Signaling Pathways in Proton and Non-proton ASIC1a Activation

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases as well as pain conditions. Classically, ASICs are described as transiently activated by a reduced pH, followed by desensitization; the activation allows sodium influx, and in the case of ASIC1a-composed channels, also calcium to some degree. Several factors are emerging and extensively analyzed as modulators, activating, inhibiting, and potentiating specific channel subunits. However, the signaling pathways triggered by channel activation are only starting to be revealed.The channel has been recently shown to be activated through a mechanism other than proton-mediated. Indeed, the large extracellular loop of these channels opens the possibility that other non-proton ligands might exist. One such molecule discovered was a toxin present in the Texas coral snake venom. The finding was associated with the activation of the channel at neutral pH via the toxin and causing intense and unremitting pain.By using different pharmacological tools, we analyzed the downstream signaling pathway triggered either by the proton and non-proton activation for human, mouse, and rat ASIC1a-composed channels in in vitro models. We show that for all species analyzed, the non-protonic mode of activation determines the activation of the ERK signaling cascade at a higher level and duration compared to the proton mode.This study adds to the growing evidence of the important role ASIC1a channels play in different physiological and pathological conditions and also hints at a possible pathological mechanism for a sustained effect.Fil: Salinas Castellanos, Libia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Ion channels and pain in Fabry disease

Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal α-galactosidase A (α-Gal A) activity which results in progressive accumulation of globotriaosylceramide (Gb3) and related metabolites. One prominent feature of Fabry disease is neuropathic pain. Accumulation of Gb3 has been documented in dorsal root ganglia (DRG) as well as other neurons, and has lately been associated with the mechanism of pain though the pathophysiology is still unclear. Small fiber (SF) neuropathy in FD differs from other entities in several aspects related to the perception of pain, alteration of fibers as well as drug therapies used in the practice with patients, with therapies far from satisfying. In order to develop better treatments, more information on the underlying mechanisms of pain is needed. Research in neuropathy has gained momentum from the development of preclinical models where different aspects of pain can be modelled and further analyzed. This review aims at describing the different in vitro and FD animal models that have been used so far, as well as some of the insights gained from their use. We focus especially in recent findings associated with ion channel alterations -that apart from the vascular alterations-, could provide targets for improved therapies in pain.Fil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Albanese, Adriana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Contreras, Natalia Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Gobetto, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Salinas Castellanos, Libia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Evaluation of Early Microstructural Changes in the R6/1 Mouse Model of Huntington's Disease by Ultra-High Field Diffusion MR Imaging

Diffusion MRI (dMRI) has been able to detect early structural changes related to neurological symptoms present in Huntington's disease (HD). However, there is still a knowledge gap to interpret the biological significance at early neuropathological stages. The purpose of this study is two-fold: (i) establish if the combination of Ultra-High Field Diffusion MRI (UHFD-MRI) techniques can add a more comprehensive analysis of the early microstructural changes observed in HD, and (ii) evaluate if early changes in dMRI microstructural parameters can be linked to cellular biomarkers of neuroinflammation. Ultra-high field magnet (16.7T), diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI) techniques were applied to fixed ex-vivo brains of a preclinical model of HD (R6/1 mice). Fractional anisotropy (FA) was decreased in deep and superficial grey matter (GM) as well as white matter (WM) brain regions with well-known early HD microstructure and connectivity pathology. NODDI parameters associated with the intracellular and extracellular compartment, such as intracellular ventricular fraction (ICVF), orientation dispersion index (ODI), and isotropic volume fractions (IsoVF) were altered in R6/1 mice GM. Further, histological studies in these areas showed that glia cell markers associated with neuroinflammation (GFAP & Iba1) were consistent with the dMRI findings. dMRI can be used to extract non-invasive information of neuropathological events present in the early stages of HD. The combination of multiple imaging techniques represents a better approach to understand the neuropathological process allowing the early diagnosis and neuromonitoring of patients affected by HD.Fil: Segatto, Rodolfo Guillermo. University Of Illinois. Deparment Of Biological Science; Estados UnidosFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Amin, Manish. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Angeles López, Quetzalli D.. Consejo Nacional de Ciencia y Tecnologia de Mexico. Centro de Investigacion Cientifica y de Educacion Superior de Ensenada Baja California.; MéxicoFil: García Lara, Lucia. Consejo Nacional de Ciencia y Tecnologia de Mexico. Centro de Investigacion Cientifica y de Educacion Superior de Ensenada Baja California.; MéxicoFil: Salinas Castellanos, Libia Catalina. Consejo Nacional de Ciencia y Tecnologia de Mexico. Centro de Investigacion Cientifica y de Educacion Superior de Ensenada Baja California.; MéxicoFil: Deyoung, Daniel. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Segovia, Jose Manuel. Consejo Nacional de Ciencia y Tecnologia de Mexico. Centro de Investigacion Cientifica y de Educacion Superior de Ensenada Baja California.; MéxicoFil: Mareci, Thomas H.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Magin, Richard L.. University Of Illinois. Deparment Of Biological Science; Estados Unido

Recombination of chl-fus gene (Plastid Origin) downstream of hop: a locus of chromosomal instability

International audienceBackground: The co-chaperone Hop [heat shock protein (HSP) organizing protein] has been shown to act as an adaptor for protein folding and maturation, in concert with Hsp70 and Hsp90. The hop gene is of eukaryotic origin. Likewise, the chloroplast elongation factor G (cEF-G) catalyzes the translocation step in chloroplast protein synthesis. The chl-fus gene, which encodes the cEF-G protein, is of plastid origin. Both proteins, Hop and cEF-G, derived from domain duplications. It was demonstrated that the nuclear chl-fus gene locates in opposite orientation to a hop gene in Glycine max. We explored 53 available plant genomes from Chlorophyta to higher plants, to determine whether the chl-fus gene was transferred directly downstream of the primordial hop in the proto-eukaryote host cell. Since both genes came from exon/module duplication events, we wanted to explore the involvement of introns in the early origin and the ensuing evolutionary changes in gene structure. Results: We reconstructed the evolutionary history of the two convergent plant genes, on the basis of their gene structure, microsynteny and microcolinearity, from 53 plant nuclear genomes. Despite a high degree (72 %) of microcolinearity among vascular plants, our results demonstrate that their adjacency was a product of chromosomal rearrangements. Based on predicted exon − intron structures, we inferred the molecular events giving rise to the current form of genes. Therefore, we propose a simple model of exon/module shuffling by intronic recombinations in which phase-0 introns were essential for domain duplication, and a phase-1 intron for transit peptide recruiting. Finally, we demonstrate a natural susceptibility of the intergenic region to recombine or delete, seriously threatening the integrity of the chl-fus gene for the future. Conclusions: Our results are consistent with the interpretation that the chl-fus gene was transferred from the chloroplast to a chromosome different from that of hop, in the primitive photosynthetic eukaryote, and much later before the appearance of angiosperms, it was recombined downstream of hop. Exon/module shuffling mediated by symmetric intron phases (i.e., phase-0 introns) was essential for gene evolution. The intergenic region is prone to recombine, risking the integrity of both genes

Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation

Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction. This method serves to identify transmembrane proteins such as channel subunits as well as PM-embedded or weakly associated proteins. This protocol uses a minute amount of cell material and typical equipment present in laboratories, and it takes approximately 3 h. The process was validated using endogenous and exogenous proteins expressed in the HEK293T cell line that were targeted to each compartment. Using a specific stimulus as a trigger, we showed and quantified the shuttling of a protein channel (ASIC1a, acid sensing ion channel) from the MO fraction to the PM fraction and the shuttling of a kinase from a cytosolic location to a nuclear location.Fil: Salinas Castellanos, Libia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Gatto, Rodolfo Gabriel. University of Illinois; Estados UnidosFil: Menchón, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Física Enrique Gaviola. Universidad Nacional de Córdoba. Instituto de Física Enrique Gaviola; ArgentinaFil: Blaustein, Matías. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Upregulation of ASIC1a channels in an in vitro model of Fabry disease

Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H+-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.Fil: Salinas Castellanos, Libia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rozenfeld, Paula Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Gatto, Rodolfo Gabriel. University of Illinois at Chicago; Estados UnidosFil: Reisin, Ricardo Claudio. Hospital Británico de Buenos Aires; ArgentinaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Segmental Upregulation of ASIC1 Channels in the Formalin Acute Pain Mouse Model

Background: Hindpaw injection of formalin in rodents is used to assess acute persistent pain. The response to formalin is biphasic. The initial response (first minutes) is thought to be linked to inflammatory, peripheral mechanisms, while the latter (around 30 min after the injection), is linked to central mechanisms. This model is useful to analyze the effect of drugs at one or both phases, and the involvement of ion channels in the response. Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in pain conditions. Recently, psalmotoxin-1 (Pctx-1), a toxin that inhibits ASIC1a-constituted channels, and antisense ASIC1a-RNA, intrathecal administered in mice were shown to affect both phases of the test. Methods: The mouse formalin test was performed on C57/BL6 7- to 9-week-old mice. Behavioral tests were conducted and tissue was extracted to detect proteins (ASIC1 and pERK) and ASIC1-mRNA and mir485-5p levels. Results: The injection of formalin was accompanied by an increase in ASIC1 levels. This was detected at the contralateral anterior cingulate cortex (ACC) compared to the ipsilateral side, and both sides of the ACC of vehicle-injected animals. At the spinal cord and dorsal root ganglia, ASIC1 levels followed a gradient stronger at lumbar (L) 3 and decreased towards L5. Gender differences were detected at the ACC; with female mice showing higher ASIC1a levels at the ACC. No significant changes in ASIC1-mRNA levels were detected. Evidence suggests ASIC1 upregulation depends on regulatory microRNAs. Conclusion: This work highlights the important role of ASIC1 in pain and the potential role of pharmacological therapies aimed at this channel