ABSTRACT NAD+ metabolism and regulation are still incompletely defined in their features. Specifi... more ABSTRACT NAD+ metabolism and regulation are still incompletely defined in their features. Specifically, several compartmentation problems, both subcellular and intercellular, have emerged in the past years. A topological paradox was identified in the CD38/NAD+/Cyclic ADP-ribose system which represents a means for achieving regulation of intracellular Calcium levels ([Ca2+]i and of Ca2+-mediated cell functions. CD38, an ectoenzyme featuring ADP-ribosyl cyclase (ADPRC) activity, is known to catalyze the generation of as many as eight Ca2+-active signal metabolites, including cADPR, ADPR, NAADP and three adenine homodinucleotides (Ap2A and two isomers thereof, designated P18 and P24). The first paradox concerns the ectocellular conversion of NAD+ to cADPR and the intracellular activity of cADPR as a potent Ca2+ mobilizer from stores expressing ryanodine receptors/channels on the endoplasmic/sarcoplasmic reticulum. This topological inconsistency was solved with the identification of hexameric Connexin 43 hemichannels (Cx43 HC) as an equilibrative transport system for NAD+, and of redundant equilibrative, and especially concentrative, transport systems for cADPR. Other transporters for NAADP+, P18 and P24 are still unknown. In addition, NAD+ itself and some of its derivatives were recognized to be agonists of purinergic receptors, e.g., P2Y11 and P2X7, thereby affecting the [Ca2+]i levels either through protein kinase A- and phospholipase C-mediated pathways or through direct influx of extracellular Ca2+, respectively. Finally, recent findings demonstrate that the enzymes involved in NAD+ biosynthesis and some of the enzymes involved in the multiple pathways of NAD+ utilization localize to distinct subcellular compartments of the same cell and, surprisingly, also to different cell types of the same organism. Therefore, NAD+ biosynthesis from several precursors, collectively defined Vitamin B3 forms, is at the same time an organismal and a cellular process, whose individual steps may occur in different cells/tissues/organs. Thus, a paracrine crosstalk is activated via the exchange of intermediate metabolites in biological fluids (e.g., Nicotinamide mononucleotide and Nicotinamide riboside), and the eventual NAD+ biosynthesis occurs in selected cells able to utilize it for the display of diverse, fundamental cell functions. These are driven by NAD+-consuming enzymes, as ADPRCs, mono ADP-ribosyltransferases, poly(ADP-ribose) polymerases and different sirtuins (NAD+-dependent protein deacetylases).
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.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progres... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and viment...
ABSTRACT NAD+ metabolism and regulation are still incompletely defined in their features. Specifi... more ABSTRACT NAD+ metabolism and regulation are still incompletely defined in their features. Specifically, several compartmentation problems, both subcellular and intercellular, have emerged in the past years. A topological paradox was identified in the CD38/NAD+/Cyclic ADP-ribose system which represents a means for achieving regulation of intracellular Calcium levels ([Ca2+]i and of Ca2+-mediated cell functions. CD38, an ectoenzyme featuring ADP-ribosyl cyclase (ADPRC) activity, is known to catalyze the generation of as many as eight Ca2+-active signal metabolites, including cADPR, ADPR, NAADP and three adenine homodinucleotides (Ap2A and two isomers thereof, designated P18 and P24). The first paradox concerns the ectocellular conversion of NAD+ to cADPR and the intracellular activity of cADPR as a potent Ca2+ mobilizer from stores expressing ryanodine receptors/channels on the endoplasmic/sarcoplasmic reticulum. This topological inconsistency was solved with the identification of hexameric Connexin 43 hemichannels (Cx43 HC) as an equilibrative transport system for NAD+, and of redundant equilibrative, and especially concentrative, transport systems for cADPR. Other transporters for NAADP+, P18 and P24 are still unknown. In addition, NAD+ itself and some of its derivatives were recognized to be agonists of purinergic receptors, e.g., P2Y11 and P2X7, thereby affecting the [Ca2+]i levels either through protein kinase A- and phospholipase C-mediated pathways or through direct influx of extracellular Ca2+, respectively. Finally, recent findings demonstrate that the enzymes involved in NAD+ biosynthesis and some of the enzymes involved in the multiple pathways of NAD+ utilization localize to distinct subcellular compartments of the same cell and, surprisingly, also to different cell types of the same organism. Therefore, NAD+ biosynthesis from several precursors, collectively defined Vitamin B3 forms, is at the same time an organismal and a cellular process, whose individual steps may occur in different cells/tissues/organs. Thus, a paracrine crosstalk is activated via the exchange of intermediate metabolites in biological fluids (e.g., Nicotinamide mononucleotide and Nicotinamide riboside), and the eventual NAD+ biosynthesis occurs in selected cells able to utilize it for the display of diverse, fundamental cell functions. These are driven by NAD+-consuming enzymes, as ADPRCs, mono ADP-ribosyltransferases, poly(ADP-ribose) polymerases and different sirtuins (NAD+-dependent protein deacetylases).
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.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progres... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and viment...
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Papers by Cesare Usai