Targeted de-repression of neuronal Nrf2 inhibits α-synuclein accumulation

Abstract

Many neurodegenerative diseases are associated with neuronal misfolded protein accumulation, indicating a need for proteostasis-promoting strategies. Here we show that de-repressing the transcription factor Nrf2, epigenetically shut-off in early neuronal development, can prevent protein aggregate accumulation. Using a paradigm of α-synuclein accumulation and clearance, we find that the classical electrophilic Nrf2 activator tBHQ promotes endogenous Nrf2-dependent α-synuclein clearance in astrocytes, but not cortical neurons, which mount no Nrf2-dependent transcriptional response. Moreover, due to neuronal Nrf2 shut-off and consequent weak antioxidant defences, electrophilic tBHQ actually induces oxidative neurotoxicity, via Nrf2-independent Jun induction. However, we find that epigenetic de-repression of neuronal Nrf2 enables them to respond to Nrf2 activators to drive α-synuclein clearance. Moreover, activation of neuronal Nrf2 expression using gRNA-targeted dCas9-based transcriptional activation complexes is sufficient to trigger Nrf2-dependent α-synuclein clearance. Thus, targeting reversal of the developmental shut-off of Nrf2 in forebrain neurons may alter neurodegenerative disease trajectory by boosting proteostasis.

Fig. 1. Endogenous Nrf2 cannot clear α-synuclein or support an Nrf2-dependent transcriptional response in neurons, unlike astrocytes.

DIV10 (Days In Vitro) mixed neuron/astrocyte cultures were transfected on DIV3 with vectors encoding eGFP, plus either α-synuclein (pCAGS- α-synuclein) or a ß-globin control. 5 days post-transfection, cells were treated ± tBHQ (10 µM) for 24 h, fixed, and subjected to immunofluorescence cytochemistry (anti- α-synuclein antibody) (BD Transduction Laboratories, #610878 (1:1000)). Immunofluorescence images were taken and quantified (blind). Neurons and astrocytes were distinguished morphologically (astrocytes have larger nuclei, thick proximal processes, and an absence of axons), a method that was validated separately using neuronal (NeuN) and astrocytic (GFAP) markers. A, B Analysis and example pictures of neurons. 43-77 cells per condition, n = 4 independent experiments. Scale bar: 25 µm. C, D Analysis and example pictures of astrocytes. *P = 0.004 2-way ANOVA plus Tukey’s post-hoc test; n = 4 independent experiments (22–30 cells analysed for each condition). Scale bar: 25 µm. E DIV10 mixed neuron/astrocyte cultures were treated ± tBHQ (10 µM,) for 8 h, RNA extracted and subjected to RNA-seq analysis (ca. 35 × 10⁶ paired-end reads/sample, 3 biological replicates; EBI ref: E-MTAB-5688). Mean data for the 11,698 genes expressed on average >2 FPKM is plotted. Differentially expressed genes (DESeq2 v1.6.3, Benjamini-Hochberg-adjusted P value < 0.05) are highlighted red (induced) or blue (repressed). Genes induced >1.5-fold are labelled. F Genes labelled in red in 1e were cross referenced to RNA-seq data from astrocytes over-expressing Nrf2 (FAC-sorted from the GFAP-Nrf2 mouse). Of the 22 genes that were expressed in this GFAP-Nrf2 RNA-seq data set >2 FPKM, the % difference in GFAP-Nrf2 astrocytes (vs. WT) is shown. *(p_adj<0.01, n = 5, see Supplemental Table S1 for exact p values). G qPCR analysis of the indicated genes in RNA extracted from mixed cultures of the indicated genotypes, treated with tBHQ as in 1e. * P value < 0.05 t test, n = 3–5 per condition. H Experiment performed as in (E) except that astrocyte-free neuronal cultures were employed. All 10,845 genes expressed on average >2 FPKM are plotted, and differentially expressed genes (DESeq2 v1.6.3, Benjamini-Hochberg-adjusted P value < 0.05) are highlighted red (induced) or blue (repressed). I Astrocyte-free Nrf2^+/+ or Nrf2^–/– cortical neurons were treated as in (F) and Jun mRNA expression analysed (by qPCR). *P = 0.005, 0.004 (reading left-to-right here and throughout the manuscript), 1-way ANOVA plus Sidak’s post-hoc test (n = 6).

Fig. 2. TBHQ promotes Jun-dependent neurotoxicity.

A, B GFP-transfected cortical neuronal cultures were imaged at the indicated times post-tBHQ treatment, fixed/DAPI-stained, and viability assessed (death = cell disappearance or neurite fragmentation). *P = 0.004, 0.021, 0.0001, 0.0001; 2-way ANOVA + Dunnett’s post-hoc test, 72-121 cells analysed per condition; n = 4 independent biological replicates. C Cells were treated ± 50 µM tBHQ ± qVD-Oph (50 µM) and cell death analysed 24 h later. *P = 0.013, 0.0056, 2-way ANOVA + Sidak’s post-hoc test (n = 4 independent experiments, 481–582 cells analysed per condition). D, E Astrocyte-free neuronal cultures were transfected with eGFP plus either a control vector (ß-globin) or TAM67. The cells were treated with GSH-EE (1 mM) for 1 h where indicated, and then ±50 µM tBHQ. Cell viability was assessed 48 h later. ^#P = 0.0012, 2-way ANOVA + Sidak’s post-hoc test (comparing Control to tBHQ conditions). NB. No significant effect of tBHQ on cell death was observed in TAM67-transfected neurons or GSH-EE treated neurons (relative to corresponding control condition). *P (left-to-right) =0.045 and 0.013, 2-way ANOVA + Sidak’s post-hoc test, n = 6 independent experiments (130-166 cells were analysed per treatment). F Neurons were treated with tBHQ (50 µM) for the indicated times and reduced glutathione levels measured using the monochlorobimane method (See and “Methods”). *P = 0.0436, 0.0003, 2-way ANOVA + Sidak’s post-hoc test (n = 4).

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).

Fig. 4. CDDO^TFEA and HDAC inhibition promote clearance of α-synuclein aggregates.

A–D Neuronal cultures were exposed where indicated to α-synuclein pre-formed fibrils (PFFs) on DIV4 until DIV15 to cause α-synuclein aggregate formation. On DIV15 the neurons were treated where indicated with TSA (1 µM, 8 h) followed by CDDO^TFEA (250 nM, 24 h). Neurons were fixed and processed for immunofluorescence using an α-synuclein aggregate conformation-specific antibody (A, B) or a phospho-(Ser-129)- α-synuclein specific antibody (C, D) A: *P = 1.5E–09, 9.5.E–06, 1-way ANOVA + Sidak’s post-hoc test (n = 5); C: *P = 1.1E–06, 1.2E–04, 1-way ANOVA + Sidak’s post-hoc test (n = 4). B and D show example pictures relating to (A) and (C) respectively. Scale bar: 50 µm. E, F Neuronal cultures were treated as per (A–D), Triton-insoluble proteins were extracted (see “Methods”) and western analysis of oligomeric α-synuclein performed, normalized to ß-actin. *P = 0.0035, 1-way ANOVA + Sidak’s post-hoc test (n = 8). G, H Neuronal cultures were treated as per (E), except that Triton-soluble proteins were extracted (see “Methods”) and α-synuclein monomer expression quantified normalized to ß-actin (n = 8).

Fig. 5. dCas9-based transcriptional activation complexes targeted to the Nrf2 promoter induce α-synuclein clearance.

A Neurons were transfected at DiV 8 with dCas9-VP64, p65 and sgRNAs targeting the Nrf2 promoter (controls were empty sgRNA vectors) with Nrf2-promoter Firefly luciferase and TK-Renilla control. Luciferase activity was measured at 96 h. *P = 0.032, Student’s t test (n = 4). B Nrf2^+/+ and Nrf2^−/− cortical neurons were transfected as in (A) in addition to α-synuclein expression vector and eGFP transfection marker. At Div 11 cells were fixed and processed for GFP and α-synuclein immunofluorescence as for Fig. 1. *P = 0.001, 2-Way ANOVA + Bonferroni’s multiple comparison test, 192–573 cells analysed per condition (n = 7 WT and 5 Nrf2 KO). C Example pictures.