Figure 1.
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.
Figure 2.
Urate's protective effect on dopaminergic neurons in neuron-enriched cultures.
A) MPP+ concentration-dependent effect on dopaminergic and total neuron viability expressed respectively as percentage of TH- and MAP-2-IR cell number in comparison to control cultures (n = 5). B) Urate concentration-dependent effect on TH-IR cell number in 3 µM MPP+-treated cultures (n = 7). C) Lack of urate effect at any concentration on TH-IR neuron number in control (MPP+-untreated) cultures (n = 5). Photomicrographs show TH-IR neurons in D) control cultures, E) MPP+/0 urate-treated cultures, F) MPP+/0.1 urate-treated cultures and G) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: **P<0.01, ***P<0.001 vs 0 MPP+ value; #P<0.05, ##P<0.01 vs MPP+/0 urate value.
Figure 3.
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.
Figure 4.
Urate's protective effect on dopaminergic neurons in mixed cultures.
A) MPP+ concentration-dependent effect on dopaminergic neuron, total neuron and astrocyte viability, expressed as percentage of TH-IR, MAP-2-IR and GFAP-IR cell number, respectively, in comparison to control cultures (n = 4). B) Urate concentration-dependent effect on TH-IR cell number in 0.5 µM MPP+-treated cultures (n = 5). C) Lack of effect of urate at any concentration on TH-IR cell number (n = 5). Urate (100 µM) effects on reductions in D) longest neurite length and E) soma size in MPP+ urate-treated TH-IR neurons. Photomicrographs show TH-IR neurons in F) control cultures, G) MPP+/0 urate-treated cultures and H) MPP+/0.1 urate-treated cultures and I) MPP+/100 µM urate-treated cultures. Scale bar = 50 µm. One-way ANOVA followed by Newman-Keuls test: *P<0.05, **p<0.01, ***P<0.001 vs 0 MPP+ value, ##P<0.01 and ###P<0.001 vs MPP+/0 urate value.
Figure 5.
Urate accumulation in cortical neurons.
A) Time-dependent effect of 100 µM exogenous urate on its intracellular content in primary cortical neurons. One-way ANOVA followed by Newman-Keuls test: **P<0.01.
Figure 6.
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.
Figure 7.
Characterization of non-Tg, Tg and Tg/Tg cortical astrocyte-enriched cultures.
A) Western blot and graph showing UOx immunostaining in non-Tg and UOx-expressing astrocytes (Tg and Tg/Tg) normalized to the β-actin level. Note that UOx was not detected in non-Tg astrocytes (n = 7). B) Effect of UOx expression on the intracellular urate content normalized to the protein level (n = 5). C) UOx activity in the media of non-Tg and UOx-expressing astrocytes (n = 9). D) Effect of UOx expression on extracellular urate concentration in astroglial cultures (n = 6). Some error bars are not visible because of their small size. Student's t test: ###P<0.0001 vs Tg value. One-way ANOVA followed by Newman-Keuls test: *P<0.05, ***P<0.001 vs non-Tg value.
Figure 8.
MPP+ effect on non-Tg, Tg and Tg/Tg neuron-enriched cultures.
A) MPP+ effect on TH-IR cell number in non-Tg (n = 18), Tg (n = 35) and Tg/Tg (n = 8) neuronal cultures. B) MPP+ effect on MAP-2-IR cell number in non-Tg (n = 18), Tg (n = 35) and Tg/Tg (n = 8) cultures. C) Two-way ANOVA followed by Bonferroni multiple comparison test: **P<0.01, ***P<0.001 vs respective non-Tg value; ###P<0.01 vs respective Tg value.
Figure 9.
MPP+ effect on non-Tg, Tg and Tg/Tg mixed neuron-astrocyte cultures.
A) MPP+ effect on TH-IR cell number in non-Tg (n = 18), Tg (n = 34) and Tg/Tg (n = 22) neuronal cultures. B) MPP+ effect on MAP-2-IR cell number in non-Tg (n = 18), Tg (n = 34) and Tg/Tg (n = 22). Two-way ANOVA followed by Bonferroni multiple comparison test: *P<0.05, **P<0.01, ***P<0.001 vs respective non-Tg value.