Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (\ub1)baclofen (3 \u3bcM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1\u3b1 with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03\u20131 \u3bcM), inactive on its own on GABA exocytosis, amplified the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA overflow. The ( \ub1 )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (\ub1)baclofen (1 \u3bcM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 \u3bcM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (\ub1)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor\u2013receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca(2+)-related mechanisms

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage

Altered glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops due to motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, while perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analysed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment. This article is protected by copyright. All rights reserved

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression

open10Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.openTiziana Bonifacino, Claudia Rebosio, Francesca Provenzano, Carola Torazza, Matilde Balbi, Marco Milanese, Luca Raiteri, Cesare Usai, Ernesto Fedele, Giambattista Bonanno.Bonifacino, Tiziana; Rebosio, Claudia; Provenzano, Francesca; Torazza, Carola; Balbi, Matilde; Milanese, Marco; Raiteri, Luca; Usai, Cesare; Fedele, Ernesto; Bonanno, Giambattist

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials

G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the \u3b1 subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation

Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 μM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 μM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03–1 μM), inactive on its own on GABA exocytosis, amplified the 3 μM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 μM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 μM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor–receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

Extracellular NAD + Is an Agonist of the Human P2Y 11 Purinergic Receptor in Human Granulocytes

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation