JNK signalling regulates antioxidant responses in neurons

Abstract

Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and as a consequence of pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. In Drosophila mutants with enhanced JNK activity, lower levels of ROS are observed and these animals are resistant to both changes in ROS and changes in synapse morphology induced by oxidative stress. In wild type flies, disrupting JNK-AP-1 signalling perturbs redox homeostasis suggesting JNK activity positively regulates neuronal antioxidant defense. We validated this hypothesis in mammalian neurons, finding that JNK activity regulates the expression of the antioxidant gene Srxn-1, in a c-Jun dependent manner. We describe a conserved 'adaptive' role for neuronal JNK in the maintenance of redox homeostasis that is relevant to several neurodegenerative diseases.

Keywords: Drosophila; Glutathione; Hydrogen Peroxide; ROS.

Fig. 1

JNK activity regulates neuronal antioxidant responsesin Drosophila. A DEM induces oxidative stress in wild type flies but not puckered (puc^E69/+) or highwire (hiw) mutants. Quantification of hydrogen peroxide levels (Amplex red fluorescence) in wild type, puc^E69/+ or hiw mutant flies administered vehicle (0.16% ethanol) or DEM (5 mM). B Quantification of hydrogen peroxide levels in hiw mutants and hiw mutants pan-neuronally expressing dominant negative jnk (hiw; nSyb > bsk^R53K), wnd (hiw; nSyb > wnd^DN), ask1 (hiw; nSyb > ask1^DN), jun (hiw; nSyb > jun^DN) and fos (hiw; nSyb > fos^DN). C Quantification of hydrogen peroxide levels in wild type flies, pan-neuronally expressing dominant negative jnk (nSyb > bsk^R53K), wnd (nSyb > wnd^DN) and ask1 (nSyb > ask1^DN). A-C Graphs show Amplex red fluorescence values normalized to the average signal of wild type flies. Data are plotted as mean ± SEM (**p < 0.01; ***p < 0.001; one-way ANOVA; minimum 15 flies per genotype). D Representative micrographs showing synaptic overgrowth at the Drosophila third instar larval NMJ (Muscle 6/7, hemi-segment A3) in wild type, puc^E69/+ mutants and wild type flies expressing dominant negative fos (fos^DN), jun (jun^DN) and ask1 (ask1^DN), reared on food containing ethanol or DEM. Scale bar = 30 μm. E Quantification of mean normalized bouton number from (D) genotypes. Data are plotted as mean ± SEM (***p < 0.001; one-way ANOVA; minimum 15 NMJ's analysed per genotype). F Schematic showing the contribution of JNK to neuronal antioxidant stress responses in Drosophila. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

ROS induced JNK phosphorylation triggers neuronal antioxidant responses. A Representative Western blot of neurons treated with Bicuculline (50 μM, Bic) and 4-Aminopyridine (500 μM, 4AP) to increase synaptic activity (Bic/4AP) or DEM (100 μM) for 1 h, to induce oxidative stress. JNK activity was inhibited by a 1 h pre-treatment with SU-3327 (SU, 700 nM). B Quantification of total phospho-JNK/pan-JNK ratios (*p < 0.05; **p < 0.01; one-way ANOVA; n = 3 biological replicates). C, D The mRNA levels of Srxn-1 and c-Fos were determined using quantitative PCR (qPCR) and were normalized to Gapdh mRNA levels. mRNA levels are expressed relative to untreated controls and shown as means ± SEM (*p < 0.05; **p < 0.01; unpaired t-Test; n = 4 biological replicates) CSrxn-1 mRNA is induced by oxidative stress in a JNK dependent manner. D JNK signalling downstream of oxidative stress does not require c-Fos. E JNK regulates SRXN-1 expression under oxidative stress but not during synaptic activity. Representative Western blot probed for SRXN-1, Pan-JNK, phospho-JNK, Pan-ERK, phospho-ERK and GAPDH antibodies. Neurons were treated with Bic/4AP (50/500 μM) or DEM (100 μM) in the presence or absence of either the JNK inhibitor SU 3327 (700 nM) or the NMDA receptor blocker MK801 (10 μM) for 30 min (lanes 2/4/6) or 4 h (lanes 3/5/7/8/9).

Fig. 3

JNK Recruits c-Jun for SRXN-1 expression. A, B Oxidative stress reduces c-Fos and increases c-Jun. Representative images showing c-Fos (top panels) and c-Jun (bottom panels) immunoreactivity in neurons left untreated (Control) or treated for 1 h with 100 μM DEM. Neurons were co-stained for the neuronal marker NeuN and nuclei were labelled with DAPI. B Quantification of immunoreactivity in A, of neurons stimulated for 1 h or 4 h with 100 μM DEM. Data are plotted as mean ± SEM. Analysis represents NeuN positive neurons. c-Fos levels were assessed in 118 neurons and c-Jun immunoreactivity was quantified in 81 neurons across 3 biological replicates (**p < 0.01; ***p < 0.001 one-way ANOVA followed by a Dunnett's post-hoc test). Scale bar = 25 μm. C Representative Western blot of neuronal lysates immunoprecipitated with a c-Jun antibody in conditions of synaptic activity (Bic/4AP) or oxidative stress (DEM 100 μM) in the presence or absence the JNK inhibitor SU 3327 (700 nM, 1 h). D Representative images of c-Jun immunofluorescence in HEK293T cells transduced with lentivirus encoding empty vector, or pLenti c-Jun. Scale bar = 25 μm. E Western blot showing c-Jun, GAPDH and SRXN-1 levels in neurons transduced with lentivirus containing empty vector (1) or c-Jun (3) overnight, or treated with the Nrf2 agonist, tert-Butylhydroquinone (TBHQ, 10 μM, 4) overnight. HEK293T cells transduced with pLenti c-Jun (2) were run as a positive control. F A schematic demonstrating JNK and c-Jun dependent regulation of SRXN-1 during oxidative stress.

Fig. 4

SRXN-1 localises to synaptic terminals in rodent neurons and rescues DEM induced retraction A SRXN-1 overexpression rescues DEM-induced dendritic retraction. Representative micrographs of mature neurons transfected with PSD95-GFP constructs alone (left panels) or in combination with Flag-tagged human SRXN-1 (right panels) ± 100 μM DEM (48hr). Cells stained with anti-GFP (green), anti SRXN-1 (Flag antibody, red) and nuclear staining with DAPI (blue). Scale bar = 100 μm. B Overexpressed human SRXN-1 co-localises with PSD95-GFP in dendritic spines. Representative images show an overlay of GFP and Flag immunofluorescence. Magnified images of dendritic spines outlined in white boxes (PSD95-GFP + SRXN-1, scale bar = 5 μm). C Endogenous SRXN-1 protein is present at synapses. Representative Western blot of synaptosome fractions prepared from mouse brain, probed with antibodies for the nuclear protein FOXO3a, the pre-synaptic marker synapsin 1 (Syn1), the mitochondrial protein ATP5A1, JNK and SRXN-1 as indicated. D Schematic representation of likely signalling pathways mediating Srxn-1 transcriptional induction in response to synaptic activity and oxidative stress. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. S1

Effects of DEM on NMJ bouton number and survival in Drosophila.A DEM (0 mM, 1 mM, 5 mM and 10 mM) was added to standard food and NMJ bouton number quantified. NMJ bouton number increases as DEM concentrations increase. (left y-axis, bars). Survival significantly decreases as DEM concentrations increase (right y-axis, red line). Data plotted as means ± SEM analysed using one-way ANOVA with a Dunnett's post hoc (**p < 0.01).

Fig. S2

DEM depletes glutathione and induces oxidative stress. A. Total glutathione content in mature neurons treated with DEM at the indicated time and concentration. Total glutathione content is relative to ethanol treated controls and data are shown as means ± SEM (**p < 0.01; ***p < 0.001, using two-way ANOVA followed by a Dunnett's post-hoc test; n = 3 biological replicates). B Mitochondrial toxicity was assessed using WST-1 assays in neurons treated with DEM, paraquat (PQ) and rotenone (RT) at the indicated concentrations, for 24 h. WST-1 turnover is normalized to ethanol treated controls (Veh) and shown as means ± SEM (***p < 0.001; one-way ANOVA; n = 3 biological replicates). C H₂O₂ production was monitored using Amplex red reagent. Neurons were treated with DEM and fluorescence measured after 24 h. Catalase (100 nM) was added to neuronal cultures 15 min before DEM treatment. Data represents means ± SEM analysed using one-way ANOVA (***p < 0.001, n = 3 biological replicates). Amplex red fluorescence is shown as a percentage of vehicle (0.1% ethanol) treated controls. D Prolonged oxidative stress causes dendritic loss. Representative micrographs of neurons transfected with PSD95-GFP and treated, with either 0.1% ethanol (control) or the indicated concentrations of DEM (100 μM) +/- Catalase (100 nM) for 48 h. Scale bar = 50 μm.

Fig. S3

DEM-induced dendritic loss is rescued by GSH overexpression. A-D Mature neurons transfected either with a PSD95-GFP expression plasmid alone (top panels) or PSD-95GFP along with plasmids expressing the catalytic (GCLC) and modifying (GCLM) subunits of Glutamate Cysteine Ligase (bottom panels). Cells were incubated with either vehicle (0.1% ethanol) or DEM (100 μM) for 48 h (scale bar = 100 μm). Insets show anti-GFP (green), anti-GCLC (red) and nuclei (blue).

Fig. S4

Synaptic activity induced c-Fos and c-Jun. A Representative western blot of protein samples from HEK293T cells transfected with a FLAG-tagged Human SRXN-1 expression vector and probed with anti-SRXN-1 mouse monoclonal antibody (top panel, SI Appendix, Table 1) or GAPDH antibody (bottom panel). B Synaptic activity induces c-Fos and c-Jun. Representative images showing c-Fos and c-Jun immunoreactivity in neurons left untreated (Control) or treated for 1 h or 4 h with Bicuculline (50 μM, Bic) and 4-Aminopyridine (500 μM, 4AP). Neurons were co-stained for the neuronal marker NeuN and nuclei were labelled with DAPI. Scale bar = 20 μm. C The mRNA levels of Srxn-1 were determined using quantitative PCR (qPCR) and were normalized to Gapdh mRNA levels. mRNA levels are expressed relative to ethanol (vehicle, 0.1%) treated controls and shown as means ± SEM (*p < 0.05; **p < 0.01; unpaired t-Test; n = 3. D Graphs showing c-Fos (left) and c-Jun (right) levels in neurons treated for 1 h or 4 h with Bic/4AP. Data are plotted as mean ± SEM. Analysis represents NeuN positive neurons and c-Fos levels were assessed in 135 neurons across 3 biological replicates (***p < 0.001 one-way ANOVA followed by a Dunnett's post-hoc test). c-Jun immunofluorescence was assessed in 78 neurons across 3 biological replicates (***p < 0.001 one-way ANOVA followed by a Dunnett's post-hoc test). CTCF = corrected total cell fluorescence analysis. E Representative immunoprecipitation of c-Jun in conditions of increased synaptic activity (Bic/4AP, 1 h) and oxidative stress (DEM, 100 μM, 1 h) and western blot showing levels of JunB (upper panel) and JunD (lower panel).