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OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p. METHODS: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p. RESULTS: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator ß-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels. CONCLUSION: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.
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MicroARNs , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Lisina , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Ácido Oléico , Perilipina-2RESUMEN
The mammary gland is a vital exocrine organ that has evolved in mammals to secrete milk and provide nutrition to ensure the growth and survival of the neonate The mouse mammary gland displays extraordinary plasticity each time the female undergoes pregnancy and lactation, including a sophisticated process of tertiary branching and alveologenesis to form a branched epithelial tree and subsequently milk-producing alveoli. Upon the cessation of lactation, the gland remodels back to a simple ductal architecture via highly regulated involution processes. At the cellular level, the plasticity is characterised by proliferation of mammary cell populations, differentiation and apoptosis, accompanied by major changes in cell function and morphology. The mammary epithelium requires a specific stromal environment to grow, known as the mammary fat pad. Mammary adipocytes are one of the most prominent cell types in the fat pad, but despite their vast proportion in the tissue and their crucial interaction with epithelial cells, their physiology remains largely unknown. Over the past decade, the need to understand the properties and contribution of mammary adipocytes has become more recognised. However, the development of adequate methods and protocols to study this cellular niche is still lagging, partially due to their fragile nature, the difficulty of isolating them, the lack of reliable cell surface markers and the heterogenous environment in this tissue, which differs from other adipocyte depots. Here, we describe a new rapid and simple flow cytometry protocol specifically designed for the analysis and isolation of mouse mammary adipocytes across mammary gland developmental stages.
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Adipocitos , Glándulas Mamarias Animales , Embarazo , Femenino , Ratones , Animales , Citometría de Flujo , Glándulas Mamarias Animales/metabolismo , Lactancia/fisiología , Biología , MamíferosRESUMEN
Recent findings in experimental models have shown that the microRNA miR-132 (mir-132) is an important regulator of liver homeostasis and lipid metabolism. We aimed to assess miR-132 expression in liver and fat tissues of obese individuals and examine its association with blood pressure (BP) and hepatic steatosis. We examined obese individuals undergoing bariatric surgery for weight loss (n = 19). Clinical and demographic information was obtained. Quantitative PCR was performed to determine tissue expression of miR-132 in liver and subcutaneous and visceral fat biopsies obtained during bariatric surgery. Liver biopsies were read by a single liver pathologist and graded for steatosis, inflammation and fibrosis. Participants (aged 39 ± 8.1 years) had a body mass index (BMI) of 42 ± 4.5 kg/m2 and presented with 2.2 ± 1.2 metabolic abnormalities. Supine BP was 127 ± 16/74 ± 11 mmHg. Hepatic and visceral fat expression of miR-132 were correlated (r = 0.59, P = 0.033). There was no correlation between subcutaneous and visceral expression of miR-132 (r = -0.31, P = 0.20). Hepatic and visceral fat miR-132 expression were associated with BMI (r = 0.62 and r = 0.68, P = 0.049 respectively) and degree of liver steatosis (r = 0.60 and r = 0.55, P < 0.05, respectively). Subcutaneous fat miRNA-132 expression was correlated to office systolic BP (r = 0.46, P < 0.05), several aspects of 24 h BP (24 h systolic BP: r = 0.52; day systolic BP: r = 0.59, P < 0.05 for all), plasma triglycerides (r = 0.51, P < 0.01) and liver enzymes (ALT: r = -0.52; AST: r = -0.48, P < 0.05 for all). We found an association between miR-132 and markers of cardiovascular and metabolic disease. Reduction of miR-132 may be a target for the regulation of liver lipid homeostasis and control of obesity-related blood pressure.
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Hígado Graso , MicroARNs , Presión Sanguínea/genética , Hígado Graso/complicaciones , Hígado Graso/genética , Hígado Graso/metabolismo , Humanos , MicroARNs/genética , Obesidad/complicaciones , Obesidad/genética , TriglicéridosRESUMEN
Intracisternal A-particles (IAPs) are endogenous retroviruses (ERVs) responsible for most insertional mutations in the mouse. Full-length IAPs harbour genes flanked by long terminal repeats (LTRs). Here, we identify a solo LTR IAP variant (Iap5-1solo) recently formed in the inbred C57BL/6J mouse strain. In contrast to the C57BL/6J full-length IAP at this locus (Iap5-1full), Iap5-1solo lacks DNA methylation and H3K9 trimethylation. The distinct DNA methylation levels between the two alleles are established during preimplantation development, likely due to loss of KRAB zinc finger protein binding at the Iap5-1solo variant. Iap5-1solo methylation increases and becomes more variable in a hybrid genetic background yet is unresponsive to maternal dietary methyl supplementation. Differential epigenetic modification of the two variants is associated with metabolic differences and tissue-specific changes in adjacent gene expression. Our characterisation of Iap5-1 as a genetically induced epiallele with functional consequences establishes a new model to study transposable element repression and host-element co-evolution.
Our genome provides a complete set of genetic instructions for life. It begins by directing the growth and development of the embryo, and subsequently supports all the cells of the adult body in their daily routines. Yet approximately 10% of the DNA in mammalian genomes is made up of sequences originating from past retroviral infections, leaving a calling card in our genetic code. While these segments of retroviral DNA can no longer produce new infectious viruses, some of them retain the ability to copy themselves and jump into new parts of the genome. This can be problematic if they jump into and disrupt an important piece of genetic code. To protect against this, our bodies have evolved the ability to chemically strap down retroviral sequences by adding methyl groups to them and by modifying the proteins they are wrapped around. However, some of these endogenous retroviruses can dodge such so-called epigenetic modifications and disrupt genome function as a result. Studying a population of widely used inbred laboratory mice, Bertozzi et al. have identified a retroviral element that evades these epigenetic restraints. They discovered that some mice carry a full-length retroviral sequence while others have a shortened version of the same element. The shorter sequence lacked the repressive epigenetic marks found on the longer version, and this affected the expression of nearby genes. Moreover, the repressive marks could be partially restored by breeding the short-version mice with a distantly related mouse strain. Bertozzi et al. highlight an important issue for research using mouse models. Inbred laboratory mouse strains are assumed to have a fixed genetic code which allows scientists to conclude that any observed differences in their experiments are not a product of background genetic variation. However, this study emphasizes that this assumption is not guaranteed, and that hidden genetic diversity may be present in ostensibly genetically identical mice, with important implications for experimental outcomes. In addition, Bertozzi et al. provide a new mouse model for researchers to study the evolution and regulation of retroviral sequences and the impact of these processes on cell function.
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Metilación de ADN/genética , Expresión Génica/genética , Retroelementos/genética , Animales , Retrovirus Endógenos , Epigénesis Genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Secuencias Repetidas Terminales , Dedos de ZincRESUMEN
Genomic imprinting is an epigenetically regulated process leading to gene expression according to its parental origin. Imprinting is essential for prenatal growth and development, regulating nutritional resources to offspring, and contributing to a favored theory about the evolution of imprinting being due to a conflict between maternal and paternal genomes for the control of prenatal resources-the so-called kinship hypothesis. Genomic imprinting has been mainly studied during embryonic and placental development; however, maternal nutrient provisioning is not restricted to the prenatal period. In this context, the mammary gland acts at the maternal-offspring interface providing milk to the newborn. Maternal care including lactation supports the offspring, delivering nutrients and bioactive molecules protecting against infections and contributing to healthy organ development and immune maturation. The normal developmental cycle of the mammary gland-pregnancy, lactation, involution-is vital for this process, raising the question of whether genomic imprinting might also play a role in postnatal nutrient transfer by controlling mammary gland development. Characterizing the function and epigenetic regulation of imprinted genes in the mammary gland cycle may therefore provide novel insights into the evolution of imprinting since the offspring's paternal genome is absent from the mammary gland, in addition to increasing our knowledge of postnatal nutrition and its relation to life-long health. This article is categorized under: Developmental Biology > Developmental Processes in Health and Disease.
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Epigénesis Genética , Evolución Molecular , Glándulas Mamarias Humanas/metabolismo , Ritmo Circadiano , Femenino , Humanos , Lactancia , Glándulas Mamarias Humanas/crecimiento & desarrollo , Placenta/metabolismo , Embarazo , Transducción de SeñalRESUMEN
RNA sequencing analyses are often limited to identifying lowest p value transcripts, which does not address polygenic phenomena. To overcome this limitation, we developed an integrative approach that combines large-scale transcriptomic meta-analysis of patient brain tissues with single-cell sequencing data of CNS neurons, short RNA sequencing of human male- and female-originating cell lines, and connectomics of transcription factor and microRNA interactions with perturbed transcripts. We used this pipeline to analyze cortical transcripts of schizophrenia and bipolar disorder patients. Although these pathologies show massive transcriptional parallels, their clinically well-known sexual dimorphisms remain unexplained. Our method reveals the differences between afflicted men and women and identifies disease-affected pathways of cholinergic transmission and gp130-family neurokine controllers of immune function interlinked by microRNAs. This approach may open additional perspectives for seeking biomarkers and therapeutic targets in other transmitter systems and diseases.
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Trastorno Bipolar/patología , Esquizofrenia/patología , Transcriptoma , Biomarcadores/metabolismo , Trastorno Bipolar/genética , Trastorno Bipolar/inmunología , Línea Celular , Neuronas Colinérgicas/metabolismo , Conectoma , Femenino , Ontología de Genes , Humanos , Masculino , MicroARNs/metabolismo , Receptores de Taquicininas/metabolismo , Esquizofrenia/genética , Esquizofrenia/inmunología , Análisis de Secuencia de ARN , Caracteres Sexuales , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Both non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes. DESIGN: We quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts. RESULTS: Transgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype. CONCLUSIONS: Our findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.
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Lipogénesis/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Anciano , Animales , Femenino , Humanos , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/etiología , Lípidos/sangre , Lipogénesis/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Obesos , Ratones Transgénicos , MicroARNs/antagonistas & inhibidores , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacologíaRESUMEN
Epilepsy is a common neurological disease, manifested in unprovoked recurrent seizures. Epileptogenesis may develop due to genetic or pharmacological origins or following injury, but it remains unclear how the unaffected brain escapes this susceptibility to seizures. Here, we report that dynamic changes in forebrain microRNA (miR)-211 in the mouse brain shift the threshold for spontaneous and pharmacologically induced seizures alongside changes in the cholinergic pathway genes, implicating this miR in the avoidance of seizures. We identified miR-211 as a putative attenuator of cholinergic-mediated seizures by intersecting forebrain miR profiles that were Argonaute precipitated, synaptic vesicle target enriched, or differentially expressed under pilocarpine-induced seizures, and validated TGFBR2 and the nicotinic antiinflammatory acetylcholine receptor nAChRa7 as murine and human miR-211 targets, respectively. To explore the link between miR-211 and epilepsy, we engineered dTg-211 mice with doxycycline-suppressible forebrain overexpression of miR-211. These mice reacted to doxycycline exposure by spontaneous electrocorticography-documented nonconvulsive seizures, accompanied by forebrain accumulation of the convulsive seizures mediating miR-134. RNA sequencing demonstrated in doxycycline-treated dTg-211 cortices overrepresentation of synaptic activity, Ca2+ transmembrane transport, TGFBR2 signaling, and cholinergic synapse pathways. Additionally, a cholinergic dysregulated mouse model overexpressing a miR refractory acetylcholinesterase-R splice variant showed a parallel propensity for convulsions, miR-211 decreases, and miR-134 elevation. Our findings demonstrate that in mice, dynamic miR-211 decreases induce hypersynchronization and nonconvulsive and convulsive seizures, accompanied by expression changes in cholinergic and TGFBR2 pathways as well as in miR-134. Realizing the importance of miR-211 dynamics opens new venues for translational diagnosis of and interference with epilepsy.
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Encéfalo/metabolismo , Epilepsia/metabolismo , MicroARNs/metabolismo , Acetilcolina/farmacología , Acetilcolinesterasa/metabolismo , Animales , Encéfalo/efectos de los fármacos , Colinérgicos/farmacología , Epilepsia/tratamiento farmacológico , Humanos , Ratones , Ratones Transgénicos , Pilocarpina/farmacología , Receptores Nicotínicos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismoRESUMEN
MicroRNA (miR)-132 brain-to-body messages suppress inflammation by targeting acetylcholinesterase (AChE), but the target specificity of 3'-AChE splice variants and the signaling pathways involved remain unknown. Using surface plasmon resonance (SPR), we identified preferential miR-132 targeting of soluble AChE-R over synaptic-bound AChE-S, potentiating miR-132-mediated brain and body cholinergic suppression of pro-inflammatory cytokines. Inversely, bacterial lipopolysaccharide (LPS) reduced multiple miR-132 targets, suppressed AChE-S more than AChE-R and elevated inflammatory hallmarks. Furthermore, blockade of peripheral miR-132 by chemically protected AM132 antisense oligonucleotide elevated muscle AChE-R 10-fold over AChE-S, and cortical miRNA-sequencing demonstrated inverse brain changes by AM132 and LPS in immune-related miRs and neurotransmission and cholinergic signaling pathways. In neuromuscular junctions, AM132 co-elevated the nicotinic acetylcholine receptor and AChE, re-balancing neurotransmission and reaching mild muscle incoordination. Our findings demonstrate preferential miR-132-induced modulation of AChE-R which ignites bidirectional brain and body anti-inflammatory regulation, underscoring splice-variant miR-132 specificity as a new complexity level in inflammatory surveillance.
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Acetilcolinesterasa/metabolismo , Corteza Cerebral/metabolismo , Citocinas/metabolismo , Acetilcolinesterasa/genética , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Citocinas/genética , Inflamación , Isoenzimas , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , MicroARNs/metabolismo , Receptores Nicotínicos/metabolismoRESUMEN
The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.
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Acetilcolina/metabolismo , Enfermedad de Alzheimer/patología , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , ARN/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/deficiencia , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Hipocampo/citología , Humanos , Discapacidades para el Aprendizaje/etiología , Discapacidades para el Aprendizaje/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/genética , Tiazoles/farmacología , Factor Nuclear Tiroideo 1/genética , Factor Nuclear Tiroideo 1/metabolismo , Urea/análogos & derivados , Urea/farmacología , Proteínas de Transporte Vesicular de Acetilcolina/genéticaRESUMEN
UNLABELLED: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD. SIGNIFICANCE STATEMENT: In Alzheimer's disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer's disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels.
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Agonistas Colinérgicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Receptor Muscarínico M1/metabolismo , Animales , Carbacol/farmacología , Células Cultivadas , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Colinérgicos/farmacología , Embrión de Mamíferos , Regulación de la Expresión Génica/genética , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA-target interaction can simultaneously affect multiple other miRNA-target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3'-untranslated region (3'UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.
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Ansiedad/genética , Hipertensión/genética , MicroARNs/metabolismo , Acetilcolinesterasa/genética , Alelos , Animales , Secuencia de Bases , Presión Sanguínea , Encéfalo/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Voluntarios Sanos , Heterocigoto , Homocigoto , Humanos , Hidrocortisona/sangre , Hipertensión/sangre , Hipertensión/fisiopatología , Interleucina-6/genética , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Primates/genética , Especificidad de la Especie , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with naïve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.
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Región CA1 Hipocampal/fisiología , Técnicas de Transferencia de Gen , Lentivirus/genética , MicroARNs/administración & dosificación , Técnicas Estereotáxicas , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/virología , Células HEK293 , Humanos , Ratones , MicroARNs/biosíntesis , MicroARNs/genética , TransfecciónRESUMEN
Genetic studies link inherited errors in RNA metabolism to familial neurodegenerative disease. Here, we report such errors and the underlying mechanism in sporadic Alzheimer's disease (AD). AD entorhinal cortices presented globally impaired exon exclusions and selective loss of the hnRNP A/B splicing factors. Supporting functional relevance, hnRNP A/B knockdown induced alternative splicing impairments and dendrite loss in primary neurons, and memory and electrocorticographic impairments in mice. Transgenic mice with disease-associated mutations in APP or Tau displayed no alterations in hnRNP A/B suggesting that its loss in AD is independent of Aß and Tau toxicity. However, cholinergic excitation increased hnRNP A/B levels while in vivo neurotoxin-mediated destruction of cholinergic neurons caused cortical AD-like decrease in hnRNP A/B and recapitulated the alternative splicing pattern of AD patients. Our findings present cholinergic-mediated hnRNP A/B loss and impaired RNA metabolism as important mechanisms involved in AD.
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Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Neuronas Colinérgicas/patología , Trastornos del Conocimiento/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/deficiencia , Animales , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Empalme del ARNRESUMEN
The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9(-/-) mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9(-/-) mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes.
Asunto(s)
FN-kappa B/metabolismo , Transducción de Señal , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Animales , Biomarcadores/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/patología , Ratones , Saliva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/fisiopatología , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/deficienciaRESUMEN
MicroRNAs (miRs) have emerged as important gene silencers affecting many target mRNAs. Here, we report the identification of 244 miRs that target the 3'-untranslated regions of different cholinesterase transcripts: 116 for butyrylcholinesterase (BChE), 47 for the synaptic acetylcholinesterase (AChE-S) splice variant, and 81 for the normally rare splice variant AChE-R. Of these, 11 and 6 miRs target both AChE-S and AChE-R, and AChE-R and BChE transcripts, respectively. BChE and AChE-S showed no overlapping miRs, attesting to their distinct modes of miR regulation. Generally, miRs can suppress a number of targets; thereby controlling an entire battery of functions. To evaluate the importance of the cholinesterase-targeted miRs in other specific biological processes we searched for their other experimentally validated target transcripts and analyzed the gene ontology enriched biological processes these transcripts are involved in. Interestingly, a number of the resulting categories are also related to cholinesterases. They include, for BChE, response to glucocorticoid stimulus, and for AChE, response to wounding and two child terms of neuron development: regulation of axonogenesis and regulation of dendrite morphogenesis. Importantly, all of the AChE-targeting miRs found to be related to these selected processes were directed against the normally rare AChE-R splice variant, with three of them, including the neurogenesis regulator miR-132, also directed against AChE-S. Our findings point at the AChE-R splice variant as particularly susceptible to miR regulation, highlight those biological functions of cholinesterases that are likely to be subject to miR post-transcriptional control, demonstrate the selectivity of miRs in regulating specific biological processes, and open new venues for targeted interference with these specific processes.
RESUMEN
Alanine-to-threonine (A to T) substitutions caused by single nucleotide polymorphisms (SNPs) occur in diverse proteins, and in certain cases these substitutions induce self-aggregation into amyloid fibrils or aggregation in other amyloidogenic proteins. This is compatible with the inverse preferences of alanine to form helices and of threonine to support beta-sheet structures, which are crucial for amyloid fibrils formation. Our interest in these mutations was initiated by studying the potential effects of the A539T substitution in the butyrylcholinesterase BChE-K variant on amyloid fibrils formation in Alzheimer's disease. Other examples are, Parkinson's disease (PD), where A53T alpha-synuclein occurs in Lewy bodies and familial amyloid polyneuropathy (FAP), where an A25T substitution appears in transthyretin (TTR). In peripheral organs, an A34T substitution is found in the light chain immunoglobulin genes of patients with systemic amyloidosis and in familial hypercholesterolemia, an A370T substitution occurs in the LDLR regulator of cholesterol homeostasis. That such substitutions appear in proteins with important cellular functions suggests that they confer antagonistic pleiotropy, providing added value at an earlier age but causing damages and inducing amyloid diseases later on. This, in turn, may explain the evolutionary selection and preservation of these substitutions. The structural effect of residue substitutions and in particular A to T substitutions in amyloidogenic diseases thus merits further attention.