RESUMO
BACKGROUND & AIMS: Niemann-Pick C2 (NPC2) is a lysosomal protein involved in the egress of low-density lipoprotein-derived cholesterol from lysosomes to other intracellular compartments. NPC2 has been detected in several tissues and is also secreted from the liver into bile. We have previously shown that NPC2-deficient mice fed a lithogenic diet showed reduced biliary cholesterol secretion as well as cholesterol crystal and gallstone formation. This study aimed to investigate the consequences of NPC2 hepatic overexpression on liver cholesterol metabolism, biliary lipid secretion, gallstone formation and the effect of NPC2 on cholesterol crystallization in model bile. METHODS: We generated NPC2 transgenic mice (Npc2.Tg) and fed them either chow or lithogenic diets. We studied liver cholesterol metabolism, biliary lipid secretion, bile acid composition and gallstone formation. We performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. RESULTS: No differences were observed in biliary cholesterol content or secretion between wild-type and Npc2.Tg mice fed the chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but did not show differences in the bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. Finally, recombinant NPC2 decreased nucleation time in model bile. CONCLUSIONS: These results suggest that NPC2 promotes cholesterol gallstone formation by decreasing the cholesterol nucleation time, indicating a pro-nucleating function of NPC2 in bile.
Assuntos
Colesterol , Cálculos Biliares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol/química , Colesterol/metabolismo , Cristalização , Modelos Animais de Doenças , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Químicos , Fatores de TempoRESUMO
Rett syndrome (RTT) is a disorder that affects patients' ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain.
Assuntos
Proteína 2 de Ligação a Metil-CpG/deficiência , Isoformas de Proteínas/metabolismo , Síndrome de Rett/genética , Transgenes , Fatores Etários , Processamento Alternativo , Animais , Comportamento Animal , Cruzamentos Genéticos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo X , Teste de Complementação Genética , Longevidade , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Síndrome de Rett/metabolismo , Aumento de PesoRESUMO
Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.
Assuntos
Expressão Gênica , Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Glomerulosclerose Segmentar e Focal/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Mutação , Especificidade de Órgãos , Canal de Cátion TRPC6RESUMO
BACKGROUND: Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) and one of the leading causes of mental retardation in females. RTT is characterized by psychomotor retardation, purposeless hand movements, autistic-like behavior and abnormal gait. We studied the effects of environmental enrichment (EE) on the phenotypic manifestations of a RTT mouse model that lacks MeCP2 (Mecp2(-/y)). PRINCIPAL FINDINGS: We found that EE delayed and attenuated some neurological alterations presented by Mecp2(-/y) mice and prevented the development of motor discoordination and anxiety-related abnormalities. To define the molecular correlate of this beneficial effect of EE, we analyzed the expression of several synaptic marker genes whose expression is increased by EE in several mouse models. CONCLUSIONS/SIGNIFICANCE: We found that EE induced downregulation of several synaptic markers, suggesting that the partial prevention of RTT-associated phenotypes is achieved through a non-conventional transcriptional program.
Assuntos
Meio Ambiente , Síndrome de Rett/genética , Síndrome de Rett/fisiopatologia , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Aprendizagem em Labirinto/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/fisiologia , Camundongos , Camundongos Mutantes , Fenótipo , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MeCP2 plays a critical role in interpreting epigenetic signatures that command chromatin conformation and regulation of gene transcription. In spite of MeCP2's ubiquitous expression, its functions have always been considered in the context of brain physiology. In this study, we demonstrate that alterations of the normal pattern of expression of MeCP2 in cardiac and skeletal tissues are detrimental for normal development. Overexpression of MeCP2 in the mouse heart leads to embryonic lethality with cardiac septum hypertrophy and dysregulated expression of MeCP2 in skeletal tissue produces severe malformations. We further show that MeCP2's expression in the heart is developmentally regulated; further suggesting that it plays a key role in regulating transcriptional programs in non-neural tissues.
Assuntos
Osso e Ossos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/embriologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , Miocárdio/metabolismo , Osteogênese/fisiologia , Azul Alciano , Animais , Antraquinonas , Osso e Ossos/embriologia , Bromodesoxiuridina , Cruzamentos Genéticos , Primers do DNA/genética , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/genéticaRESUMO
MeCP2 is an abundant protein that binds to methylated cytosine residues in DNA and regulates transcription. Mutations in MECP2 cause Rett syndrome, a severe neurological disorder that affects approximately 1:10 000 females. Mice lacking MeCP2 have been generated and constitute important models of Rett syndrome. However, it is yet unclear whether certain physiological events are sensitive to a decrease, rather than a complete lack of MeCP2. Here we report that a Mecp2 floxed allele (Mecp2(lox)) that was generated to allow conditional mutagenesis behaves as a hypomorph and the corresponding mutant mice exhibit phenotypical alterations including body weight gain, motor abnormalities and altered social behavior. Our data reinforce the view that the central nervous system is extremely sensitive to MeCP2 expression levels and suggest that the 3'-UTR of Mecp2 might contain important elements that contribute to the regulation of its stability or processing.
Assuntos
Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/genética , Síndrome de Rett/fisiopatologia , Regiões 3' não Traduzidas/metabolismo , Animais , Peso Corporal , Feminino , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Desempenho Psicomotor , RNA Mensageiro/metabolismo , Síndrome de Rett/psicologia , Comportamento SocialRESUMO
Rett syndrome (RTT), a leading cause of mental retardation with autistic features in females, is caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). RTT is characterized by a diverse set of neurological features that includes cognitive, motor, behavioral and autonomic disturbances. The diverse features suggest that specific neurons contribute to particular phenotypes and raise the question whether restoring MeCP2 function in a cell-specific manner will rescue some of the phenotypes seen in RTT. To address this, we generated transgenic mice expressing inducible MeCP2 under the control of the brain-specific promoters calcium/calmodulin-dependent protein kinase II (CamKII) or neuron-specific enolase (Eno2) and bred them onto mouse models lacking functional MeCP2. Expression of normal MeCP2 in either CamKII or Eno2 distribution was unable to prevent the appearance of most of the phenotypes of the RTT mouse models. These results suggest that most RTT phenotypes are caused either by disruption of complex neural networks involving neurons throughout the brain or by disruption of the function of specific neurons outside of the broad CamKII or Eno2 distribution.
Assuntos
Perfilação da Expressão Gênica , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Animais , Comportamento Animal , Western Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteína 2 de Ligação a Metil-CpG/fisiologia , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios/metabolismo , Fenótipo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de SobrevidaRESUMO
We used beta-endorphin-deficient mice as a novel approach to confirm the physiological role that opioid peptides play in the development or regulation of the immune system. We found that mice lacking beta-endorphin possessed an enhanced immune response, measured in terms of splenocyte proliferation and interleukin (IL)-2 mRNA levels, in vitro production of the splenic macrophage inflammatory cytokines IL-6 and Tumor Necrosis Factor (TNF)-alpha and plasma IL-6 following lipopolysaccharide (LPS) administration. beta-Endorphin-deficient mice had attenuated increases of plasma ACTH and corticosterone levels in response to LPS. These results are consistent with a postulated inhibitory role of endogenous beta-endorphin on the immune system at multiple levels.
Assuntos
Citocinas/biossíntese , Baço/imunologia , beta-Endorfina/fisiologia , Hormônio Adrenocorticotrópico/sangue , Animais , Divisão Celular , Células Cultivadas , Corticosterona/sangue , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-6/sangue , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossíntese , Baço/citologia , Fator de Necrose Tumoral alfa/análise , beta-Endorfina/genéticaRESUMO
The prefrontal cortex receives a major dopaminergic input from the ventral tegmental area, which plays an important role in the integration of neuronal signals influencing behavioural responses to stressful environmental stimuli. The dopamine D4 receptor (D4R) is expressed at highest levels in the prefrontal cortex and is the predominant D2-like receptor localized in this brain area. To investigate the functional significance of D4Rs in dopamine-mediated responses we have analysed a strain of mice lacking this receptor subtype (Drd4-/-). Wild-type and Drd4-/- mice were challenged in two different approach/avoidance conflict paradigms: the elevated plus maze and the light/dark preference exploration test. By these behavioural measures Drd4-/- mice showed heightened avoidance to the more fear-provoking areas of each maze as demonstrated by reduced exploration of the open arms of the plus maze and longer latencies to explore the illuminated compartment of the light/dark shuttle box. These exaggerated avoidance behaviours were further enhanced by an additional handling stress but completely prevented by anxiolytic agents such as the benzodiazepine midazolam and ethanol. Although Drd4-/- mice displayed heightened anxiety, they exhibited normal ethanol preference and consumption in a two-bottle choice test. Learned fear responses evaluated by contextual, cued and instrumental fear-conditioning tests showed no difference between wild-type and Drd4-/- mice. Taken together these results indicate that the absence of D4Rs increases avoidance behaviour to unconditioned stimuli and does not impair behavioural reactions to Pavlovian fear-conditioning, suggesting that the D4R could play a key role in the dopaminergic modulation of cortical signals triggered by environmental stimuli.