Gethin J. McBean

Cerebral cystine uptake: a tale of two transporters

Transport of cystine across the cell membrane is essential for synthesis of the major cellular antioxidant glutathione. Cystine uptake in the brain occurs by both the Na(+)-independent x(c)(-) cystine-glutamate exchanger and the X(AG)(-) family of high-affinity, Na(+)-dependent glutamate transporters. New evidence concerning the role of cystine transport in the defence against oxidative stress is described.

Neurotoxicity of L‐Glutamate and DL‐Threo‐3‐Hydroxyaspartate in the Rat Striatum

Destruction of the glutamatergic corticostriatal pathway potentiates the neurotoxic action of 1 μmol L‐glutamate injected into the rat striatum, whereas the toxic effects of 10 nmol kainate are markedly attenuated. Injection of 170 nmol of the glutamate uptake inhibitor, DL‐threo‐3‐hydroxyaspartate, into the intact striatum also causes neuronal degeneration, which is accompanied by a reduction in markers for cholinergic and GABAergic neurones. Prior removal of the corticostriatal pathway destroys the ability of DL‐threo‐3‐hydroxyaspartate to cause lesions in the striatum. These results indicate that removal, or blockade, of uptake sites for glutamate increase the vulnerability of striatal neurones to the toxic effects of synaptically released glutamate.

Striatal glutamatergic function: Modifications following specific lesions

The effects of specific lesions of the striatum: (a) hemidecortication; (b) striatal injection of (+/-) ibotenate; and (c) 6-hydroxydopamine injections into the substantia nigra, were investigated on specific [3H]glutamate binding to striatal membranes. One month after decortication, there was a substantial reduction of calcium-dependent, stimulated glutamate release from striatal slices, indicating effective loss of glutamatergic fibres. Striatal glutamate binding increased by approximately 30% and this supersensitivity could be attributed solely to an increased receptor density. Ibotenate lesions which destroy target neurones for the glutamatergic fibres (sparing terminals), reduced glutamate binding in the striatum, as did nigral 6-OHDA lesions which delete striatal dopaminergic terminals. This finding supports the concept of there being glutamate receptors on pre-synaptic dopamine terminals in the striatum, involved in regulation of dopamine release. 6-OHDA lesions also result in a supersensitivity of the dopamine receptors localized on the cortico-striatal afferent terminals, as evidenced by the enhanced ability of dopamine to inhibit the K+-evoked, calcium-dependent release of endogenous striatal glutamate.

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.

The transsulfuration pathway: a source of cysteine for glutathione in astrocytes.

Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (γ-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the γ-glutamylcycle leading to formation of glutathione (l-γ-glutamyl-l-cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the xc− cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from the indispensable amino acid, methionine, has recently been identified as a significant contributor to GSH synthesis in astrocytes. The purpose of this review is to evaluate the importance of the transsulfuration pathway in these cells, particularly in the context of a reserve pathway that channels methionine towards cysteine when the demand for glutathione is high, or under conditions in which the supply of cystine by the xc− exchanger may be compromised.

Chronic infusion ofl-glutamate causes neurotoxicity in rat striatum

Chronic infusion of a high dose of monosodium glutamate (approximately 8 nmol/min) into the rat left striatum, over a period of 1 week, caused degeneration of striatal neurones. This was accompanied by a significant loss of neurochemical markers for both GABAergic and cholinergic interneurones. These results indicate that the sustained presence of elevated concentrations of glutamate will, in time, give rise to changes similar to those seen in human neurodegenerative disorders, such as Huntingtons disease.

Reduction of striatal N-methyl-d-aspartate toxicity by inhibition of nitric oxide synthase

Coronal slices of rat brain were incubated for 40 min in 300 microM kainate (KA) or 500 microM N-methyl-D-aspartate (NMDA). Histological examination showed neuronal degeneration accompanied by significant losses in the activity of neuron-specific enolase (NSE; EC 4.2.1.11) (-23% KA; -26% NMDA). The activity of the glial enzyme glutamine synthetase (GS; EC 6.3.1.2) was also reduced (-32% KA; -27% NMDA). Pre-incubation with 100 microM L-NG-nitroarginine (L-N-ARG), an inhibitor of nitric oxide (NO) synthase (EC 1.14.23.-), for 20 min attenuated the toxicity of toxicity of NMDA, but not KA. NSE levels after successive incubation in L-N-ARG and NMDA were 95% of controls incubated in Krebs bicarbonate medium only (GS activity 89% of controls). In contrast, pre-incubation with L-N-ARG prior to the addition of KA resulted in neuronal degeneration and significant reductions in NSE levels and GS activities. These observations suggest that the unrestricted function of NO synthase is significant in mediating NMDA neurotoxicity whereas KA toxicity is associated with alternative mechanisms not linked to NO production.

A glutamatergic innervation of the nucleus basalis/substantia innominata

The possibility of there being a glutamatergic innervation of the nucleus basalis/substantia innominata, was investigated in the rat. High affinity glutamate uptake, and a calcium-dependent, potassium-evoked release of endogenous glutamate in this tissue, were demonstrated. In contrast to the striatum, however (which is known to receive a major, probably glutamatergic, corticofugal input), removal of the fronto-parietal cortex failed to modify these parameters. Thus, while the nucleus basalis/substantia innominata appears to possess an important glutamatergic innervation, its origins are as yet unknown. The existence of such an innervation, however, may be of relevance for the degeneration in Alzheimers disease of the magno-cellular cholinergic neurones, which show a particular sensitivity to excitotoxic agents.

Astroglial plasticity and glutamate function in a chronic mouse model of Parkinson's disease.

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases, astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1. However, in Parkinsons disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse model of Parkinsons disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8 weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the progressive nature of the pathophysiology associated with Parkinsons disease.

Inhibition of the glutamate transporter and glial enzymes in rat striatum by the gliotoxin, ocaminoadipate

1 The effect of the gliotoxic analogue of glutamate, ocaminoadipate, on the high affinity transport of D‐[3H]‐aspartate into a crude striatal P2 preparation, and on the activity of two enzymes of which glutamate is the substrate has been examined. 2 The L‐isomer of ocaminoadipate competitively inhibited the transport protein, with a Ki value of 192 μm, whereas the D‐isomer of ocaminoadipate was ineffective. The potent convulsant, L‐methionine‐S‐sulphoximine, was also without effect on the activity of the gluatmate transport protein. 3 L‐αAminoadipate was a competitive inhibitor of both glutamine synthetase, and γ‐glutamylcysteine synthetase, with Ki values of 209 μm and 7 mm respectively. Once again, the D‐isomer of ocaminoadipate was a far weaker inhibitor of either enzyme. 4 The results are discussed in terms of the mechanism of action of ocaminoadipate in causing toxicity of glial cells.