Browsing by Author "Estela Andres, Maria"

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    Corticotropin-releasing factor binding protein enters the regulated secretory pathway in neuroendocrine cells and cortical neurons
    (2011) Blanco, Elias H.; Pablo Zuniga, Juan; Estela Andres, Maria; Alvarez, Alejandra R.; Gysling, Katia
    Corticotropin releasing factor binding protein (CRF-BP) is a 37 kDa glycoprotein that binds CRF with high affinity. CRF-BP controls CRF levels within plasma during human pregnancy. It has also been shown that CRF-BP is expressed in various brain nuclei. Main actions that have been proposed for brain CRF-BP are either decreasing available CRF or facilitating CRF ligand-induced activation of CRF-R2 receptors. For both actions, it is necessary the release of CRF-BP from CRF-BP expressing neurons. However, the secretion mode of CRF-BP is currently unknown. We used heterologous expression of CRF-BP-Flag in PC12 cells and in primary culture of rat cortical neurons to study CRF-BP secretion mode. We observed that CRF-BP-Flag immunoreactivity presents the typical cytoplasmatic punctuate pattern that has been described for neuropeptides and proteins that enter the regulated secretory pathway in PC12 cells. Quantitative analysis of double immunofluorescence confocal images showed that CRF-BP-Flag colocalizes with secretogranin II, marker of secretory granules, both in PC12 and in primary-cultured rat neurons. Furthermore, CRF-BP-Flag is released from PC12 cells upon high K(+)-depolarization. Thus, our results show that CRF-BP is efficiently sorted to the regulated secretory pathway in two cellular contexts, suggesting that the extracellular levels of CRF-BP in the central nervous system depends on neuronal activity. (C) 2011 Elsevier Ltd. All rights reserved.
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    Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons
    (2021) Zarate, Rafaella, V; Arancibia, Duxan; Fernandez, Anllely; Signorelli, Janetti R.; Larrondo, Luis F.; Estela Andres, Maria; Zamorano, Pedro
    Background: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO.
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    Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus
    (2021) Noches, Veronica; Rivera, Carlos; Gonzalez, Marcela P.; Merello, Gianluca; Olivares-Costa, Montserrat; Estela Andres, Maria
    Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a nonselective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.
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    Repeated Treatment With the κ-Opioid Agonist U-69593 Increases K+-Stimulated Dopamine Release in the Rat Medial Prefrontal Cortex
    (2010) Antonio Fuentealba, Jose; Gysling, Katia; Estela Andres, Maria
    Acute activation of K-opioid receptors (KOR) decreases dopamine (DA) extracellular levels in both the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). Also, the acute activation of KOR prevents alterations in behavior and neurochemistry occurring after repeated use of psychostimulants. Opposing to the acute effects, repeated administration of the KOR agonist, U-6593, potentiates both high-potassium and amphetamine induced DA release in the NAc, suggesting that repeated activation of KOR sensitizes mesolimbic dopaminergic neurotransmission. This study investigated the effect of repeated treatment with U-69593 on basal and stimulated DA and serotonin (5HT) extracellular levels in the rat mPFC. Rats were injected once daily with U-69593 (0.16-0.32 mg/kg) or vehicle for 4 days. One day after the last injection, microdialysis experiments assessing DA and 5HT extracellular levels in mPFC were conduced. The repeated treatment with U-69593 significantly augmented potassium-stimulated DA extracellular levels, without affecting potassium-stimulated 5HT extracellular levels, suggesting an increase in DA releasability. Synapse 64:898 904, 2010. (C)2010 Wiley-Liss, Inc.
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    Type 1 Corticotropin-Releasing Factor Receptor Differentially Modulates Neurotransmitter Levels in the Nucleus Accumbens of Juvenile versus Adult Rats
    (2022) Zegers-Delgado, Juan; Aguilera-Soza, Alejandro; Calderon, Florencia; Davidson, Harley; Verbel-Vergara, Daniel; Yarur, Hector E.; Novoa, Javier; Blanlot, Camila; Bastias, Cristian P.; Estela Andres, Maria; Gysling, Katia
    Adversity is particularly pernicious in early life, increasing the likelihood of developing psychiatric disorders in adulthood. Juvenile and adult rats exposed to social isolation show differences in anxiety-like behaviors and significant changes in dopamine (DA) neurotransmission in the nucleus accumbens (NAc). Brain response to stress is partly mediated by the corticotropin-releasing factor (CRF) system, composed of CRF and its two main receptors, CRF-R1 and CRF-R2. In the NAc shell of adult rats, CRF induces anxiety-like behavior and changes local DA balance. However, the role of CRF receptors in the control of neurotransmission in the NAc is not fully understood, nor is it known whether there are differences between life stages. Our previous data showed that infusion of a CRF-R1 antagonist into the NAc of juvenile rats increased DA levels in response to a depolarizing stimulus and decreased basal glutamate levels. To extend this analysis, we now evaluated the effect of a CRF-R1 antagonist infusion in the NAc of adult rats. Here, we describe that the opposite occurred in the NAc of adult compared to juvenile rats. Infusion of a CRF-R1 antagonist decreased DA and increased glutamate levels in response to a depolarizing stimulus. Furthermore, basal levels of DA, glutamate, and gamma-Aminobutyric acid (GABA) were similar in juvenile animals compared to adults. CRF-R1 protein levels and localization were not different in juvenile compared to adult rats. Interestingly, we observed differences in the signaling pathways of CRF-R1 in the NAc of juveniles compared to adult rats. We propose that the function of CRF-R1 receptors is differentially modulated in the NAc according to life stage.
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    Type 2β Corticotrophin Releasing Factor Receptor Forms a Heteromeric Complex With Dopamine D1 Receptor in Living Cells
    (2020) Yarur, Hector E.; Estela Andres, Maria; Gysling, Katia
    Corticotrophin releasing factor (CRF) and its related peptides differentially bind to CRF receptors to modulate stress-related behaviors. CRF receptors comprise two G-protein coupled receptors (GPCR), type-1 CRF receptors (CRF1), and type-2 CRF receptors (CRF2). CRF2 encompasses three spliced variants in humans, alpha (CRF2 alpha), beta (CRF2 beta), and gamma (CRF2 gamma), which differ in their N-terminal extracellular domains and expression patterns. Previously, we showed that CRF2 alpha form a heteromeric protein complex with dopamine D1 receptors (D1R), leading to changes in the signaling of D1R. Based on the high sequence identity between CRF2 alpha and CRF2 beta, we hypothesized that CRF2 beta also heteromerize with D1R. To test the hypothesis, we compared the expression and localization of both CRF2 isoforms and whether CRF2 beta form stable protein complexes with D1R in HEK293 and ATR75 cell lines. We observed that the immunoreactivity for CRF2 beta was similar to that of CRF2 alpha in the endoplasmic compartment but significantly higher in the Golgi compartment. Immunoprecipitation analysis showed that CRF2 beta forms a heteromeric protein complex with D1R. Furthermore, the protein complex formed by CRF2 beta and D1R was stable enough to change the sub-cellular localization of CRF2 beta when it was co-expressed with a construct of D1R bearing a nuclear localization signal. Immunofluorescence in A7R5 cells, which endogenously express CRF2 beta and D1R, shows significant colocalization of CRF2 beta with D1R. In conclusion, our results show that CRF2 beta forms a stable heteromeric protein complex with D1R, a potential new therapeutic target in tissues where both receptors are co-expressed, such as the septum in the brain, and heart, kidney, and skeletal muscle in the periphery.