Browsing by Author "Cifuentes, Manuel"
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Publication Basal Sodium-Dependent Vitamin C Transporter 2 polarization in choroid plexus explant cells in normal or scorbutic conditions.(2019-10-08) Ulloa, Viviana; Saldivia, Natalia; Ferrada, Luciano; Salazar, Katterine; Martínez, Fernando; Silva-Alvarez, Carmen; Magdalena, Rocio; Oviedo, María José; Montecinos, Hernán; Torres-Vergara, Pablo; Cifuentes, Manuel; Nualart, FranciscoVitamin C is incorporated into the cerebrospinal fluid (CSF) through choroid plexus cells. While the transfer of vitamin C from the blood to the brain has been studied functionally, the vitamin C transporter, SVCT2, has not been detected in the basolateral membrane of choroid plexus cells. Furthermore, it is unknown how its expression is induced in the developing brain and modulated in scurvy conditions. We concluded that SVCT2 is intensely expressed in the second half of embryonic brain development and postnatal stages. In postnatal and adult brain, SVCT2 is highly expressed in all choroidal plexus epithelial cells, shown by colocalization with GLUT1 in the basolateral membranes and without MCT1 colocalization, which is expressed in the apical membrane. We confirmed that choroid plexus explant cells (in vitro) form a sealed epithelial structure, which polarized basolaterally, endogenous or overexpressed SVCT2. These results are reproduced in vivo by injecting hSVCT2wt-EYFP lentivirus into the CSF. Overexpressed SVCT2 incorporates AA (intraperitoneally injected) from the blood to the CSF. Finally, we observed in Guinea pig brain under scorbutic condition, that normal distribution of SVCT2 in choroid plexus may be regulated by peripheral concentrations of vitamin C. Additionally, we observed that SVCT2 polarization also depends on the metabolic stage of the choroid plexus cells.Publication IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment(Springer, 2021-09-17) Baeza, Victor; Cifuentes, Manuel; Martínez, Fernando; Ramírez, Eder; Nualart, Francisco; Ferrada, Luciano; Oviedo, María José; De Lima, Isabelle; Troncoso, Ninoschka; Saldivia, Natalia; Salazar, Katterine; [Baeza,V; Martínez,F; Ramírez,E; Nualart,F; Oviedo,MJ; De Lima,I; Troncoso,N; Saldivia,N; Salazar,K] Laboratory of Neurobiology and Stem Cells, NeuroCellT, Department of Cellular Biology, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile. [Nualart,F; Ferrada,L; Salazar,K] Faculty of Biological Sciences, Center for Advanced Microscopy CMA BIOBIO, University of Concepcion, Concepcion, Chile. [Cifuentes,M] Department of Cell Biology, Genetics and Physiology, University of Malaga, IBIMA, Malaga, Spain. [Cifuentes,M] Andalusian Center for Nanomedicine and Biotechnology, BIONAND, Malaga, Spain. [Cifuentes,M] Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, Malaga, Spain.Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.Publication Is IIIG9 a New Protein with Exclusive Ciliary Function? Analysis of Its Potential Role in Cancer and Other Pathologies.(2022-10-21) Oviedo, María José; Ramírez, Eder; Cifuentes, Manuel; Farkas, Carlos; Mella, Andy; Bertinat, Romina; Gajardo, Roberto; Ferrada, Luciano; Jara, Nery; De Lima, Isabelle; Martínez, Fernando; Nualart, Francisco; Salazar, KatterineThe identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.Publication Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.(Frontiers Media, 2014-06-27) Rivera, Patricia; Arrabal, Sergio; Cifuentes, Manuel; Grondona, Jesús M; Pérez-Martín, Margarita; Rubio, Leticia; Vargas, Antonio; Serrano, Antonia; Pavón, Francisco-Javier; Suárez, Juan; Rodríguez de Fonseca, Fernando; [Rivera,P; Arrabal,S; Vargas,A; Serrano,A; Pavón,FJ; Suárez,J; Rodriguez de Fonseca,F] Laboratorio de Investigación, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga-Hospital Regional Universitario de Málaga (UGC Salud Mental), Málaga, Spain. [Rivera,P; Arrabal,S; Serrano,A; Pavón,FJ; Suárez,J; Rodriguez de Fonseca,F] CIBER OBN, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain. [Cifuentes,M; Grondona,JM; Pérez-Martín,M] Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga, Málaga, Spain. [Cifuentes,M] CIBER BBN, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain. [Rubio,L] Departamento de Anatomía y Medicina Legal, Facultad de Medicina, Universidad de Málaga, Málaga, Spain.The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.Publication Microbial Neuraminidase Induces a Moderate and Transient Myelin Vacuolation Independent of Complement System Activation.(2017-03-07) Granados-Durán, Pablo; López-Ávalos, María Dolores; Cifuentes, Manuel; Pérez-Martín, Margarita; Fernández-Arjona, María Del Mar; Hughes, Timothy R; Johnson, Krista; Morgan, B Paul; Fernández-Llebrez, Pedro; Grondona, Jesús MSome central nervous system pathogens express neuraminidase (NA) on their surfaces. In the rat brain, a single intracerebroventricular (ICV) injection of NA induces myelin vacuolation in axonal tracts. Here, we explore the nature, the time course, and the role of the complement system in this damage. The spatiotemporal analysis of myelin vacuolation was performed by optical and electron microscopy. Myelin basic protein-positive area and oligodendrocyte transcription factor (Olig2)-positive cells were quantified in the damaged bundles. Neuronal death in the affected axonal tracts was assessed by Fluoro-Jade B and anti-caspase-3 staining. To evaluate the role of the complement, membrane attack complex (MAC) deposition on damaged bundles was analyzed using anti-C5b9. Rats ICV injected with the anaphylatoxin C5a were studied for myelin damage. In addition, NA-induced vacuolation was studied in rats with different degrees of complement inhibition: normal rats treated with anti-C5-blocking antibody and C6-deficient rats. The stria medullaris, the optic chiasm, and the fimbria were the most consistently damaged axonal tracts. Vacuolation peaked 7 days after NA injection and reverted by day 15. Olig2+ cell number in the damaged tracts was unaltered, and neurodegeneration associated with myelin alterations was not detected. MAC was absent on damaged axonal tracts, as revealed by C5b9 immunostaining. Rats ICV injected with the anaphylatoxin C5a displayed no myelin injury. When the complement system was experimentally or constitutively inhibited, NA-induced myelin vacuolation was similar to that observed in normal rats. Microbial NA induces a moderate and transient myelin vacuolation that is not caused either by neuroinflammation or complement system activation.Publication Neocortical tissue recovery in severe congenital obstructive hydrocephalus after intraventricular administration of bone marrow-derived mesenchymal stem cells(BioMed Central (BMC), 2020) García-Bonilla, María; Ojeda-Pérez, Betsaida; García-Martín, María L.; Muñoz-Hernández, M Carmen; Vitorica, Javier; Jiménez, Sebastián; Cifuentes, Manuel; Santos-Ruíz, Leonor; Shumilov, Kirill; Claros, Silvia; Gutiérrez, Antonia; Páez-González, Patricia; Jiménez, Antonio J.; [García-Bonilla,M; Ojeda-Pérez,B; Cifuentes,M; Santos-Ruíz,L; Shumilov,K; Claros,S; Gutiérrez,A; Páez-González,P; Jiménez,AJ] Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga, Malaga, Spain. [García-Bonilla,M; Ojeda-Pérez,B; Cifuentes,M; Santos-Ruíz,L; Shumilov,K; Claros,S; Gutiérrez,A; Páez-González,P; Jiménez,AJ] Instituto de Investigación Biomédica de Málaga (IBIMA), Malaga, Spain. [García-Martín,ML; Muñoz-Hernández,MC] BIONAND, Andalusian Centre for Nanomedicine & Biotechnology (Junta de Andalucía-Universidad de Málaga), Malaga, Spain. [Vitorica,J; Jiménez,S] Department of Molecular Biology and Biochemistry, University of Seville, Seville, Spain. [Vitorica,J; Jiménez,S; Gutiérrez,A] Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.Background: In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure and the presence of periventricular edema, ischemia/hypoxia, damage of the white matter, and glial reactions in the neocortex. The viability and short time effects of a therapy based on bone marrow-derived mesenchymal stem cells (BM-MSC) have been evaluated in such pathological conditions in the hyh mouse model. Methods: BM-MSC obtained from mice expressing fluorescent mRFP1 protein were injected into the lateral ventricle of hydrocephalic hyh mice at the moment they present a very severe form of the disease. The effect of transplantation in the neocortex was compared with hydrocephalic hyh mice injected with the vehicle and non-hydrocephalic littermates. Neural cell populations and the possibility of transdifferentiation were analyzed. The possibility of a tissue recovering was investigated using 1H High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, thus allowing the detection of metabolites/osmolytes related with hydrocephalus severity and outcome in the neocortex. An in vitro assay to simulate the periventricular astrocyte reaction conditions was performed using BM-MSC under high TNFα level condition. The secretome in the culture medium was analyzed in this assay. Results: Four days after transplantation, BM-MSC were found undifferentiated and scattered into the astrocyte reaction present in the damaged neocortex white matter. Tissue rejection to the integrated BM-MSC was not detected 4 days after transplantation. Hyh mice transplanted with BM-MSC showed a reduction in the apoptosis in the periventricular neocortex walls, suggesting a neuroprotector effect of the BM-MSC in these conditions. A decrease in the levels of metabolites/osmolytes in the neocortex, such as taurine and neuroexcytotoxic glutamate, also indicated a tissue recovering. Under high TNFα level condition in vitro, BM-MSC showed an upregulation of cytokine and protein secretion that may explain homing, immunomodulation, and vascular permeability, and therefore the tissue recovering. Conclusions: BM-MSC treatment in severe congenital hydrocephalus is viable and leads to the recovery of the severe neurodegenerative conditions in the neocortex. NMR spectroscopy allows to follow-up the effects of stem cell therapy in hydrocephalus.Publication Regenerative Therapies in Dry Eye Disease: From Growth Factors to Cell Therapy.(2017-10-28) Villatoro, Antonio J; Fernández, Viviana; Claros, Silvia; Alcoholado, Cristina; Cifuentes, Manuel; Merayo-Lloves, Jesús; Andrades, José A; Becerra, José; [Villatoro, Antonio J.] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Fernandez, Viviana] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Claros, Silvia] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Alcoholado, Cristina] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Cifuentes, Manuel] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Andrades, Jose A.] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Becerra, Jose] Univ Malaga, Dept Cell Biol Genet & Physiol, IBIMA, Malaga, Spain; [Claros, Silvia] CIBER, BBN, Networking Res Ctr Bioengn Biomat & Nanomed, Malaga, Spain; [Alcoholado, Cristina] CIBER, BBN, Networking Res Ctr Bioengn Biomat & Nanomed, Malaga, Spain; [Cifuentes, Manuel] CIBER, BBN, Networking Res Ctr Bioengn Biomat & Nanomed, Malaga, Spain; [Andrades, Jose A.] CIBER, BBN, Networking Res Ctr Bioengn Biomat & Nanomed, Malaga, Spain; [Becerra, Jose] CIBER, BBN, Networking Res Ctr Bioengn Biomat & Nanomed, Malaga , Spain; [Merayo-Lloves, Jesus] Fdn Invest Oftalmol, Oviedo, Spain; [Becerra, Jose] Andalusian Ctr Nanomed & Biotechnol BIONAND, Lab Bioingn & Tissue Regenerat, Malaga, SpainDry eye syndrome is a complex and insidious pathology with a high level of prevalence among the human population and with a consequently high impact on quality of life and economic cost. Currently, its treatment is symptomatic, mainly based on the control of lubrication and inflammation, with significant limitations. Therefore, the latest research is focused on the development of new biological strategies, with the aim of regenerating affected tissues, or at least restricting the progression of the disease, reducing scar tissue, and maintaining corneal transparency. Therapies range from growth factors and cytokines to the use of different cell sources, in particular mesenchymal stem cells, due to their multipotentiality, trophic, and immunomodulatory properties. We will review the state of the art and the latest advances and results of these promising treatments in this pathology.