Dysregulation of system xc(-) expression induced by mutant huntingtin in a striatal neuronal cell line and in R6/2 mice

Abstract

Oxidative stress has been implicated in the pathogenesis of Huntington's disease (HD), however, the origin of the oxidative stress is unknown. System xc(-) plays a role in the import of cystine to synthesize the antioxidant glutathione. We found in the STHdh(Q7/Q7) and STHdh(Q111/Q111) striatal cell lines, derived from neuronal precursor cells isolated from knock-in mice containing 7 or 111 CAG repeats in the huntingtin gene, that there is a decrease in system xc(-) function. System xc(-) is composed of two proteins, the substrate specific transporter, xCT, and an anchoring protein, CD98. The decrease in function in system xc(-) that we observed is caused by a decrease in xCT mRNA and protein expression in the STHdh(Q111/Q111) cells. In addition, we found a decrease in protein and mRNA expression in the transgenic R6/2 HD mouse model at 6weeks of age. STHdh(Q111/Q111) cells have lower basal levels of GSH and higher basal levels of ROS. Acute inhibition of system xc(-) causes greater increase in oxidative stress in the STHdh(Q111/Q111) cells than in the STHdh(Q7/Q7) cells. These results suggest that a defect in the regulation of xCT may be involved in the pathogenesis of HD by compromising xCT expression and increasing susceptibility to oxidative stress.

Keywords: Glutamate uptake; Glutathione; Huntington’s disease; Oxidative stress; STHdh cells; xCT.

Figure 1

Na⁺-dependent and independent L-glutamate uptake in STHdh cells, showing the relative contribution of each to total glutamate uptake. A, Glutamate uptake was measured using ³[H] L-glutamate in the presence and absence of sodium, in which case sodium was replaced by choline. Sodium dependent uptake represents total uptake with uptake in the absence of sodium subtracted. Sodium independent uptake is the uptake in the choline medium with background subtracted. A representative saturation analysis is shown. Best fit line was established using the Michaelis-Menten equation. Error bars represent SEM, n=3. B, Na-independent glutamate uptake. Data from A, expanded scale. C. The V_max values for Na-independent glutamate uptake in the Q7 and Q111 cells were determined from the saturation analysis, error bars represent SEM; unpaired Student's t test: t(4)=12.2 *** p<0.001, n=3; Mann-Whitney test, p<0.05. D, The K_m values for Q7 and Q111 cells were also determined from the saturation analysis. Error bars represent SEM, n=3; unpaired Student's t test t(4)=1.5, p>0.05; Mann-Whitney test, p>0.05.

Figure 2

System x_c inhibitors inhibited Na⁺-independent L-glutamate. A, Glutamate uptake was measured using ³[H] L-glutamate in the absence of sodium (choline buffer). Inhibitors or vehicle were added to the uptake buffer: L-homocysteic acid (HCA; 1 mM), sulfasalazine (SSZ; 250 μM), 4-(S)-carboxyphenylglycine (CPG; 10 μM), L-cystine (L-CySS) at 100 and 200 μM (blue and green bars). Error bars represent SEM; two-way ANOVA main effect of treatment: F(5, 132)=212.3, p<0.001; main effect of cell type: F(1, 132)=835.4, p<0.001; interaction: F(5, 132)=125.2, p<0.001; followed by Tukey's multiple comparison test comparing each treatment to control for both cell types: *** p<0.001; Kruskal-Wallis followed by Dunn's post hoc test, p<0.0001. B, Percent of Na⁺-independent uptake inhibited compared to control, error bars represent SEM, two-way ANOVA main effect of cell type: F(1, 132)=0.4, p>0.05; followed by Tukey's multiple comparison test comparing between cell types of each treatment, p=0.5218, n=3.

Figure 3

xCT protein expression was decreased in STHdh^Q111/Q111 cells. A, Western blot of xCT protein expression in STHdh^Q7/Q7 (lanes 1-3) and STHdh^Q111/Q111 (lanes 4-6); specific bands (see “B”) are indicated by arrows at ∼50 and ∼100 kDa. B, Western blot with anti- xCT antibody pre-adsorbed using the immunogenic peptide; arrows indicate the position of bands at ∼50 and ∼100 kDa lost with pre-adsorption. C, Densitometry of total xCT protein (bands at ∼50 and ∼100 kDa) normalized to β-actin in the STHdh cells. Error bars represent SEM; unpaired Student's t test: t(4)=11.8, *** p<0.001, n=3; Mann-Whitney test, p<0.05.

Figure 4

xCT protein expression was decreased in the striatum of 6 week old R6/2 mice. Western blot of xCT protein expression in the cortex (A) and striatum (D), of WT (lanes 1-3) and HD (lanes 4-6) littermates. Bands corresponding to xCT are indicated by arrows at ∼50 and ∼100 kDa. B,E, Western blot with pre-absorbed xCT antibody using immunogenic peptide for the antibody; arrows indicate the loss of xCT specific bands at ∼50 and ∼100 kDa. Densitometry of total xCT protein levels normalized to β-III tubulin in the cortex (C) and the striatum (F). Error bars represent SEM: unpaired Student's t test: striatum t(4)=4.8, * p<0.05; cortex t(4)=1.1, p>0.05, n=3; Mann-Whitney test for striatum, p<0.05.

Figure 5

xCT mRNA expression was decreased in STHdh^Q111/Q111 cells and in 6 week old R6/2 mice. A, xCT mRNA expression normalized to HPRT in STHdh cells. Error bars represent SEM; unpaired Student's t test: t(7)=7.4, *** p<0.001, n=8; Mann-Whitney test p<0.05. B, xCT mRNA expression in the cortex (lanes 1-2) and striatum (lanes 3-4) in WT and HD littermates. Error bars represent SEM; unpaired Student's t test: striatum t(12)=2.6, * p<0.05; cortex t(12)=1.5, p>0.05, n=6; Mann-Whitney test for striatum, p<0.05.

Figure 6

GSH content was decreased and ROS expression was increased in STHdh^Q111/Q111 cells. A, STHdh cells treated with vehicle (control), 1 mM HCA, 100 μM SSZ, and 100 μM tBHQ for 2, 8, and 24 hours. Nuclei were stained with DAPI and ROS with CellROX Deep Red Reagent; scale bar 25 μm. B, Quantification of fluorescence intensity from cells treated with CellROX Deep Red Reagent. Errors bars represent SEM; two-way ANOVA main effect of treatment by cell type: F(7, 785)=532.3, p<0.001; main effect of time: F(2, 785)=160.4, p<0.001; interaction: F(14, 785)=30.4, p<0.001) followed by Tukey's multiple comparison test: * p<0.05 (comparing STHdh^Q7/Q7 control cells to STHdh^Q111/Q111 control cells), ** p<0.01 (comparing STHdh^Q7/Q7 control to tBHQ treated cells; and STHdh^Q111/Q111 control to HCA, SSZ, and tBHQ treated cells), n=50; Kruskal-Wallis followed by Dunn's post hoc test, p<0.001. C, Total GSH levels in STHdh cells treated with vehicle (control), 1 mM HCA, 100 μM SSZ, and 100 μM tBHQ for 2, 8, and 24 hours. Error bars represent SEM; n=3 two-way ANOVA main effect of treatment by cell type: F(7, 24)=281.5, p<0.001; main effect of time: F(2, 24)=75.6, p<0.001; interaction: F(14, 24)=59.4, p<0.001) followed by Tukey's multiple comparison test, **p<0.01 (comparing basal GSH levels in control STHdh cells; and treated and control STHdh cells at 24 hrs); Kruskal-Wallis followed by Dunn's post hoc test, p<0.01.

Figure 7

STHdh^Q111/Q111 are more susceptible to oxidative cell death. A, STHdh cells treated with 0, 1, 3, 10, 30, 100, 300, 1000 μM DEM for 24 hours. Error bars represent SEM; n=4. B, LD₅₀ of STHdh cells treated with DEM. Error bars represent SEM; unpaired Student's t-test: t(6)=6.1, p<0.001, n=4; Mann-Whitney test, p<0.05.