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Urate and its transgenic depletion modulate neuronal vulnerability in a cellular model of Parkinson's disease.

Cipriani S, Desjardins CA, Burdett TC, Xu Y, Xu K, Schwarzschild MA - PLoS ONE (2012)

Bottom Line: In this study we investigated the effects of modulating intracellular urate concentration on 1-methyl-4-phenyl-pyridinium (MPP(+))-induced degeneration of dopaminergic neurons in cultures of mouse ventral mesencephalon prepared to contain low (neuron-enriched cultures) or high (neuron-glial cultures) percentage of astrocytes.To assess the effect of reducing cellular urate content on MPP(+)-induced toxicity, mesencephalic neurons were prepared from mice over-expressing urate oxidase (UOx).Dopaminergic neurons expressing UOx were more susceptible to MPP(+) in mesencephalic neuron-enriched cultures and to a greater extent in mesencephalic neuron-astrocyte cultures.

View Article: PubMed Central - PubMed

Affiliation: Neurology Department, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, Massachusetts, United States of America. scipriani@partners.org

ABSTRACT
Urate is a major antioxidant as well as the enzymatic end product of purine metabolism in humans. Higher levels correlate with a reduced risk of developing Parkinson's disease (PD) and with a slower rate of PD progression. In this study we investigated the effects of modulating intracellular urate concentration on 1-methyl-4-phenyl-pyridinium (MPP(+))-induced degeneration of dopaminergic neurons in cultures of mouse ventral mesencephalon prepared to contain low (neuron-enriched cultures) or high (neuron-glial cultures) percentage of astrocytes. Urate, added to the cultures 24 hours before and during treatment with MPP(+), attenuated the loss of dopaminergic neurons in neuron-enriched cultures and fully prevented their loss and atrophy in neuron-astrocyte cultures. Exogenous urate was found to increase intracellular urate content in cortical neuronal cultures. To assess the effect of reducing cellular urate content on MPP(+)-induced toxicity, mesencephalic neurons were prepared from mice over-expressing urate oxidase (UOx). Transgenic UOx expression decreased endogenous urate content both in neurons and astrocytes. Dopaminergic neurons expressing UOx were more susceptible to MPP(+) in mesencephalic neuron-enriched cultures and to a greater extent in mesencephalic neuron-astrocyte cultures. Our findings correlate intracellular urate content in dopaminergic neurons with their toxin resistance in a cellular model of PD and suggest a facilitative role for astrocytes in the neuroprotective effect of urate.

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Urate's protective effect on dopaminergic neurons in mixed cultures.A) MPP+ concentration-dependent effect on dopaminergic neuron, total neuron and astrocyte viability, expressed as percentage of TH-IR, MAP-2-IR and GFAP-IR cell number, respectively, in comparison to control cultures (n = 4). B) Urate concentration-dependent effect on TH-IR cell number in 0.5 µM MPP+-treated cultures (n = 5). C) Lack of effect of urate at any concentration on TH-IR cell number (n = 5). Urate (100 µM) effects on reductions in D) longest neurite length and E) soma size in MPP+ urate-treated TH-IR neurons. Photomicrographs show TH-IR neurons in F) control cultures, G) MPP+/0 urate-treated cultures and H) MPP+/0.1 urate-treated cultures and I) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: *P<0.05, **p<0.01, ***P<0.001 vs 0 MPP+ value, ##P<0.01 and ###P<0.001 vs MPP+/0 urate value.
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pone-0037331-g004: Urate's protective effect on dopaminergic neurons in mixed cultures.A) MPP+ concentration-dependent effect on dopaminergic neuron, total neuron and astrocyte viability, expressed as percentage of TH-IR, MAP-2-IR and GFAP-IR cell number, respectively, in comparison to control cultures (n = 4). B) Urate concentration-dependent effect on TH-IR cell number in 0.5 µM MPP+-treated cultures (n = 5). C) Lack of effect of urate at any concentration on TH-IR cell number (n = 5). Urate (100 µM) effects on reductions in D) longest neurite length and E) soma size in MPP+ urate-treated TH-IR neurons. Photomicrographs show TH-IR neurons in F) control cultures, G) MPP+/0 urate-treated cultures and H) MPP+/0.1 urate-treated cultures and I) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: *P<0.05, **p<0.01, ***P<0.001 vs 0 MPP+ value, ##P<0.01 and ###P<0.001 vs MPP+/0 urate value.

Mentions: Previous data [35] have shown that urate's protective effect against toxin-induced neuronal cell death can be dependent on the presence of astrocytes in cultures. In our study urate treatment in neuron-enriched cultures only partially attenuated MPP+ toxicity on dopaminergic neurons. To assess whether astrocytes might potentiate the protective effect of urate in our cells, urate was tested in MPP+-treated mixed neuron-astrocyte cultures (Fig. 3A–D). To obtain selective degeneration of dopaminergic neurons without toxic effect on non-TH-IR cells, cultures were treated with relatively low concentrations of MPP+ for four days as previously described [36]. MPP+ induced selective loss of TH-IR neurons in a concentration-dependent manner (P = 0.0005) with no statistically significant effect on MAP-2-IR or glial fibrillary acid protein-immunoreactive (GFAP-IR) cells (Fig. 4A). To assess the effect of urate, neuron-astrocyte cultures were pretreated with urate 24 hours before and during exposure to 0.5 µM MPP+. Urate increased the number of TH-IR neurons over a concentration range of 0.1–100 µM (P<0.0001). The maximum effect was seen at 100 µM with a 97% increase in the number of TH-IR neurons in comparison to cultures treated with MPP+ only (P<0.01; Fig. 4B, F–I), corresponding to a complete blockade of MPP+ toxicity. Urate on its own did not affect TH-IR cell number (Fig. 4C). No statistically significant difference was seen at the estimated EC50's for urate in neuron-enriched and neuron-astrocytes cultures (∼1 µM in both; F1,53 = 0.01, P = 0.9).


Urate and its transgenic depletion modulate neuronal vulnerability in a cellular model of Parkinson's disease.

Cipriani S, Desjardins CA, Burdett TC, Xu Y, Xu K, Schwarzschild MA - PLoS ONE (2012)

Urate's protective effect on dopaminergic neurons in mixed cultures.A) MPP+ concentration-dependent effect on dopaminergic neuron, total neuron and astrocyte viability, expressed as percentage of TH-IR, MAP-2-IR and GFAP-IR cell number, respectively, in comparison to control cultures (n = 4). B) Urate concentration-dependent effect on TH-IR cell number in 0.5 µM MPP+-treated cultures (n = 5). C) Lack of effect of urate at any concentration on TH-IR cell number (n = 5). Urate (100 µM) effects on reductions in D) longest neurite length and E) soma size in MPP+ urate-treated TH-IR neurons. Photomicrographs show TH-IR neurons in F) control cultures, G) MPP+/0 urate-treated cultures and H) MPP+/0.1 urate-treated cultures and I) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: *P<0.05, **p<0.01, ***P<0.001 vs 0 MPP+ value, ##P<0.01 and ###P<0.001 vs MPP+/0 urate value.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351394&req=5

pone-0037331-g004: Urate's protective effect on dopaminergic neurons in mixed cultures.A) MPP+ concentration-dependent effect on dopaminergic neuron, total neuron and astrocyte viability, expressed as percentage of TH-IR, MAP-2-IR and GFAP-IR cell number, respectively, in comparison to control cultures (n = 4). B) Urate concentration-dependent effect on TH-IR cell number in 0.5 µM MPP+-treated cultures (n = 5). C) Lack of effect of urate at any concentration on TH-IR cell number (n = 5). Urate (100 µM) effects on reductions in D) longest neurite length and E) soma size in MPP+ urate-treated TH-IR neurons. Photomicrographs show TH-IR neurons in F) control cultures, G) MPP+/0 urate-treated cultures and H) MPP+/0.1 urate-treated cultures and I) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: *P<0.05, **p<0.01, ***P<0.001 vs 0 MPP+ value, ##P<0.01 and ###P<0.001 vs MPP+/0 urate value.
Mentions: Previous data [35] have shown that urate's protective effect against toxin-induced neuronal cell death can be dependent on the presence of astrocytes in cultures. In our study urate treatment in neuron-enriched cultures only partially attenuated MPP+ toxicity on dopaminergic neurons. To assess whether astrocytes might potentiate the protective effect of urate in our cells, urate was tested in MPP+-treated mixed neuron-astrocyte cultures (Fig. 3A–D). To obtain selective degeneration of dopaminergic neurons without toxic effect on non-TH-IR cells, cultures were treated with relatively low concentrations of MPP+ for four days as previously described [36]. MPP+ induced selective loss of TH-IR neurons in a concentration-dependent manner (P = 0.0005) with no statistically significant effect on MAP-2-IR or glial fibrillary acid protein-immunoreactive (GFAP-IR) cells (Fig. 4A). To assess the effect of urate, neuron-astrocyte cultures were pretreated with urate 24 hours before and during exposure to 0.5 µM MPP+. Urate increased the number of TH-IR neurons over a concentration range of 0.1–100 µM (P<0.0001). The maximum effect was seen at 100 µM with a 97% increase in the number of TH-IR neurons in comparison to cultures treated with MPP+ only (P<0.01; Fig. 4B, F–I), corresponding to a complete blockade of MPP+ toxicity. Urate on its own did not affect TH-IR cell number (Fig. 4C). No statistically significant difference was seen at the estimated EC50's for urate in neuron-enriched and neuron-astrocytes cultures (∼1 µM in both; F1,53 = 0.01, P = 0.9).

Bottom Line: In this study we investigated the effects of modulating intracellular urate concentration on 1-methyl-4-phenyl-pyridinium (MPP(+))-induced degeneration of dopaminergic neurons in cultures of mouse ventral mesencephalon prepared to contain low (neuron-enriched cultures) or high (neuron-glial cultures) percentage of astrocytes.To assess the effect of reducing cellular urate content on MPP(+)-induced toxicity, mesencephalic neurons were prepared from mice over-expressing urate oxidase (UOx).Dopaminergic neurons expressing UOx were more susceptible to MPP(+) in mesencephalic neuron-enriched cultures and to a greater extent in mesencephalic neuron-astrocyte cultures.

View Article: PubMed Central - PubMed

Affiliation: Neurology Department, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, Massachusetts, United States of America. scipriani@partners.org

ABSTRACT
Urate is a major antioxidant as well as the enzymatic end product of purine metabolism in humans. Higher levels correlate with a reduced risk of developing Parkinson's disease (PD) and with a slower rate of PD progression. In this study we investigated the effects of modulating intracellular urate concentration on 1-methyl-4-phenyl-pyridinium (MPP(+))-induced degeneration of dopaminergic neurons in cultures of mouse ventral mesencephalon prepared to contain low (neuron-enriched cultures) or high (neuron-glial cultures) percentage of astrocytes. Urate, added to the cultures 24 hours before and during treatment with MPP(+), attenuated the loss of dopaminergic neurons in neuron-enriched cultures and fully prevented their loss and atrophy in neuron-astrocyte cultures. Exogenous urate was found to increase intracellular urate content in cortical neuronal cultures. To assess the effect of reducing cellular urate content on MPP(+)-induced toxicity, mesencephalic neurons were prepared from mice over-expressing urate oxidase (UOx). Transgenic UOx expression decreased endogenous urate content both in neurons and astrocytes. Dopaminergic neurons expressing UOx were more susceptible to MPP(+) in mesencephalic neuron-enriched cultures and to a greater extent in mesencephalic neuron-astrocyte cultures. Our findings correlate intracellular urate content in dopaminergic neurons with their toxin resistance in a cellular model of PD and suggest a facilitative role for astrocytes in the neuroprotective effect of urate.

Show MeSH
Related in: MedlinePlus