Cesare Usai - Academia.edu (original) (raw)
Papers by Cesare Usai
Blood, 2015
Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare ... more Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Com...
PloS one, 2018
Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the... more Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by ...
Frontiers in Immunology, 2021
Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor... more Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes ...
Reverse Micelles, 1984
Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one ... more Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one or several residues of sialic acid, a negatively charged sugar (Figure 1). In animal tissues the most abundant gangliosides are monosialoganglioside GM1, disialogangliosides, GD1a and GD1b, and trisialoganglioside, GT. Most of them are localized on the outer surface of the plasma membrane and are ubiquitous but minor components of the cell surface, with the major exception of the mammalian central nervous system where they comprise up to 5–10% of the total lipid, and where lipid-bound sialic acid often exceeds that bound to glycoproteins1,2. Gangliosides have been shown to interact with several bacterial toxins3, peptide hormones and other external ligands4,5 probably through association with membrane proteins6. Most likely they are involved in functional activities of the cell surface, such as regulation of growth and structural plasticity. All these functions are likely to be mediated by ganglioside head group dynamics with a strong tendency for cooperative associations with each other and with glycoproteins6,8,9,10. This behavior appears to be strongly enhanced by divalent cations and affected by crosslinking agents6,8, as observed with both native and articial membrances.
Experimental cell research, Dec 10, 2003
In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a ... more In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca2+ influx, and the addition of drugs that block the Ca2+ current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABAB receptors in P. primaurelia swimming control. The presence of GABAB immunoanalogue in Paramecium ...
Journal of Biological Chemistry, 2005
Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD ؉ by the ADP-r... more Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD ؉ by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38 ؉ feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1 ؉ stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1 ؉ stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1 ؉ human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1 ؉ feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine-and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1 ؉ feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, Jan 20, 2015
Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-... more Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analyzed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 hours after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was h...
International Journal of Molecular Sciences
Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) a... more Glutamate (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 ...
Neuropharmacology, May 1, 2018
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the fi... more Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H]D-aspartate ([H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HTheteroreceptors. Actually, the 15 mM KCl-evoked [H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HTagonist (±)DOI, an effect reversed by the 5-HTantagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HTreceptors colocalize and cross-talk in these terminals and if 5-HTligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HTreceptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HTreceptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord ...
British journal of pharmacology, 2017
We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals an... more We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are pr...
Neuropharmacology, 2017
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper an... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1(G93A) mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1(G93A) mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1(G93A) mice with reduced expression of mGluR5 (SOD1(G93A)Grm5(-/+)) by crossing the SOD1(G93A) mutant mouse with the mGluR5 heterozigous Grm5(-/+) mouse. SOD1(G93A)Grm5(-/+) mice showed prolonged s...
Brain research bulletin, 2017
Brain Research Bulletin, 2016
There is increasing evidence for the neuronal coexistence of classical transmitters. Implications... more There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their synthetic enzymes and/or their vesicular transporters. In contrast, coexpression of neurotransmitter transporters on the plasma membrane of the same nerve terminal, although a potentially important indication for cotransmission, has received poor attention. We here used preparations of isolated nerve endings to functionally identify transporters coexpressed on the plasma membrane of the same terminal, in order to verify if such transporter coexpression indeed exists in neuronal systems in which cotransmission has already been established or reasonably suspected through other technical approaches. We could observe that functional transporters for glycine and glutamate are coexpressed on nerve terminals in the cerebellum; transporters for dopamine and GABA coexist on striatal terminals; transporters for glycine and GABA, previously found to coexist as cotransmission markers on nerve terminals of spinal cord and cerebellum, are not coexpressed in neocortex and hippocampus, where cotransmission has not been proposed to occur; transporters for GABA, glycine and glutamate are colocalized on nerve terminals of the spinal cord. Confocal microscopy experiments were performed to substantiate functional data, highlighting the presence of the co-existing transporters under study on MAP-2 positive synaptosomes. It is concluded that investigating the colocalization of functional neurotransmitter transporters on the plasma membrane of nerve terminals can provide useful information on the possibility of cotransmission.
Cell and Tissue Research, 1991
Scientific Reports, 2016
Abscisic acid (ABA) , a long known phytohormone, has been recently demonstrated to be present als... more 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 α subunit of a G i 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. The human genome encodes three distinct LANCL proteins, LANCL1, LANCL2 and LANCL3 1 , which share a high structural homology with the lanthionine synthetase component C, a cyclase involved in the synthesis of lantibiotics in Prokaryotes 2. LANCL3 has been suggested to be a pseudogene 3. LANCL1 has been hypothesized to be implicated in the metabolism of lanthionine metabolites in the central nervous system 4. LANCL2 proved to be the human receptor of abscisic acid (ABA) 5-10. ABA, a long-known plant hormone 11,12 , has been shown to be present also in mammals, where it affects several key functions in different cell types 9,10,13,14. ABA behaves as a pro-inflammatory modulator of cells of the innate immune response 7,15-17 , stimulates the proliferation of human mesenchymal and hemopoietic stem cells 18,19 , and is involved in the control of systemic glucose homeostasis 5,20-24. LANCL2-mediated ABA signaling in mammals requires a pertussis toxin (PTX)-sensitive G protein 5 , eventually leading to an increase of intracellular Ca 2+ levels ([Ca 2+ ] i). The signaling pathway downstream of ABA binding to LANCL2 involves the activation of adenylate cyclase (AC), followed by overproduction of cAMP, PKA-catalyzed phosphorylation and stimulation of the plasmamembrane-bound ADP-ribosyl cyclase CD38, which converts NAD + to cADPR and ADPR, leading to an increase of both extracellular Ca 2+ entry and Calcium-induced calcium release (CICR)-mediated intracellular Ca 2+ mobilization 5,9,15,21. Several indirect lines of evidence point to a G i as the G protein coupled to LANCL2: i) the sensitivity of the ABA signaling pathway to PTX in human granulocytes and in insulin-releasing cells 15,21 ; ii) the accumulation of inositol 1,4,5-P3 (IP 3) in human cells co-transfected with LANCL2 and a chimeric G protein, Gα q/i , upon stimulation with ABA 5 , and, iii) inhibition of the ABA-induced cAMP increase in ABA-sensitive human cells by overexpression of transducin, a β γ-subunit scavenger 5. However, conclusive identification of the nature of the G protein coupled to LANCL2 has yet to be provided. For instance, the role of G βγ in AC signaling is exceedingly complex, as witnessed by both AC-activating and inhibiting effects related to wide heterogeneity of the coupling receptors and of the various membrane-associated AC isoforms 25,26. Moreover, the mechanism of the LANCL2-G protein coupling, specifically whether it is direct or mediated by other proteins, remains to be defined. Interestingly, LANCL2 is not a transmembrane protein, as predicted in silico from its sequence 27-29 and confirmed in vitro by the observation that it can be removed from the plasmamembrane without the use of detergents, either by mild chemical treatments 30 or by inhibition of its post-translational N-terminal myristoylation 28 .
Scientific Reports, 2016
Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genet... more 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 ([Ca 2+ ] 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. Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease characterized by pancreatic insufficiency, skeletal abnormalities, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) 1,2. SDS is caused by mutations in the SBDS (Shwachman-Bodian-Diamond syndrome) gene, which account for approximately 90% of affected individuals 3. Consistent with the hypothesis that at least one SBDS variant is hypomorphic, knockout Sbds− /− mice are embryonic lethal 4. The SBDS protein localizes in the nucleolus where it plays an important role in ribosome biogenesis, as was shown in several studies 5-7. SBDS is also involved in other molecular processes that may be independent of ribosome biogenesis, such as chemotaxis 8 , mitotic spindle formation 9 and cellular stress responses. Regarding the latter aspect, in SBDS depleted human cells, endoplasmic reticulum (ER) stress is associated with caspase 3 cleavage and activation of the intrinsic apoptotic pathway, as well as eukaryotic initiation factor 2-alpha (eIF2-alpha) phosphorylation, which rapidly reduces mRNA translation initiation 10. Finally, the silencing of the SBDS gene in human cells and a yeast model depleted of its SBDS orthologue (Sdo1) display reduced mitochondrial functionality 11. In turn, ER and mitochondrial stress may induce reactive oxygen species (ROS) production
British Journal of Pharmacology, 2016
Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in c... more Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in central nervous system (CNS). They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows to further characterize these autoreceptors. This study aims at investigating the pharmacological profile of mGlu2/3 receptors in selected CNS regions and at evaluating their functions in mice suffering from experimental autoimmune encephalomyelitis (EAE). The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by analyzing the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3) H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. Experiments were also carried out to analyze mGlu2/3 autoreceptor-mediated releasing functions in EAE animals and whether in vivo LY379268 administration can restore impaired glutamate release in these mice. Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possess LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 exist in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors lost completely their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 (1-0.01 mg kg(-1) ) administration restored glutamate exocytosis capability in spinal cord but not in cortical terminals. We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord, respectively. The mGlu3-preferring autoreceptors could represent a target for new pharmacological approach for demyelinating diseases.
Experimental biology
ABSTRACT
Blood, 2015
Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare ... more Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Com...
PloS one, 2018
Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the... more Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by ...
Frontiers in Immunology, 2021
Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor... more Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes ...
Reverse Micelles, 1984
Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one ... more Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one or several residues of sialic acid, a negatively charged sugar (Figure 1). In animal tissues the most abundant gangliosides are monosialoganglioside GM1, disialogangliosides, GD1a and GD1b, and trisialoganglioside, GT. Most of them are localized on the outer surface of the plasma membrane and are ubiquitous but minor components of the cell surface, with the major exception of the mammalian central nervous system where they comprise up to 5–10% of the total lipid, and where lipid-bound sialic acid often exceeds that bound to glycoproteins1,2. Gangliosides have been shown to interact with several bacterial toxins3, peptide hormones and other external ligands4,5 probably through association with membrane proteins6. Most likely they are involved in functional activities of the cell surface, such as regulation of growth and structural plasticity. All these functions are likely to be mediated by ganglioside head group dynamics with a strong tendency for cooperative associations with each other and with glycoproteins6,8,9,10. This behavior appears to be strongly enhanced by divalent cations and affected by crosslinking agents6,8, as observed with both native and articial membrances.
Experimental cell research, Dec 10, 2003
In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a ... more In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca2+ influx, and the addition of drugs that block the Ca2+ current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABAB receptors in P. primaurelia swimming control. The presence of GABAB immunoanalogue in Paramecium ...
Journal of Biological Chemistry, 2005
Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD ؉ by the ADP-r... more Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD ؉ by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38 ؉ feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1 ؉ stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1 ؉ stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1 ؉ human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1 ؉ feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine-and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1 ؉ feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, Jan 20, 2015
Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-... more Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analyzed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 hours after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was h...
International Journal of Molecular Sciences
Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) a... more Glutamate (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 ...
Neuropharmacology, May 1, 2018
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the fi... more Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H]D-aspartate ([H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HTheteroreceptors. Actually, the 15 mM KCl-evoked [H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HTagonist (±)DOI, an effect reversed by the 5-HTantagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HTreceptors colocalize and cross-talk in these terminals and if 5-HTligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HTreceptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HTreceptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord ...
British journal of pharmacology, 2017
We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals an... more We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are pr...
Neuropharmacology, 2017
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper an... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1(G93A) mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1(G93A) mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1(G93A) mice with reduced expression of mGluR5 (SOD1(G93A)Grm5(-/+)) by crossing the SOD1(G93A) mutant mouse with the mGluR5 heterozigous Grm5(-/+) mouse. SOD1(G93A)Grm5(-/+) mice showed prolonged s...
Brain research bulletin, 2017
Brain Research Bulletin, 2016
There is increasing evidence for the neuronal coexistence of classical transmitters. Implications... more There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their synthetic enzymes and/or their vesicular transporters. In contrast, coexpression of neurotransmitter transporters on the plasma membrane of the same nerve terminal, although a potentially important indication for cotransmission, has received poor attention. We here used preparations of isolated nerve endings to functionally identify transporters coexpressed on the plasma membrane of the same terminal, in order to verify if such transporter coexpression indeed exists in neuronal systems in which cotransmission has already been established or reasonably suspected through other technical approaches. We could observe that functional transporters for glycine and glutamate are coexpressed on nerve terminals in the cerebellum; transporters for dopamine and GABA coexist on striatal terminals; transporters for glycine and GABA, previously found to coexist as cotransmission markers on nerve terminals of spinal cord and cerebellum, are not coexpressed in neocortex and hippocampus, where cotransmission has not been proposed to occur; transporters for GABA, glycine and glutamate are colocalized on nerve terminals of the spinal cord. Confocal microscopy experiments were performed to substantiate functional data, highlighting the presence of the co-existing transporters under study on MAP-2 positive synaptosomes. It is concluded that investigating the colocalization of functional neurotransmitter transporters on the plasma membrane of nerve terminals can provide useful information on the possibility of cotransmission.
Cell and Tissue Research, 1991
Scientific Reports, 2016
Abscisic acid (ABA) , a long known phytohormone, has been recently demonstrated to be present als... more 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 α subunit of a G i 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. The human genome encodes three distinct LANCL proteins, LANCL1, LANCL2 and LANCL3 1 , which share a high structural homology with the lanthionine synthetase component C, a cyclase involved in the synthesis of lantibiotics in Prokaryotes 2. LANCL3 has been suggested to be a pseudogene 3. LANCL1 has been hypothesized to be implicated in the metabolism of lanthionine metabolites in the central nervous system 4. LANCL2 proved to be the human receptor of abscisic acid (ABA) 5-10. ABA, a long-known plant hormone 11,12 , has been shown to be present also in mammals, where it affects several key functions in different cell types 9,10,13,14. ABA behaves as a pro-inflammatory modulator of cells of the innate immune response 7,15-17 , stimulates the proliferation of human mesenchymal and hemopoietic stem cells 18,19 , and is involved in the control of systemic glucose homeostasis 5,20-24. LANCL2-mediated ABA signaling in mammals requires a pertussis toxin (PTX)-sensitive G protein 5 , eventually leading to an increase of intracellular Ca 2+ levels ([Ca 2+ ] i). The signaling pathway downstream of ABA binding to LANCL2 involves the activation of adenylate cyclase (AC), followed by overproduction of cAMP, PKA-catalyzed phosphorylation and stimulation of the plasmamembrane-bound ADP-ribosyl cyclase CD38, which converts NAD + to cADPR and ADPR, leading to an increase of both extracellular Ca 2+ entry and Calcium-induced calcium release (CICR)-mediated intracellular Ca 2+ mobilization 5,9,15,21. Several indirect lines of evidence point to a G i as the G protein coupled to LANCL2: i) the sensitivity of the ABA signaling pathway to PTX in human granulocytes and in insulin-releasing cells 15,21 ; ii) the accumulation of inositol 1,4,5-P3 (IP 3) in human cells co-transfected with LANCL2 and a chimeric G protein, Gα q/i , upon stimulation with ABA 5 , and, iii) inhibition of the ABA-induced cAMP increase in ABA-sensitive human cells by overexpression of transducin, a β γ-subunit scavenger 5. However, conclusive identification of the nature of the G protein coupled to LANCL2 has yet to be provided. For instance, the role of G βγ in AC signaling is exceedingly complex, as witnessed by both AC-activating and inhibiting effects related to wide heterogeneity of the coupling receptors and of the various membrane-associated AC isoforms 25,26. Moreover, the mechanism of the LANCL2-G protein coupling, specifically whether it is direct or mediated by other proteins, remains to be defined. Interestingly, LANCL2 is not a transmembrane protein, as predicted in silico from its sequence 27-29 and confirmed in vitro by the observation that it can be removed from the plasmamembrane without the use of detergents, either by mild chemical treatments 30 or by inhibition of its post-translational N-terminal myristoylation 28 .
Scientific Reports, 2016
Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genet... more 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 ([Ca 2+ ] 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. Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease characterized by pancreatic insufficiency, skeletal abnormalities, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) 1,2. SDS is caused by mutations in the SBDS (Shwachman-Bodian-Diamond syndrome) gene, which account for approximately 90% of affected individuals 3. Consistent with the hypothesis that at least one SBDS variant is hypomorphic, knockout Sbds− /− mice are embryonic lethal 4. The SBDS protein localizes in the nucleolus where it plays an important role in ribosome biogenesis, as was shown in several studies 5-7. SBDS is also involved in other molecular processes that may be independent of ribosome biogenesis, such as chemotaxis 8 , mitotic spindle formation 9 and cellular stress responses. Regarding the latter aspect, in SBDS depleted human cells, endoplasmic reticulum (ER) stress is associated with caspase 3 cleavage and activation of the intrinsic apoptotic pathway, as well as eukaryotic initiation factor 2-alpha (eIF2-alpha) phosphorylation, which rapidly reduces mRNA translation initiation 10. Finally, the silencing of the SBDS gene in human cells and a yeast model depleted of its SBDS orthologue (Sdo1) display reduced mitochondrial functionality 11. In turn, ER and mitochondrial stress may induce reactive oxygen species (ROS) production
British Journal of Pharmacology, 2016
Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in c... more Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in central nervous system (CNS). They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows to further characterize these autoreceptors. This study aims at investigating the pharmacological profile of mGlu2/3 receptors in selected CNS regions and at evaluating their functions in mice suffering from experimental autoimmune encephalomyelitis (EAE). The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by analyzing the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3) H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. Experiments were also carried out to analyze mGlu2/3 autoreceptor-mediated releasing functions in EAE animals and whether in vivo LY379268 administration can restore impaired glutamate release in these mice. Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possess LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 exist in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors lost completely their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 (1-0.01 mg kg(-1) ) administration restored glutamate exocytosis capability in spinal cord but not in cortical terminals. We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord, respectively. The mGlu3-preferring autoreceptors could represent a target for new pharmacological approach for demyelinating diseases.
Experimental biology
ABSTRACT