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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-astrocyte cultures.Composite fluorescence photomicrographs of neuron-astrocyte cultures that were immuno-stained with A–C) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red) or C) the oligondendrocyte marker CNPase (red, not detected). D) Dopaminergic neurons were stained with the dopaminergic neuron marker TH (red). Nuclei were counterstained with DAPI; scale bar is 100 µm.
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pone-0037331-g003: Cellular composition of neuron-astrocyte cultures.Composite fluorescence photomicrographs of neuron-astrocyte cultures that were immuno-stained with A–C) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red) or C) the oligondendrocyte marker CNPase (red, not detected). D) Dopaminergic neurons were stained with the dopaminergic neuron marker TH (red). Nuclei were counterstained with DAPI; scale bar is 100 µm.

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)

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

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getmorefigures.php?uid=PMC3351394&req=5

pone-0037331-g003: Cellular composition of neuron-astrocyte cultures.Composite fluorescence photomicrographs of neuron-astrocyte cultures that were immuno-stained with A–C) the neuronal marker MAP-2 (green) together with A) astrocyte marker GFAP (red) or B) the microglia marker CD11b (red) or C) the oligondendrocyte marker CNPase (red, not detected). D) Dopaminergic neurons were stained with the dopaminergic neuron marker TH (red). Nuclei were counterstained with DAPI; scale bar is 100 µm.
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