L-DOPA Neurotoxicity Is Mediated by Up-Regulation of DMT1−IRE Expression

2008-2009 > Academic research: refereed > Publication in refereed journalpublished_fina

Effects of astrocyte-conditioned medium on the expression of DMT1−IRE, DMT1+IRE, Fpn1 and TfR1 proteins in cortical neurons treated with L-DOPA.

<p>Neurons were treated with L-DOPA (0, 100 or 200 µM) in ACM for 16 hours and Western blot analysis was then conducted. A, representative Western blots of DMT1−IRE and β-actin. B, the relative values of DMT1−IRE protein expression. C, representative Western blots of DMT1+IRE and β-actin. D, the relative values of DMT1+IRE protein expression. E, representative Western blots of Fpn1 and β-actin. F, the relative values of Fpn1 protein expression. G, representative Western blots of TfR1 and β-actin. H, the relative values of TfR1 protein expression. Data are means±SEM (percentage of control) of four independent experiments. There were no significant differences in the levels of all four proteins we investigated between the neurons treated with or without L-DOPA.</p

Effects of L-DOPA on the expression of DMT1−IRE, DMT1+IRE, Fpn1 and TfR1 proteins in cortical neurons.

<p>Neurons were treated with L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS for 16 hours and the expression of proteins was determined using Western blot analysis. A, representative Western blots of DMT1−IRE and β-actin. B, the relative values of DMT1−IRE protein expression. C, representative Western blots of DMT1+IRE and β-actin. D, the relative values of DMT1+IRE protein expression. E, representative Western blots of Fpn1 and β-actin. F, the relative values of Fpn1 protein expression. G, representative Western blots of TfR1 and β-actin. H, the relative values of TfR1 protein expression. Data are means±SEM (percentage of control) of four independent experiments. L-DOPA treatment induced a dose-dependent decrease in TfR. There were no significant differences in the levels of DMT1+IRE and Fpn1 protein between neurons treated with and without L-DOPA. * P<0.05, ** P<0.01 vs. the control.</p

Immunocytochemistry of MAP2 in cortical neurons treated with or without L-DOPA.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004593#s2" target="_blank">Materials and Methods</a>.</p

Effects of L-DOPA on neuronal viability.

<p>Neurons were treated with 0, 1, 5, 10, 100 or 200 µM of L-DOPA in DMEM+5%FBS for 16 hours (A) or incubated with 0, 10, 100 or 200 µM of L-DOPA in DMEM +5%FBS medium (L-DOPA) or astrocyte-conditioned medium (L-DOPA+ACM) for 16 hours (B). The neuronal viability was then determined using an MTT assay. Data are the mean±SEM (percentage of control) of three independent experiments performed in triplicate. A: *P<0.01, **P<0.001, vs. the control (0 µM). B: **P<0.001, ***P<0.0001 vs. the corresponding controls.</p

Effects of L-DOPA on neuronal iron.

<p>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 iron staining was conducted. Total iron was also measured using a GFAAS method in the neurons treated with or without different concentrations of L-DOPA in the presence or absence of ACM (C). Data are the mean±SEM (percentage of control) of three independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. the control (0 µM); #<0.01 vs. 200 µM of L-DOPA.</p

Hoechst 33342 staining in cortical neurons treated with or without L-DOPA.

<p>Neurons were exposed to 0, 1, 5, 10, 100 or 200 µM of L-DOPA in DMEM+5%FBS (A) or 0 or 200 µM of L-DOPA in astrocyte-conditioned medium (ACM) (B) for 16 hours and then Hoechst 33342 staining was performed. The nuclei with fluorescing intensely, shrunken in size and irregular in shape are indicated in arrows.</p

L–DOPA induced morphological changes in cortical neurons.

<p>Neurons were treated with 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 morphological changes were observed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004593#s2" target="_blank">Materials and Methods</a>.</p

Effects of L-DOPA on 55Fe(II) uptake by cortical cells.

<p>Neurons were treated with L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS for 16 hours and then incubated with 2 µM of 55Fe(II) in 0.27 M sucrose (pH 6.5) at 37°C for 30 min. The 55Fe(II) taken into neurons was then measured. Data were presented as mean±SEM of 6 independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. the control.</p

Effects of siRNA DMT−IRE on DMT1−IRE expression and L-DOPA-induced neurontoxicity in cortical neurons.

<p>A and B: Neurons were pre-incubated with siRNA DMT−IRE for 24 hours and Western blots analysis was then performed (A: representative Western blots of DMT1−IRE and β-actin, and B: the relative values of DMT1−IRE protein in neurons infected with or without siRNA DMT−IRE). Data are means±SEM (percentage of control) of four independent experiments performed in triplicate. **P<0.01 vs. the control. C: Neurons were pre-incubated with siRNA DMT−IRE for 24 hours and then treated with different concentrations of L-DOPA (0, 1, 5, 10, 100, 200 µM) in DMEM+5%FBS medium fro 16 hours. The neuronal viability was then determined using an MTT assay. Data are the mean±SEM (percentage of control) of four independent experiments performed in triplicate. *P<0.05, ***P<0.001 vs. the corresponding controls.</p