Davide Trotti

SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivatea glial glutamate transporter

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.

High Sensitivity of Glutamate Uptake to Extracellular Free Arachidonic Acid Levels in Rat Cortical Synaptosomes and Astrocytes

Abstract: By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high‐affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1–100 μM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2–1 mg/ml), a phospholipase A2 activator, or thimerosal (50–200 μM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate. whereas inhibition ≥60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1–10 mg/ml), a free fatty acid‐binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.

Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism

Glutamate uptake into nerve cells and astrocytes via high‐affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox‐sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5′‐dithio‐bis(2–nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole‐cell patch‐clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT‐reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH‐based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo

Amyotrophic Lateral Sclerosis-linked Glutamate Transporter Mutant Has Impaired Glutamate Clearance Capacity

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.

The Competitive Transport Inhibitor L‐trans‐pyrrolidine‐2,4‐dicarboxylate Triggers Excitotoxicity in Rat Cortical Neuron‐Astrocyte Co‐cultures via Glutamate Release rather than Uptake Inhibition

We studied the early and late effects of L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC), a competitive inhibitor of glutamate uptake with low affinity for glutamate receptors, in co‐cultures of rat cortical neurons and glia expressing spontaneous excitatory amino acid (EAA) neurotransmission. At 100 or 200 μM, PDC induced different patterns of electrical changes: 100 μM prolonged tetrodotoxin‐sensitive excitation triggered by synaptic glutamate release; 200 μM produced sustained, tetrodotoxin‐insensitive and EAA‐mediated neuronal depolarization, overwhelming synaptic activity. At 200 μM, but not at 100 μM, PDC caused rapid elevation of the glutamate concentration ([Glu]0) in the culture medium, resulting in NMDA receptor‐mediated excitotoxic death of neurons 24 h later. The increase in [Glu]0 was largely insensitive to tetrodotoxin, independent of extracellular Ca2+, and present also in astrocyte‐pure cultures. By the use of glutamate transporters functionally reconstituted in liposomes, we showed directly that PDC activates carrier‐mediated release of glutamate via heteroexchange. Glutamate release and delayed neurotoxicity in our cultures were suppressed if PDC was applied in a Na+‐free medium containing Li+. However, replacement of Na+ with choline instead of Li+ did not result in an identical effect, suggesting that Li+ does not act simply as an external Na+ substitute. In conclusion, our data indicate that alteration of glutamate transport by PDC has excitotoxic consequences and that active release of glutamate rather than just uptake inhibition is responsible for the generation of neuronal injury.

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre‐ or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co‐localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non‐synaptic function of this high‐affinity glutamate carrier, not restricted to glutamatergic fibres.

Reactive Oxygen Species Inhibit High‐Affinity Glutamate Uptake: Molecular Mechanism and Neuropathological Implications

Two types of events encountered in several acute and long-term neurodegenerativc brain diseases (including Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, as well as ischemia, trauma, and seizures) are thought to play a major role in triggering neuronal injury: (a) abnormal formation of oxygen free radicals or related reactive oxygen species (ROS) (named “oxidative stress”); and (b) dysfunction of excitatory amino acid (EAA) transmission (named “excitotoxicity”) (reviewed in reference 1). Several lines of experimental evidence in the last few years have brought to the idea that oxidative stress and excitotoxicity may be interdependent events, acting in a sequential and reinforcing manner to eventually produce neuronal degeneration (reviewed in reference 2). Thus, studies utilizing spin-trapping and electron paramagnetic resonance (EPR) detection have recently shown that ROS can be generated in neuronal cell cultures in response to potent stimulation of glutamate receptors. Both N-methyla-aspartate (NMDA)3 and kainate4 seem able to activate ROS formation. Ca++ entry through EAA receptors has been implicated as the trigger event. ROS might then be generated either by Ca+ +-dependent activation of phospholipase via oxidative metabolism of arachidonic acid, or by activation of calpain, a protease able to cleave and activate xanthine oxidase to produce superoxide anion (0;) by reaction with its substrate xanthine.6 ROS might also be a coproduct of Ca++/calmodulin-dependent activation of nitric oxide synthase.’ A different mechanism of ROS generation by glutamate was described in a neuroblastoma cell line: glutamate competitively inhibits the transport of cystine. an essential substrate for the synthesys of glutathione (GSH), which plays an important role in the antioxidant cell defenses. As a consequence, GSH depletion slowly occurs, resulting in oxidative strewx Interestingly ROS, on turn, have feedback actions on glutamatergic transmission (see also Pellmar et a/., this volume). In particular, they have been shown to cause

Differential modulation of the uptake currents by redox interconversion of cysteine residues in the human neuronal glutamate transporter EAAC1

Control of extrasynaptic glutamate concentration in the central nervous system is an important determinant of neurotransmission and excitotoxicity. Mechanisms that modulate glutamate transporter function are therefore critical factors in these processes. The redox modulation of glutamate uptake was examined by measuring transporter‐mediated electrical currents and radiolabelled amino acid influx in voltage‐clamped Xenopus oocytes expressing the human neuronal glutamate transporter EAAC1. Up and down changes of the glutamate uptake currents in response to treatment with dithiothreitol and 5,5′‐dithio‐bis‐(2‐nitrobenzoic) acid (DTNB) were observed in oocytes clamped at ‐60 mV. The redox interconversion of cysteines induced by dithiothreitol/DTNB influenced the Vmax (Imax) of transport, while the apparent affinity for glutamate was not affected. Formation or breakdown of disulphide groups did not affect the pre‐steady‐state currents, suggesting that these manipulations do not interfere with the Na+ binding/unbinding and/or the charge distribution on the transporter molecule. The glutamate‐evoked net uptake current of EAAC1 was composed of the inward current from electrogenic glutamate transport and the current arising from the glutamate‐activated CI‐ conductance. The structural rearrangement produced by the formation or breakdown of disulphide groups only affected the current from electrogenic giutamate transport. The electrogenic currents of EAAC1 were significantly reduced by peroxynitrite, an endogenously occurring oxidant formed in certain pathological brain processes, and the mechanism of inhibition partially depended on the formation of disulphide groups.

Symmetry of H+ Binding to the Intra- and Extracellular Side of the H+-coupled Oligopeptide Cotransporter PepT1

Ion-coupled solute transporters exhibit pre-steady-tate 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- (pHi) and extracellular pH (pHo). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pHi and pHo. At a pHo of 7.0 and a holding potential (Vh) of −50 mV, the charge movements were mostly inwardly directed, whereas reduction of pHo to below 7.0 resulted in outwardly directed charge movements. When pHi 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 (V0.5) of the charge distribution shifted linearly as a function of pHi and pHo. 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 (ΔμH) or increasing the membrane potential (Vm) 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.

Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters

Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.