Disrupted and transgenic urate oxidase alter urate and dopaminergic neurodegeneration

Abstract

Urate is the end product of purine metabolism in humans, owing to the evolutionary disruption of the gene encoding urate oxidase (UOx). Elevated urate can cause gout and urolithiasis and is associated with cardiovascular and other diseases. However, urate also possesses antioxidant and neuroprotective properties. Recent convergence of epidemiological and clinical data has identified urate as a predictor of both reduced risk and favorable progression of Parkinson's disease (PD). In rodents, functional UOx catalyzes urate oxidation to allantoin. We found that UOx KO mice with a constitutive mutation of the gene have increased concentrations of brain urate. By contrast, UOx transgenic (Tg) mice overexpressing the enzyme have reduced brain urate concentrations. Effects of the complementary UOx manipulations were assessed in a mouse intrastriatal 6-hydroxydopamine (6-OHDA) model of hemiparkinsonism. UOx KO mice exhibit attenuated toxic effects of 6-OHDA on nigral dopaminergic cell counts, striatal dopamine content, and rotational behavior. Conversely, Tg overexpression of UOx exacerbates these morphological, neurochemical, and functional lesions of the dopaminergic nigrostriatal pathway. Together our data support a neuroprotective role of endogenous urate in dopaminergic neurons and strengthen the rationale for developing urate-elevating strategies as potential disease-modifying therapy for PD.

Fig. 1.

Altered urate in serum and brain in UOx KO and Tg mice. (A) Western blot of UOx showing the absence of UOx in liver, heart, and brain in a UOx KO mouse (10 mo old). UOx is expressed in liver, heart, and brain in a UOx Tg mouse (12 mo old), and it is not detectable in heart and brain in WT mice. HPLC analysis indicates elevated urate levels in blood (B) and brain (C) in UOx KO mice. (D) Changes in urate levels are not accompanied by changes in concentrations of urate precursors adenosine (Ado), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), or urate metabolite allantoin (Alt) in brain in UOx KO mice. Conversely, UOx Tg mice have lower urate levels in blood (E) and brain (F). Overexpression of UOx also leads to an increase in striatal Alt in the Tg mice (G). There are no significant differences in striatal Ado, Ino, Hyp, or Xan between UOx Tg mice and the non-Tg WT littermate controls (G). Data are expressed as mean ± SEM. n = 6, WT and UOx KO (8–10 mo old); n = 9, WT and UOx Tg (4–5 mo old). *P < 0.05 vs. WT; **P < 0.01 vs. WT.

Fig. 2.

Kidney to body weight ratios and urea levels in UOx KO and Tg mice. (A) UOx KO have significantly lower kidney to body weight ratio than WT mice (both kidneys from each animal; 4 mo old; n = 11 and 8 WT and UOx KO, respectively). (B) HPLC demonstrates a marked increase in brain urea level in UOx KO mice (3–4 mo old; n = 5, both WT and UOx KO). (C) Kidney to body weight ratio in UOx Tg mice (both kidneys from each animal. 6 mo old; n = 11 and 14 for WT and UOx Tg, respectively). (D) Brain urea is not changed in UOx Tg mice (4–5 mo old; n = 5, WT and UOx Tg). Data are expressed as mean ± SEM. **P < 0.01 vs. WT.

Fig. 3.

UOx disruption or overexpression changes levels of protein carbonyls in mice. Protein carbonyls were assessed by Oxyblot. Band density was normalized with Ponceau staining of the proteins. (A) A trend toward increased levels of protein carbonyls in liver in UOx KO animals. (B) Protein carbonyls are higher in brain in UOx KO mice. Overexpression of UOx leads to increased protein carbonyl content in both liver (C) and brain (D) in Tg mice. Data are expressed as mean ± SEM n = 6, WT and UOx KO (3–4 mo old); n = 5, WT and UOx Tg (4–5 mo old). *P < 0.05; **P < 0.01 vs. WT.

Fig. 4.

UOx KO mice are more resistant to 6-OHDA neurotoxicity. (A) 6-OHDA (15 μg) was infused into the left striatum of UOx KO mice (average age, 3 mo). Spontaneous and 5 mg/kg amphetamine-induced rotational behavior were recorded at 3 and 4 wk after the lesion. Animals were killed at 5 wk after 6-OHDA lesion. (B) Spontaneous net ipsilateral rotations in UOx KO mice are attenuated (*P < 0.05 vs. WT), with a similar trend for attenuated amphetamine-induced net ipsilateral turns (WT n = 11; UOx KO n = 9). (C) UOx KO animals have significantly higher levels of DA and HVA on the experimental (lesion) side compared with their WT littermates (WT n = 11; UOx KO n = 8). *P < 0.05; **P < 0.01 vs. WT experimental side. (D) HPLC-electrochemical detection (ECD) confirms an increased level of urate in the UOx KO group. Injection of 6-OHDA induces an increase in urate in WT mice (n = 11 and 9 WT and UOx KO, respectively). *P < 0.05; **P < 0.01 vs. WT control side. (E) Preservation of SN dopaminergic neurons (TH positive) on the lesion side in a UOx KO mouse and disruption of SN TH neurons in a WT mouse. (F) Stereological quantification of TH neurons in the SN demonstrates more surviving dopaminergic neurons in the KO mice (n = 8 both WT and UOx KO groups). *P < 0.05 vs. WT experimental side. Data are expressed as mean ± SEM. CON, control nonlesion side; EXP, experimental lesion side.

Fig. 5.

UOx Tg mice are more susceptible to 6-OHDA neurotoxicity. UOx Tg mice (average age, 5 mo) received intrastriatal 6-OHDA infusion. Behavioral tests were performed and animals killed at time points indicated in Fig. 4A. (A) Marked increases in both spontaneous and amphetamine-induced net ipsilateral rotations in UOx Tg mice after 6-OHDA infusion compared with WT non-Tg mice (n = 11 WT; n = 14 UOx Tg). *P < 0.05; **P < 0.01 vs. WT. (B) UOx Tg mice had lower urate levels in the striatum, and 6-OHDA induces local increases in urate in both WT and Tg mice (n = 11 and 14 WT and UOx Tg, respectively). **P < 0.01 vs. WT nonlesion control side; ^#P < 0.05 vs. WT experimental side, and vs. UOx Tg control side. (C) Significant further reductions in DA and DOPAC on experimental side in UOx Tg animals after 6-OHDA lesion (n = 11 and 14 WT and UOx Tg, respectively). *P < 0.05 vs. WT experimental side. (D) A significant decrease in the number of nigral TH-positive neurons on the experimental side in UOx Tg mice, compared with WT littermates. The difference is still significant statistically when expressing as percentage of CON to normalize for the difference on control side between the two genotypes (n = 6 and 7 WT and UOx Tg, respectively). *P < 0.05 vs. WT nonlesion control side; ^##P < 0.01 vs. WT experimental side. (E) Few remaining TH-positive neurons in the SN in a UOx Tg mouse after intrastriatal 6-OHDA. Data are expressed as mean ± SEM. CON, control nonlesion side; EXP, experimental lesion side.