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L-DOPA neurotoxicity is mediated by up-regulation of DMT1-IRE expression.

Du F, Qian ZM, Zhu L, Wu XM, Yung WH, Tsim TY, Ke Y - PLoS ONE (2009)

Bottom Line: Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability.Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1-IRE expression.In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1-IRE expression as well as L-DOPA neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong.

ABSTRACT

Background: The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1-IRE) induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM) and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons.

Methods and findings: We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay) and increase in iron content (using a graphite furnace atomic absorption spectrophotometer), DMT1-IRE expression (Western blot analysis) and ferrous iron (55Fe(II)) uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1-IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1-IRE expression as well as L-DOPA neurotoxicity.

Conclusion: The up-regulation of DMT1-IRE and the increase in DMT1-IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.

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Related in: MedlinePlus

Immunocytochemistry of MAP2 in cortical neurons treated with or without L-DOPA.Neurons were exposed to L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS (A) or astrocyte-conditioned medium (ACM) (B) for 16 hours and then immunostained for MAP-2 antibody as described in Materials and Methods.
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pone-0004593-g002: Immunocytochemistry of MAP2 in cortical neurons treated with or without L-DOPA.Neurons were exposed to L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS (A) or astrocyte-conditioned medium (ACM) (B) for 16 hours and then immunostained for MAP-2 antibody as described in Materials and Methods.

Mentions: Figure 1A presented the morphological observations in neurons treated with L-DOPA (0, 1, 5, 10, 100 or 200 µM) in DMEM+5%FBS for 16 hours. As compared to the controls, neurons pre-treated with L-DOPA displayed a dose-dependent morphological change. It was characterized by a progressive increase in the number of irregular cell bodies with disrupted and shrunken neuronal processes with the increase in the concentrations of L-DOPA in the medium. Pre-treatment with 200 µM of L-DOPA induced a significant reduction in or disappearance of the neuronal number or processes. The observations under phase contrast microscopy corresponded to the findings of immunostaining of cortical neurons for MAP2 (Figure 2A). Hoechst 33342 staining (Figure 3A) displayed a gradual increase in the number of condensed or fragmented nuclei (apoptosis) in cortical cells with the increase in the concentrations of L-DOPA. The number of condensed nuclei was the highest in the neurons pretreated with 200 µM of L-DOPA. The neuronal viability was determined using an MTT assay. As shown in Figure 4A, treatment of neurons with L-DOPA in DMEM+5%FBS for 16 hours led to a progressive decrease in the cell viability with the increase in the concentrations of L-DOPA. A significant decrease in viability was found in the neurons treated with 10, 100 or 200 µM of L-DOPA (P<0.01 or 0.001).


L-DOPA neurotoxicity is mediated by up-regulation of DMT1-IRE expression.

Du F, Qian ZM, Zhu L, Wu XM, Yung WH, Tsim TY, Ke Y - PLoS ONE (2009)

Immunocytochemistry of MAP2 in cortical neurons treated with or without L-DOPA.Neurons were exposed to L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS (A) or astrocyte-conditioned medium (ACM) (B) for 16 hours and then immunostained for MAP-2 antibody as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2643485&req=5

pone-0004593-g002: Immunocytochemistry of MAP2 in cortical neurons treated with or without L-DOPA.Neurons were exposed to L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS (A) or astrocyte-conditioned medium (ACM) (B) for 16 hours and then immunostained for MAP-2 antibody as described in Materials and Methods.
Mentions: Figure 1A presented the morphological observations in neurons treated with L-DOPA (0, 1, 5, 10, 100 or 200 µM) in DMEM+5%FBS for 16 hours. As compared to the controls, neurons pre-treated with L-DOPA displayed a dose-dependent morphological change. It was characterized by a progressive increase in the number of irregular cell bodies with disrupted and shrunken neuronal processes with the increase in the concentrations of L-DOPA in the medium. Pre-treatment with 200 µM of L-DOPA induced a significant reduction in or disappearance of the neuronal number or processes. The observations under phase contrast microscopy corresponded to the findings of immunostaining of cortical neurons for MAP2 (Figure 2A). Hoechst 33342 staining (Figure 3A) displayed a gradual increase in the number of condensed or fragmented nuclei (apoptosis) in cortical cells with the increase in the concentrations of L-DOPA. The number of condensed nuclei was the highest in the neurons pretreated with 200 µM of L-DOPA. The neuronal viability was determined using an MTT assay. As shown in Figure 4A, treatment of neurons with L-DOPA in DMEM+5%FBS for 16 hours led to a progressive decrease in the cell viability with the increase in the concentrations of L-DOPA. A significant decrease in viability was found in the neurons treated with 10, 100 or 200 µM of L-DOPA (P<0.01 or 0.001).

Bottom Line: Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability.Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1-IRE expression.In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1-IRE expression as well as L-DOPA neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong.

ABSTRACT

Background: The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1-IRE) induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM) and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons.

Methods and findings: We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay) and increase in iron content (using a graphite furnace atomic absorption spectrophotometer), DMT1-IRE expression (Western blot analysis) and ferrous iron (55Fe(II)) uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1-IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1-IRE expression as well as L-DOPA neurotoxicity.

Conclusion: The up-regulation of DMT1-IRE and the increase in DMT1-IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.

Show MeSH
Related in: MedlinePlus