RESUMEN
We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring.
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Dieta con Restricción de Proteínas , Dopamina , Animales , Femenino , Ratones , Embarazo , Alelos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Neuronas , Conducta AnimalRESUMEN
PURPOSE: There is robust evidence that people with schizophrenia show elevated dopamine (DA) synthesis capacity in the striatum. This finding comes from positron emission tomography (PET) studies using radiolabelled l-3,4-dihydroxyphenylalanine (18F-DOPA). DA synthesis capacity also appears to be elevated in the midbrain of people with schizophrenia compared to healthy controls. We therefore aimed to optimise a method to quantify 18F-DOPA uptake in the midbrain of mice, and to utilise this method to quantify DA synthesis capacity in the midbrain of the sub-chronic ketamine model of schizophrenia-relevant hyperdopaminergia. PROCEDURES: Adult male C57Bl6 mice were treated daily with either ketamine (30 mg/kg, i.p.) or vehicle (saline) for 5 days. On day 7, animals were administered 18F-DOPA (i.p.) and scanned in an Inveon PET/CT scanner. Data from the saline-treated group were used to optimise an atlas-based template to position the midbrain region of interest and to determine the analysis parameters which resulted in the greatest intra-group consistency. These parameters were then used to compare midbrain DA synthesis capacity (KiMod) between ketamine- and saline-treated animals. RESULTS: Using an atlas-based template to position the 3.7 mm3 midbrain ROI with a T*-Tend window of 15-140 min to estimate KiMod resulted in the lowest intra-group variability and moderate test-retest agreement. Using these parameters, we found that KiMod was elevated in the midbrain of ketamine-treated animals in comparison to saline-treated animals (t(22) = 2.19, p = 0.048). A positive correlation between DA synthesis capacity in the striatum and the midbrain was also evident in the saline-treated animals (r2 = 0.59, p = 0.005) but was absent in ketamine-treated animals (r2 = 0.004, p = 0.83). CONCLUSIONS: Using this optimised method for quantifying 18F-DOPA uptake in the midbrain, we found that elevated striatal DA synthesis capacity in the sub-chronic ketamine model extends to the midbrain. Interestingly, the dysconnectivity between the midbrain and striatum seen in this model is also evident in the clinical population. This model may therefore be ideal for assessing novel compounds which are designed to modulate pre-synaptic DA synthesis capacity.
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Dopamina , Ketamina , Humanos , Adulto , Masculino , Animales , Ratones , Ketamina/farmacología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Ratones Endogámicos C57BL , Dihidroxifenilalanina , Tomografía de Emisión de Positrones/métodos , Cuerpo Estriado , Mesencéfalo/diagnóstico por imagenRESUMEN
Positron emission tomography (PET) using the radiotracer [18F]-FDOPA provides a tool for studying brain dopamine synthesis capacity in animals and humans. We have previously standardised a micro-PET methodology in mice by intravenously administering [18F]-FDOPA via jugular vein cannulation and assessment of striatal dopamine synthesis capacity, indexed as the influx rate constant K i Mod of [18F]-FDOPA, using an extended graphical Patlak analysis with the cerebellum as a reference region. This enables a direct comparison between preclinical and clinical output values. However, chronic intravenous catheters are technically difficult to maintain for longitudinal studies. Hence, in this study, intraperitoneal administration of [18F]-FDOPA was evaluated as a less-invasive alternative that facilitates longitudinal imaging. Our experiments comprised the following assessments: (i) comparison of [18F]-FDOPA uptake between intravenous and intraperitoneal radiotracer administration and optimisation of the time window used for extended Patlak analysis, (ii) comparison of Ki Mod in a within-subject design of both administration routes, (iii) test-retest evaluation of Ki Mod in a within-subject design of intraperitoneal radiotracer administration, and (iv) validation of Ki Mod estimates by comparing the two administration routes in a mouse model of hyperdopaminergia induced by subchronic ketamine. Our results demonstrate that intraperitoneal [18F]-FDOPA administration resulted in good brain uptake, with no significant effect of administration route on Ki Mod estimates (intraperitoneal: 0.024 ± 0.0047 min-1, intravenous: 0.022 ± 0.0041 min-1, p = 0.42) and similar coefficient of variation (intraperitoneal: 19.6%; intravenous: 18.4%). The technique had a moderate test-retest validity (intraclass correlation coefficient (ICC) = 0.52, N = 6) and thus supports longitudinal studies. Following subchronic ketamine administration, elevated K i Mod as compared to control condition was measured with a large effect size for both methods (intraperitoneal: Cohen's d = 1.3; intravenous: Cohen's d = 0.9), providing further evidence that ketamine has lasting effects on the dopamine system, which could contribute to its therapeutic actions and/or abuse liability.
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Dopamina , Ketamina , Humanos , Animales , Ratones , Tomografía de Emisión de Positrones , Encéfalo , Modelos Animales de EnfermedadRESUMEN
Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. The mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we develop a translational mouse model of the dopamine pathophysiology seen in schizophrenia and test approaches to reverse the dopamine changes. Mice were treated with sub-chronic ketamine (30 mg/kg) or saline and then received in vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open-field test. In vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen's d = 2.5) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of PV interneurons in pre-limbic cortex and ventral subiculum of the hippocampus. Sub-chronic ketamine reduced PV expression in these cortical and hippocampal regions. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1) and 5-HT1A receptors but no appreciable action at dopamine D2 receptors, significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, that this requires activation of midbrain dopamine neurons, and can be ameliorated by activating PV interneurons and by a TAAR1/5-HT1A agonist. This identifies novel therapeutic approaches for targeting presynaptic dopamine dysfunction in patients with schizophrenia and effects of ketamine relevant to its therapeutic use for treating major depression.
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Ketamina , Esquizofrenia , Animales , Dopamina , Humanos , Ketamina/farmacología , Ratones , Piranos , Receptores de N-Metil-D-Aspartato , Esquizofrenia/tratamiento farmacológicoRESUMEN
Cyclin-dependent kinase-like 5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females who typically present with severe early epileptic encephalopathy, global developmental delay, motor dysfunction, autistic features and sleep disturbances. To develop a gene replacement therapy, we initially characterized the human CDKL5 transcript isoforms expressed in the brain, neuroblastoma cell lines, primary astrocytes and embryonic stem cell-derived cortical interneurons. We found that the isoform 1 and to a lesser extent the isoform 2 were expressed in human brain, and both neuronal and glial cell types. These isoforms were subsequently cloned into recombinant adeno-associated viral (AAV) vector genome and high-titre viral vectors were produced. Intrajugular delivery of green fluorescence protein via AAV vector serotype PHP.B in adult wild-type male mice transduced neurons and astrocytes throughout the brain more efficiently than serotype 9. Cdkl5 knockout male mice treated with isoform 1 via intrajugular injection at age 28-30 days exhibited significant behavioural improvements compared to green fluorescence protein-treated controls (1012 vg per animal, n = 10 per group) with PHP.B vectors. Brain expression of the isoform 1 transgene was more abundant in hindbrain than forebrain and midbrain. Transgene brain expression was sporadic at the cellular level and most prominent in hippocampal neurons and cerebellar Purkinje cells. Correction of postsynaptic density protein 95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 knockout mice, was found in regions of high transgene expression within the cerebellum. AAV vector serotype DJ efficiently transduced CDKL5-mutant human induced pluripotent stem cell-derived neural progenitors, which were subsequently differentiated into mature neurons. When treating CDKL5-mutant neurons, isoform 1 expression led to an increased density of synaptic puncta, while isoform 2 ameliorated the calcium signalling defect compared to green fluorescence protein control, implying distinct functions of these isoforms in neurons. This study provides the first evidence that gene therapy mediated by AAV vectors can be used for treating CDKL5 disorder.
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Terapia Genética , Isoformas de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Adenoviridae , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Células Cultivadas , Homólogo 4 de la Proteína Discs Large/biosíntesis , Femenino , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Sinapsis/metabolismo , TransfecciónRESUMEN
Arcuate nucleus agouti-related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs, and other behaviors, implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here, we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. The activity of these neurons increased upon food availability, and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST GABAergic nociceptin neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and nonhomeostatic neuronal inputs.
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Proteína Relacionada con Agouti/fisiología , Núcleo Arqueado del Hipotálamo/fisiología , Conducta Alimentaria/fisiología , Neuronas GABAérgicas/fisiología , Péptidos Opioides/fisiología , Núcleos Septales/fisiología , Animales , Peso Corporal , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptido Y/fisiología , NociceptinaRESUMEN
Senescence is a cellular stress response that results in the stable arrest of old, damaged or preneoplastic cells. Oncogene-induced senescence is tumor suppressive but can also exacerbate tumorigenesis through the secretion of pro-inflammatory factors from senescent cells. Drugs that selectively kill senescent cells, termed senolytics, have proved beneficial in animal models of many age-associated diseases. Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity. Senescent cells are sensitized to ouabain-induced apoptosis, a process mediated in part by induction of the pro-apoptotic Bcl2-family protein NOXA. We show that cardiac glycosides synergize with anti-cancer drugs to kill tumor cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent preneoplastic cells. Our findings suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanism. Given the broad range of senescent cells targeted by cardiac glycosides their use against age-related diseases warrants further exploration.
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Glicósidos Cardíacos/farmacología , Senescencia Celular/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Humanos , Ratones , Ouabaína/farmacología , Quercetina/farmacología , RatasRESUMEN
Huntington's disease (HD) is a fatal inherited autosomal dominant neurodegenerative disorder caused by an expansion in the number of CAG trinucleotide repeats in the huntingtin gene. The disease is characterized by motor, behavioural and cognitive symptoms for which at present there are no disease altering treatments. It has been shown that manipulating the mTOR (mammalian target of rapamycin) pathway using rapamycin or its analogue CCI-779 can improve the cellular and behavioural phenotypes of HD models. Ribosomal protein S6 kinase 1 (S6K1) is a major downstream signalling molecule of mTOR, and its activity is reduced by rapamycin suggesting that deregulation of S6K1 activity may be beneficial in HD. Furthermore, S6k1 knockout mice have increased lifespan and improvement in age-related phenotypes. To evalute the potential benefit of S6k1 loss on HD-related phenotypes, we crossed the R6/2 HD model with the long-lived S6k1 knockout mouse line. We found that S6k1 knockout does not ameliorate behavioural or physiological phenotypes in the R6/2 mouse model. Additionally, no improvements were seen in brain mass reduction or mutant huntingtin protein aggregate levels. Therefore, these results suggest that while a reduction in S6K1 signalling has beneficial effects on ageing it is unlikely to be a therapeutic strategy for HD patients.
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Eliminación de Gen , Enfermedad de Huntington/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
OBJECTIVE: Sympathetic nervous system and immune cell interactions play key roles in the regulation of metabolism. For example, recent convergent studies have shown that macrophages regulate obesity through brown adipose tissue (BAT) activation and beiging of white adipose tissue (WAT) via effects upon local catecholamine availability. However, these studies have raised issues about the underlying mechanisms involved including questions regarding the production of catecholamines by macrophages, the role of macrophage polarization state and the underlying intracellular signaling pathways in macrophages that might mediate these effects. METHODS: To address such issues we generated mice lacking Irs2, which mediates the effects of insulin and interleukin 4, specifically in LyzM expressing cells (Irs2LyzM-/- mice). RESULTS: These animals displayed obesity resistance and preservation of glucose homeostasis on high fat diet feeding due to increased energy expenditure via enhanced BAT activity and WAT beiging. Macrophages per se did not produce catecholamines but Irs2LyzM-/- mice displayed increased sympathetic nerve density and catecholamine availability in adipose tissue. Irs2-deficient macrophages displayed an anti-inflammatory transcriptional profile and alterations in genes involved in scavenging catecholamines and supporting increased sympathetic innervation. CONCLUSIONS: Our studies identify a critical macrophage signaling pathway involved in the regulation of adipose tissue sympathetic nerve function that, in turn, mediates key neuroimmune effects upon systemic metabolism. The insights gained may open therapeutic opportunities for the treatment of obesity.
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Tejido Adiposo Pardo/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Células Precursoras de Monocitos y Macrófagos/metabolismo , Obesidad/genética , Sistema Nervioso Simpático/metabolismo , Tejido Adiposo Pardo/fisiología , Animales , Catecolaminas/metabolismo , Células Cultivadas , Metabolismo Energético , Eliminación de Gen , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Sistema Nervioso Simpático/fisiologíaRESUMEN
GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.
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Óxido Nítrico Sintasa de Tipo I/metabolismo , Porción Compacta de la Sustancia Negra/metabolismo , Sustancia Negra/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Neuronas GABAérgicas/metabolismo , Ratones Endogámicos C57BL , Núcleo Accumbens/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECTIVE: Imprinted genes are crucial for the growth and development of fetal and juvenile mammals. Altered imprinted gene dosage causes a variety of human disorders, with growth and development during these crucial early stages strongly linked with future metabolic health in adulthood. Neuronatin (Nnat) is a paternally expressed imprinted gene found in neuroendocrine systems and white adipose tissue and is regulated by the diet and leptin. Neuronatin expression is downregulated in obese children and has been associated with stochastic obesity in C57BL/6 mice. However, our recent studies of Nnat null mice on this genetic background failed to display any body weight or feeding phenotypes but revealed a defect in glucose-stimulated insulin secretion due to the ability of neuronatin to potentiate signal peptidase cleavage of preproinsulin. Nnat deficiency in beta cells therefore caused a lack of appropriate storage and secretion of mature insulin. METHODS: To further explore the potential role of Nnat in the regulation of body weight and adiposity, we studied classical imprinting-related phenotypes such as placental, fetal, and postnatal growth trajectory patterns that may impact upon subsequent adult metabolic phenotypes. RESULTS: Here we find that, in contrast to the lack of any body weight or feeding phenotypes on the C57BL/6J background, deletion of Nnat in mice on 129S2/Sv background causes a postnatal growth restriction with reduced adipose tissue accumulation, followed by catch up growth after weaning. This was in the absence of any effect on fetal growth or placental development. In adult 129S2/Sv mice, Nnat deletion was associated with hyperphagia, reduced energy expenditure, and partial leptin resistance. Lack of neuronatin also potentiated obesity caused by either aging or high fat diet feeding. CONCLUSIONS: The imprinted gene Nnat plays a key role in postnatal growth, adult energy homeostasis, and the pathogenesis of obesity via catch up growth effects, but this role is dependent upon genetic background.
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Trastornos del Crecimiento/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Obesidad/genética , Adiposidad/genética , Animales , Peso Corporal/genética , Metabolismo Energético , Eliminación de Gen , Impresión Genómica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Obesidad/metabolismoRESUMEN
Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone- and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in ß cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat-null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in ß cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of ß cell function and whole-animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
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Regulación de la Expresión Génica , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Glucosa/genética , Glucosa/metabolismo , Insulina/genética , Células Secretoras de Insulina/citología , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genéticaRESUMEN
Salt intake is an essential dietary requirement, but excessive consumption is implicated in hypertension and associated conditions. Little is known about the neural circuit mechanisms that control motivation to consume salt, although the midbrain dopamine system, which plays a key role in other reward-related behaviors, has been implicated. We, therefore, examined the effects on salt consumption of either optogenetic excitation or chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons in male mice. Strikingly, optogenetic excitation of dopamine neurons decreased salt intake in a rapid and reversible manner, despite a strong salt appetite. Importantly, optogenetic excitation was not aversive, did not induce hyperactivity, and did not alter salt concentration preferences in a need-free state. In addition, we found that chemogenetic inhibition of dopamine neurons had no effect on salt intake. Lastly, optogenetic excitation of dopamine neurons reduced consumption of sucrose following an overnight fast, suggesting a more general role of VTA dopamine neuron excitation in organizing motivated behaviors.
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Apetito/fisiología , Neuronas Dopaminérgicas/fisiología , Fenómenos Electrofisiológicos , Conducta Alimentaria/fisiología , Cloruro de Sodio Dietético , Área Tegmental Ventral/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , OptogenéticaRESUMEN
Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans.
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Ansiedad/fisiopatología , Emociones , Conducta Alimentaria , Hipotálamo/fisiología , Neuronas/fisiología , Animales , Ansiedad/metabolismo , Apetito , Calcio/metabolismo , Conducta Exploratoria , Femenino , Hipotálamo/citología , Hipotálamo/metabolismo , Locomoción , Masculino , Ratones , Neuronas/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
Current therapies for Alzheimer's disease (AD) are symptomatic and do not target the underlying Aß pathology and other important hallmarks including neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a cofactor for transcription factors including the peroxisome proliferator-activated receptor-γ (PPARγ), and it is involved in the regulation of metabolic genes, oxidative phosphorylation, and mitochondrial biogenesis. We previously reported that PGC-1α also regulates the transcription of ß-APP cleaving enzyme (BACE1), the main enzyme involved in Aß generation, and its expression is decreased in AD patients. We aimed to explore the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α and target it by stereotaxic delivery to hippocampus and cortex of APP23 transgenic mice at the preclinical stage of the disease. Four months after injection, APP23 mice treated with hPGC-1α showed improved spatial and recognition memory concomitant with a significant reduction in Aß deposition, associated with a decrease in BACE1 expression. hPGC-1α overexpression attenuated the levels of proinflammatory cytokines and microglial activation. This effect was accompanied by a marked preservation of pyramidal neurons in the CA3 area and increased expression of neurotrophic factors. The neuroprotective effects were secondary to a reduction in Aß pathology and neuroinflammation, because wild-type mice receiving the same treatment were unaffected. These results suggest that the selective induction of PGC-1α gene in specific areas of the brain is effective in targeting AD-related neurodegeneration and holds potential as therapeutic intervention for this disease.
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Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Agregación Patológica de Proteínas/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Animales , Regulación de la Expresión Génica/genética , Vectores Genéticos/uso terapéutico , Humanos , Lentivirus/genética , Memoria/fisiología , Ratones Transgénicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/administración & dosificación , Agregación Patológica de Proteínas/terapia , Células Piramidales/metabolismo , Células Piramidales/patologíaRESUMEN
OBJECTIVE: We have previously described the generation of coxsackievirus and adenovirus receptor (α CAR)-targeted vector, and shown that intramuscular delivery in mouse leg muscles resulted in specific retrograde transduction of lumbar-motor neurons (MNs). Here, we utilized the α CAR-targeted vector to investigate the in vivo neuroprotective effects of lentivirally expressed IGF-1 for inducing neuronal survival and ameliorating the neuropathology and behavioral phenotypes of the SOD1G93A mouse model of ALS. METHODS: We produced cell factories of IGF-1 expressing lentiviral vectors (LVs) bearing α CAR or Vesicular Stomatitis Virus glycoprotein (VSV-G) on their surface so as to compare neuroprotection from MN transduced versus muscle transduced cells. We performed intramuscular delivery of either α CAR IGF-1 or VSVG IGF-1 LVs into key muscles of SOD1G93A mice prior to disease onset at day 28. Motor performance, coordination and gait analysis were assessed weekly. RESULTS: We observed substantial therapeutic efficacy only with the α CAR IGF-1 LV pretreatment with up to 50% extension of survival compared to controls. α CAR IGF-1 LV-treated animals retained muscle tone and had better motor performance during their prolonged survival. Histological analysis of spinal cord samples at end-stage further confirmed that α CAR IGF-1 LV treatment delays disease onset by increasing MN survival compared with age-matched controls. Intrastriatal injection of α CAR eGFP LV in rats leads to transduction of neurons and glia locally and neurons in olfactory bulb distally. INTERPRETATION: Our data are indicative of the efficacy of the α CAR IGF-1 LV in this model and support its candidacy for early noninvasive neuroprotective therapy in ALS.
RESUMEN
The mechanistic target of rapamycin (mTOR) signalling pathway plays a highly conserved role in aging; mice lacking ribosomal protein S6 kinase 1 (S6K1-/-) have extended lifespan and healthspan relative to wild type (WT) controls. Exactly how reduced mTOR signalling induces such effects is unclear, although preservation of stem cell function may be important. We show, using gene expression analyses, that there was a reduction in expression of cell cycle genes in young (12 week) and aged (80 week) S6K1-/- BM-derived c-Kit+ cells when compared to age-matched WT mice, suggesting that these cells are more quiescent in S6K1-/- mice. In addition, we investigated hematopoietic stem cell (HSC) frequency and function in young and aged S6K1-/-and WT mice. Young, but not aged, S6K1-/-mice had more LSK (lineage-, c-Kit+, Sca-1+) cells (% of bone marrow (BM)), including the most primitive long-term repopulating HSCs (LT-HSC) relative to WT controls. Donor-derived engraftment of LT-HSCs in recipient mice was unaffected by genotype in young mice, but was enhanced in transplants using LT-HSCs derived from aged S6K1-/- mice. Our results are the first to provide evidence that age-associated HSC functional decline is ameliorated in a long-lived mTOR mutant mouse.
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Envejecimiento/genética , Células Madre Hematopoyéticas/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Envejecimiento/metabolismo , Animales , Antígenos Ly/metabolismo , Médula Ósea/metabolismo , Femenino , Perfilación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-kit/metabolismoRESUMEN
Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K+ channel function. Phosphorylation of K()channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies of vertebrates and invertebrates, the contribution to memory of single phosphorylation sites on K+ channels has never been reported. We have used gene targeting in mice to inactivate protein kinase A substrate residues in the fast-inactivating subunit Kv4.2 (T38A mutants), and in the small-conductance Ca2+ -activated subunit SK1 (S105A mutants). Both manipulations perturbed a specific form of memory, leaving others intact. T38A mutants had enhanced spatial memory for at least 4 wk after training, whereas performance in three tests of fear memory was unaffected. S105A mutants were impaired in passive avoidance memory, sparing fear, and spatial memory. Together with recent findings that excitability governs the participation of neurons in a memory circuit, this result suggests that the memory type supported by neurons may depend critically on the phosphorylation of specific K+ channels at single residues.