Fig. 3: The triterpenoid CDDO^TFEA can drive α-synuclein in neurons after epigenetic derepression of Nrf2.

A Astrocyte-free neuron cultures were treated ± 1 µM TSA for 16 h and Nrf2 mRNA expression analysed. *P = 0.035 (Student’s t test, n = 3). B Neuronal cultures were transfected on DIV3 with eGFP, plus vectors encoding either α-synuclein or a ß-globin control. 5 days post-transfection, cells were treated ± TSA (1 µM) for 8 h, and subsequently (where indicated) with 10 µM tBHQ for 24 h. Cells were then fixed and processed for α-synuclein immunofluorescence (n = 3-5). C Experiment performed as in Fig. 1c except that CDDO^TFEA (250 nM) was employed instead of tBHQ. *P = 0.033, 2-way ANOVA + Sidak’s post-hoc test (n = 4). D Experiment performed as in Fig. 3c except that western blotting was performed to study α-synuclein levels, rather than immunofluorescence, and only WT astrocytes studied. *P = 0.0003, 2-way ANOVA + Sidak’s post-hoc test (n = 5). Cortical astrocyte cultures (E) or astrocyte-free neuronal cultures (F) were treated with different concentrations of tBHQ or CDDO^TFEA for 8 h, RNA harvested and expression of the Srxn1 measured by qPCR, normalized to Rpl13a. *P values: 0.003, 0.0009, <0.0001, 0.001,0.006, <0.0001; 2-way ANOVA + Dunnett’s post-hoc test (n = 5). Concentrations 1, 2, 3 of tBHQ are 1,10, and 50 µM; concentrations 1, 2, 3 of CDDO^TFEA are 5, 50, and 250 nM as shown in the table inset in (F), as are the structures of tBHQ and CDDO^TFEA. G Same samples as in (E), analysed for Jun levels. ^@P = 0.0045 (major drug effect, 2-way ANOVA); ^#P < 0.0001 (Sidak’s posthoc test); *P <0.0001, <0.0001, 0.001 (Sidak’s posthoc test, n = 5). H Same samples as in (F), analysed for Jun levels. ^@P = 0.0027 (major drug effect, 2-way ANOVA); ^#P < 0.0001 ⁽Sidak’s posthoc test); *P < 0.0001, <0.0001, ^<0.0001 (Sidak’s posthoc test), (n = 5). I GFP-transfected astrocyte-free cortical neuronal cultures (DIV10 ¹⁷) were imaged before and 24 h post-tBHQ (50 µM) or post- CDDO^TFEA (250 nM) treatment, fixed/DAPI-stained, and viability assessed. *P = 0.0001, <0.0001, 1-way ANOVA plus Tukey’s post-hoc test, 76–86 cells analysed per condition (n = 4). J, K Neuronal cultures (WT and Nrf2 KO) were transfected on DIV3 with eGFP, plus vectors encoding either α-synuclein or a ß-globin control. 5 days post-transfection, cells were treated ± TSA (1 µM) for 8 h, and subsequently (where indicated) with 250 nM CDDO^TFEA for 24 h. Cells were then fixed and processed for α-synuclein immunofluorescence as for Fig. 1. *P < 0.0001, 1-way ANOVA + Dunnett’s post-hoc test 68–162 cells analysed per condition across n = 4 independent experiments. K shows example pictures. L Example pictures showing the co-localisation of synapsin and endogenous α-synuclein in puncta of a size consistent with being pre-synaptic boutons (scale bar=10 µm). M Neurons were treated as indicated, similarly to Fig. 3h, and after 24 h cells were fixed and endogenous synapsin and endogenous α-synuclein analysed by immunofluorescence, and Pearson’s colocalization coefficient calculated (ImageJ JACoP plugin, n = 3).