RESUMEN
The renin-angiotensin system (RAS) is a key player in the control of the cardiovascular system and hydroelectrolyte balance, with an influence on organs and functions throughout the body. The classical view of this system saw it as a sequence of many enzymatic steps that culminate in the production of a single biologically active metabolite, the octapeptide angiotensin (ANG) II, by the angiotensin converting enzyme (ACE). The past two decades have revealed new functions for some of the intermediate products, beyond their roles as substrates along the classical route. They may be processed in alternative ways by enzymes such as the ACE homolog ACE2. One effect is to establish a second axis through ACE2/ANG-(1-7)/MAS, whose end point is the metabolite ANG-(1-7). ACE2 and other enzymes can form ANG-(1-7) directly or indirectly from either the decapeptide ANG I or from ANG II. In many cases, this second axis appears to counteract or modulate the effects of the classical axis. ANG-(1-7) itself acts on the receptor MAS to influence a range of mechanisms in the heart, kidney, brain, and other tissues. This review highlights the current knowledge about the roles of ANG-(1-7) in physiology and disease, with particular emphasis on the brain.
Asunto(s)
Angiotensina I/metabolismo , Encéfalo/metabolismo , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina , Enzima Convertidora de Angiotensina 2 , Animales , Humanos , Proto-Oncogenes Mas , Transducción de SeñalRESUMEN
Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.
Asunto(s)
Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Serotonina/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Diferenciación Celular , Línea Celular , Femenino , Proteínas de Unión al GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Neuronas Serotoninérgicas/citología , Transglutaminasas/metabolismoRESUMEN
Serotonin is an essential neuromodulator for mental health and animals' socio-cognitive abilities. However, we previously found that a constitutive depletion of central serotonin did not impair rat cognitive abilities in stand-alone tests. Here, we investigated how a mild and acute decrease in brain serotonin would affect rats' cognitive abilities. Using a novel rat model of inducible serotonin depletion via the genetic knockdown of tryptophan hydroxylase 2 (TPH2), we achieved a 20% decrease in serotonin levels in the hypothalamus after three weeks of non-invasive oral doxycycline administration. Decision making, cognitive flexibility, and social recognition memory were tested in low-serotonin (Tph2-kd) and control rats. Our results showed that the Tph2-kd rats were more prone to choose disadvantageously in the long term (poor decision making) in the Rat Gambling Task and that only the low-serotonin poor decision makers were more sensitive to probabilistic discounting and had poorer social recognition memory than other low-serotonin and control individuals. Flexibility was unaffected by the acute brain serotonin reduction. Poor social recognition memory was the most central characteristic of the behavioral network of low-serotonin poor decision makers, suggesting a key role of social recognition in the expression of their profile. The acute decrease in brain serotonin appeared to specifically amplify the cognitive impairments of the subgroup of individuals also identified as poor decision makers in the population. This study highlights the great opportunity the Tph2-kd rat model offers to study inter-individual susceptibilities to develop cognitive impairment following mild variations of brain serotonin in otherwise healthy individuals. These transgenic and differential approaches together could be critical for the identification of translational markers and vulnerabilities in the development of mental disorders.
Asunto(s)
Toma de Decisiones , Serotonina , Triptófano Hidroxilasa , Animales , Ratas , Conducta Animal , Cognición , Técnicas de Silenciamiento del Gen , Hipotálamo/metabolismo , Serotonina/metabolismo , Conducta Social , Triptófano Hidroxilasa/metabolismo , Triptófano Hidroxilasa/genéticaRESUMEN
Chronic kidney disease is one of the leading causes of morbidity and mortality especially among the aged population. A decline in kidney function with ageing comparable to ageing-related processes in human kidneys has also been described in Sprague-Dawley (SD) rats. The renin-angiotensin-system (RAS) plays a pivotal role in the pathophysiology of cardiovascular and kidney disease and is a successful therapeutic target. The discovery of angiotensin-(1-7) (Ang(1-7)), mainly produced by angiotensin-converting enzyme 2 (ACE2), and its receptor MAS offered a new view on the RAS. This ACE2/Ang(1-7)/MAS axis counteracts most deleterious actions of the RAS in the kidney. In order to evaluate if activation of this axis has a protective effect in ageing-induced kidney disease we generated a transgenic rat model (TGR(SM22hACE2)) overexpressing human ACE2 in vascular smooth muscle cells. These animals showed a specific transgene expression pattern and increased ACE2 activity in the kidney. Telemetric recording of cardiovascular parameters and evaluation of kidney function by histology and urine analysis revealed no alterations in blood pressure regulation and basal kidney function in young transgenic rats when compared to young SD rats. However, with ageing, SD rats developed a decline in kidney function characterized by severe albuminuria which was significantly less pronounced in TGR(SM22hACE2) rats. Concomitantly, we detected lower mRNA expression levels of kidney damage markers in aged transgenic animals. Thus, our results indicate that vascular ACE2-overexpression protects the kidney against ageing-induced decline in kidney function, supporting the kidney-protective role of the ACE2/Ang(1-7)/MAS axis.
Asunto(s)
Peptidil-Dipeptidasa A , Insuficiencia Renal Crónica , Ratas , Animales , Humanos , Anciano , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Ratas Sprague-Dawley , Sistema Renina-Angiotensina , Riñón/metabolismo , Fragmentos de Péptidos/metabolismo , Ratas Transgénicas , Insuficiencia Renal Crónica/metabolismo , Envejecimiento/genética , Angiotensina I/metabolismo , Receptores Acoplados a Proteínas GRESUMEN
BACKGROUND: An unbalance in the renin-angiotensin (Ang) system (RAS) between the Ang II/AT1 and Ang-(1-7)/Mas axis appears to be involved in preeclampsia (PE), in which a reduction in Ang-(1-7) was observed. Here, we tested whether the reduction in the activity of the Ang-(1-7)/Mas axis could be a contributing factor for the development of PE, using Mas-deficient (Mas-/-) mice. METHODS AND RESULTS: Cardiovascular parameters were evaluated by telemetry before, during pregnancy and 4 days postpartum in 20-week-old Mas-/- and wild-type (WT) female mice. Mas-/- mice presented reduced arterial blood pressure (BP) at baseline (91.3 ± 0.8 in Mas-/- vs. 94.0 ± 0.9 mmHg in WT, Diastolic, P<0.05). However, after the 13th day of gestation, BP in Mas-/- mice started to increase, time-dependently, and at day 19 of pregnancy, these animals presented a higher BP in comparison with WT group (90.5 ± 0.7 in Mas-/- vs. 80.3 ± 3.5 mmHg in WT, Diastolic D19, P<0.0001). Moreover, pregnant Mas-/- mice presented fetal growth restriction, increase in urinary protein excretion as compared with nonpregnant Mas-/-, oliguria, increase in cytokines, endothelial dysfunction and reduced ACE, AT1R, ACE2, ET-1A, and eNOS placental mRNA, similar to some of the clinical manifestations found in the development of PE. CONCLUSIONS: These results show that Mas-deletion produces a PE-like state in FVB/N mice.
Asunto(s)
Peptidil-Dipeptidasa A , Preeclampsia , Embarazo , Femenino , Ratones , Animales , Humanos , Peptidil-Dipeptidasa A/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proto-Oncogenes Mas , Preeclampsia/genética , Preeclampsia/metabolismo , Placenta/metabolismo , Sistema Renina-Angiotensina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina II/metabolismo , Fenotipo , Angiotensina I/metabolismo , Fragmentos de Péptidos/metabolismoRESUMEN
The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes , Serotonina , Triptófano Hidroxilasa , Animales , Ratones , Fibroblastos/metabolismo , Serotonina/biosíntesis , Triptófano/metabolismo , Triptófano Hidroxilasa/metabolismoRESUMEN
Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.
Asunto(s)
Encéfalo/metabolismo , Hepatocitos/metabolismo , Serotonina/biosíntesis , Triptófano Hidroxilasa/metabolismo , Animales , Encéfalo/citología , Hepatocitos/citología , RatonesRESUMEN
Serotonin is synthetized through the action of tryptophan hydroxylase (TPH) enzymes. While the TPH2 isoform is responsible for the production of serotonin in the brain, TPH1 is expressed in peripheral organs. Interestingly, despite its peripheral localization, alterations of the gene coding for TPH1 have been related to stress sensitivity and an increased susceptibility for psychiatric pathologies. On these bases, we took advantage of newly generated TPH1-/- rats, and we evaluated the impact of the lack of peripheral serotonin on the behavior and expression of brain plasticity-related genes under basal conditions and in response to stress. At a behavioral level, TPH1-/- rats displayed reduced anxiety-like behavior. Moreover, we found that neuronal activation, quantified by the expression of Bdnf and the immediate early gene Arc and transcription of glucocorticoid responsive genes after 1 h of acute restraint stress, was blunted in TPH1-/- rats in comparison to TPH1+/+ animals. Overall, we provided evidence for the influence of peripheral serotonin levels in modulating brain functions under basal and dynamic situations.
Asunto(s)
Serotonina , Triptófano Hidroxilasa , Animales , Ansiedad/genética , Ansiedad/metabolismo , Encéfalo/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Serotonina/genética , Serotonina/metabolismo , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
AIMS/HYPOTHESIS: The impact of diabetic pregnancy has been investigated extensively regarding offspring metabolism; however, little is known about the influence on the heart. We aimed to characterise the effects of a diabetic pregnancy on male adult offspring cardiac health after feeding a high-fat diet in an established transgenic rat model. METHODS: We applied our rat model for maternal type 2 diabetes characterised by maternal insulin resistance with hyperglycaemia and hyperinsulinaemia. Diabetes was induced preconceptionally via doxycycline-induced knock down of the insulin receptor in transgenic rats. Male wild-type offspring of diabetic and normoglycaemic pregnancies were raised by foster mothers, followed up into adulthood and subgroups were challenged by a high-fat diet. Cardiac phenotype was assessed by innovative speckle tracking echocardiography, circulating factors, immunohistochemistry and gene expression in the heart. RESULTS: When feeding normal chow, we did not observe differences in cardiac function, gene expression and plasma brain natriuretic peptide between adult diabetic or normoglycaemic offspring. Interestingly, when being fed a high-fat diet, adult offspring of diabetic pregnancy demonstrated decreased global longitudinal (-14.82 ± 0.59 vs -16.60 ± 0.48%) and circumferential strain (-23.40 ± 0.57 vs -26.74 ± 0.34%), increased relative wall thickness (0.53 ± 0.06 vs 0.37 ± 0.02), altered cardiac gene expression, enlarged cardiomyocytes (106.60 ± 4.14 vs 87.94 ± 1.67 µm), an accumulation of immune cells in the heart (10.27 ± 0.30 vs 6.48 ± 0.48 per fov) and higher plasma brain natriuretic peptide levels (0.50 ± 0.12 vs 0.12 ± 0.03 ng/ml) compared with normoglycaemic offspring on a high-fat diet. Blood pressure, urinary albumin, blood glucose and body weight were unaltered between groups on a high-fat diet. CONCLUSIONS/INTERPRETATION: Diabetic pregnancy in rats induces cardiac dysfunction, left ventricular hypertrophy and altered proinflammatory status in adult offspring only after a high-fat diet. A diabetic pregnancy itself was not sufficient to impair myocardial function and gene expression in male offspring later in life. This suggests that a postnatal high-fat diet is important for the development of cardiac dysfunction in rat offspring after diabetic pregnancy. Our data provide evidence that a diabetic pregnancy is a novel cardiac risk factor that becomes relevant when other challenges, such as a high-fat diet, are present.
Asunto(s)
Diabetes Mellitus Tipo 2 , Cardiopatías , Efectos Tardíos de la Exposición Prenatal , Animales , Diabetes Mellitus Tipo 2/genética , Dieta Alta en Grasa/efectos adversos , Femenino , Desarrollo Fetal , Masculino , Miocitos Cardíacos , Embarazo , Ratas , Ratas Sprague-Dawley , Factores de RiesgoRESUMEN
Experiments aimed to evaluate the tissue distribution of Mas-related G protein-coupled receptor D (MrgD) revealed the presence of immunoreactivity for the MrgD protein in the rostral insular cortex (rIC), an important area for autonomic and cardiovascular control. To investigate the relevance of this finding, we evaluated the cardiovascular effects produced by the endogenous ligand of MrgD, alamandine, in this brain region. Mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in urethane anesthetized rats. Unilateral microinjection of equimolar doses of alamandine (40 pmol/100 nL), angiotensin-(1-7), angiotensin II, angiotensin A, and Mas/MrgD antagonist d-Pro7-Ang-1-7 (50 pmol/100 nL), Mas antagonist A779 (100 pmol/100 nL), or vehicle (0.9% NaCl) were made in different rats (n = 4-6/group) into rIC. To verify the specificity of the region, a microinjection of alamandine was also performed into intermediate insular cortex (iIC). Microinjection of alamandine in rIC produced an increase in MAP (Δ = 15 ± 2 mmHg), HR (Δ = 36 ± 4 beats/min), and RSNA (Δ = 31 ± 4%), but was without effects at iIC. Strikingly, an equimolar dose of angiotensin-(1-7) at rIC did not produce any change in MAP, HR, and RSNA. Angiotensin II and angiotensin A produced only minor effects. Alamandine effects were not altered by A-779, a Mas antagonist, but were completely blocked by the Mas/MrgD antagonist d-Pro7-Ang-(1-7). Therefore, we have identified a brain region in which alamandine/MrgD receptor but not angiotensin-(1-7)/Mas could be involved in the modulation of cardiovascular-related neuronal activity. This observation also suggests that alamandine might possess unique effects unrelated to angiotensin-(1-7) in the brain.
Asunto(s)
Angiotensina I/farmacología , Presión Arterial/efectos de los fármacos , Sistema Cardiovascular/inervación , Corteza Cerebral/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Riñón/inervación , Proteínas del Tejido Nervioso/agonistas , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Corteza Cerebral/fisiología , Ligandos , Masculino , Microinyecciones , Proteínas del Tejido Nervioso/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/metabolismo , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Sistema Nervioso Simpático/fisiologíaRESUMEN
In spite of the fact that the modulatory effects of angiotensin II (Ang II) on the sympathetic nerve activity to targeted organs involved in blood pressure (BP) regulation is well acknowledged, the local production of this peptide in the brain and the consequences of enhanced central Ang II beyond the cardiovascular system are not yet well comprehended. In the present study, we generated and validated a new transgenic mouse line overexpressing the rat full-length angiotensinogen (Agt) protein specifically in the brain (Agt-Tg). Adult Agt-Tg mice presented overall increased gene expression of total Agt in the brain including brainstem and hypothalamus. In addition, the excess of Agt led to abundantly detectable brain Ang II levels as well as increased circulating copeptin levels. Agt-Tg displayed raised BP in acute recordings, while long-term telemetrically measured basal BP was indistinguishable from wild-types. Agt-Tg has altered peripheral renin-angiotensin system and vasomotor sympathetic tone homeostasis because renal gene expression analysis, plasma Ang II measurements and ganglionic blockade experiments revealed suppressed renin expression and reduced Ang II and higher neurogenic pressure response, respectively. Plasma and urine screens revealed apparently normal fluid and electrolyte handling in Agt-Tg. Interestingly, hematological analyses showed increased hematocrit in Agt-Tg caused by enhanced erythropoiesis, which was reverted by submitting the transgenic mice to a long-term peripheral sympathectomy protocol. Collectively, our findings suggest that Agt-Tg is a valuable tool to study not only brain Ang II formation and its modulatory effects on cardiovascular homeostasis but also its role in erythropoiesis control via autonomic modulation.
Asunto(s)
Angiotensina II/metabolismo , Eritropoyesis/fisiología , Homeostasis/fisiología , Sistema Renina-Angiotensina/fisiología , Animales , Encéfalo/metabolismo , Hipertensión/metabolismo , Riñón/metabolismo , Ratones , Ratones Transgénicos , Receptor de Angiotensina Tipo 1/metabolismo , Renina/metabolismoRESUMEN
Activation of the angiotensin (Ang)-converting enzyme (ACE) 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system (RAS) induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay (RIA) were similar to control Sprague-Dawley (SD) rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure (MAP), assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma atrial natriuretic peptide (ANP) and higher urinary sodium concentration. Moreover, evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decrease in total peripheral resistance (TPR). TG7371 rats, on the other hand, also presented increased cardiac and global sympathetic tone, increased plasma vasopressin (AVP) levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower blood pressure (BP). Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.
Asunto(s)
Angiotensina I/metabolismo , Sistema Cardiovascular/metabolismo , Hemodinámica , Hipertensión/prevención & control , Fragmentos de Péptidos/metabolismo , Sistema Nervioso Simpático/metabolismo , Angiotensina I/genética , Animales , Arginina Vasopresina/metabolismo , Factor Natriurético Atrial/metabolismo , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Sistema Cardiovascular/fisiopatología , Modelos Animales de Enfermedad , Genotipo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemodinámica/genética , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Fragmentos de Péptidos/genética , Fenotipo , Ratas Sprague-Dawley , Ratas Transgénicas , Proteínas Recombinantes de Fusión/metabolismo , Flujo Sanguíneo Regional , Sistema Nervioso Simpático/fisiopatología , Factores de Tiempo , Resistencia VascularRESUMEN
INTRODUCTION: Brain-derived neurotrophic factor (BDNF) has been implicated in the pro-neurogenic effect of selective serotonin reuptake inhibitors. In this study, we used Tph2 -/- mice lacking brain serotonin to dissect the interplay between BDNF and the serotonin system in mediating the effects of antidepressant pharmacotherapy on adult neurogenesis in the hippocampus. METHODS: Besides citalopram (CIT), we tested tianeptine (TIA), an antidepressant whose mechanism of action is not well understood. Specifically, we examined cell survival and endogenous concentrations of BDNF following daily injection of the drugs. RESULTS: Twenty-one days of CIT, but not of TIA, led to a significant increase in the survival of newly generated cells in the dentate gyrus of wild-type mice, without a significant effect on BDNF protein levels by either treatment. In Tph2 -/- mice, adult neurogenesis was consistently increased. Furthermore, Tph2 -/- mice showed increased BDNF protein levels, which were not affected by TIA but were significantly reduced by CIT. DISCUSSION: We conclude that the effects of CIT on adult neurogenesis are not explained by changes in BDNF protein concentrations in the hippocampus.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Citalopram , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Citalopram/farmacología , Hipocampo/metabolismo , Ratones , Ratones Noqueados , NeurogénesisRESUMEN
Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.
Asunto(s)
Envejecimiento/genética , Angiotensina II/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Progeria/genética , Insuficiencia Renal Crónica/genética , Renina/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Endonucleasas/deficiencia , Femenino , Regulación de la Expresión Génica , Tasa de Filtración Glomerular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Riñón/metabolismo , Riñón/patología , Losartán/farmacología , Masculino , Ratones , Ratones Noqueados , Progeria/metabolismo , Progeria/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Transducción de SeñalRESUMEN
In the last two decades, interest has grown significantly in the investigation of the role of trace amines and their receptors in mammalian physiology and pathology. Trace amine-associated receptor 9 (TAAR9) is one of the least studied members of this receptor family with unidentified endogenous ligands and an unknown role in the central nervous system and periphery. In this study, we generated two new TAAR9 knockout (TAAR9-KO) rat strains by CRISPR-Cas9 technology as in vivo models to evaluate the role of TAAR9 in mammalian physiology. In these mutant rats, we performed a comparative analysis of a number of hematological and biochemical parameters in the blood. Particularly, we carried out a complete blood count, erythrocyte osmotic fragility test, and screening of a panel of basic biochemical parameters. No significant alterations in any of the hematological and most biochemical parameters were found between mutant and WT rats. However, biochemical studies revealed a significant decrease in total and low-density lipoprotein cholesterol levels in the blood of both strains of TAAR9-KO rats. Such role of TAAR9 in cholesterol regulation not only brings a new understanding of mechanisms and biological pathways of lipid exchange but also provides a new potential drug target for disorders involving cholesterol-related pathology, such as atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Colesterol/sangre , Receptores Acoplados a Proteínas G/genética , Animales , Aterosclerosis/sangre , Aterosclerosis/patología , Sistemas CRISPR-Cas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Colesterol/genética , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Eritrocitos/patología , Técnicas de Inactivación de Genes , Humanos , Ligandos , Fragilidad Osmótica/genética , RatasRESUMEN
The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.
Asunto(s)
Proliferación Celular , Cromatina/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Supervivencia Celular , Cromatina/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ratones , Factores de Transcripción/genéticaRESUMEN
This Editorial, written by Guest Editors Professor Michael Bader, Professor Anthony J. Turner and Dr Natalia Alenina, proudly introduces the Clinical Science-themed collection on angiotensin-converting enzyme 2 (ACE2), a multifunctional protein - from cardiovascular regulation to coronavirus disease 2019 (COVID-19).
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Fenómenos Fisiológicos Cardiovasculares , Sistema Cardiovascular/enzimología , SARS-CoV-2/patogenicidad , Biomarcadores/metabolismo , COVID-19/enzimología , COVID-19/etiología , COVID-19/fisiopatología , Homeostasis , HumanosRESUMEN
Serotonin (5-HT) signaling pathways are thought to be involved in colorectal tumorigenesis (CRT), but the role of 5-HT synthesis in the early steps of this process is presently unknown. In this study, we used carcinogen treatment in the tryptophan hydroxylase 1 knockout (Tph1KO) and transgenic (Tph1fl/fl VillinCre ) mouse models defective in 5-HT synthesis to investigate the early mutagenic events associated with CRT. Our observations of the colonic crypt post-treatment followed a timeline designed to understand how disruption of 5-HT synthesis affects the initial steps leading to CRT. We found Tph1KO mice had decreased development of both allograft tumors and colitis-related CRT. Interestingly, carcinogenic exposure alone induced multiple colon tumors and increased cyclooxygenase-2 (Ptgs2) expression in Tph1KO mice. Deletion of interleukin 6 (Il6) in Tph1KO mice confirmed that inflammation was a part of the process. 5-HT deficiency increased colonic DNA damage but inhibited genetic repair of specific carcinogen-related damage, leading to CRT-related inflammatory reactions and dysplasia. To validate a secondary effect of 5-HT deficiency on another DNA repair pathway, we exposed Tph1KO mice to ionizing radiation and found an increase in DNA damage associated with reduced levels of ataxia telangiectasia and Rad3 related (Atr) gene expression in colonocytes. Restoring 5-HT levels with 5-hydroxytryptophan treatment decreased levels of DNA damage and increased Atr expression. Analysis of Tph1fl/fl VillinCre mice with intestine-specific loss of 5-HT synthesis confirmed that DNA repair was tissue specific. In this study, we report a novel protective role for 5-HT synthesis that promotes DNA repair activity during the early stages of colorectal carcinogenesis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Colon/metabolismo , Neoplasias Colorrectales/prevención & control , Daño del ADN , Reparación del ADN , Lesiones Precancerosas/prevención & control , Serotonina/biosíntesis , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Interleucina-6/deficiencia , Interleucina-6/genética , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Transducción de Señal , Factores de Tiempo , Triptófano Hidroxilasa/deficiencia , Triptófano Hidroxilasa/genéticaRESUMEN
Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III ß-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Triptófano Hidroxilasa/genética , Tubulina (Proteína)/genética , Animales , Diferenciación Celular , Células Cultivadas , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Neuronas/citología , Recombinación Genética , TransgenesRESUMEN
This study focuses on analyzing long-term potentiation (LTP) changes in the lateral nucleus of the amygdala (LA) and in the CA1 region of the hippocampus in slices derived from mice deficient in tryptophan hydroxylase 2 (TPH2-/- ), the rate-limiting enzyme for 5-HT synthesis in the brain. We found a reduced LTP in both brain structures in TPH2-/- mice. However, we found no changes in the magnitude of LTP in TPH2-/- mice compared to wildtype mice when it was preceded by a paired pulse protocol. Whereas the magnitude of long-term depression (LTD) did not differ between wildtype and TPH2-/- mice, priming synapses by LTD-induction facilitated subsequent CA1-LTP in wildtype mice to a greater extent than in TPH2-/- mice. In the LA we found no differences between the genotypes in this protocol of metaplasticity. These data show that, unlike exogenous 5-HT application, lack of 5-HT in the brain impairs cellular mechanisms responsible for induction of LTP. It is supposed that suppression of LTP observed in TPH2-/- mice might be compensated by mechanisms of metaplasticity induced by paired pulse stimulation or low frequency stimulation before the induction of LTP.