We recently demonstrated that interleukin-1β (IL-1β) increases system xc− (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes and microglia all express system xc− subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT— the light chain that confers substrate specificity— in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system xc− inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system xc− activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.

The myelin sheath is an extension of the oligoddendrocyte (OL) plasma membrane enriched in lipids which ensheaths the axons of the central and peripheral nervous system. Here we review the involvement of glycosphingolipid in myelin/OL functions; including the regulation of OL differentiation, lipid raft-mediated trafficking and signaling, and neuron-glia interactions.

Astrocytes produce and export the antioxidant glutathione (GSH). Previously, we found that interleukin-1β (IL-1β) enhanced the expression of astrocyte system xc−, the transporter that delivers the rate-limiting substrate for GSH synthesis —cyst(e)ine. Herein, we demonstrate directly that IL-1β mediates a time-dependent increase in extracellular GSH levels in cortical astrocyte cultures, suggesting both enhanced synthesis and export. This increased GSH production was blocked by inhibition of nuclear factor κB (NFκB) activity but not by inhibition of p38 MAPK. To determine whether this increase could provide protection against oxidative stress, the oxidants tert-butyl hydroperoxide (tBOOH) and ferrous sulfate (FeSO4) were employed. IL-1β treatment prevented the increase in reactive oxygen species produced in astrocytes following tBOOH exposure. Additionally, the toxicity induced by tBOOH or FeSO4 exposure was significantly attenuated following treatment with IL-1β, an effect reversed by concomitant exposure to L-buthionine-S,R-sulfoximine (BSO), which prevented the IL-1β-mediated rise in GSH production. IL-1β failed to increase GSH or to provide protection against t-BOOH toxicity in astrocyte cultures derived from IL-1R1 null mutant mice. Overall, our data indicate that under certain conditions IL-1β may be an important stimulus for increasing astrocyte GSH production, and potentially, total antioxidant capacity in brain, via an NFκB-dependent process.

Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc− (xCT). Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

Microglia, resident phagocytic cells of the central nervous system, are frequent contaminants of astrocyte cultures. Unfortunately and not always fully appreciated, contamination by microglia can confound results of studies designed to elucidate the molecular mechanisms underlying astrocyte-specific responses. The paradigm described herein employs the mitotic inhibitor, cytosine β-D: -arabinofuranoside, followed by the lysosomotropic agent, leucine methylester, to maximally deplete microglia, thereby generating highly enriched astrocyte monolayers that remain viable and functional. Successful removal of microglia from confluent monolayers of primary astrocyte cultures is achieved without the need for cell passage and successful reduction is confirmed by depletion of microglial-specific markers.