RESUMO
BACKGROUND: Müller glial cells are important regulators of physiological function of retina. In a model disease of retinal inflammation and spontaneous recurrent uveitis in horses (ERU), we could show that retinal Müller glial cells significantly change potassium and water channel protein expression during autoimmune pathogenesis. The most significantly changed channel protein in neuroinflammatory ERU was aquaporin 11 (AQP11). Aquaporins (AQP, 13 members) are important regulators of water and small solute transport through membranes. AQP11 is an unorthodox member of this family and was assigned to a third group of AQPs because of its difference in amino acid sequence (conserved sequence is only 11 %) and especially its largely unknown function. METHODS: In order to gain insight into the distribution, localization, and function of AQP11 in the retina, we first developed a novel monoclonal antibody for AQP11 enabling quantification, localization, and functional studies. RESULTS: In the horse retina, AQP11 was exclusively expressed at Müller glial cell membranes. In uveitic condition, AQP11 disappeared from gliotic Müller cells concomitant with glutamine synthase. Since function of AQP11 is still under debate, we assessed the impact of AQP11 channel on cell volume regulation of primary Müller glial cells under different osmotic conditions. We conclude a concomitant role for AQP11 with AQP4 in water efflux from these glial cells, which is disturbed in ERU. This could probably contribute to swelling and subsequent severe complication of retinal edema through impaired intracellular fluid regulation. CONCLUSIONS: Therefore, AQP11 is important for physiological Müller glia function and the expression pattern and function of this water channel seems to have distinct functions in central nervous system. The significant reduction in neuroinflammation points to a crucial role in pathogenesis of autoimmune uveitis.
Assuntos
Aquaporinas/metabolismo , Doenças Autoimunes/veterinária , Células Ependimogliais/metabolismo , Gliose/veterinária , Uveíte/veterinária , Animais , Aquaporinas/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Western Blotting , Gliose/imunologia , Gliose/metabolismo , Doenças dos Cavalos , Cavalos , Imuno-Histoquímica , Pressão Osmótica , Uveíte/metabolismo , Uveíte/patologiaRESUMO
The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43(A315TKi) mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43(A315TKi) animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Mitocôndrias/patologia , Alelos , Esclerose Lateral Amiotrófica/genética , Animais , Comportamento Animal , Glicemia/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , HDL-Colesterol/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Ácidos Graxos/metabolismo , Feminino , Técnicas de Introdução de Genes , Genoma , Genótipo , Heterozigoto , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Diarrheal diseases represent a major health burden in developing countries. Parenteral immunization typically does not induce efficient protection against enteropathogens because it does not stimulate migration of immune cells to the gut. Retinoic acid (RA) is critical for gut immunity, inducing upregulation of gut-homing receptors on activated T cells. In this study, we have demonstrated that RA can redirect immune responses elicited by s.c. vaccination of mice from skin-draining inguinal LNs (ingLNs) to the gut. When present during priming, RA induced robust upregulation of gut-homing receptors in ingLNs, imprinting gut-homing capacity on T cells. Concurrently, RA triggered the generation of gut-tropic IgA+ plasma cells in ingLNs and raised the levels of antigen-specific IgA in the intestinal lumen and blood. RA applied s.c. in vivo induced autonomous RA production in ingLN DCs, further driving efficient induction of gut-homing molecules on effector cells. Importantly, RA-supplemented s.c. immunization elicited a potent immune response in the small intestine that protected mice from cholera toxininduced diarrhea and diminished bacterial loads in Peyer patches after oral infection with Salmonella. Thus, the use of RA as a gut-homing navigator represents a powerful tool to induce protective immunity in the intestine after s.c. immunization, offering what we believe to be a novel approach for vaccination against enteropathogens.
Assuntos
Linfócitos B/metabolismo , Linfócitos T/metabolismo , Tretinoína/metabolismo , Animais , Linfócitos B/citologia , Técnicas de Cocultura , Sistema Imunitário , Imunização , Injeções Subcutâneas , Mucosa Intestinal/metabolismo , Excisão de Linfonodo/métodos , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mucosa/patologia , Nódulos Linfáticos Agregados , Salmonella/metabolismo , Infecções por Salmonella/metabolismo , Pele/metabolismo , Linfócitos T/citologiaRESUMO
All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/metabolismo , Proteínas de Homeodomínio/biossíntese , Neurogênese/fisiologia , Proteínas de Peixe-Zebra/biossíntese , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/citologia , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hipotálamo/citologia , Hipotálamo/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Especificidade da Espécie , Telencéfalo/citologia , Telencéfalo/metabolismo , Fatores de Transcrição HES-1 , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.