RESUMO
Social and economic impacts of neurodegenerative diseases (NDs) become more prominent in our constantly aging population. Currently, due to the lack of knowledge about the aetiology of most NDs, only symptomatic treatment is available for patients. Hence, researchers and clinicians are in need of solid studies on pathological mechanisms of NDs. Autophagy promotes degradation of pathogenic proteins in NDs, while microRNAs post-transcriptionally regulate multiple signalling networks including autophagy. This chapter will critically discuss current research advancements in the area of microRNAs regulating autophagy in NDs. Moreover, we will introduce basic strategies and techniques used in microRNA research. Delineation of the mechanisms contributing to NDs will result in development of better approaches for their early diagnosis and effective treatment.
Assuntos
MicroRNAs , Doenças Neurodegenerativas , Idoso , Autofagia/genética , Humanos , MicroRNAs/genética , Doenças Neurodegenerativas/genética , Transdução de Sinais/genéticaRESUMO
More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.
Assuntos
Proliferação de Células , MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Ribonuclease III/genética , TranscriptomaRESUMO
The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.
Assuntos
Hiperfagia/complicações , Hipotálamo/metabolismo , MicroRNAs/metabolismo , Obesidade/etiologia , Obesidade/patologia , Absorciometria de Fóton , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Serina-Treonina Quinases TOR/metabolismo , Transdução GenéticaRESUMO
Absence of the leucine zipper transcription factor p45NF-E2 results in thrombocytopenia, impaired placental vascularization and intrauterine growth restriction (IUGR) in mice. The mechanism underlying the p45NF-E2-dependent placental defect and IUGR remains unknown. Here, we show that the placental defect and IUGR of p45NF-E2 (Nfe2) null mouse embryos is unrelated to thrombocytopenia, establishing that embryonic platelets and platelet-released mediators are dispensable for placentation. Rather, p45NF-E2, which was hitherto thought to be specific to hematopoietic cells, is expressed in trophoblast cells, where it is required for normal syncytiotrophoblast formation, placental vascularization and embryonic growth. Expression of p45NF-E2 in labyrinthine trophoblast cells colocalizes with that of Gcm1, a transcription factor crucial for syncytiotrophoblast formation. In the absence of p45NF-E2, the width of syncytiotrophoblast layer 2 and the expression of Gcm1 and Gcm1-dependent genes (Synb and Cebpa) are increased. In vitro, p45NF-E2 deficiency results in spontaneous syncytiotrophoblast formation, which can be reversed by Gcm1 knockdown. Increased Gcm1 expression in the absence of p45NF-E2 is dependent on enhanced protein acetylation, including post-translational modification of Gcm1. Increasing and inhibiting acetylation in the placenta of wild-type control embryos phenocopies and corrects, respectively, the changes observed in p45NF-E2-deficient embryos. These studies identify a novel function of p45NF-E2 during placental development: in trophoblast cells, p45NF-E2 represses Gcm1 and syncytiotrophoblast formation via acetylation.
Assuntos
Desenvolvimento Embrionário , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Neovascularização Fisiológica , Neuropeptídeos/metabolismo , Placenta/irrigação sanguínea , Trofoblastos/metabolismo , Acetilação , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Retardo do Crescimento Fetal , Técnicas de Introdução de Genes , Células Gigantes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Subunidade p45 do Fator de Transcrição NF-E2/genética , Neuropeptídeos/genética , Placenta/metabolismo , Placentação , Reação em Cadeia da Polimerase , Gravidez , Processamento de Proteína Pós-Traducional , Trombocitopenia , Fatores de TranscriçãoRESUMO
Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.
Assuntos
Apoptose , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/prevenção & controle , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Endotélio Vascular/patologia , Podócitos/patologia , Proteína C/fisiologia , Substituição de Aminoácidos/genética , Animais , Apoptose/genética , Linhagem Celular Transformada , Células Cultivadas , Citoproteção/genética , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Endotélio Vascular/enzimologia , Ativação Enzimática/genética , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Microcirculação/enzimologia , Microcirculação/patologia , Podócitos/enzimologia , Proteína C/biossíntese , Proteína C/genética , Transdução de Sinais/genética , Trombomodulina/fisiologiaRESUMO
The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive. â¢All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.â¢Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.
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Vitamin A (VA) is a micronutrient essential for the physiology of many organisms, but its role in longevity and age-related diseases remains unclear. In this work, we used Caenorhabditis elegans to study the impact of various bioactive compounds on lifespan. We demonstrate that VA extends lifespan and reduces lipofuscin and fat accumulation while increasing resistance to heat and oxidative stress. This resistance can be attributed to high levels of detoxifying enzymes called glutathione S-transferases, induced by the transcription factor skinhead-1 (SKN-1). Notably, VA upregulated the transcript levels of skn-1 or its mammalian ortholog NRF2 in both C. elegans, human cells, and liver tissues of mice. Moreover, the loss-of-function genetic models demonstrated a critical involvement of the SKN-1 pathway in longevity extension by VA. Our study thus provides novel insights into the molecular mechanism of anti-aging and anti-oxidative effects of VA, suggesting that this micronutrient could be used for the prevention and/or treatment of age-related disorders.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Humanos , Camundongos , Caenorhabditis elegans/metabolismo , Longevidade/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vitamina A/farmacologia , Vitamina A/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para Cima , Proteínas de Caenorhabditis elegans/metabolismo , Estresse Oxidativo , Mamíferos/metabolismo , Micronutrientes/metabolismo , Micronutrientes/farmacologiaRESUMO
Whereas it is generally perceived to be harmful, enhanced coagulation activation can also convey salutary effects. The high prevalence of the prothrombotic factor V Leiden (FVL) mutation in whites has been attributed to a positive selection pressure (eg, resulting from reduced blood loss or improved survival in sepsis). The consequences of enhanced coagulation activation, as observed in FVL carriers, on microvascular diabetic complications remain unknown. We therefore investigated the role of FVL in diabetic nephropathy. In heterozygous or homozygous diabetic FVL mice, albuminuria and indices of diabetic nephropathy were reduced compared with diabetic wild-type mice. This was associated with reduced glomerular apoptosis and preservation of podocytes in diabetic FVL-positive mice. In vitro, low-dose thrombin (50pM) prevented, whereas high-dose thrombin (20nM) aggravated, glucose-induced apoptosis in podocytes. In diabetic patients, the FVL mutation, but not the plasminogen activator inhibitor-1 4G/5G polymorphism, is associated with reduced albuminuria, which is consistent with a nephroprotective role of low but sustained thrombin generation. Consistently, anticoagulation of diabetic FVL-positive mice with hirudin abolished the nephroprotective effect. These results identify a nephroprotective function of low but sustained thrombin levels in FVL carriers, supporting a dual, context-dependent function of thrombin in chronic diseases.
Assuntos
Apoptose/genética , Coagulação Sanguínea/fisiologia , Nefropatias Diabéticas/genética , Fator V/genética , Podócitos/patologia , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Fator V/metabolismo , Genótipo , Glucose/efeitos adversos , Humanos , Hiperglicemia/complicações , Immunoblotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido IncorretoRESUMO
Parkinson's disease (PD) is a progressive age-related movement disorder that results primarily from the selective loss of midbrain dopaminergic (DA) neurons. Symptoms of PD can be induced by genetic mutations or by DA neuron-specific toxins. A specific ablation of an essential factor controlling ribosomal RNA transcription, TifIa, in adult mouse DA neurons represses mTOR signaling and leads to progressive neurodegeneration and PD-like phenotype. Using an inducible Cre system in adult mice, we show here that the specific ablation of Pten in adult mouse DA neurons leads to activation of mTOR pathway and is neuroprotective in genetic (TifIa deletion) and neurotoxin-induced (MPTP or 6OHDA) mouse models of PD. Adult mice with DA neuron-specific Pten deletion exhibit elevated expression of tyrosine hydroxylase, a rate-limiting enzyme in the dopamine biosynthesis pathway, associated with increased striatal dopamine content, and increased mRNA levels of Foxa2, Pitx3, En1, Nurr1, and Lmx1b-the essential factors for maintaining physiological functions of adult DA neurons. Pten deletion attenuates the loss of tyrosine hydroxylase-positive cells after 6OHDA treatment, restores striatal dopamine in TifIa-knockout and MPTP-treated mice, and rescues locomotor impairments caused by TifIa loss. Inhibition of Pten-dependent functions in adult DA neurons may represent a promising PD therapy.
Assuntos
Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Doença de Parkinson/prevenção & controle , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Animais , Corpo Estriado/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/toxicidade , Modelos Animais de Doenças , Dopamina/metabolismo , Dopaminérgicos/toxicidade , Deleção de Genes , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Doença de Parkinson/etiologia , Doença de Parkinson/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Obesity is a growing medical and social problem worldwide. The control of energy homeostasis in the brain is achieved by various regions including the arcuate hypothalamic nucleus (ARH). The latter comprises a number of neuronal populations including the first order metabolic neurons, appetite-stimulating agouti-related peptide (AgRP) neurons and appetite-suppressing proopiomelanocortin (POMC) neurons. Using an in vivo reductionist approach and POMCCre-dependent CRISPR-Cas9, we demonstrate that miR-15a-5p protects from obesity. Moreover, we have identified Bace1, a gene previously linked to energy metabolism imbalance, as a direct target of miR-15a-5p. This work warrants further investigations of non-coding RNA-mediated regulation of energy homeostasis and might contribute to the development of novel therapeutic approaches to treat metabolic diseases.
Assuntos
MicroRNAs , Pró-Opiomelanocortina , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Camundongos , MicroRNAs/genética , Obesidade/genética , Obesidade/metabolismo , Pró-Opiomelanocortina/genéticaRESUMO
OBJECTIVE: Obesity, a growing threat to the modern society, represents an imbalance of metabolic queues that normally signal to the arcuate hypothalamic nucleus, a critical brain region sensing and regulating energy homeostasis. This is achieved by various neurons many of which developmentally originate from the proopiomelanocortin (POMC)-expressing lineage. Within the mature neurons originating from this lineage, we aimed to identify non-coding genes in control of metabolic function in the adulthood. METHODS: In this work, we used microRNA mimic delivery and POMCCre-dependent CRISPR-Cas9 knock-out strategies in young or aged mice. Importantly, we also used CRISPR guides directing suicide cleavage of Cas9 to limit the off-target effects. RESULTS: Here we found that mature neurons originating from the POMC lineage employ miR-29a to protect against insulin resistance obesity, hyperphagia, decreased energy expenditure and obesity. Moreover, we validated the miR-29 family as a prominent regulator of the PI3K-Akt-mTOR pathway. Within the latter, we identified a direct target of miR-29a-3p, Nras, which was up-regulated in those and only those mature POMCCreCas9 neurons that were effectively transduced by anti-miR-29 CRISPR-equipped construct. Moreover, POMCCre-dependent co-deletion of Nras in mature neurons attenuated miR-29 depletion-induced obesity. CONCLUSIONS: Thus, the first to our knowledge case of in situ Cre-dependent CRISPR-Cas9-mediated knock-out of microRNAs in a specific hypothalamic neuronal population helped us to decipher a critical metabolic circuit in adult mice. This work significantly extends our understanding about the involvement of neuronal microRNAs in homeostatic regulation.
Assuntos
MicroRNAs , Pró-Opiomelanocortina , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Obesidade/genética , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Opiomelanocortina/metabolismoRESUMO
Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.
Assuntos
Citrus paradisi/química , Vesículas Extracelulares/química , Nanocápsulas/química , Células Cultivadas , Vesículas Extracelulares/ultraestrutura , Células HCT116 , Proteínas de Choque Térmico HSP70/administração & dosagem , Humanos , Leucócitos Mononucleares/metabolismo , Nanocápsulas/ultraestrutura , Soroalbumina Bovina/administração & dosagemRESUMO
BACKGROUND: Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. METHODS AND RESULTS: De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM(Pro/Pro)ApoE(-/-) and FVL(Q/Q)ApoE(-/-) mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. CONCLUSIONS: Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.
Assuntos
Aterosclerose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C beta/metabolismo , Receptor PAR-1/metabolismo , Trombofilia/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Movimento Celular/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Feminino , Hiperlipidemias/imunologia , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/citologia , Monócitos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Trombina/metabolismo , Trombofilia/imunologia , Vasculite/imunologia , Vasculite/metabolismo , Vasculite/patologiaRESUMO
MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (DicerHET) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (DicerCKO). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.
Assuntos
Envelhecimento/metabolismo , Neurônios Dopaminérgicos/metabolismo , MicroRNAs/metabolismo , Neuroproteção , Ribonuclease III/metabolismo , Alelos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Neurônios Dopaminérgicos/patologia , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Deleção de Genes , Mesencéfalo/metabolismo , Camundongos Knockout , MicroRNAs/genética , Atividade Motora/efeitos dos fármacos , Degeneração Neural/genética , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neuroproteção/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tapsigargina/farmacologiaRESUMO
The proteins Foxa1 and Foxa2 belong to the forkhead family of transcription factors and are involved in the development of several tissues, including liver, pancreas, lung, prostate, and the neural system. Both Foxa1 and Foxa2 are also crucial for the specification and differentiation of dopamine (DA) neurons during embryonic development, while about 30% of mice with an embryonic deletion of a single allele of the Foxa2 gene exhibit an age-related asymmetric loss of DA neurons and develop locomotor symptoms resembling Parkinson's disease (PD). Notably, both Foxa1 and Foxa2 factors continue to be expressed in the adult dopamine system. To directly assess their functions selectively in adult DA neurons, we induced genetic deletions of Foxa1/2 transcription factors in mice using a tamoxifen inducible tissue-specific CreERT2 recombinase expressed under control of the dopamine transporter (DAT) promoter (DATCreERT2). The conditional DA neurons-specific ablation of both genes, but not of Foxa2 alone, in early adulthood, caused a decline of striatal dopamine and its metabolites, along with locomotor deficits. At early pre-symptomatic stages, we observed a decline in aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1) protein expression in DA neurons. Further analyses revealed a decline of aromatic amino acid decarboxylase (AADC) and a complete loss of DAT expression in these neurons. These molecular changes ultimately led to a reduction of DA neuron numbers in the substantia nigra pars compacta (SNpc) of aged cFoxa1/2 (-/-) mice, resembling the progressive course of PD in humans. Altogether, in this study, we address the molecular, cellular, and functional role of both Foxa1 and Foxa2 factors in the maintenance of the adult dopamine system which may help to find better approaches for PD treatment.
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Coagulation and complement regulators belong to two interactive systems constituting emerging mechanisms of diabetic nephropathy. Thrombomodulin (TM) regulates both coagulation and complement activation, in part through discrete domains. TM's lectin like domain dampens complement activation, while its EGF-like domains independently enhance activation of the anti-coagulant and cytoprotective serine protease protein C (PC). A protective effect of activated PC in diabetic nephropathy is established. We hypothesised that TM controls diabetic nephropathy independent of PC through its lectin-like domain by regulating complement. Diabetic nephropathy was analysed in mice lacking TM's lectin-like domain (TMLeD/LeD) and controls (TMwt/wt). Albuminuria (290 µg/mg vs. 166 µg/mg, p=0.03) and other indices of experimental diabetic nephropathy were aggravated in diabetic TMLeD/LeD mice. Complement deposition (C3 and C5b-9) was markedly increased in glomeruli of diabetic TMLeD/LeD mice. Complement inhibition with enoxaparin ameliorated diabetic nephropathy in TMLeD/LeD mice (e.g. albuminuria 85 µg/mg vs. 290 µg/mg, p<0.001). In vitro TM's lectin-like domain cell-autonomously prevented glucose-induced complement activation on endothelial cells and - notably - on podocytes. Podocyte injury, which was enhanced in diabetic TMLeD/LeD mice, was reduced following complement inhibition with enoxaparin. The current study identifies a novel mechanism regulating complement activation in diabetic nephropathy. TM's lectin-like domain constrains glucose-induced complement activation on endothelial cells and podocytes and ameliorates albuminuria and glomerular damage in mice.