D. Trotti - Academia.edu (original) (raw)
Papers by D. Trotti
Journal of lipid research, 2000
Compared with classic essential fatty acid deficiency or the feeding of a fat-free diet, little i... more Compared with classic essential fatty acid deficiency or the feeding of a fat-free diet, little is known about specific linoleate deficiency in the rat. Carbon recycling into de novo lipogenesis has been reported to be an obligatory feature of linoleate metabolism in the liver, even in extreme linoleate deficiency (LA-D). The present study had two objectives: 1) to report a brief summary of the tissue n-6 polyunsaturated fatty acid (PUFA) profiles in specific LA-D, and 2) to quantify whole body carbon recycling from [(14)C]linoleate in specific LA-D. Rats consumed a linoleate-deficient diet for 12 weeks and then received a bolus of [1-(14)C]linoleate by gavage. In linoleate-deficient rats, the triene/tetraene ratio in several organs increased by 18- to 100-fold. The amount of (14)C appearing in organ sterols (dpm/g) of linoleate-deficient rats was 2- to 10-fold higher than in the controls and equaled 16.3% of the [(14)C]linoleate dose given, compared with 7.4% in the controls. We co...
Progress in Brain Research, 2001
... system. Curr: Pharm. Des., 5: 363-379. Brooks-Kayal,AR,Munir,M.,Jin, H. and Robinson,MB ( 199... more ... system. Curr: Pharm. Des., 5: 363-379. Brooks-Kayal,AR,Munir,M.,Jin, H. and Robinson,MB ( 1998) The glutamate transporter, GLT-1, is expressed in cul-tured hippocampal neurons. Neurochem. Znt., 33: 95-100. Carlsson ...
Pharmacological Research Communications, 1988
Down regulation of 8-receptors coupled to desensitization of ME-dependent adenylate~cycJase l is/... more Down regulation of 8-receptors coupled to desensitization of ME-dependent adenylate~cycJase l is/one of the most common effect of repeated exposure to antidepressant drugs . In order to clarify the molecular basis of receptor regulatlon we have performed a series of studies with an "in vitro ~ model of 8-adrenoceptor homologous desensitization. Rat cortical sllces incubated for 30' in ~ the presence of isoproterenol (ISO) show dose-dependent reversible down regulation of B-adrenoceptors para]ie]ed by a reduction in cAMP accumu]ation following NE exposure. The role of alpha-I receptors in modulating the response of B-adrenoceptors was investigated by studying the effect of methoxamine on ISO induced desensitization of 8-receptors. Incubation with the alpha-! agonist reduced the number of 8-receptors and the ISO-induced desensitization was somehow reduced after incubation with methoxamlne, lhese findings clearly indicate that multlple synaptic and transynaptic mechanisms may be involved in the regulatlon of 8-adrenoceptor function.
Advances in Experimental Medicine and Biology, 1992
ABSTRACT
Nature neuroscience, 1999
The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degener... more The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the glutamate transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.
The Journal of experimental biology, 1997
In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and pa... more In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has p...
Molecular pharmacology, 1994
Reuptake of glutamate in astrocytes, a critical mechanism involved in the maintenance of physiolo... more Reuptake of glutamate in astrocytes, a critical mechanism involved in the maintenance of physiological excitatory amino acid neurotransmission, is inhibited by both arachidonic acid (AA) and reactive oxygen species (ROS), via incompletely defined molecular mechanisms. Because ROS are generated during AA metabolism and AA can be released as a result of ROS-mediated phospholipase A2 activation, it seems likely that their effects on uptake are mediated by a common mechanism. However, here we show that rapid (10-min) uptake inhibitions by AA or by ROS generated by the xanthine plus xanthine oxidase (XO) reaction are selectively abolished by distinct agents; bovine serum albumin (BSA) acts only on AA, whereas the scavenger enzymes superoxide dismutase (SOD) and catalase (CAT) and the disulfide-reducing agent dithiothreitol (DTT) act only on ROS. Moreover, when added together, xanthine/XO and AA decrease uptake in a fully additive manner. In particular, the effect of xanthine/XO is seen a...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1994
Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system ar... more Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximately 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase+catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic antioxidants or...
Advances in Insect Physiology, 2001
... Acid Absorption in the Midgut of Lepidopteran Larvae V. Franca Sacchia, Michela Castagnab,Dav... more ... Acid Absorption in the Midgut of Lepidopteran Larvae V. Franca Sacchia, Michela Castagnab,Davide Trottib, Chairat Shayakulb, and Matthias A. Hedigerb stituto di Fisiologia Generale e di Chimica Biologica, Facolt& di Farmacia, Universit& di Milano, Via Trentacoste 2, 20134 ...
Journal of Neuroscience, 2013
Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of fa... more Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.
Proceedings of the National Academy of Sciences, 1998
Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotranspo... more Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na ؉ or H ؉ gradients. The present work extends the Na ؉ and H ؉ dogma by including the H ؉ and K ؉ paradigm. Lepidopteran insect larvae have a high K ؉ and a low Na ؉ content, and their midgut cells lack Na ؉ ͞K ؉ ATPase. Instead, an H ؉ translocating, vacuolar-type ATPase generates a voltage of approximately ؊240 mV across the apical plasma membrane of so-called goblet cells, which drives H ؉ back into the cells in exchange for K ؉ , resulting in net K ؉ secretion into the lumen. The resulting inwardly directed K ؉ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K ؉ -coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes ␣-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K ؉ , Na ؉ , and to a lesser extent Li ؉ were accepted as cotransported ions, but K ؉ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl ؊ -dependent, and the K ؉ ͞Na ؉ selectivity increased with hyperpolarization of oocytes, ref lecting the increased K ؉ ͞Na ؉ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na ؉ and Cl ؊ -coupled neurotransmitter transporter family.
Proceedings of the National Academy of Sciences, 2012
Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familia... more Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALSlinked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iperoxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patientderived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.
Neuroscience Letters, 1995
While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of... more While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of hippocampal astrocytes, we observed that all the HPLC peaks corresponding to AA metabolites displayed significantly different levels depending on the presence or not of free Ca 2+ in the incubation medium. A specific pattern was noticed, i.e. lipoxygenase (LOX) derivatives, in particular 12hydroxyeicosatetraenoic acid (12-HETE), showed higher levels in medium containing 1 mM Ca 2+, while cycloxygenase (COX) products including prostaglandins (PG) F2a, E 2 and D 2 and 12-hydroxyheptadecatrienoic acid (12-HHT), were higher in Ca2÷-free medium. COX metabolism exceeded LOX metabolism by threefold in Ca2+-free medium, while it was only 60% of it in 1 mM Ca 2+. The total amount of AA processed under the two conditions was identical. These data suggest that free [Ca 2+] influences the pattern of AA metabolites formed in hippocampal astrocytes, with possible important implications in view of the distinct roles played by COX and LOX eicosanoids in synaptic transmission and neurotoxicity in this area.
Journal of Biological Chemistry, 2001
We have investigated the functional impact of a naturally occurring mutation of the human glutama... more We have investigated the functional impact of a naturally occurring mutation of the human glutamate transporter GLT1 (EAAT2), which had been detected in a patient with sporadic amyotrophic lateral sclerosis. The mutation involves a substitution of the putative N-linked glycosylation site asparagine 206 by a serine residue (N206S) and results in reduced glycosylation of the transporter and decreased uptake activity. Electrophysiological analysis of N206S revealed a pronounced reduction in transport rate compared with wild-type, but there was no alteration in the apparent affinities for glutamate and sodium. In addition, no change in the sensitivity for the specific transport inhibitor dihydrokainate was observed. However, the decreased rate of transport was associated with a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a dominant negative effect on the wild-type GLT1 activity, whereas it did not affect wild-type EAAC1. These findings provide evidence for a role of the N-linked glycosylation in both cellular trafficking and transport function. The resulting alteration in glutamate clearance capacity likely contributes to excitotoxicity that participates in motor neuron degeneration in amyotrophic lateral sclerosis.
Journal of Biological Chemistry, 2010
Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeos... more Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process since K + is believed to equilibrate freely between cytosol and inter-mitochondrial membrane space. By patchclamping mitochondria isolated from the central nervous system of adult mitoCFP transgenic mice, we report the existence of I OMMKi , a novel voltage-dependent inward rectifying K + conductance located in the OMM. I OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K + to enter the mitochondrial intermembrane space in a controlled, regulated fashion. The identification of I OMMKi in the outer mitochondrial membrane supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the outer mitochondria membrane possesses regulated pathways for K + uptake.
Journal of Biological Chemistry, 1997
Ion-coupled solute transporters exhibit pre-steadytate currents that resemble those of voltage-de... more Ion-coupled solute transporters exhibit pre-steadytate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H ؉ -coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra-(pH i ) and extracellular pH (pH o ). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pH i and pH o . At a pH o of 7.0 and a holding potential (V h ) of ؊50 mV, the charge movements were mostly inwardly directed, whereas reduction of pH o to below 7.0 resulted in outwardly directed charge movements. When pH i was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 ؎ 0.03 (n ؍ 7). The midpoint voltage (V 0.5 ) of the charge distribution shifted linearly as a function of pH i and pH o . Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H ؉ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H ؉ chemical potential (⌬ H ) or increasing the membrane potential (V m ) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.
Journal of Biological Chemistry, 2007
4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu 2ϩ / Zn 2ϩ superoxide ... more 4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu 2ϩ / Zn 2ϩ superoxide dismutase; mutSOD1, mutated SOD1; WT-SOD1, wild type SOD1; SUMO, small ubiquitin modifier protein; PML, promyelocytic leukemia; PML-NB, PML nuclear body; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; CTE, COOH terminus of EAAT2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; HD, Huntington disease; EGFP, enhanced green fluorescent protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPI, 4Ј,6-diamidino-2phenylindole.
Journal of Biological Chemistry, 1996
The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents th... more The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H 2 O 2 ). Here we show that another biological oxidant, peroxynitrite (ONOO ؊ ), formed by combination of superoxide ( ⅐ O 2 ؊ ) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO ؊ reduces selectively the V max of transport; its action is fast (reaching 90% within 20 s), dose-dependent (50% inhibition at 50 M), persistent upon ONOO ؊ (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO ؊ with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO ؊ . H 2 O 2 also inhibits reconstituted transport, and its action matches that of ONOO ؊ on all respects; however, this is observed only with 5-10 mM H 2 O 2 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO ؊ and other oxidants.
Journal of Biological Chemistry, 2006
4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid tra... more 4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid transporter; (Tr)EAAT2, truncated EAAT2 derived from caspase-3 cleavage at the consensus site; CTE, C-terminal domain of EAAT2; SOD1, Cu 2ϩ /Zn 2ϩ -superoxide dismutase; mutSOD1, mutant human SOD1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.; DTT, dithiothreitol; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PARP, poly(ADP-ribose) polymerase; WT, wild type.
Journal of Biological Chemistry, 1995
Journal of lipid research, 2000
Compared with classic essential fatty acid deficiency or the feeding of a fat-free diet, little i... more Compared with classic essential fatty acid deficiency or the feeding of a fat-free diet, little is known about specific linoleate deficiency in the rat. Carbon recycling into de novo lipogenesis has been reported to be an obligatory feature of linoleate metabolism in the liver, even in extreme linoleate deficiency (LA-D). The present study had two objectives: 1) to report a brief summary of the tissue n-6 polyunsaturated fatty acid (PUFA) profiles in specific LA-D, and 2) to quantify whole body carbon recycling from [(14)C]linoleate in specific LA-D. Rats consumed a linoleate-deficient diet for 12 weeks and then received a bolus of [1-(14)C]linoleate by gavage. In linoleate-deficient rats, the triene/tetraene ratio in several organs increased by 18- to 100-fold. The amount of (14)C appearing in organ sterols (dpm/g) of linoleate-deficient rats was 2- to 10-fold higher than in the controls and equaled 16.3% of the [(14)C]linoleate dose given, compared with 7.4% in the controls. We co...
Progress in Brain Research, 2001
... system. Curr: Pharm. Des., 5: 363-379. Brooks-Kayal,AR,Munir,M.,Jin, H. and Robinson,MB ( 199... more ... system. Curr: Pharm. Des., 5: 363-379. Brooks-Kayal,AR,Munir,M.,Jin, H. and Robinson,MB ( 1998) The glutamate transporter, GLT-1, is expressed in cul-tured hippocampal neurons. Neurochem. Znt., 33: 95-100. Carlsson ...
Pharmacological Research Communications, 1988
Down regulation of 8-receptors coupled to desensitization of ME-dependent adenylate~cycJase l is/... more Down regulation of 8-receptors coupled to desensitization of ME-dependent adenylate~cycJase l is/one of the most common effect of repeated exposure to antidepressant drugs . In order to clarify the molecular basis of receptor regulatlon we have performed a series of studies with an "in vitro ~ model of 8-adrenoceptor homologous desensitization. Rat cortical sllces incubated for 30' in ~ the presence of isoproterenol (ISO) show dose-dependent reversible down regulation of B-adrenoceptors para]ie]ed by a reduction in cAMP accumu]ation following NE exposure. The role of alpha-I receptors in modulating the response of B-adrenoceptors was investigated by studying the effect of methoxamine on ISO induced desensitization of 8-receptors. Incubation with the alpha-! agonist reduced the number of 8-receptors and the ISO-induced desensitization was somehow reduced after incubation with methoxamlne, lhese findings clearly indicate that multlple synaptic and transynaptic mechanisms may be involved in the regulatlon of 8-adrenoceptor function.
Advances in Experimental Medicine and Biology, 1992
ABSTRACT
Nature neuroscience, 1999
The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degener... more The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the glutamate transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.
The Journal of experimental biology, 1997
In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and pa... more In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has p...
Molecular pharmacology, 1994
Reuptake of glutamate in astrocytes, a critical mechanism involved in the maintenance of physiolo... more Reuptake of glutamate in astrocytes, a critical mechanism involved in the maintenance of physiological excitatory amino acid neurotransmission, is inhibited by both arachidonic acid (AA) and reactive oxygen species (ROS), via incompletely defined molecular mechanisms. Because ROS are generated during AA metabolism and AA can be released as a result of ROS-mediated phospholipase A2 activation, it seems likely that their effects on uptake are mediated by a common mechanism. However, here we show that rapid (10-min) uptake inhibitions by AA or by ROS generated by the xanthine plus xanthine oxidase (XO) reaction are selectively abolished by distinct agents; bovine serum albumin (BSA) acts only on AA, whereas the scavenger enzymes superoxide dismutase (SOD) and catalase (CAT) and the disulfide-reducing agent dithiothreitol (DTT) act only on ROS. Moreover, when added together, xanthine/XO and AA decrease uptake in a fully additive manner. In particular, the effect of xanthine/XO is seen a...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1994
Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system ar... more Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximately 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase+catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic antioxidants or...
Advances in Insect Physiology, 2001
... Acid Absorption in the Midgut of Lepidopteran Larvae V. Franca Sacchia, Michela Castagnab,Dav... more ... Acid Absorption in the Midgut of Lepidopteran Larvae V. Franca Sacchia, Michela Castagnab,Davide Trottib, Chairat Shayakulb, and Matthias A. Hedigerb stituto di Fisiologia Generale e di Chimica Biologica, Facolt& di Farmacia, Universit& di Milano, Via Trentacoste 2, 20134 ...
Journal of Neuroscience, 2013
Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of fa... more Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.
Proceedings of the National Academy of Sciences, 1998
Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotranspo... more Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na ؉ or H ؉ gradients. The present work extends the Na ؉ and H ؉ dogma by including the H ؉ and K ؉ paradigm. Lepidopteran insect larvae have a high K ؉ and a low Na ؉ content, and their midgut cells lack Na ؉ ͞K ؉ ATPase. Instead, an H ؉ translocating, vacuolar-type ATPase generates a voltage of approximately ؊240 mV across the apical plasma membrane of so-called goblet cells, which drives H ؉ back into the cells in exchange for K ؉ , resulting in net K ؉ secretion into the lumen. The resulting inwardly directed K ؉ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K ؉ -coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes ␣-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K ؉ , Na ؉ , and to a lesser extent Li ؉ were accepted as cotransported ions, but K ؉ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl ؊ -dependent, and the K ؉ ͞Na ؉ selectivity increased with hyperpolarization of oocytes, ref lecting the increased K ؉ ͞Na ؉ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na ؉ and Cl ؊ -coupled neurotransmitter transporter family.
Proceedings of the National Academy of Sciences, 2012
Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familia... more Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALSlinked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iperoxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patientderived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.
Neuroscience Letters, 1995
While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of... more While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of hippocampal astrocytes, we observed that all the HPLC peaks corresponding to AA metabolites displayed significantly different levels depending on the presence or not of free Ca 2+ in the incubation medium. A specific pattern was noticed, i.e. lipoxygenase (LOX) derivatives, in particular 12hydroxyeicosatetraenoic acid (12-HETE), showed higher levels in medium containing 1 mM Ca 2+, while cycloxygenase (COX) products including prostaglandins (PG) F2a, E 2 and D 2 and 12-hydroxyheptadecatrienoic acid (12-HHT), were higher in Ca2÷-free medium. COX metabolism exceeded LOX metabolism by threefold in Ca2+-free medium, while it was only 60% of it in 1 mM Ca 2+. The total amount of AA processed under the two conditions was identical. These data suggest that free [Ca 2+] influences the pattern of AA metabolites formed in hippocampal astrocytes, with possible important implications in view of the distinct roles played by COX and LOX eicosanoids in synaptic transmission and neurotoxicity in this area.
Journal of Biological Chemistry, 2001
We have investigated the functional impact of a naturally occurring mutation of the human glutama... more We have investigated the functional impact of a naturally occurring mutation of the human glutamate transporter GLT1 (EAAT2), which had been detected in a patient with sporadic amyotrophic lateral sclerosis. The mutation involves a substitution of the putative N-linked glycosylation site asparagine 206 by a serine residue (N206S) and results in reduced glycosylation of the transporter and decreased uptake activity. Electrophysiological analysis of N206S revealed a pronounced reduction in transport rate compared with wild-type, but there was no alteration in the apparent affinities for glutamate and sodium. In addition, no change in the sensitivity for the specific transport inhibitor dihydrokainate was observed. However, the decreased rate of transport was associated with a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a dominant negative effect on the wild-type GLT1 activity, whereas it did not affect wild-type EAAC1. These findings provide evidence for a role of the N-linked glycosylation in both cellular trafficking and transport function. The resulting alteration in glutamate clearance capacity likely contributes to excitotoxicity that participates in motor neuron degeneration in amyotrophic lateral sclerosis.
Journal of Biological Chemistry, 2010
Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeos... more Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process since K + is believed to equilibrate freely between cytosol and inter-mitochondrial membrane space. By patchclamping mitochondria isolated from the central nervous system of adult mitoCFP transgenic mice, we report the existence of I OMMKi , a novel voltage-dependent inward rectifying K + conductance located in the OMM. I OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K + to enter the mitochondrial intermembrane space in a controlled, regulated fashion. The identification of I OMMKi in the outer mitochondrial membrane supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the outer mitochondria membrane possesses regulated pathways for K + uptake.
Journal of Biological Chemistry, 1997
Ion-coupled solute transporters exhibit pre-steadytate currents that resemble those of voltage-de... more Ion-coupled solute transporters exhibit pre-steadytate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H ؉ -coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra-(pH i ) and extracellular pH (pH o ). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pH i and pH o . At a pH o of 7.0 and a holding potential (V h ) of ؊50 mV, the charge movements were mostly inwardly directed, whereas reduction of pH o to below 7.0 resulted in outwardly directed charge movements. When pH i was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 ؎ 0.03 (n ؍ 7). The midpoint voltage (V 0.5 ) of the charge distribution shifted linearly as a function of pH i and pH o . Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H ؉ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H ؉ chemical potential (⌬ H ) or increasing the membrane potential (V m ) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.
Journal of Biological Chemistry, 2007
4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu 2ϩ / Zn 2ϩ superoxide ... more 4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu 2ϩ / Zn 2ϩ superoxide dismutase; mutSOD1, mutated SOD1; WT-SOD1, wild type SOD1; SUMO, small ubiquitin modifier protein; PML, promyelocytic leukemia; PML-NB, PML nuclear body; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; CTE, COOH terminus of EAAT2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; HD, Huntington disease; EGFP, enhanced green fluorescent protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPI, 4Ј,6-diamidino-2phenylindole.
Journal of Biological Chemistry, 1996
The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents th... more The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H 2 O 2 ). Here we show that another biological oxidant, peroxynitrite (ONOO ؊ ), formed by combination of superoxide ( ⅐ O 2 ؊ ) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO ؊ reduces selectively the V max of transport; its action is fast (reaching 90% within 20 s), dose-dependent (50% inhibition at 50 M), persistent upon ONOO ؊ (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO ؊ with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO ؊ . H 2 O 2 also inhibits reconstituted transport, and its action matches that of ONOO ؊ on all respects; however, this is observed only with 5-10 mM H 2 O 2 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO ؊ and other oxidants.
Journal of Biological Chemistry, 2006
4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid tra... more 4 The abbreviations used are: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid transporter; (Tr)EAAT2, truncated EAAT2 derived from caspase-3 cleavage at the consensus site; CTE, C-terminal domain of EAAT2; SOD1, Cu 2ϩ /Zn 2ϩ -superoxide dismutase; mutSOD1, mutant human SOD1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.; DTT, dithiothreitol; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PARP, poly(ADP-ribose) polymerase; WT, wild type.
Journal of Biological Chemistry, 1995