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. 2011;6(7):e21907.
doi: 10.1371/journal.pone.0021907. Epub 2011 Jul 1.

NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease

Anthony L Cook  1 , Alejandra M Vitale, Sugandha Ravishankar, Nicholas Matigian, Greg T Sutherland, Jiangou Shan, Ratneswary Sutharsan, Chris Perry, Peter A Silburn, George D Mellick, Murray L Whitelaw, Christine A Wells, Alan Mackay-Sim, Stephen A Wood
Affiliations

NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease

Anthony L Cook et al. PLoS One. 2011.

Abstract

Background: Without appropriate cellular models the etiology of idiopathic Parkinson's disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson's disease.

Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson's Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson's disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment.

Conclusions: Our results confirmed NRF2 as a potential therapeutic _target for Parkinson's disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. NRF2 depletion recapitulates PD-associated changes in metabolic function.
A. Immunoblot analysis of 3 Control hONS cell lines transiently transfected with either negative control (Neg Cont) siRNAs or siRNAs _targeting NRF2 (NRF2 #09 and NRF2#10). The protein being detected is indicated to the left of the gel panels. B–C. Quantification of NRF2 and NQO1 protein levels relative to GAPDH protein in cultures described in A. D–G. Total GSH content (D) and MTS metabolism (E) were both statistically significantly decreased in Control hONS transfected with NRF2 _targeting siRNAs compared to negative control siRNAs. LDH activity (F) and DNA content (G) were not affected by decreased NRF2 function. For B–G, statistical significance was determined using ANOVA with Dunnett's multiple comparison post-test with the following thresholds: *, p<0.05; **, p<0.01, ***, p<0.001.
Figure 2
Figure 2. NRF2 pathway components are expressed in hONS cultures.
A. Immunoblot analysis of total protein lysates from 6 Control donor-derived and 6 PD patient-derived hONS cell lines. The protein being immunodetected is indicated to the left of each gel panel. B–C. Quantitation of NRF2 (B) and NQO1 (C) protein levels relative to GAPDH.
Figure 3
Figure 3. Dosage effects of L-SUL on cell metabolic function and NRF2 in PD-derived hONS cell metabolism.
A–F. PD patient-derived hONS cultures (N = 4) were treated with the indicated doses of L-SUL for 24 hr and assessed for MTS metabolism (A), total glutathione (GSH) content (B), reduced GSH content (C), DNA content (D), caspase-3/7 activity (E) and membrane integrity (LDH activity, F). Bars show mean and s.e.m., with data presented as fold change compared to vehicle treated cultures for each cell line. For A-F, data was analysed by one-way ANOVA using Dunnett's multiple comparison test. The p value at the bottom of each panel indicates the ANOVA result with p<0.05 considered significant. The asterisks above show the results of Dunnett's multiple comparison post-tests comparing all L-SUL treated cultures to vehicle (Veh) treated cultures; * p<0.05, *** p<0.001. G. Total protein lysates from three hONS cell lines were analysed by immunoblot for NRF2 and KEAP1. GAPDH and β-tubulin were used as loading controls. The protein being immunodetected is indicated to the left of each gel, and the L-SUL dose and cell line are indicated above the blot panels. H. Nuclear extracts of three hONS cell lines were analysed by immunoblot for NRF2 levels using Histone H3 as a loading control. The protein being immunodetected is indicated to the left of each gel, and the L-SUL dose and cell line are indicated above the blot panels. I. Quantitation of NRF2 nuclear accumulation in PD hONS cultures relative to Histone H3 protein levels; p = 0.021 using one-tailed Student's t-test. J. NRF2 DNA binding to the ARE motif was quantified in nuclear fractions of DMSO and L-SUL treated PD hONS cell lines; p = 0.041 using one-tailed Student's t-test.
Figure 4
Figure 4. Induction of Glutathione and MTS metabolism in PD-derived hONS cell cultures.
A–I. PD patient-derived hONS cultures (N = 14) were treated with 2.5 µM L-SUL for 24 hr prior to assessment of cell metabolic functions, including (A) MTS metabolism, (B–C) total and reduced glutathione (GSH) content, (D–F) chymotrypsin-like, trypsin-like and caspase-like proteasome activities, (G) ATP content, (H) caspase3/7 activity and (I) membrane integrity/LDH activity. Each circle (veh) or square (L-SUL treatment) represents data obtained from triplicate plates for an individual cell line. Error bars show mean and s.e.m.. Statistical significance was determined by two-tailed t-test with Welch's correction of vehicle versus L-SUL treated values, with p<0.05 considered significant. Resulting p-values are shown above each graph.
Figure 5
Figure 5. Basal and L-SUL induced levels of NRF2 _targets genes in hONS cells.
A–D. hONS cultures established from Control donors (N = 16) or from PD patients (N = 13) were treated either with vehicle (circles) or 2.5 µM L-SUL (squarers) for 24 hr prior to RNA extraction. The mRNA levels of NRF2 (A), or the NRF2 _target genes NQO1, GCLC and GCLM (B–D) were measured by real-time PCR of cDNA and are presented relative to EEF1A1. Statistical significance was determined by 2-way ANOVA. The p value at the bottom of each panel indicates the ANOVA results comparing Control to PD hONS cultures; p<0.05 was considered significant. The asterisks above show the results of Bonferroni post-tests comparing DMSO and L-SUL treated cultures for either Control or PD hONS cell; * p<0.05, *** p<0.001.
Figure 6
Figure 6. Transcriptomic analysis identified discrete mRNA sets induced by L-SUL in Control versus PD hONS cultures.
A. Graphical representation of the ‘NRF2-mediated oxidative stress response’ Ingenuity pathway annotation mRNA set indicating mRNAs either induced (red icons) or repressed (green icons) by L-SUL treatment. Three discrete clusters of mRNA expression level changes were able to be identified: i) PD-specific (blue shading), ii) Control hONS-specific (red shading) and iii) those common to both PD and Control hONS cultures (yellow shading). B. Graphical representation of the ‘PI3K / AKT signalling’ Ingenuity pathway annotation mRNA set indicating mRNAs either induced (red icons) or repressed (green icons) by L-SUL treatment. Three discrete clusters of mRNA expression level changes were able to be identified: i) PD-specific (blue shading), ii) Control hONS-specific (red shading) and iii) those common to both PD and Control hONS cultures (yellow shading).
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