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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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Cellular composition of neuron-enriched cultures.Composite fluorescence photomicrographs of neuron-enriched cultures that were immuno-stained with A–D) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red, not detected) or C) the oligondendrocyte marker CNPase (red, not detected) or D) the dopaminergic neuron marker TH (yellow). Nuclei were counterstained with DAPI; scale bar length represents 100 µm.
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pone-0037331-g001: Cellular composition of neuron-enriched cultures.Composite fluorescence photomicrographs of neuron-enriched cultures that were immuno-stained with A–D) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red, not detected) or C) the oligondendrocyte marker CNPase (red, not detected) or D) the dopaminergic neuron marker TH (yellow). Nuclei were counterstained with DAPI; scale bar length represents 100 µm.

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)

Cellular composition of neuron-enriched cultures.Composite fluorescence photomicrographs of neuron-enriched cultures that were immuno-stained with A–D) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red, not detected) or C) the oligondendrocyte marker CNPase (red, not detected) or D) the dopaminergic neuron marker TH (yellow). Nuclei were counterstained with DAPI; scale bar length represents 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351394&req=5

pone-0037331-g001: Cellular composition of neuron-enriched cultures.Composite fluorescence photomicrographs of neuron-enriched cultures that were immuno-stained with A–D) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red, not detected) or C) the oligondendrocyte marker CNPase (red, not detected) or D) the dopaminergic neuron marker TH (yellow). Nuclei were counterstained with DAPI; scale bar length represents 100 µm.
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