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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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Characterization of non-Tg, Tg and Tg/Tg cortical neuron-enriched cultures.A) Western blot and graph showing UOx expression in wild-type (non-Tg) and UOx-expressing neurons (Tg and Tg/Tg) normalized to the β-actin level. Note that UOx was not detected in wild-type neurons (n = 3). B) Effect of UOx expression on intracellular urate content in neurons normalized to the protein level (n = 3). C) UOx activity in the media of non-Tg and UOx-expressing neurons (n = 6). Student's t test: ##P = 0.005 vs Tg value; one-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs non-Tg value and ###P<0.001 vs Tg value.
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pone-0037331-g006: Characterization of non-Tg, Tg and Tg/Tg cortical neuron-enriched cultures.A) Western blot and graph showing UOx expression in wild-type (non-Tg) and UOx-expressing neurons (Tg and Tg/Tg) normalized to the β-actin level. Note that UOx was not detected in wild-type neurons (n = 3). B) Effect of UOx expression on intracellular urate content in neurons normalized to the protein level (n = 3). C) UOx activity in the media of non-Tg and UOx-expressing neurons (n = 6). Student's t test: ##P = 0.005 vs Tg value; one-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs non-Tg value and ###P<0.001 vs Tg value.

Mentions: To assess whether intracellular urate content affects dopaminergic neuron resistance to MPP+ we prepared ventral mesencephalon cultures from a mouse line expressing transgenic uricase (UOx) [37], the enzyme that converts urate to allantoin. Intracellular urate content was measured in cortical neurons and astrocytes prepared from non-transgenic UOx (non-Tg), hemizygous transgenic UOx (Tg) and homozygous (double) transgenic UOx (Tg/Tg) mice. In Tg/Tg neurons UOx expression was about 6 times higher than in Tg neurons as assessed by western blotting; in non-Tg neurons UOx was not detected (Fig. 6A). UOx expression reduced intracellular urate content by 50% (P<0.01) and 60% (P<0.01) in Tg and Tg/Tg neurons, respectively (Fig. 6B). UOx activity was significantly increased in Tg/Tg (p<0.001) but not in Tg cell medium in comparison to non-Tg samples (Fig. 6C). In Tg/Tg astrocytes intracellular UOx expression was about 15 times higher than in Tg astrocytes; in non-Tg astrocytes UOx was not detected (Fig. 7A). UOx expression reduced intracellular urate content by 30% both in Tg and Tg/Tg (P<0.01) astrocytes (Fig. 7B). UOx activity was detected in the cell media of Tg and Tg/Tg astrocytes (Fig. 7C) where medium urate concentration was significantly reduced in comparison to that from non-Tg astrocytes (P<0.001) (Fig. 7D).


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)

Characterization of non-Tg, Tg and Tg/Tg cortical neuron-enriched cultures.A) Western blot and graph showing UOx expression in wild-type (non-Tg) and UOx-expressing neurons (Tg and Tg/Tg) normalized to the β-actin level. Note that UOx was not detected in wild-type neurons (n = 3). B) Effect of UOx expression on intracellular urate content in neurons normalized to the protein level (n = 3). C) UOx activity in the media of non-Tg and UOx-expressing neurons (n = 6). Student's t test: ##P = 0.005 vs Tg value; one-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs non-Tg value and ###P<0.001 vs Tg value.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037331-g006: Characterization of non-Tg, Tg and Tg/Tg cortical neuron-enriched cultures.A) Western blot and graph showing UOx expression in wild-type (non-Tg) and UOx-expressing neurons (Tg and Tg/Tg) normalized to the β-actin level. Note that UOx was not detected in wild-type neurons (n = 3). B) Effect of UOx expression on intracellular urate content in neurons normalized to the protein level (n = 3). C) UOx activity in the media of non-Tg and UOx-expressing neurons (n = 6). Student's t test: ##P = 0.005 vs Tg value; one-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs non-Tg value and ###P<0.001 vs Tg value.
Mentions: To assess whether intracellular urate content affects dopaminergic neuron resistance to MPP+ we prepared ventral mesencephalon cultures from a mouse line expressing transgenic uricase (UOx) [37], the enzyme that converts urate to allantoin. Intracellular urate content was measured in cortical neurons and astrocytes prepared from non-transgenic UOx (non-Tg), hemizygous transgenic UOx (Tg) and homozygous (double) transgenic UOx (Tg/Tg) mice. In Tg/Tg neurons UOx expression was about 6 times higher than in Tg neurons as assessed by western blotting; in non-Tg neurons UOx was not detected (Fig. 6A). UOx expression reduced intracellular urate content by 50% (P<0.01) and 60% (P<0.01) in Tg and Tg/Tg neurons, respectively (Fig. 6B). UOx activity was significantly increased in Tg/Tg (p<0.001) but not in Tg cell medium in comparison to non-Tg samples (Fig. 6C). In Tg/Tg astrocytes intracellular UOx expression was about 15 times higher than in Tg astrocytes; in non-Tg astrocytes UOx was not detected (Fig. 7A). UOx expression reduced intracellular urate content by 30% both in Tg and Tg/Tg (P<0.01) astrocytes (Fig. 7B). UOx activity was detected in the cell media of Tg and Tg/Tg astrocytes (Fig. 7C) where medium urate concentration was significantly reduced in comparison to that from non-Tg astrocytes (P<0.001) (Fig. 7D).

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