed in the ACC recapitulated the pattern of ASR and PPI behaviors. Tat exposure also increased frontal cortex GFAP. Pretreatment with indomethacin attenuated the behavioral effects of brief (but not prolonged) Tat-exposure. Overall, exposure to HIV-1 Tat protein induced sensorimotor deficits associated with acute and persistent neuroinflammation in limbic/extralimbic brain regions.
Assuntos
Encéfalo/imunologia , Microglia/fisiologia , Filtro Sensorial/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Doxiciclina , Proteína Glial Fibrilar Ácida , HIV-1 , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
RATIONALE: Human immunodeficiency virus (HIV) infection is associated with substantial increases in generalized anxiety. The HIV regulatory protein, transactivator of transcription (Tat), has been implicated in the neuropathogenesis related to HIV-1 infection. However, direct examination of the effect of Tat on behavioral measures of anxiety has not been demonstrated. OBJECTIVE: To identify whether expression of the Tat1-86 protein exerts dose-dependent and persistent anxiety-like effects in a whole animal model, the GT-tg bigenic mouse. METHODS: GT-tg mice and C57BL/6J controls were administered doxycycline in a dose- (0, 50, 100, or 125 mg/kg, i.p., for 7 days) or duration- (100 mg/kg, i.p., for 0, 1, 3, 5, or 14 days) dependent manner to induce Tat1-86 in brain. Mice were assessed for anxiety-like behavior in an open field, social interaction, or marble burying task 0, 7, and/or 14 days later. Central expression of Tat1-86 protein was verified with Western blot analyses. RESULTS: Doxycycline produced no effects on C57BL/6J controls that lacked the Tat1-86 transgene. Among GT-tg mice, doxycycline (100 mg/kg for 3, 5, or 7 days) significantly increased anxiety-like behavior in all tasks, commensurate with enhanced Western blot labeling of Tat1-86 protein in brain, displaying optimal effects with the 7-day regimen. Greater exposure to doxycycline (either 125 mg/kg for 7 days or 100 mg/kg for 14 days) impaired locomotor behavior; whereas lower dosing (below 100 mg/kg) produced only transient increases in anxiety-like behavior. CONCLUSIONS: Expression of HIV-1-Tat1-86 in GT-tg mouse brain produces exposure-dependent, persistent increases in anxiety-like behavior.
Assuntos
Transtornos de Ansiedade/fisiopatologia , Encéfalo/fisiopatologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Transtornos de Ansiedade/induzido quimicamente , Western Blotting , Encéfalo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes Neuropsicológicos , Psicotrópicos/farmacologia , Fatores de Tempo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of( 14)C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of (14)C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.
Assuntos
Ésteres/química , Mycobacterium tuberculosis/metabolismo , Acil Coenzima A/química , Aldeído Redutase/genética , Aldeídos/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Clonagem Molecular , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Ácidos Graxos/química , Álcoois Graxos/química , Teste de Complementação Genética , Glicerol/química , Humanos , Lipídeos/química , Modelos Químicos , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
HIV-Tat protein has been implicated in the pathogenesis of HIV-1 neurological complications (i.e., neuroAIDS), but direct demonstrations of the effects of Tat on behavior are limited. GT-tg mice with a doxycycline (Dox)-inducible and brain-selective tat gene coding for Tat protein were used to test the hypothesis that the activity of Tat in brain is sufficient to impair learning and memory processes. Western blot analysis of GT-tg mouse brains demonstrated an increase in Tat antibody labeling that seemed to be dependent on the dose and duration of Dox pretreatment. Dox-treated GT-tg mice tested in the Barnes maze demonstrated longer latencies to find an escape hole and displayed deficits in probe trial performance versus uninduced GT-tg littermates, suggesting Tat-induced impairments of spatial learning and memory. Reversal learning was also impaired in Tat-induced mice. Tat-induced mice additionally demonstrated long-lasting (up to one month) deficiencies in novel object recognition learning and memory performance. Furthermore, novel object recognition impairment was dependent on the dose and duration of Dox exposure, suggesting that Tat exposure progressively mediated deficits. These experiments provide evidence that Tat protein expression is sufficient to mediate cognitive abnormalities seen in HIV-infected individuals. Moreover, the genetically engineered GT-tg mouse may be useful for improving our understanding of the neurological underpinnings of neuroAIDS-related behaviors.