RESUMO
GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.
Assuntos
Glutamato Descarboxilase/deficiência , Palato/embriologia , Prosencéfalo/embriologia , Retina/embriologia , Animais , Glutamato Descarboxilase/metabolismo , Ratos , Ratos MutantesRESUMO
The taiep rat undergoes hypomyelination and progressive demyelination caused by an abnormal microtubule accumulation in oligodendrocytes, which elicits neuroinflammation and motor behavior dysfunction. Based on taurine antioxidant and proliferative actions, this work explored whether its sustained administration from the embryonic age to adulthood could prevent neuroinflammation, stimulate cell proliferation, promote myelination, and relieve motor impairment. Taurine (50 mg/L of drinking water = 50 ppm) was given to taiep pregnant rats on gestational day 15 and afterward to the male offspring until eight months of age. We measured the levels of nitric oxide (NO), malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA), CXCL1, CXCR2 receptor, growth factors (BNDF and FGF2), cell proliferation, and myelin content over time. Integral motor behavior was also evaluated. Our results showed that taurine administration significantly decreased NO and MDA + 4-HDA levels, increased cell proliferation, and promoted myelination in an age- and brain region-dependent fashion compared with untreated taiep rats. Taurine effect on chemokines and growth factors was also variable. Taurine improved vestibular reflexes and limb muscular strength in perinatal rats and fine movements and immobility episodes in adult rats. These results show that chronic taurine administration partially alleviates the taiep neuropathology.
Assuntos
Destreza Motora , Taurina , Animais , Masculino , Doenças Neuroinflamatórias , Estresse Oxidativo , Ratos , Ratos Mutantes , Ratos Sprague-DawleyRESUMO
Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.
Assuntos
Modelos Animais de Doenças , Fenantrenos/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Poli Adenosina Difosfato Ribose/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/transplante , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Transplante de Células , Células Cultivadas , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Células Fotorreceptoras de Vertebrados/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Mutantes , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologiaRESUMO
Anxiety disorders are associated with a failure to sufficiently extinguish fear memories. The serotonergic system (5-hydroxytryptamine, 5-HT) with the 5-HT transporter (5-HTT, SERT) is strongly implicated in the regulation of anxiety and fear. In the present study, we examined the effects of SERT deficiency on fear extinction in a differential fear conditioning paradigm in male and female rats. Fear-related behavior displayed during acquisition, extinction, and recovery, was measured through quantification of immobility and alarm 22-kHz ultrasonic vocalizations (USV). Trait-like inter-individual differences in novelty-seeking, anxiety-related behavior, habituation learning, cognitive performance, and pain sensitivity were examined for their predictive value in forecasting fear extinction. Our results show that SERT deficiency strongly affected the emission of 22-kHz USV during differential fear conditioning. During acquisition, extinction, and recovery, SERT deficiency consistently led to a reduction in 22-kHz USV emission. While SERT deficiency did not affect immobility during acquisition, genotype differences started to emerge during extinction, and during recovery rats lacking SERT showed higher levels of immobility than wildtype littermate controls. Recovery was reflected in increased levels of immobility but not 22-kHz USV emission. Prominent sex differences were evident. Among several measures for trait-like inter-individual differences, anxiety-related behavior had the best predictive quality.
Assuntos
Comportamento Animal , Medo , Locos de Características Quantitativas , Proteínas de Ligação a RNA/genética , Animais , Ratos , Ratos MutantesRESUMO
Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Dependovirus/fisiologia , Terapia Genética/métodos , Proteínas do Tecido Nervoso/genética , Distrofias Retinianas/genética , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Dependovirus/genética , Células Ependimogliais/metabolismo , Proteínas do Olho/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacologia , Injeções Intravítreas , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Ratos , Ratos Mutantes , Retina/fisiopatologia , Distrofias Retinianas/etiologia , Distrofias Retinianas/terapia , Epitélio Pigmentado da Retina/metabolismo , Tropismo ViralRESUMO
Recent studies suggest that treatment with SGLT-2 inhibitors can reduce hepatic lipid storage and ameliorate non-alcoholic fatty liver disease (NAFLD) development beyond their glycemic benefits. However, the exact mechanism involved is still unclear. We investigated the hepatic metabolic effect of empagliflozin (10 mg/kg/day for eight weeks) on the development of NAFLD and its complications using HHTg rats as a non-obese prediabetic rat model. Empagliflozin treatment reduced neutral triacylglycerols and lipotoxic diacylglycerols in the liver and was accompanied by significant changes in relative mRNA expression of lipogenic enzymes (Scd-1, Fas) and transcription factors (Srebp1, Pparγ). In addition, alterations in the gene expression of cytochrome P450 proteins, particularly Cyp2e1 and Cyp4a, together with increased Nrf2, contributed to the improvement of hepatic lipid metabolism after empagliflozin administration. Decreased circulating levels of fetuin-A improved lipid metabolism and attenuated insulin resistance in the liver and in peripheral tissues. Our results highlight the beneficial effect of empagliflozin on hepatic lipid metabolism and lipid accumulation independent of obesity, with the mechanisms understood to involve decreased lipogenesis, alterations in cytochrome P450 proteins, and decreased fetuin-A. These changes help to alleviate NAFLD symptoms in the early phase of the disease and before the onset of diabetes.
Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estado Pré-Diabético/tratamento farmacológico , Estado Pré-Diabético/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Hiperglicemia/tratamento farmacológico , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Hiperlipoproteinemia Tipo IV/complicações , Hiperlipoproteinemia Tipo IV/tratamento farmacológico , Hiperlipoproteinemia Tipo IV/metabolismo , Mediadores da Inflamação/metabolismo , Resistência à Insulina , Masculino , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/complicações , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estado Pré-Diabético/complicações , Ratos , Ratos Mutantes , Ratos WistarRESUMO
The angiotensin receptor/neprilysin inhibitor Sacubitril/Valsartan (Sac/Val) has been shown to be beneficial in patients suffering from heart failure with reduced ejection fraction (HFrEF). However, the impact of Sac/Val in patients presenting with heart failure with preserved ejection fraction (HFpEF) is not yet clearly resolved. The present study aimed to reveal the influence of the drug on the functionality of the myocardium, the skeletal muscle, and the vasculature in a rat model of HFpEF. Female obese ZSF-1 rats received Sac/Val as a daily oral gavage for 12 weeks. Left ventricle (LV) function was assessed every four weeks using echocardiography. Prior to organ removal, invasive hemodynamic measurements were performed in both ventricles. Vascular function of the carotid artery and skeletal muscle function were monitored. Sac/Val treatment reduced E/é ratios, left ventricular end diastolic pressure (LVEDP) and myocardial stiffness as well as myocardial fibrosis and heart weight compared to the obese control group. Sac/Val slightly improved endothelial function in the carotid artery but had no impact on skeletal muscle function. Our results demonstrate striking effects of Sac/Val on the myocardial structure and function in a rat model of HFpEF. While vasodilation was slightly improved, functionality of the skeletal muscle remained unaffected.
Assuntos
Aminobutiratos/farmacologia , Compostos de Bifenilo/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Músculo Esquelético/efeitos dos fármacos , Valsartana/farmacologia , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Conectina/metabolismo , GMP Cíclico/sangue , Diástole/efeitos dos fármacos , Diástole/fisiologia , Modelos Animais de Doenças , Combinação de Medicamentos , Eletrocardiografia , Feminino , Fibrose , Hemoglobinas Glicadas/análise , Músculo Esquelético/fisiologia , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/fisiopatologia , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Fosforilação/efeitos dos fármacos , Ratos Mutantes , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Eukaryotic cell metabolism consists of processes that generate available energy, such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxphos), and those that consume it, including macromolecular synthesis, the maintenance of ionic gradients, and cellular detoxification. By converting pyruvate to acetyl-CoA (AcCoA), the pyruvate dehydrogenase (PDH) complex (PDC) links glycolysis and the TCA cycle. Surprisingly, disrupting the connection between glycolysis and the TCA cycle by inactivation of PDC has only minor effects on cell replication. However, the molecular basis for this metabolic re-equilibration is unclear. We report here that CRISPR/Cas9-generated PDH-knockout (PDH-KO) rat fibroblasts reprogrammed their metabolism and their response to short-term c-Myc (Myc) oncoprotein overexpression. PDH-KO cells replicated normally but produced surprisingly little lactate. They also exhibited higher rates of glycolysis and Oxphos. In addition, PDH-KO cells showed altered cytoplasmic and mitochondrial pH, redox states, and mitochondrial membrane potential (ΔΨM). Conditionally activated Myc expression affected some of these parameters in a PDH-dependent manner. PDH-KO cells had increased oxygen consumption rates in response to glutamate, but not to malate, and were depleted in all TCA cycle substrates between α-ketoglutarate and malate despite high rates of glutaminolysis, as determined by flux studies with isotopically labeled glutamine. Malate and pyruvate were diverted to produce aspartate, thereby potentially explaining the failure to accumulate lactate. We conclude that PDH-KO cells maintain proliferative capacity by utilizing glutamine to supply high rates of AcCoA-independent flux through the bottom portion of the TCA cycle while accumulating pyruvate and aspartate that rescue their redox defects.
Assuntos
Ciclo do Ácido Cítrico , Fibroblastos/metabolismo , Potencial da Membrana Mitocondrial , Consumo de Oxigênio , Complexo Piruvato Desidrogenase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Fibroblastos/patologia , Humanos , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos MutantesRESUMO
BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.
Assuntos
Pressão Arterial/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Mutação , Contração Miocárdica/genética , Artéria Pulmonar/fisiopatologia , Função Ventricular Direita/genética , Potenciais de Ação , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Predisposição Genética para Doença , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Fosforilação , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Artéria Pulmonar/metabolismo , Ratos Mutantes , Proteínas Smad/metabolismoRESUMO
Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH.NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 (Klk10), and Jun Proto-Oncogene (Jun) were identified as potential mediators in ERα-regulated pathways in RV pressure overload.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Hipertrofia Ventricular Direita/prevenção & controle , Miocárdio/metabolismo , Disfunção Ventricular Direita/prevenção & controle , Função Ventricular Direita , Remodelação Ventricular , Animais , Modelos Animais de Doenças , Receptor alfa de Estrogênio/genética , Feminino , Colágenos Fibrilares/metabolismo , Fibrose , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mutação , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos Mutantes , Ratos Sprague-Dawley , Fatores Sexuais , Transdução de Sinais , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/patologia , Disfunção Ventricular Direita/fisiopatologiaRESUMO
Children with Rett syndrome show abnormal cutaneous sensitivity. The precise nature of sensory abnormalities and underlying molecular mechanisms remain largely unknown. Rats with methyl-CpG binding protein 2 (MeCP2) mutation, characteristic of Rett syndrome, show hypersensitivity to pressure and cold, but hyposensitivity to heat. They also show cutaneous hyperinnervation by nonpeptidergic sensory axons, which include subpopulations encoding noxious mechanical and cold stimuli, whereas peptidergic thermosensory innervation is reduced. MeCP2 knockdown confined to dorsal root ganglion sensory neurons replicated this phenotype in vivo, and cultured MeCP2-deficient ganglion neurons showed augmented axonogenesis. Transcriptome analysis revealed dysregulation of genes associated with cytoskeletal dynamics, particularly those controlling actin polymerization and focal-adhesion formation necessary for axon growth and mechanosensory transduction. Down-regulation of these genes by topoisomerase inhibition prevented abnormal axon sprouting. We identified eight key affected genes controlling actin signaling and adhesion formation, including members of the Arhgap, Tiam, and cadherin families. Simultaneous virally mediated knockdown of these genes in Rett rats prevented sensory hyperinnervation and reversed mechanical hypersensitivity, indicating a causal role in abnormal outgrowth and sensitivity. Thus, MeCP2 regulates ganglion neuronal genes controlling cytoskeletal dynamics, which in turn determines axon outgrowth and mechanosensory function and may contribute to altered pain sensitivity in Rett syndrome.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Citoesqueleto/metabolismo , Regulação para Baixo , Cistos Glanglionares/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Mutação , Síndrome de Rett/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Proteínas do Citoesqueleto/genética , Citoesqueleto/genética , Cistos Glanglionares/patologia , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Ratos , Ratos Mutantes , Síndrome de Rett/genética , Síndrome de Rett/patologiaRESUMO
Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Ataxia Telangiectasia/complicações , Inflamação/etiologia , Doenças Neurodegenerativas/etiologia , Neurônios/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Betametasona/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Inflamação/prevenção & controle , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/prevenção & controle , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Ratos , Ratos MutantesRESUMO
MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , MicroRNAs/fisiologia , Retina/metabolismo , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/metabolismo , Animais , Autofagia , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Retina/fisiopatologia , Degeneração Retiniana/enzimologia , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Transdução de SinaisRESUMO
Preserving of vision is the main goal in vision research. The presented research evaluates the preservation of visual function in Royal College of Surgeon (RCS) rats using a depth perception test. Rats were placed on a stage with one side containing an illusory steep drop ("cliff") and another side with a minimal drop ("table"). Latency of stage dismounting and the percentage of rats that set their first foot on the "cliff" side were determined. Nondystrophic Long-Evans (LE) rats were tested as control. Electroretinogram and histology analysis were used to determine retinal function and structure. Four-week-old RCS rats presented a significantly shorter mean latency to dismount the stage compared with 6-week-old rats (mean ± standard error, 13.7 ± 1.68 vs. 20.85 ± 6.5 s, P = 0.018). Longer latencies were recorded as rats aged, reaching 45.72 s in 15-week-old rats (P < 0.00001 compared with 4-week-old rats). All rats at the age of 4 weeks placed their first foot on the table side. By contrast, at the age of 8 weeks, 28.6% rats dismounted on the cliff side and at the age of 10 and 15 weeks, rats randomly dismounted the stage to either table or cliff side. LE rats dismounted the stage faster than 4-week-old RCS rats, but the difference was not statistically significant (7 ± 1.58 s, P = 0.057) and all LE rats dismounted on the table side. The latency to dismount the stage in RCS rats correlated with maximal electroretinogram b-wave under dark and light adaptation (Spearman's rho test = -0.603 and -0.534, respectively, all P < 0.0001), outer nuclear layer thickness (Spearman's rho test = -0.764, P = 0.002), and number of S- and M-cones (Spearman's rho test = -0.763 [P = 0.002], and -0.733 [P = 0.004], respectively). The cliff avoidance test is an objective, quick, and readily available method for the determination of RCS rats' visual function.
Assuntos
Percepção de Profundidade/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Acuidade Visual/fisiologia , Envelhecimento/fisiologia , Animais , Opsinas dos Cones/metabolismo , Eletrorretinografia , Microscopia de Fluorescência , Ratos , Ratos Long-Evans , Ratos Mutantes , Retina/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Testes VisuaisRESUMO
Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.
Assuntos
Condroitina ABC Liase/administração & dosagem , Terapia Genética/métodos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Condroitina ABC Liase/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Regeneração Nervosa/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Mutantes , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Transgenes/genéticaRESUMO
Sirtuin 2 (SIRT2) is a deacetylase that belongs to class III family of histone deacetylases (HDACs). Although it is the most abundantly expressed member of HDAC-III in human bone tissues, it is unclear whether SIRT2 plays a role in bone metabolism. In this study, the role of SIRT2 in bone metabolism, and the underlying mechanism were investigated. In in vivo experiments, micro-CT analysis revealed that there were no differences in bone microstructures between SIRT2-KO and WT rats at 12 weeks of age. However, in 36-week-old rats, increased Tb. BMD, bone volume fraction (BV/TV) and trabecular number (Tb. N) of distal femurs were observed in SIRT2-KO rats, when compared with those of WT rats. Moreover, reduced serum ß-CTX was identified in the 36-week old rats. In in vitro studies, inhibition of SIRT2 with its specific inhibitor, AGK2, suppressed the differentiation of bone marrow-derived mononuclear cells (BMMs) into osteoclasts via reduction of the expressions of c-Fos and NFATc1. These results suggest that SIRT2 plays a role in age-related bone loss, probably by regulating osteoclastogenesis.
Assuntos
Osteogênese/fisiologia , Osteoporose/metabolismo , Osteoporose/prevenção & controle , Sirtuína 2/deficiência , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Feminino , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese/genética , Osteoporose/genética , Ratos , Ratos Mutantes , Sirtuína 2/genéticaRESUMO
The hemochorial placenta develops from the coordinated multilineage differentiation of trophoblast stem (TS) cells. An invasive trophoblast cell lineage remodels uterine spiral arteries, facilitating nutrient flow, failure of which is associated with pathological conditions such as preeclampsia, intrauterine growth restriction, and preterm birth. Hypoxia plays an instructive role in influencing trophoblast cell differentiation and regulating placental organization. Key downstream hypoxia-activated events were delineated using rat TS cells and tested in vivo, using trophoblast-specific lentiviral gene delivery and genome editing. DNA microarray analyses performed on rat TS cells exposed to ambient or low oxygen and pregnant rats exposed to ambient or hypoxic conditions showed up-regulation of genes characteristic of an invasive/vascular remodeling/inflammatory phenotype. Among the shared up-regulated genes was matrix metallopeptidase 12 (MMP12). To explore the functional importance of MMP12 in trophoblast cell-directed spiral artery remodeling, we generated an Mmp12 mutant rat model using transcription activator-like nucleases-mediated genome editing. Homozygous mutant placentation sites showed decreased hypoxia-dependent endovascular trophoblast invasion and impaired trophoblast-directed spiral artery remodeling. A link was established between hypoxia/HIF and MMP12; however, evidence did not support Mmp12 as a direct target of HIF action. Lysine demethylase 3A (KDM3A) was identified as mediator of hypoxia/HIF regulation of Mmp12 Knockdown of KDM3A in rat TS cells inhibited the expression of a subset of the hypoxia-hypoxia inducible factor (HIF)-dependent transcripts, including Mmp12, altered H3K9 methylation status, and decreased hypoxia-induced trophoblast cell invasion in vitro and in vivo. The hypoxia-HIF-KDM3A-MMP12 regulatory circuit is conserved and facilitates placental adaptations to environmental challenges.
Assuntos
Fator 1 Induzível por Hipóxia , Hipóxia/metabolismo , Histona Desmetilases com o Domínio Jumonji , Metaloproteinase 12 da Matriz , Placenta/metabolismo , Animais , Linhagem Celular , Plasticidade Celular , Feminino , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Gravidez , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Trofoblastos/fisiologiaRESUMO
The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 µm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.
Assuntos
Conectina/fisiologia , Coração/fisiologia , Animais , Sinalização do Cálcio , Conectina/química , Conectina/genética , Modelos Cardiovasculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miofibrilas/fisiologia , Miosinas/metabolismo , Ratos , Ratos Mutantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Estresse Mecânico , Troponina C/genética , Troponina C/metabolismo , Difração de Raios XRESUMO
Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression.
Assuntos
Depressão/tratamento farmacológico , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/genética , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Estresse Psicológico/genética , Animais , Antidepressivos/farmacologia , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Depressão/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/metabolismo , Humanos , Inflamação/complicações , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxilipinas/metabolismo , Compostos de Fenilureia/farmacocinética , Piperidinas/farmacocinética , Ratos Mutantes , Comportamento Social , Estresse Psicológico/tratamento farmacológicoRESUMO
Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/ß (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.