Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death

Abstract

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.

Figure 1. GD injures neurons, but not astrocytes in vitro

Mixed cortical cultures or purified astrocytes (inset) were washed into an incubated medium containing (hatched bars) or lacking (black bars) glucose. The percentage of total cell death was determined at the times indicated. Between group differences (*) were determined by one-way (astrocytes) or two-way ANOVA (mixed cultures) followed by Bonferroni's post-hoc test (n = 11–12 cultures from four independent experiments).

Figure 2. Hypoglycaemic neuronal cell death is attenuated by glutamate receptor antagonism

Mixed cortical cultures were washed into a BSS₀ containing vehicle, MK-801 (10 μM) or MK-801 plus CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 μM) for 8 h (GD). Neuronal cell death was determined 20–24 h later. (*) Indicates values significantly different from control conditions ( = 10.56±3.02%) determined 24 h following wash of cells into BSS₀ followed by immediate addition of glucose. (#) Represents a significant diminution of GD-induced cell death as determined by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 11 cultures from three experiments).

Figure 3. Inhibition of synaptic release of glutamate partially attenuates GD-induced neuronal cell death

Mixed cultures were incubated overnight with tetanus toxin (TeNT; 0.3–3 μg/ml) or its vehicle then washed into a BSS₀ (GD). Eight hour later, glucose (10 mM) was added and the cultures were placed back into the incubator, after which neuronal cell death was assessed 20–24 h later. NB: Values for GD indicate that all the neurons (100%) plus some of the astrocytes (7–8%) were killed. (*) Indicates values significantly different from control conditions ( = 2.06±0.8%) determined by washing cells into BSS₀ followed by immediate addition of glucose, whereas (#) represents a significant diminution from GD-induced neuronal cell death as determined by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 3).

Figure 4. System x_c⁻ antagonism prevents neuronal cell death

Mixed cortical cultures were washed into BSS₀ in the presence or absence of the dual system x_c⁻/mGluR1 antagonists 4-CPG (50 μM) and LY367385 (50 μM), or the selective mGluR1 inhibitor, YM298198 (10 μM). Eight hours later, supernatant was collected for measurement of neuronal cell death. (*) Indicates values that are significantly different from control conditions ( = 7.98±0.89%) determined by washing cells into BSS₀ followed by immediate addition of glucose, while (#) represents a significant diminution from GD-induced neuronal death as assessed by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 11–12 cultures from three independent experiments). Right: Representative phase micrographs of mixed cortical cultures: (a) control; (b) 8 h GD; (c) GD+4-CPG (50 μM); (d) GD+YM298198 (10 μM).

Figure 5. Hypoglycaemic neuronal death is dependent on l-cystine

(A) Mixed cultures were deprived of glucose (4 h) in a medium containing (GD) or lacking (−AA) MEM amino acids, l-cystine (−CYSS) alone or l-methionine (−MET) alone. (*) Indicates values significantly different from control ( = 0.55±0.28%); (#) represents a significant diminution from GD-induced neuronal death as determined by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 24 cultures from six independent experiments). (B) Cultures were deprived of glucose (4 h) in a medium containing (GD) or lacking (−AA) MEM amino acids save for supplementation with 100 μM l-cystine (+CYSS) or 100 μM l-methionine (+MET). (*) Indicates values different from control ( = 2.87±0.38% death), while (#) represents a significant diminution from GD-induced neuronal death as determined by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 4). (C) Cultures were deprived of glucose for 8 h in a medium containing MEM amino acids and various concentrations of cystine (0–100 μM), after which neuronal death was assessed. (*) Indicates values significantly different from control conditions ( = 7.11±1.27%) as determined by one-way ANOVA followed by Dunnett's post-hoc test (n = 4).

Figure 6. System x_c⁻ expression and activity is higher in astrocytes than in neurons

(A) Total RNA was isolated from unstimulated pure astrocytes and pure neurons (n = 3 cultures each from three independent experiments), reverse transcribed and relative basal expression of xCT mRNA was assessed via qPCR. Data are expressed as means±S.E.M. fold change in mRNA compared with pure astrocyte cultures (set at 1). No statistical difference was noted. (B) Pure astrocytes (n = 12 cultures from three independent experiments) and pure neurons (n = 8 cultures from two independent experiments) were washed and incubated with an uptake buffer containing ¹⁴C-l-cystine (3 μM) for 30 min. Data are expressed as means±S.E.M. ¹⁴C-l-cystine uptake in pmol/30 min/mg protein. An asterisk (*) denotes values different from neurons as assessed by a Student's t test. Significance was set at P<0.05.

Figure 7. Hypoglycaemic neuronal death is dependent on astrocyte system x_c⁻

Chimaeric cultures were obtained by plating WT neurons on astrocytes derived from sut mice (hatched white bars). These and control cultures (WT neurons on WT astrocytes; black bars) were washed into BSS₀, glucose added (final = 10 mM) immediately to control cultures (0 h) or 8 h later to previously glucose-deprived cells (8 h), and neuronal cell death determined 20–24 h later. (*) Indicates a significant within-group difference, while (#) indicates a significant between-group difference as determined by two-way ANOVA followed by Bonferroni's post-hoc test (n = 15–18 cultures from three independent experiments). Inset: LDH absorbance values for chimaeric and control cultures treated with 250 μM NMDA for 20–24 h.

Figure 8. Enhanced astrocyte system x_c⁻ activity potentiates hypoglycaemic neuronal death

(A) Purified astrocytes (n = 3 from three independent experiments) were incubated with IL-1β or vehicle (media stock supplemented with 0.1% fatty-acid-free BSA) for 6 h, after which xCT mRNA expression was assessed. (*) Denotes values different from 0 h as determined by one-way ANOVA followed by Dunnett's post-hoc test. (B) Mixed cultures were incubated with IL-1β for 20–24 h then washed into BSS₀. Glucose was added after 3.5 h and neuronal cell death determined 20–24 h later. (*) Indicates values different from control (0 ng/ml IL-1β) as determined by one-way ANOVA followed by Dunnett's post-hoc test (n = 16 cultures from four independent experiments). (C) Mixed cultures were incubated with IL-1β (GD+IL-1β) or vehicle (GD) for 20–24 h, then washed into BSS₀ containing 4-CPG (50 μM) or LY367385 (50 μM) or one lacking cystine (−CYSS) for 4 h. (*) Indicates values different from GD. (#) Represents a significant diminution from the IL-1β-mediated potentiation of GD-induced neuronal death as determined by one-way ANOVA followed by Student–Newman–Keul's post-hoc test (n = 6–16 cultures from four independent experiments).