Examinando por Autor "De Ferrari, Giancarlo V."
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Ítem Copper-uptake is critical for the down regulation of synapsin and dynamin induced by neocuproine: Modulation of synaptic activity in hippocampal neurons(Frontiers Research Foundation, 2014) Castro, Patricio A.; Ramirez, Alejandra; Sepúlveda, Fernando J.; Peters, Christian; Fierro, Humberto; Waldron, Javier; Luza, Sandra; Fuentealba, Jorge; Muñoz, Francisco J.; De Ferrari, Giancarlo V.; Bush, Ashley I.; Aguayo, Luis G.; Opazo, CarlosExtracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin 3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer’s and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.Ítem Early Transcriptional Changes Induced by Wnt/𝛽-Catenin Signaling in Hippocampal Neurons(Hindawi Publishing Corporation, 2016) Pérez-Palma, Eduardo; Andrade, Víctor; Caracci, Mario O.; Bustos, Bernabé I.; Villaman, Camilo; Medina, Matías A.; Ávila, Miguel E.; Ugarte, Giorgia D.; De Ferrari, Giancarlo V.Wnt/𝛽-catenin signaling modulates brain development and function and its deregulation underlies pathological changes occurring in neurodegenerative and neurodevelopmental disorders. Since one of the main effects ofWnt/𝛽-catenin signaling is the modulation of target genes, in the present work we examined global transcriptional changes induced by short-term Wnt3a treatment (4 h) in primary cultures of rat hippocampal neurons. RNAseq experiments allowed the identification of 170 differentially expressed genes, including known Wnt/𝛽-catenin target genes such as Notum, Axin2, and Lef1, as well as novel potential candidates Fam84a, Stk32a, and Itga9. Main biological processes enriched with differentially expressed genes included neural precursor (GO:0061364, 𝑝-adjusted = 2.5 × 10−7), forebrain development (GO:0030900, 𝑝-adjusted = 7.3 × 10−7), and stem cell differentiation (GO:0048863 𝑝-adjusted = 7.3 × 10−7). Likewise, following activation of the signaling cascade, the expression of a significant number of genes with transcription factor activity (GO:0043565, 𝑝-adjusted = 4.1 × 10−6) was induced. We also studied molecular networks enriched upon Wnt3a activation and detected three highly significant expression modules involved in glycerolipid metabolic process (GO:0046486, 𝑝-adjusted = 4.5 × 10−19), learning or memory (GO:0007611, 𝑝-adjusted = 4.0 × 10−5), and neurotransmitter secretion (GO:0007269, 𝑝-adjusted = 5.3 × 10−12). Our results indicate that Wnt/𝛽-catenin mediated transcription controls multiple biological processes related to neuronal structure and activity that are affected in synaptic dysfunction disorders.Wnt/𝛽-catenin signaling modulates brain development and function and its deregulation underlies pathological changes occurring in neurodegenerative and neurodevelopmental disorders. Since one of the main effects ofWnt/𝛽-catenin signaling is the modulation of target genes, in the present work we examined global transcriptional changes induced by short-term Wnt3a treatment (4 h) in primary cultures of rat hippocampal neurons. RNAseq experiments allowed the identification of 170 differentially expressed genes, including known Wnt/𝛽-catenin target genes such as Notum, Axin2, and Lef1, as well as novel potential candidates Fam84a, Stk32a, and Itga9. Main biological processes enriched with differentially expressed genes included neural precursor (GO:0061364, 𝑝-adjusted = 2.5 × 10−7), forebrain development (GO:0030900, 𝑝-adjusted = 7.3 × 10−7), and stem cell differentiation (GO:0048863 𝑝-adjusted = 7.3 × 10−7). Likewise, following activation of the signaling cascade, the expression of a significant number of genes with transcription factor activity (GO:0043565, 𝑝-adjusted = 4.1 × 10−6) was induced. We also studied molecular networks enriched upon Wnt3a activation and detected three highly significant expression modules involved in glycerolipid metabolic process (GO:0046486, 𝑝-adjusted = 4.5 × 10−19), learning or memory (GO:0007611, 𝑝-adjusted = 4.0 × 10−5), and neurotransmitter secretion (GO:0007269, 𝑝-adjusted = 5.3 × 10−12). Our results indicate that Wnt/𝛽-catenin mediated transcription controls multiple biological processes related to neuronal structure and activity that are affected in synaptic dysfunction disorders.Ítem Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells(Frontiers Research Foundation, 2014-04) Avilés, Evelyn C.; Pinto, Cristina; Hanna, Patricia; Ojeda, Jorge; Pérez, Viviana; De Ferrari, Giancarlo V.; Zamorano, Pedro; Albistur, Miguel; Sandoval, Daniel; Henríquez, Juan P.Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ.Ítem Heterogeneous contribution of microdeletions in the development of common generalised and focal epilepsies(BMJ Publishing Group, 2017-09) Pérez-Palma, Eduardo; Helbig, Ingo; Klein, Karl Martin; Anttila, Verneri; Horn, Heiko; Reinthaler, Eva Maria; Gormley, Padhraig; Ganna, Andrea; Byrnes, Andrea; Pernhorst, Katharina; Toliat, Mohammad R.; Saarentaus, Elmo; Howrigan, Daniel P.; Hoffman, Per; Miquel, Juan Francisco; De Ferrari, Giancarlo V.; Nürnberg, Peter; Lerche, Holger; Zimprich, Fritz; Neubauer, Bern A.; Becker, Albert J.; Rosenow, Felix; Perucca, Emilio; Zara, Federico; Weber, Yvonne G.; Lal, DennisBackground Microdeletions are known to confer risk to epilepsy, particularly at genomic rearrangement 'hotspot' loci. However, microdeletion burden not overlapping these regions or within different epilepsy subtypes has not been ascertained. Objective T o decipher the role of microdeletions outside hotspots loci and risk assessment by epilepsy subtype. Methods We assessed the burden, frequency and genomic content of rare, large microdeletions found in a previously published cohort of 1366 patients with genetic generalised epilepsy (GGE) in addition to two sets of additional unpublished genome-wide microdeletions found in 281 patients with rolandic epilepsy (RE) and 807 patients with adult focal epilepsy (AFE), totalling 2454 cases. Microdeletions were assessed in a combined and subtype-specific approaches against 6746 controls. Results When hotspots are considered, we detected an enrichment of microdeletions in the combined epilepsy analysis (adjusted p=1.06×10-6,OR 1.89, 95% CI 1.51 to 2.35). Epilepsy subtype-specific analyses showed that hotspot microdeletions in the GGE subgroup contribute most of the overall signal (adjusted p=9.79×10-12, OR 7.45, 95% CI 4.20-13.5). Outside hotspots , microdeletions were enriched in the GGE cohort for neurodevelopmental genes (adjusted p=9.13×10-3,OR 2.85, 95% CI 1.62-4.94). No additional signal was observed for RE and AFE. Still, gene-content analysis identified known (NRXN1, RBFOX1 and PCDH7) and novel (LOC102723362) candidate genes across epilepsy subtypes that were not deleted in controls. Conclusions Our results show a heterogeneous effect of recurrent and non-recurrent microdeletions as part of the genetic architecture of GGE and a minor contribution in the aetiology of RE and AFE. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017.Ítem Overrepresentation of glutamate signaling in alzheimer's disease: Network-based pathway enrichment using meta-analysis of genome-wide association studies(Public Library of Science, 2014-04) Pérez-Palma, Eduardo; Bustos, Bernabé I.; Villamán, Camilo F.; Alarcón, Marcelo A.; Avila, Miguel E.; Ugarte, Giorgia D.; Reyes, Ariel E.; Opazo, Carlos; De Ferrari, Giancarlo V.Genome-wide association studies (GWAS) have successfully identified several risk loci for Alzheimer’s disease (AD). Nonetheless, these loci do not explain the entire susceptibility of the disease, suggesting that other genetic contributions remain to be identified. Here, we performed a meta-analysis combining data of 4,569 individuals (2,540 cases and 2,029 healthy controls) derived from three publicly available GWAS in AD and replicated a broad genomic region (.248,000 bp) associated with the disease near the APOE/TOMM40 locus in chromosome 19. To detect minor effect size contributions that could help to explain the remaining genetic risk, we conducted network-based pathway analyses either by extracting gene wise p-values (GW), defined as the single strongest association signal within a gene, or calculated a more stringent gene based association p-value using the extended Simes (GATES) procedure. Comparison of these strategies revealed that ontological sub-networks (SNs) involved in glutamate signaling were significantly overrepresented in AD (p,2.7610211, p, 1.9610211; GW and GATES, respectively). Notably, glutamate signaling SNs were also found to be significantly overrepresented (p,5.161028 ) in the Alzheimer’s disease Neuroimaging Initiative (ADNI) study, which was used as a targeted replication sample. Interestingly, components of the glutamate signaling SNs are coordinately expressed in disease-related tissues, which are tightly related to known pathological hallmarks of AD. Our findings suggest that genetic variation within glutamate signaling contributes to the remaining genetic risk of AD and support the notion that functional biological networks should be targeted in future therapies aimed to prevent or treat this devastating neurological disorder.Ítem Variants in ABCG8 and TRAF3 genes confer risk for gallstone disease in admixed Latinos with Mapuche Native American ancestry(Nature Publishing Group, 2019-12-01) Bustos, Bernabé I.; Pérez-Palma, Eduardo; Buch, Stephan; Azócar, Lorena; Riveras, Eleodoro; Ugarte, Giorgia D.; Toliat, Mohammad; Nürnberg, Peter; Lieb, Wolfgang; Franke, Andre; Hinz, Sebastian; Burmeister, Greta; von Schönfels, Witigo; Schafmayer, Clemens; Völzke, Henry; Völker, Uwe; Homuth, Georg; Lerch, Markus M.; Santos, José Luis; Puschel, Klaus; Bambs, Claudia; Roa, Juan Carlos; Gutiérrez, Rodrigo A.; Hampe, Jochen; De Ferrari, Giancarlo V.; Miquel, Juan FranciscoLatin Americans and Chilean Amerindians have the highest prevalence of gallstone disease (GSD) and gallbladder cancer (GBC) in the world. A handful of loci have been associated with GSD in populations of predominantly European ancestry, however, they only explain a small portion of the genetic component of the disease. Here, we performed a genome-wide association study (GWAS) for GSD in 1,095 admixed Chilean Latinos with Mapuche Native American ancestry. Disease status was assessed by cholecystectomy or abdominal ultrasonography. Top-10 candidate variants surpassing the suggestive cutoff of P < 1 × 10−5 in the discovery cohort were genotyped in an independent replication sample composed of 1,643 individuals. Variants with positive replication were further examined in two European GSD populations and a Chilean GBC cohort. We consistently replicated the association of ABCG8 gene with GSD (rs11887534, P = 3.24 × 10−8, OR = 1.74) and identified TRAF3 (rs12882491, P = 1.11 × 10−7, OR = 1.40) as a novel candidate gene for the disease in admixed Chilean Latinos. ABCG8 and TRAF3 variants also conferred risk to GBC. Gene expression analyses indicated that TRAF3 was significantly decreased in gallbladder (P = 0.015) and duodenal mucosa (P = 0.001) of GSD individuals compared to healthy controls, where according to GTEx data in the small intestine, the presence of the risk allele contributes to the observed effect. We conclude that ABCG8 and TRAF3 genes are associated with GSD and GBC in admixed Latinos and that decreased TRAF3 levels could enhance gallbladder inflammation as is observed in GSD and GSD-associated GBC.Ítem Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells(American Society of Hematology, 2015-10) Ugarte, Giorgia D.; Vargas, Macarena F.; Medina, Matías A.; León, Pablo; Necuñir, David; Elorza, Alvaro A.; Gutiérrez, Soraya E.; Moon, Randall T.; Loyola, Alejandra; De Ferrari, Giancarlo V.Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia. © 2015 by The American Society of Hematology.