Ross D. O'Shea

Transporters for L-glutamate: An update on their molecular pharmacology and pathological involvement

L‐Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high‐affinity Glu transporters (EAAT1–5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntingtons disease, Alzheimers disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post‐translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.

Preproneuropeptide Y messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat is increased by food deprivation or dehydration

The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro‐NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro‐NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro‐NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro‐GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.

Food or Water Deprivation Modulate Nitric Oxide Synthase (NOS) Activity and Gene Expression in Rat Hypothalamic Neurones: Correlation with Neurosecretory Activity?

Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P<0.05 and 261±34% of control, P<0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P<0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment‐induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH‐diaphorase histochemistry, and NADPH‐diaphorase staining was observed in neurones both positive and negative for oxytocin‐like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity‐dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.

The Rho kinase inhibitor Fasudil up-regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

BACKGROUND AND PURPOSE Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

SummaryThe possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate ([3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and “granule” membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 μM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5–0.9 μM and Bmax 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [3H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and “granule” membranes the Bmax values for the binding of [3H]GLU and [3H]glycine had a stoichiometry of 1∶:1, whilst in cerebellar synaptic membranes this ratio was 4∶:1. Receptor autoradiography of NMDA-related [3H]GLU and [3H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [3H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.

Astrocyte mGlu2/3-mediated cAMP potentiation is calcium sensitive: studies in murine neuronal and astrocyte cultures

Signal transduction mechanisms of group II metabotropic glutamate receptors (mGlu(2/3)) remains a matter of some controversy, therefore we sought to gain new insights into its regulation by studying cAMP production in cultured neurons and astrocytes, and by examining inter-relationships of mGlu(2/3)-induced signalling with cellular calcium and various signalling cascades. mGlu(2/3) agonists 2R,4R-4-aminopyrrolidine-2,4-dicarboxylic acid (2R,4R-APDC) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited 10 microM forskolin-stimulated production of cAMP in murine cortical neurons, striatal neurons and forebrain astrocytes in the absence of extracellular Ca(2+). These agonists potentiated cAMP production in the presence of 1.8 mM Ca(2+) in astrocytes only. This potentiation was dependent on the extracellular Ca(2+) concentration (0.001-10 mM) and inhibited by the mGlu(2/3) antagonist LY341495 (1 microM), adenosine deaminase (1 U/ml) and the adenosine A(2A) receptor antagonist ZM241385 (1 microM). Pre-incubation with the phospholipase C (PLC) inhibitor U73122 (10 microM), L-type Ca(2+)-channel blockers nifedipine (1 microM) and nimodipine (1 microM), the calmodulin kinase II (CaMKII) inhibitor KN-62 (10 microM) or pertussis toxin (100 ng/ml) inhibited this potentiation. In the absence of 1.8 mM Ca(2+), thapsigargin (1 microM) facilitated the potentiation of cAMP production. Measurement of the Ca(2+)-binding dye Fluo-3/AM showed that, compared to Ca(2+)-free conditions, thapsigargin and 1.8 mM Ca(2+) elevated [Ca(2+)](i) in astrocytes; the latter effect being prevented by L-type Ca(2+)-channel blockers. Potentiation of cAMP production was also demonstrated when astrocytes were stimulated with the beta-adrenoceptor agonist isoprenaline (10 microM) in the presence of 1.8 mM Ca(2+), but not with the adenosine agonist NECA (10 microM) or the group I mGlu receptor agonist DHPG (100 microM). BaCl(2) (1.8 mM) in place of Ca(2+) did not facilitate forskolin-stimulated mGlu(2/3)-potentiation of cAMP. In short, this study in astrocytes demonstrates that under physiological Ca(2+) and adenylate cyclase stimulation an elevation of cAMP production is achieved that is mediated by PLC/IP(3)- and CaMKII-dependent pathways and results in the release of endogenous adenosine which acts at G(s) protein-coupled A(2A) receptors. These findings provide new insights into mGlu(2/3) signalling in astrocytes versus neurons, and which could determine the functional phenotypy of astrocytes under physiological and pathological conditions.

Hypoxic preconditioning in neonatal rat brain involves regulation of excitatory amino acid transporter 2 and estrogen receptor alpha

Exposure of the brain to a sublethal insult can protect against a subsequent brain injury. Hypoxic preconditioning induces tolerance to hypoxic--ischemic injury in neonatal rat brain and is associated with changes in gene and protein expression. To study the involvement of excitatory amino acid transporters (EAAT1 and EAAT2) and estrogen receptors (ERalpha and ERbeta) in neonatal hypoxia--induced ischemic tolerance, we examined changes in expression of these proteins in the cortex, hippocampus and striatum of newborn rats at different time points after exposure to sublethal hypoxia (8% O(2), 3h). Preconditioning with hypoxia 24h before hypoxia-ischemia afforded marked brain protection compared with littermate control animals as determined by morphological assessment. Immunoblot analysis showed that EAAT2 and ERalpha were significantly increased by 55% and 49%, respectively, in cortex at 24h after hypoxic-preconditioning. Surprisingly, at the same time point, a significant decrease of EAAT2 by 48% in striatum was observed. In contrast, hypoxic preconditioning had no effect on the levels of EAAT1 and ERbeta in any of the brain regions studied at any of the time points analyzed. The similar pattern of changes in EAAT2 and ERalpha levels suggests that ERalpha might interact with EAAT2 in producing preconditioning. The endogenous molecular mechanisms modulated by hypoxia preconditioning may contribute to the development of hypoxia-induced ischemic tolerance, and may provide novel therapeutic targets for the treatment of cerebral ischemia.

Transcriptomic profiling of astrocytes treated with the Rho kinase inhibitor Fasudil reveals cytoskeletal and pro-survival responses†

Inhibitors of Rho kinase (ROCK) have potential for management of neurological disorders by inhibition of glial scarring. Since astrocytes play key roles in brain physiology and pathology, we determined changes in the astrocytic transcriptome produced by the ROCK inhibitor Fasudil to obtain mechanistic insights into its beneficial action during brain injury. Cultured murine astrocytes were treated with Fasudil (100 µM) and morphological analyses revealed rapid stellation by 1 h and time‐dependent (2–24 h) dissipation of F‐actin‐labelled stress fibres. Microarray analyses were performed on RNA and the time‐course of global gene profiling (2, 6, 12 and 24 h) provided a comprehensive description of transcriptomic changes. Hierarchical clustering of differentially expressed genes and analysis for over‐represented gene ontology groups using the DAVID database focused attention on Fasudil‐induced changes to major biological processes regulating cellular shape and motility (actin cytoskeleton, axon guidance, transforming growth factor‐β (TGFβ) signalling and tight junctions). Bioinformatic analyses of transcriptomic changes revealed how these biological processes contributed to changes in astrocytic motility and cytoskeletal reorganisation. Here genes associated with extracellular matrix were also involved, but unexpected was a subset of alterations (EAAT2, BDNF, anti‐oxidant species, metabolic and signalling genes) indicative of adoption by astrocytes of a pro‐survival phenotype. Expression profiles of key changes with Fasudil and another ROCK inhibitor Y27632 were validated by real‐time PCR. Although effects of ROCK inhibition have been considered to be primarily cytoskeletal via reduction of glial scarring, we demonstrate additional advantageous actions likely to contribute to their ameliorative actions in brain injury. J. Cell. Physiol. 227: 1199–1211, 2012.

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS) non‐neuronal cells play key roles in disease etiology and loss of motoneurons via noncell‐autonomous mechanisms. Reactive astrogliosis and dysfunctional transporters for L‐glutamate [excitatory amino acid transporters, (EAATs)] are hallmarks of ALS pathology. Here, we describe mechanistic insights into ALS pathology involving EAAT‐associated homeostasis in response to a destructive milieu, in which oxidative stress and excitotoxicity induce respectively astrogliosis and motoneuron injury. Using an in vitro neuronal‐glial culture of embryonic mouse spinal cord, we demonstrate that EAAT activity was maintained initially, despite a loss of cellular viability induced by exposure to oxidative [3‐morpholinosydnonimine chloride (SIN‐1)] and excitotoxic [(S)‐5‐fluorowillardiine (FW)] conditions. This homeostatic response of EAAT function involved no change in the cell surface expression of EAAT1/2 at 0.5–4 h, but rather alterations in kinetic properties. Over this time‐frame, EAAT1/2 both became more widespread across astrocytic arbors in concert with increased expression of glial fibrillary acidic protein (GFAP), although at 8–24 h there was gliotoxicity, especially with SIN‐1 rather than FW. An opposite picture was found for motoneurons where FW, not SIN‐1, produced early and extensive neuritic shrinkage and blebbing (≥0.5 h) with somata loss from 2 h. We postulate that EAATs play an early homeostatic and protective role in the pathologic milieu. Moreover, the differential profiles of injury produced by oxidative and excitotoxic insults identify two distinct phases of injury which parallel important aspects of the pathology of ALS.