Protection of dopaminergic cells by urate requires its accumulation in astrocytes

Abstract

Urate is the end product of purine metabolism and a major antioxidant circulating in humans. Recent data link higher levels of urate with a reduced risk of developing Parkinson's disease and with a slower rate of its progression. In this study, we investigated the role of astrocytes in urate-induced protection of dopaminergic cells in a cellular model of Parkinson's disease. In mixed cultures of dopaminergic cells and astrocytes oxidative stress-induced cell death and protein damage were reduced by urate. By contrast, urate was not protective in pure dopaminergic cell cultures. Physical contact between dopaminergic cells and astrocytes was not required for astrocyte-dependent rescue as shown by conditioned medium experiments. Urate accumulation in dopaminergic cells and astrocytes was blocked by pharmacological inhibitors of urate transporters expressed differentially in these cells. The ability of a urate transport blocker to prevent urate accumulation into astroglial (but not dopaminergic) cells predicted its ability to prevent dopaminergic cell death. Transgenic expression of uricase reduced urate accumulation in astrocytes and attenuated the protective influence of urate on dopaminergic cells. These data indicate that urate might act within astrocytes to trigger release of molecule(s) that are protective for dopaminergic cells.

Fig. 1

Protection of MES 23.5 cells by urate is mediated by astrocytes. (A) Cell viability in MES 23.5 cultures after 24 hours of H₂O₂ treatment at indicated concentrations. One-way ANOVA: n = 3, *p < 0.05, **p < 0.001, ***p < 0.001 vs 0 value. (B) Effect of urate (n = 4) treatment on 200 μM H₂O₂-induced cell death in MES 23.5 cultures. (C) Cell viability of MES 23.5 cells cultured for 24 hours with increasing H₂O₂ concentrations and astrocyte densities. Ratio between astrocytes and MES 23.5 cells is shown in symbol key. Two-way ANOVA: n = 3; **p < 0.01 vs 0 and 1:5. (D) Effect of urate treatment on 200 μM H₂O₂-induced cell death in co-cultures (1:5::astrocytes/MES 23.5). One-way ANOVA: n = 13; *p < 0.05, **p < 0.01, ***p < 0.001 vs respective 0 value. Photomicrograph of (E) untreated, (F) H₂O₂-treated, (G) H₂O₂+100 μM urate-treated and MES 23.5 cells (green) cultured on astrocytes (red), DAPI staining was used to label nuclei (blue). Scale bar is 50 μm. The dashed line indicates the control value (100%) against which the other values were measured.

Fig. 2

Urate reduced ROS and protein oxidation in MES 23.5 cells cultured with astrocytes. (A) NO₂⁻ release in co-culture medium after 200 μM H₂O₂ treatment for the indicated times. One- way ANOVA: n = 15; *p < 0.05 vs 0′ value. (B) Effect of urate treatment on H₂O₂-induced NO₂ release at 24 hours of treatment. One-way ANOVA: n = 16; **P < 0.01 and ***P < 0.001 vs control and urate values, ^##p < 0.01 vs H₂O₂ value. (C) Protein carbonyl content in MES 23.5 cells cultured with astrocytes after 200 μM H₂O₂ treatment for the indicated times. One-way ANOVA: n = 5; *p < 0.05 vs 0 value. (D) Effect of urate treatment on H₂O₂-induced protein carbonylation at 3 hours of treatment with H₂O₂. One-way ANOVA: n = 7; **p < 0.01 and ***p < 0.001 vs control and urate values; ^##p < 0.05 vs H₂O₂ value.

Fig. 3

(A) Effect of increasing percentages of conditioned medium from control or urate-treated astrocytes on MES 23.5 cell viability. Two-way ANOVA: n = 15; **p < 0.01 and ***p < 0.001 vs respective control value. (B) Effect of uricase (UOx; 0.12 U/l) on conditioned medium from urate-treated astrocytes on MES 23.5 cell viability (n = 4). UOx or vehicle was added to astroglial cultures after 24 hours of urate treatment; conditioned media were collected after 15 more hours.

Fig. 4

Urate’s protective effect depends on its accumulation in astrocytes. (A) Western blotting shows URAT1, GLUT9 and OAT1 urate transporter expression in extracts of MES 23.5 cells and astrocytes. Kidney extract was used as positive control. (B) Urate concentration in MES 23.5 cells and (C) astrocytes treated with urate transporter inhibitors: pyrazinoate (PZO), hydrochlorothiazide (HCTZ) or probenecid (PBN), at the indicated concentrations (mM) (n = 4–7). (D) Effects of urate transporter inhibitors: PZO, HCTZ and PBN, at the indicated concentrations (mM), on MES 23.5 cell viability in H₂O₂-treated co-cultures expressed as percentage of control (n = 4–6). One-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 vs 0 value.

Fig. 5

UOx expression in transgenic (Tg) astrocytes reduced urate intracellular content. (A) Uricase (UOx) expression in Tg UOx astrocytes with none detected (ND) in wild-type (WT) astrocytes. (B) Western blot of UOx immunoreactivity in 50 μg of WT and Tg astrocytes; liver extract (10 μg) was used as positive control. Actin was used as loading control. (C) Basal intracellular urate content in WT and Tg astrocytes. Student’s t test: n = 5; *p = 0.02. (D) Basal UOx activity in media from WT and Tg astrocytes. Student’s t test: n = 10, *p = 0.015.

Fig. 6

UOx expression in transgenic astrocytes reduced astrocyte-mediated protection of H₂O₂- treated MES 23.5 cells. (A) Medium urate concentration in 100 μM urate-treated WT and Tg astroglial cultures over the time (n = 3; ***P < 0.001 vs respective WT value). Error bars were smaller than symbols for all data points. (B) Intracellular urate content in WT and Tg astrocytes after 8 hours of treatment with 100 μM urate; Student’s t test (n = 4, *p = 0.02). (C) Effect of conditioned medium collected from urate-treated WT and Tg astrocytes on viability of 200 μM H₂O₂-treated cells (n = 4; *p < 0.05 vs respective Tg value).