Fig. 4: Tyrosine induces oxidative DNA damage in neurons, and cis-RSV protects neurons against it.

a Cis-RSV and trans-RSV have opposite effects on tyrosine-mediated accumulation of γ-H2AX. Immunostaining images (scale bar, 10 µm) for DNA damage marker, pSer139-H2AX foci (γ-H2AX, green; DAPI – nuclear marker, blue) in cortical neurons (DIV 10) after treatment with cis- and trans-RSV (50 µM) alone or in combination with L-tyrosine (250 μM) for 24 hr. The graph represents the average number of γ-H2AX foci per n = 30 neurons per treatment condition for n = 3 experiments. b cis- and trans-RSV have opposite effects on tyrosine-mediated induction of the oxidative damage in the neuronal DNA. Quantification of the levels of 8-oxo-2′-dG using immunofluorescence (IF) in rat primary cortical neurons (DIV 9/10) after treatment with L-tyrosine (500 μM) either alone or in combination with cis or trans-RSV (50 μM) for 16 hr for n = 3 experiments. c D-tyrosine and trans-RSV induce neurite degeneration. Representative images (scale bar, 20 µm) for cortical neurons following D-tyrosine or cis-RSV and trans-RSV (50 µM) for 24 hr treatment (MAP2 – neurite marker, magenta and DAPI – nuclear marker, blue). Neurons were immunoassayed with anti-MAP2 antibody and quantified for neurite degeneration. d Comet assay measuring the extent of DNA strand breaks induced by tyrosine and trans-RSV. Cortical neurons (DIV 9/10) were treated with cis-RSV (50 µM), trans-RSV (50 µM), L-tyrosine (1 mM) either alone or in combination for 1 hr, and the percentage of DNA in the comet tails was quantified as described in the Methods. All data represent mean ± SEM for n = 3 independent experiments with significance measured using two-way ANOVA with Tukey’s test for multiple comparisons.