Sarjubhai A. Patel

System xc⁻ cystine/glutamate antiporter: an update on molecular pharmacology and roles within the CNS.

System xc‐ is an amino acid antiporter that typically mediates the exchange of extracellular l‐cystine and intracellular l‐glutamate across the cellular plasma membrane. Studied in a variety of cell types, the import of l‐cystine through this transporter is critical to glutathione production and oxidative protection. The exchange‐mediated export of l‐glutamate takes on added significance within the CNS, as it represents a non‐vesicular route of release through which this excitatory neurotransmitter can participate in either neuronal signalling or excitotoxic pathology. When both the import of l‐cystine and the export of l‐glutamate are taken into consideration, system xc‐ has now been linked to a wide range of CNS functions, including oxidative protection, the operation of the blood–brain barrier, neurotransmitter release, synaptic organization, viral pathology, drug addiction, chemosensitivity and chemoresistance, and brain tumour growth. The ability to selectively manipulate system xc‐, delineate its function, probe its structure and evaluate it as a therapeutic target is closely linked to understanding its pharmacology and the subsequent development of selective inhibitors and substrates. Towards that goal, this review will examine the current status of our understanding of system xc‐ pharmacology and the structure–activity relationships that have guided the development of an initial pharmacophore model, including the presence of lipophilic domains adjacent to the substrate binding site. A special emphasis is placed on the roles of system xc‐ within the CNS, as it is these actions that are among the most exciting as potential long‐range therapeutic targets.

Differentiation of substrate and non-substrate inhibitors of transport system xc−: an obligate exchanger of L-glutamate and L-cystine

In addition to the well-characterized sodium-dependent excitatory amino acid transporters (EAATs) present in the mammalian CNS, a chloride-dependent, sodium-independent transporter has also been identified that is capable of mediating the uptake of L-glutamate. Named system x(c)(-), this transporter is an obligate exchanger that normally couples the export of intracellular L-glutamate with the import of extracellular L-cystine. Two cell lines that express high levels of system x(c)(-) are used to delineate the pharmacology of the transporter and demonstrate that it is distinct from both the EAATs and EAA ionotropic receptors. Potent competitive inhibitors of system x(c)(-) include: L-homocysteate, ibotenate, L-serine-O-sulphate, (RS)-4-bromohomoibotenate, quisqualate, and (S)-4-carboxyphenylglycine. A fluorescent-based assay that allows system x(c)(-)-mediated exchange of L-glutamate and L-cystine to be followed in real time is used to assess substrate activity. Interestingly, those compounds that proved to be the most potent competitive inhibitors (e.g. L-quisqualate and 4-S-CPG) also proved to be the least active as substrates, suggesting that distinct structural features may control binding and translocation. Lastly, the finding that a number of system x(c)(-) inhibitors are also commonly used as probes of excitotoxic pathology (e.g., L-quisqualate, ibotenate and L-homocysteate) raises some interesting questions regarding the mechanisms through which these analogues produce CNS damage.

Regulation of the system x(C)- cystine/glutamate exchanger by intracellular glutathione levels in rat astrocyte primary cultures.

The system x  C− (Sx  C− ) transporter functions to mediate the exchange of extracellular cystine (L‐Cys2) and intracellular glutamate (L‐Glu). Internalized L‐Cys2 serves as a rate‐limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L‐Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl‐cAMP) and GSH levels on the expression of Sx  C− were investigated in primary rat astrocyte cultures. Sx  C− activity in dbcAMP‐treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (∼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx  C− triggered by GSH depletion was only observed in the dbcAMP‐treated phenotype and was distinct from the Nrf2‐mediated response initiated by exposure to electrophiles. Changes in Sx  C− activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx  C− heterodimer, an increase in the Vmax for L‐Glu uptake and was linked temporally to GSH levels. This induction of Sx  C− was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co‐administration of the cell‐permeant thiols GSH‐ethyl ester and N‐acetylcysteine. These findings demonstrate that the expression of Sx  C− on astrocytes is dynamically regulated by intracellular GSH levels in a cell‐ and phenotype‐dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx  C− activity in CNS injury and disease.

Plasma micro-RNA biomarkers for diagnosis and prognosis after traumatic brain injury: A pilot study.

Prediction of post-concussive syndrome after apparent mild traumatic brain injury (TBI) and subsequent cognitive recovery remains challenging, with substantial limitations of current methods of cognitive testing. This pilot study aimed to determine if levels of micro ribonucleic acids (RNAs) circulating in plasma are altered following TBI, and if changes to levels of such biomarkers over time could assist in determination of prognosis after TBI. Patients were enrolled after TBI on presentation to the Emergency Department and allocated to three groups: A - TBI (physical trauma to the head), witnessed loss of consciousness, amnesia, GCS=15, a normal CT Brain and a recorded first pass after post-traumatic amnesia (PTA) scale; B TBI, witnessed LOC, amnesia, GCS=15, a normal CT brain and a PTA scale test fail and: C - TBI and initial GCS <13 on arrival to the ED. Venous blood was collected at three time points (arrival, day 5 and day 30). Isolation of cell-free total RNA was then assayed using a custom miRNA PCR array. Two micro-RNAs, mir142-3p and mir423-3p demonstrated potential clinical utility differentiating patients after mild head injury into those at greater risk of developing amnesia and therefore, post-concussive syndromes. In addition, these miRNA demonstrated a decrease in expression over time, possibly indicative of brain healing after the injury. Further evaluation of these identified miRNA markers with larger patient cohorts, correlation with clinical symptoms and analysis over longer time periods are essential next steps in developing objective markers of severity of TBI.

The effects of mild traumatic brain injury on postural control

ABSTRACT Primary objective: The purpose of this study was to investigate the effects of mild traumatic brain injury (mTBI) on multiple postural indices that characterize body sway behaviour. Methods and procedures: The body’s centre of pressure (COP) displacement was recorded from 11 individuals with a history of mTBI (29.4 ± 6.7 years old) and 11 healthy controls (26.8 ± 3.7 years old) performing bipedal stance on a force platform for 120 seconds. Spatio-temporal (area, amplitude and mean velocity of the COP displacement) and frequency characteristics (frequency containing 80% of the power spectral density) of the body oscillation, as well as its dynamic characteristics (sample entropy estimate of the COP displacement) were extracted from COP signals. Main outcomes and results: All postural indices studied were significantly affected by mTBI (p < 0.010). Participants with a history of mTBI presented a larger, slower, and more random body oscillation compared to controls. Conclusion: The results suggest that (a) balance deficits can be recognized as an effect of mTBI; (b) balance deficits induced by mTBI are multi-dimensional, affecting all three domains included in this study; and (c) the postural indices employed in this study are potential markers to detect changes in postural control following mTBI.

Use of the hydantoin isostere to produce inhibitors showing selectivity toward the vesicular glutamate transporter versus the obligate exchange transporter system x(c)(

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pK(a)∼9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system x(c)(-)). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5mM but few inhibited system x(c)(-) greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system x(c)(-) inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT.

Increased excitatory amino acid transport into murine prion protein knockout astrocytes cultured in vitro

Prion protein (PrP) is expressed on a wide variety of cells and plays an important role in the pathogenesis of transmissible spongiform encephalopathies. However, its normal function remains unclear. Mice that do not express PrP exhibit deficits in spatial memory and abnormalities in excitatory neurotransmission suggestive that PrP may function in the glutamatergic synapse. Here, we show that transport of D‐aspartate, a nonmetabolized L‐glutamate analog, through excitatory amino acid transporters (EAATs) was faster in astrocytes from PrP knockout (PrPKO) mice than in astrocytes from C57BL/10SnJ wild‐type (WT) mice. Experiments using EAAT subtype‐specific inhibitors demonstrated that in both WT and PrPKO astrocytes, the majority of transport was mediated by EAAT1. Furthermore, PrPKO astrocytes were more effective than WT astrocytes at alleviating L‐glutamate‐mediated excitotoxic damage in both WT and PrPKO neuronal cultures. Thus, in this in vitro model, PrPKO astrocytes exerted a functional influence on neuronal survival and may therefore influence regulation of glutamatergic neurotransmission in vivo.

Efficient digestion and mass spectral analysis of vesicular glutamate transporter 1: a recombinant membrane protein expressed in yeast.

Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.

Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of ExcitatoryAmino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c −

As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamateparticipates not only in standard fast synaptic communication, but also contributes to higher order signalprocessing, as well as neuropathology. Given this variety of functional roles, interest has been growingas to how the extracellular concentrations of l-glutamate surroundingneurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, eachof which appears capable of influencing both the signaling and pathological actions of l-glutamatewithin the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystineexchanger, system x c −(Sx c −). Whilethe family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequesteringl-glutamate in neurons and glia, Sxc −provides a route for the export of glutamate from cells into the extracellular environment. The primaryintent of this work is to provide an overview of the inhibitors and substrates that have been developedto delineate the pharmacological specificity of these transport systems, as well as be exploited as probeswith which to selectively investigate function. Particular attention is paid to the development of smallmolecule templates that mimic the structural properties of the endogenous substrates, l-glutamate,l-aspartate and l-cystine andhow strategic control of functional group position and/or the introduction of lipophilic R-groups can impactmultiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrateactivity.

Novel di-aryl-substituted isoxazoles act as noncompetitive inhibitors of the system Xc(-) cystine/glutamate exchanger.

The system x c antiporter is a plasma membrane transporter that mediates the exchange of extracellular L-cystine with intracellular L-glutamate. This exchange is significant within the context of the CNS because the import of L-cystine is required for the synthesis of the antioxidant glutathione, while the efflux of L-glutamate has the potential to contribute to either excitatory signaling or excitotoxic pathology. Changes in the activity of the transport system have been linked to the underlying pathological mechanisms of a variety of CNS disorders, one of the most prominent of which is its highly enriched expression in glial brain tumors. In an effort to produce more potent system x c blockers, we have been using amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) as a scaffold for inhibitor development. We previously demonstrated that the addition of lipophilic aryl groups to either the #4 or #5 position on the isoxazole ring markedly increased the inhibitory activity at system x c . In the present work a novel series of analogues has been prepared in which aryl groups have been introduced at both the #4 and #5 positions. In contrast to the competitive action of the mono-substituted analogues, kinetic analyses indicate that the di-substituted isoxazoles block system x c -mediated uptake of H-L-glutamate into SNB-19 cells by a noncompetitive mechanism. These new analogues appear to be the first noncompetitive inhibitors identified for this transport system, as well as being among the most potent blockers identified to date. These diaryl-isoxazoles should be of value in assessing the physiological roles and molecular pharmacology of system x c . 2013 Published by Elsevier Ltd.The system xc(-) antiporter is a plasma membrane transporter that mediates the exchange of extracellular l-cystine with intracellular l-glutamate. This exchange is significant within the context of the CNS because the import of l-cystine is required for the synthesis of the antioxidant glutathione, while the efflux of l-glutamate has the potential to contribute to either excitatory signaling or excitotoxic pathology. Changes in the activity of the transport system have been linked to the underlying pathological mechanisms of a variety of CNS disorders, one of the most prominent of which is its highly enriched expression in glial brain tumors. In an effort to produce more potent system xc(-) blockers, we have been using amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) as a scaffold for inhibitor development. We previously demonstrated that the addition of lipophilic aryl groups to either the #4 or #5 position on the isoxazole ring markedly increased the inhibitory activity at system xc(-). In the present work a novel series of analogues has been prepared in which aryl groups have been introduced at both the #4 and #5 positions. In contrast to the competitive action of the mono-substituted analogues, kinetic analyses indicate that the di-substituted isoxazoles block system xc(-)-mediated uptake of (3)H-l-glutamate into SNB-19 cells by a noncompetitive mechanism. These new analogues appear to be the first noncompetitive inhibitors identified for this transport system, as well as being among the most potent blockers identified to date. These diaryl-isoxazoles should be of value in assessing the physiological roles and molecular pharmacology of system xc(-).