RESUMEN
Glucocorticoids (GCs) are involved in stress and circadian regulation, and produce many actions via the GC receptor (GR), which is classically understood to function as a nuclear transcription factor. However, the nuclear genome is not the only genome in eukaryotic cells. The mitochondria also contain a small circular genome, the mitochondrial DNA (mtDNA), that encodes 13 polypeptides. Recent work has established that, in the brain and other systems, the GR is translocated from the cytosol to the mitochondria and that stress and corticosteroids have a direct influence on mtDNA transcription and mitochondrial physiology. To determine if stress affects mitochondrially transcribed mRNA (mtRNA) expression, we exposed adult male rats to both acute and chronic immobilization stress and examined mtRNA expression using quantitative RT-PCR. We found that acute stress had a main effect on mtRNA expression and that expression of NADH dehydrogenase 1, 3, and 6 (ND-1, ND-3, ND-6) and ATP synthase 6 (ATP-6) genes was significantly down-regulated. Chronic stress induced a significant up-regulation of ND-6 expression. Adrenalectomy abolished acute stress-induced mtRNA regulation, demonstrating GC dependence. ChIP sequencing of GR showed that corticosterone treatment induced a dose-dependent association of the GR with the control region of the mitochondrial genome. These findings demonstrate GR and stress-dependent transcriptional regulation of the mitochondrial genome in vivo and are consistent with previous work linking stress and GCs with changes in the function of brain mitochondria.
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Corticosterona/farmacología , ADN Mitocondrial/genética , Regulación de la Expresión Génica , Hipocampo/metabolismo , Receptores de Glucocorticoides/fisiología , Estrés Psicológico/metabolismo , Animales , Masculino , Mitocondrias/fisiología , NADH Deshidrogenasa/genética , ARN Mensajero/análisis , ARN Mitocondrial , Ratas , Ratas Sprague-DawleyRESUMEN
Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.
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Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Genoma/genética , Receptores de Esteroides/genética , Transducción de Señal/genética , Animales , Receptor alfa de Estrógeno/genética , Hipocampo/metabolismo , Hibridación in Situ , Hibridación Fluorescente in Situ , Masculino , Ratones Endogámicos C57BL , Receptores de Progesterona/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glucocorticoid receptor (GR) antagonism may be of considerable therapeutic value in stress-related psychopathology such as depression. However, blockade of all GR-dependent processes in the brain will lead to unnecessary and even counteractive effects, such as elevated endogenous cortisol levels. Selective GR modulators are ligands that can act both as agonist and as antagonist and may be used to separate beneficial from harmful treatment effects. We have discovered that the high-affinity GR ligand C108297 is a selective modulator in the rat brain. We first demonstrate that C108297 induces a unique interaction profile between GR and its downstream effector molecules, the nuclear receptor coregulators, compared with the full agonist dexamethasone and the antagonist RU486 (mifepristone). C108297 displays partial agonistic activity for the suppression of hypothalamic corticotropin-releasing hormone (CRH) gene expression and potently enhances GR-dependent memory consolidation of training on an inhibitory avoidance task. In contrast, it lacks agonistic effects on the expression of CRH in the central amygdala and antagonizes GR-mediated reduction in hippocampal neurogenesis after chronic corticosterone exposure. Importantly, the compound does not lead to disinhibition of the hypothalamus-pituitary-adrenal axis. Thus, C108297 represents a class of ligands that has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain, while retaining beneficial aspects of GR signaling.
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Encéfalo/metabolismo , Regulación de la Expresión Génica , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Animales , Encéfalo/embriología , Encéfalo/fisiología , Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Dexametasona/farmacología , Hipocampo/metabolismo , Ligandos , Masculino , Mifepristona/farmacología , Coactivador 1 de Receptor Nuclear/metabolismo , Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Esteroides/metabolismo , Factores de Tiempo , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The hippocampus is a highly plastic brain region particularly susceptible to the effects of environmental stress; it also shows dynamic changes in epigenetic marks in response to stress and learning. We have previously shown that, in the rat, acute (30 min) restraint stress induces a substantial, regionally specific, increase in hippocampal levels of the repressive histone H3 lysine 9 trimethylation (H3K9me3). Because of the large magnitude of this effect and the fact that stress can induce the expression of endogenous retroviruses and transposable elements in many systems, we hypothesized that the H3K9me3 response was targeted to these elements as a means of containing potential genomic instability. We used ChIP coupled with next generation sequencing (ChIP-Seq) to determine the genomic localization of the H3K9me3 response. Although there was a general increase in this response across the genome, our results validated this hypothesis by demonstrating that stress increases H3K9me3 enrichment at transposable element loci and, using RT-PCR, we demonstrate that this effect represses expression of intracisternal-A particle endogenous retrovirus elements and B2 short interspersed elements, but it does not appear to have a repressive effect on long interspersed element RNA. In addition, we present data showing that the histone H3K9-specific methyltransferases Suv39h2 is up-regulated by acute stress in the hippocampus, and that this may explain the hippocampal specificity we observe. These results are a unique demonstration of the regulatory effect of environmental stress, via an epigenetic mark, on the vast genomic terra incognita represented by transposable elements.
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Silenciador del Gen , Hipocampo/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Retroelementos , Estrés Fisiológico , Animales , Inmunoprecipitación de Cromatina , Corticosterona/administración & dosificación , ADN/metabolismo , Histonas/química , Masculino , Metilación , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismoRESUMEN
OBJECTIVE: Huntington's disease (HD) is an autosomal dominant, fully penetrant, neurodegenerative disease that most commonly affects middle-aged adults. HD is caused by a CAG repeat expansion in the HTT gene, resulting in the expression of mutant huntingtin (mHTT). Our aim was to detect and quantify mHTT in tear fluid, which, to our knowledge, has never been measured before. METHODS: We recruited 20 manifest and 13 premanifest HD gene expansion carriers, and 20 age-matched controls. All patients underwent detailed assessments, including the Unified Huntington's Disease Rating Scale (UHDRS) total motor score (TMS) and total functional capacity (TFC) score. Tear fluid was collected using paper Schirmer's strips. The level of tear mHTT was determined using single-molecule counting SMCxPRO technology. RESULTS: The average tear mHTT levels in manifest (67,223 ± 80,360 fM) and premanifest patients (55,561 ± 45,931 fM) were significantly higher than those in controls (1,622 ± 2,179 fM). We noted significant correlations between tear mHTT levels and CAG repeat length, "estimated years to diagnosis," disease burden score and UHDRS TMS and TFC. The receiver operating curve demonstrated an almost perfect score (area under the curve [AUC] = 0.9975) when comparing controls to manifest patients. Similarly, the AUC between controls and premanifest patients was 0.9846. The optimal cutoff value for distinguishing between controls and manifest patients was 4,544 fM, whereas it was 6,596 fM for distinguishing between controls and premanifest patients. CONCLUSION: Tear mHTT has potential for early and noninvasive detection of alterations in HD patients and could be integrated into both clinical trials and clinical diagnostics.
RESUMEN
Migraine is a disabling common brain disorder typically characterized by attacks of severe headache and associated with autonomic and neurological symptoms. Its etiology is far from resolved. This review will focus on evidence that epigenetic mechanisms play an important role in disease etiology. Epigenetics comprise both DNA methylation and post-translational modifications of the tails of histone proteins, affecting chromatin structure and gene expression. Besides playing a role in establishing cellular and developmental stage-specific regulation of gene expression, epigenetic processes are also important for programming lasting cellular responses to environmental signals. Epigenetic mechanisms may explain how non-genetic endogenous and exogenous factors such as female sex hormones, stress hormones and inflammation trigger may modulate attack frequency. Developing drugs that specifically target epigenetic mechanisms may open up exciting new avenues for the prophylactic treatment of migraine.
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Epigénesis Genética , Trastornos Migrañosos/etiología , Trastornos Migrañosos/fisiopatología , Regulación de la Expresión Génica , HumanosRESUMEN
BACKGROUND: Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon. METHODS: For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants. RESULTS: We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression. CONCLUSIONS: We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.
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Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacología , Isoformas de Proteínas/metabolismo , Animales , Proteína Ácida Fibrilar de la Glía/metabolismo , Captura por Microdisección con Láser , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Coactivador 1 de Receptor Nuclear/genética , Fosfopiruvato Hidratasa/metabolismo , Isoformas de Proteínas/genética , Empalme del ARN/efectos de los fármacos , Factores de TiempoRESUMEN
INTRODUCTION: In a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5-HT)-1A agonist R-(+)-8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) inhibits sexual receptivity in female marmoset monkeys and increases aggression toward the male pairmate. AIM: The aims of this study are to investigate gene expression changes induced by 8-OH-DPAT in laser-microdissected brain areas that regulate female sexual function and to identify genes, functional gene classes, and pathways associated with 8-OH-DPAT-mediated inhibition of female sexual receptivity. METHODS: Gene expression was measured in the medial prefrontal cortex (mPFC), medial preoptic area (mPOA), cornu ammonis-1 (CA1) area of the hippocampus (CA1), and dorsal raphé nucleus (DRN) of four 8-OH-DPAT-treated (0.1 mg/kg; daily administration for 16 weeks) and four vehicle-treated female marmosets using a marmoset-specific microarray (European Marmoset Microarray [EUMAMA]) and validated by real-time quantitative polymerase chain reaction (RTqPCR). Enriched functional gene classes were determined. In a parallel candidate gene approach, the expression of serotonergic candidate genes, i.e., the 5-HT1A, 5-HT2A, and 5-HT7 receptors and the 5-HT transporter (5-HTT), was measured by RTqPCR. MAIN OUTCOME MEASURES: The main outcome is the differential expression of genes between 8-OH-DPAT- and vehicle-treated marmosets. RESULTS: 8-OH-DPAT affected the gene classes important to neural development (mPFC, mPOA, and DRN), neurotransmission (mPOA), energy production (mPFC and mPOA), learning and memory (CA1), and intracellular signal transduction (DRN). Oxytocin (OXT) in the mPOA and 5-HTT in the DRN were strongly increased by 8-OH-DPAT. 5-HT1A tended to increase in the mPFC, while 5-HT7 was decreased in the CA1. CONCLUSIONS: Brain region-specific alterations of gene expression regulating neural circuitries, energy demands, and learning processes are associated with 8-OH-DPAT-induced decrease in female sexual receptivity and increase in pairmate aggression. The role of OXT in the serotonergic regulation of female sexual behavior and partner interactions warrants attention in future studies.
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8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Encéfalo/efectos de los fármacos , Callithrix/fisiología , Receptor de Serotonina 5-HT1A/metabolismo , Rechazo en Psicología , Agonistas de Receptores de Serotonina/farmacología , Conducta Sexual Animal/efectos de los fármacos , 8-Hidroxi-2-(di-n-propilamino)tetralin/administración & dosificación , Agresión/efectos de los fármacos , Animales , Encéfalo/metabolismo , Esquema de Medicación , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Estudios de Asociación Genética , Masculino , Reproducibilidad de los Resultados , Agonistas de Receptores de Serotonina/administración & dosificación , Factores Sexuales , Factores de Tiempo , TranscriptomaRESUMEN
In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.
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Distrofia Muscular de Duchenne , Ratones , Animales , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofina/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Ratones Endogámicos mdx , Terapia Genética/métodos , Exones/genéticaRESUMEN
The dentate gyrus (DG) of the hippocampus plays a crucial role in learning and memory. This subregion is unique in its ability to generate new neurons throughout life and integrate these new neurons into the hippocampal circuitry. Neurogenesis has further been implicated in hippocampal plasticity and depression. Exposure to chronic stress affects DG function and morphology and suppresses neurogenesis and long-term potentiation (LTP) with consequences for cognition. Previous studies demonstrated that glucocorticoid receptor (GR) blockade by a brief treatment with the GR antagonist mifepristone (RU486) rapidly reverses the stress and glucocorticoid effects on neurogenesis. The molecular pathways underlying both the stress-induced effects and the RU486 effects on the DG are, however, largely unknown. The aim of this study was therefore (1) to investigate by microarray analysis which genes and pathways in the DG are sensitive to chronic stress and (2) to investigate to what extent blockade of GR can normalize these stress-induced effects on DG gene expression. Chronic stress exposure affected the expression of 90 genes in the DG (P < 0.01), with an overrepresentation of genes involved in brain development and morphogenesis and synaptic transmission. RU486 treatment of stressed animals affected expression of 107 genes; however, mostly different genes than those responding to stress. Interestingly, we found CREBBP to be normalized by RU486 treatment to levels observed in control animals, suggesting that CREB-signaling may play a central role in mediating the chronic stress effects on neurogenesis, LTP and calcium currents. The identified genetic pathways provide insight into the stress-induced adaptive plasticity of the hippocampal DG that is so central in learning and memory and will direct future studies on the functional outcome and modulation of these stress effects.
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Giro Dentado/fisiopatología , Perfilación de la Expresión Génica , Potenciación a Largo Plazo/genética , Neurogénesis/genética , Receptores de Glucocorticoides/antagonistas & inhibidores , Estrés Psicológico/genética , Animales , Giro Dentado/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Captura por Microdisección con Láser , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Mifepristona/farmacología , Neurogénesis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR) that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs) or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq). RESULTS: In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58%) of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context. CONCLUSIONS: Here we present the first genome-wide inventory of GR-binding sites in a neuronal context. These results provide an exciting first global view into neuronal GR targets and the neuron-specific modes of GR action and potentially contributes to our understanding of glucocorticoid action in the brain.
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Sitios de Unión/genética , Genómica , Unión Proteica/genética , Receptores de Glucocorticoides/metabolismo , Animales , Inmunoprecipitación de Cromatina , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Reproducibilidad de los Resultados , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
INTRODUCTION: Psychopathological origins of personally distressing, hypoactive sexual desire disorder (HSDD) in women are unknown, but are generally attributed to an inhibitory neural regulator, serotonin (5-HT). Flibanserin, a 5-HT(1A) agonist and 5-HT(2A) antagonist, shows promise as a treatment for HSDD. AIM: To test the hypothesis that female marmoset sexual behavior is enhanced by flibanserin and diminished by 8-OH-DPAT, in order to evaluate the efficacy of serotonergic modulation of female sexual behavior in a pairmate social setting comparable to humans. METHODS: Sexual and social behavior were examined in eight female marmoset monkeys receiving daily flibanserin (15 mg/kg), 8-OH-DPAT (0.1 mg/kg), or corresponding vehicle for 15-16 weeks in a counterbalanced, within-subject design, while housed in long-term, stable male-female pairs. MAIN OUTCOME MEASURES: Marmoset pairmate interactions, including sexual and social behavior, were scored during weeks 5-6 of daily flibanserin, 8-OH-DPAT or vehicle treatment. 24-hour pharmacokinetic profiles of the drugs and their metabolites, as well as drug-induced acute symptoms of the 5-HT behavioral syndrome were also assessed. RESULTS: Two-way analysis of variance reveals that flibanserin-treated females attract more male sexual interest (P=0.020) and trigger increased grooming (P=0.001) between partners. In contrast, 8-OH-DPAT-treated females show increased rejection of male sexual advances (P=0.024), a tendency for decreased male sexual interest (P=0.080), and increased aggression with their male pairmates (P=0.049). CONCLUSIONS: While 8-OH-DPAT-treated female marmosets display decreased sexual receptivity and increased aggressive interactions with their male pairmates, flibanserin-treated female marmosets demonstrate increased affiliative behavior with their male pairmates. Such pro-affiliation attributes may underlie flibanserin's effectiveness in treating HSDD in women.
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8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Bencimidazoles/farmacología , Apareamiento , Serotoninérgicos/farmacología , Serotonina/metabolismo , Conducta Sexual Animal/efectos de los fármacos , Animales , Callithrix , Femenino , Masculino , Modelos AnimalesRESUMEN
Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.
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Pruebas Genéticas/métodos , Hipocampo/metabolismo , Individualidad , Memoria a Corto Plazo/efectos de los fármacos , Receptores AMPA/metabolismo , Estrés Psicológico/metabolismo , Animales , Corticosterona/sangre , Trastorno Depresivo Mayor/etiología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Masculino , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple , Análisis por Matrices de Proteínas/métodos , Receptores AMPA/agonistas , Receptores AMPA/genética , Resiliencia Psicológica/efectos de los fármacos , Factores de Riesgo , Estrés Psicológico/sangre , Estrés Psicológico/genética , Estrés Psicológico/psicología , Sulfonamidas/farmacologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.
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Modelos Animales de Enfermedad , Distrofina/genética , Eliminación de Gen , Distrofia Muscular de Duchenne/genética , Fenotipo , Transgenes , Animales , Fenómenos Biomecánicos , Distrofina/metabolismo , Exones , Marcha , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/patologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx-specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.
Asunto(s)
Exones/efectos de los fármacos , Terapia Genética , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/farmacología , Animales , Modelos Animales de Enfermedad , Exones/genética , Humanos , Ratones , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Oligonucleótidos Antisentido/genéticaRESUMEN
Delivery to the target site and adversities related to off-target exposure have made the road to clinical success and approval of antisense oligonucleotide (AON) therapies challenging. Various classes of AONs have distinct chemical features and pharmacological properties. Understanding the similarities and differences in pharmacokinetics (PKs) among AON classes is important to make future development more efficient and may facilitate regulatory guidance of AON development programs. For the class of 2'-O-methyl phosphorothioate (2OMe PS) RNA AONs, most nonclinical and clinical PK data available today are derived from development of exon skipping therapies for Duchenne muscular dystrophy (DMD). While some publications have featured PK aspects of these AONs, no comprehensive overview is available to date. This article presents a detailed review of absorption, distribution, metabolism, and excretion of 2OMe PS AONs, compiled from publicly available data and previously unpublished internal data on drisapersen and related exon skipping candidates in preclinical species and DMD patients. Considerations regarding drug-drug interactions, toxicokinetics, and pharmacodynamics are also discussed. From the data presented, the picture emerges of consistent PK properties within the 2OMe PS class, predictable behavior across species, and a considerable overlap with other single-stranded PS AONs. A level of detail on muscle as a target tissue is provided, which was not previously available. Furthermore, muscle biopsy samples taken in DMD clinical trials allowed confirmation of the applicability of interspecies scaling approaches commonly applied in the absence of clinical target tissue data.
Asunto(s)
Terapia Genética/tendencias , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/genética , Distrofina/genética , Exones/efectos de los fármacos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/farmacocinética , Oligonucleótidos Fosforotioatos/uso terapéutico , Empalme del ARN/efectos de los fármacosRESUMEN
Spinocerebellar ataxia type 3 (SCA3) and type 1 (SCA1) are dominantly inherited neurodegenerative disorders that are currently incurable. Both diseases are caused by a CAG-repeat expansion in exon 10 of the Ataxin-3 and exon 8 of the Ataxin-1 gene, respectively, encoding an elongated polyglutamine tract that confers toxic properties to the resulting proteins. We have previously shown lowering of the pathogenic polyglutamine protein in Huntington's disease mouse models using (CUG)7, a CAG repeat-targeting antisense oligonucleotide. Here we evaluated the therapeutic capacity of (CUG)7 for SCA3 and SCA1, in vitro in patient-derived cell lines and in vivo in representative mouse models. Repeated intracerebroventricular (CUG)7 administration resulted in a significant reduction of mutant Ataxin-3 and Ataxin-1 proteins throughout the brain of SCA3 and SCA1 mouse models, respectively. Furthermore, in both a SCA3 patient cell line and the MJD84.2 mouse model, (CUG)7 induced formation of a truncated Ataxin-3 protein species lacking the polyglutamine stretch, likely arising from (CUG)7-mediated exon 10 skipping. In contrast, skipping of exon 8 of Ataxin-1 did not significantly contribute to the Ataxin-1 protein reduction observed in (CUG)7-treated SCA1154Q/2Q mice. These findings support the therapeutic potential of a single CAG repeat-targeting AON for the treatment of multiple polyglutamine disorders.
RESUMEN
Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample.
Asunto(s)
Técnicas de Laboratorio Clínico , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Animales , Técnicas de Laboratorio Clínico/métodos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismoRESUMEN
Although many of the physiological effects of corticosteroid stress hormones on neuronal function are well recognised, the underlying genomic mechanisms are only starting to be elucidated. Linking physiology and genomics has proven to be a complicated task, despite the emergence of large-scale gene expression profiling technology in the last decade. This is in part due to the complexity of glucocorticoid-signaling, in part due to the complexity of the brain itself. The presence of a binary receptor system for glucocorticoid hormones in limbic brain structures, the coexistence of membrane and intracellular receptors and the highly contextual action of glucocorticoids contribute to this complexity. In addition, the anatomical complexity, extensive cellular heterogeneity of brain and the modest changes in gene expression (mostly in the range of 10-30%) hamper detection of responsive genes, in particular of low abundant transcripts, such as many neurotransmitter receptors and growth factors. Nonetheless, ongoing research into central targets of glucocorticoids has identified many different functional gene classes that underlie the diverse effects of glucocorticoids on brain function. These functional classes include genes involved in energy metabolism, signal transduction, neuronal structure, vesicle dynamics, neurotransmitter catabolism, cell adhesion, genes encoding neurotrophic factors and their receptors and genes involved in regulating glucocorticoid-signalling. The aim of this review is to give an overview of the current status of the field on identification of central corticosteroid targets, discuss the opportunities and pitfalls and highlight new developments in understanding central corticosteroid action.
Asunto(s)
Regulación de la Expresión Génica , Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Animales , Encéfalo/metabolismo , Perfilación de la Expresión Génica , Genómica , Humanos , Receptores de Esteroides/genética , Transducción de Señal , Transcripción Genética/fisiologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe, neuromuscular disorder caused by mutations in the DMD gene, precluding synthesis of functional dystrophin protein. Antisense oligonucleotide (AON)-mediated exon skipping has been developed as a method to restore the reading frame, which allows the synthesis of internally truncated, but partially functional dystrophin proteins, as found in the less severe Becker muscular dystrophy (BMD). This approach is species specific, since AONs targeting human exons often will not have full homology to mouse exons. As such, mouse models with mutations in the murine Dmd gene are of limited use to study human specific AONs in vivo. However, our del52hDMD/mdx mouse model contains mutated copies of both the mouse (nonsense mutation in exon 23) and human (deletion of exon 52) dystrophin-encoding genes. This model allows for testing effects of treatment with human specific exon 51 or 53 targeting AONs on RNA, protein, histological, and functional levels. Therefore, the model can be used to optimize human specific AONs, e.g., by comparing dystrophin protein and exon skipping levels.Absolute quantification of exon skipping levels can be obtained by digital droplet PCR (ddPCR). This method compartmentalizes samples into thousands of droplets that represent individual micro PCR reactions, and can be either positive or negative after amplification depending on whether there was a template molecule present or not. This allows for precise determination of the copy numbers of template molecules. The protocol described here uses probes binding to exon-exon junctions (EEJs) of human DMD transcripts with and without skipping of exon 51. We report that this method is specific for human transcripts so that exon skipping levels can be quantified accurately by ddPCR in del52hDMD/mdx mice.