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. 2020 Nov 4;40(45):8734–8745. doi: 10.1523/JNEUROSCI.0940-20.2020

Figure 4.

Figure 4.

4E-BP1-overexpressing neurons are protected against PD-associated environmental toxins. A, DIV14 primary cortical neurons from control and 4E-BP1 transgenic mice were treated with rotenone (10 nm), maneb (1 μm), or paraquat (10 μm), or left untreated. We then measured neurotoxicity by propidium iodide exclusion. n ≥ 3 biological replicates. ANOVA with post hoc Tukey test: ***p = 0.0001 for WT untreated versus WT rotenone-treated (df = 63, EF = 3.1). ***p = 0.0001 for WT untreated versus WT maneb-treated (df = 63, EF = 20.4). ***p = 0.0001 for WT untreated versus WT paraquat-treated (df = 63, EF = 22.1). ***p = 0.0001 for WT rotenone-treated versus 4EBP1 rotenone-treated (df = 69, EF = 1.8). ***p = 0.0001 for WT maneb-treated versus 4EBP1 maneb-treated (df = 71, EF = 11.6). ***p = 0.0001 for WT paraquat-treated versus 4EBP1 paraquat-treated (df = 70, EF = 10.7). B, DIV14 primary cortical neurons from control and 4E-BP1 transgenic mice were treated with rotenone (10 nm), maneb (1 μm), or paraquat (10 μm). We then measured neurotoxicity by quantifying LDH release into the culture media. n ≥ 3 biological replicates. Two-tailed t test: *p = 0.041 for WT rotenone-treated versus 4EBP1 rotenone-treated (df = 7, EF = 1.7). *p = 0.027 for WT maneb-treated versus 4EBP1 maneb-treated (df = 7, EF = 1.0). *p = 0.018 for WT paraquat-treated versus 4EBP1 paraquat-treated (df = 7, EF = 0.4). C, DIV14 primary cortical neurons from control and 4E-BP1 transgenic mice were treated with rotenone (10 nm), immunostained with antibodies against activated caspase-3 and MAP2, and counterstained with DAPI. Cell death is visibly decreased in primary neurons from 4E-BP1 transgenic mice, based on decreased activated caspase-3 nuclear staining and increased MAP2 neurite staining. Scale bar, 10 µm. D, DIV14 primary cortical neurons from control and 4E-BP1 transgenic mice were treated with rotenone (10 nm), and counterstained with DAPI as in C. We then counted the number of neurons showing nuclear condensation. n = 3 biological replicates; n ≥ 50 neurons. *p = 0.048 (two-tailed t test; df = 4; EF = 2.8). E, DIV14 primary cortical neurons from control and 4E-BP1 transgenic mice were treated with rotenone (10 nm), immunostained with antibodies against activated caspase-3 and MAP2, and counterstained with DAPI as in C. We then counted the number of neurons showing activated caspase-3 nuclear staining. n = 3 biological replicates; n ≥ 50 neurons. p = 0.083 (two-tailed t test; df = 4; EF = 2.4). Error bars indicate SEM.