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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 neuron-enriched cultures.A) MPP+ concentration-dependent effect on dopaminergic and total neuron viability expressed respectively as percentage of TH- and MAP-2-IR cell number in comparison to control cultures (n = 5). B) Urate concentration-dependent effect on TH-IR cell number in 3 µM MPP+-treated cultures (n = 7). C) Lack of urate effect at any concentration on TH-IR neuron number in control (MPP+-untreated) cultures (n = 5). Photomicrographs show TH-IR neurons in D) control cultures, E) MPP+/0 urate-treated cultures, F) MPP+/0.1 urate-treated cultures and G) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs 0 MPP+ value; #P<0.05, ##P<0.01 vs MPP+/0 urate value.
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pone-0037331-g002: Urate's protective effect on dopaminergic neurons in neuron-enriched cultures.A) MPP+ concentration-dependent effect on dopaminergic and total neuron viability expressed respectively as percentage of TH- and MAP-2-IR cell number in comparison to control cultures (n = 5). B) Urate concentration-dependent effect on TH-IR cell number in 3 µM MPP+-treated cultures (n = 7). C) Lack of urate effect at any concentration on TH-IR neuron number in control (MPP+-untreated) cultures (n = 5). Photomicrographs show TH-IR neurons in D) control cultures, E) MPP+/0 urate-treated cultures, F) MPP+/0.1 urate-treated cultures and G) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs 0 MPP+ value; #P<0.05, ##P<0.01 vs MPP+/0 urate value.

Mentions: To identify an MPP+ concentration with selective toxicity for dopaminergic neurons, mesencephalic neuron-enriched cultures (Fig. 1A–D) were treated for 24 hours with increasing concentrations of MPP+. Toxicant treatment reduced the number of dopaminergic neurons, which were identified by their immunoreactivity for tyrosine hydroxylase (TH), in a concentration-dependent manner (P<0.0001). There was no change in the total number of neurons, which were scored as microtubule-associated protein 2-immunoreactive (MAP-2-IR) cells (Fig. 2A), due to the selectively toxic effect of MPP+ on dopaminergic neurons and their low number in ventral mesencephalon cultures (2–3% of MAP-2-IR cells; see also Materials and Methods). To assess the effect of urate on dopaminergic neuron viability, neuron-enriched cultures were pretreated with urate 24 hours before and during exposure to 3 µM MPP+. In MPP+-treated cultures urate increased TH-IR viability over a concentration range of 0.1–100 µM (P<0.0001). The maximum effect was achieved at 100 µM with a 51% increase in TH-IR cell number in comparison to cells treated with MPP+ only (P<0.01). Half-maximally effective concentration (EC50) was achieved at a concentration of 1 µM [95% confidence interval (95%CI): 0.096–5.9] (Fig. 2B, D–G). Urate on its own produced no significant effect on dopaminergic neuron viability (Fig. 2C).


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 neuron-enriched cultures.A) MPP+ concentration-dependent effect on dopaminergic and total neuron viability expressed respectively as percentage of TH- and MAP-2-IR cell number in comparison to control cultures (n = 5). B) Urate concentration-dependent effect on TH-IR cell number in 3 µM MPP+-treated cultures (n = 7). C) Lack of urate effect at any concentration on TH-IR neuron number in control (MPP+-untreated) cultures (n = 5). Photomicrographs show TH-IR neurons in D) control cultures, E) MPP+/0 urate-treated cultures, F) MPP+/0.1 urate-treated cultures and G) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs 0 MPP+ value; #P<0.05, ##P<0.01 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-g002: Urate's protective effect on dopaminergic neurons in neuron-enriched cultures.A) MPP+ concentration-dependent effect on dopaminergic and total neuron viability expressed respectively as percentage of TH- and MAP-2-IR cell number in comparison to control cultures (n = 5). B) Urate concentration-dependent effect on TH-IR cell number in 3 µM MPP+-treated cultures (n = 7). C) Lack of urate effect at any concentration on TH-IR neuron number in control (MPP+-untreated) cultures (n = 5). Photomicrographs show TH-IR neurons in D) control cultures, E) MPP+/0 urate-treated cultures, F) MPP+/0.1 urate-treated cultures and G) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs 0 MPP+ value; #P<0.05, ##P<0.01 vs MPP+/0 urate value.
Mentions: To identify an MPP+ concentration with selective toxicity for dopaminergic neurons, mesencephalic neuron-enriched cultures (Fig. 1A–D) were treated for 24 hours with increasing concentrations of MPP+. Toxicant treatment reduced the number of dopaminergic neurons, which were identified by their immunoreactivity for tyrosine hydroxylase (TH), in a concentration-dependent manner (P<0.0001). There was no change in the total number of neurons, which were scored as microtubule-associated protein 2-immunoreactive (MAP-2-IR) cells (Fig. 2A), due to the selectively toxic effect of MPP+ on dopaminergic neurons and their low number in ventral mesencephalon cultures (2–3% of MAP-2-IR cells; see also Materials and Methods). To assess the effect of urate on dopaminergic neuron viability, neuron-enriched cultures were pretreated with urate 24 hours before and during exposure to 3 µM MPP+. In MPP+-treated cultures urate increased TH-IR viability over a concentration range of 0.1–100 µM (P<0.0001). The maximum effect was achieved at 100 µM with a 51% increase in TH-IR cell number in comparison to cells treated with MPP+ only (P<0.01). Half-maximally effective concentration (EC50) was achieved at a concentration of 1 µM [95% confidence interval (95%CI): 0.096–5.9] (Fig. 2B, D–G). Urate on its own produced no significant effect on dopaminergic neuron viability (Fig. 2C).

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