NRF2 Activation Restores Disease Related Metabolic Deficiencies in Olfactory Neurosphere-Derived Cells from Patients with Sporadic Parkinson's Disease

BackgroundWithout 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.
ResultsWe 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 H2O2 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.
ConclusionsOur 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.

背景：缺乏合适的细胞模型，特发性帕金森病（idiopathic Parkinson's disease）的病因至今仍不明晰。我们此前报道了一种新型患者来源细胞模型，该模型取自嗅黏膜活检组织，被命名为嗅觉神经球衍生细胞（olfactory neurosphere-derived cells，简称hONS细胞），其功能与遗传特征会以疾病特异性方式产生差异。对患者与健康对照者的hONS细胞进行转录组分析后发现，核因子E2相关因子2（NRF2）转录因子信号通路是帕金森病中差异表达最为显著的通路。
结果：我们通过引入额外细胞系验证了初始发现的可靠性，并证实：与健康对照供者的细胞培养物相比，帕金森病患者的hONS细胞的还原型谷胱甘肽水平及MTS[3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓内盐]代谢活性降低了20%。我们还证实，帕金森病患者的hONS细胞处于氧化应激状态，其过氧化氢（H₂O₂）生成量高于健康对照细胞培养物。通过小干扰RNA（siRNA）介导敲除健康对照供者细胞中的NRF2，可使总谷胱甘肽含量及MTS代谢活性降低至帕金森病患者细胞中的水平。相反，更重要的是，我们证实：在帕金森病患者的hONS细胞培养物中激活NRF2通路，可将谷胱甘肽水平及MTS代谢活性恢复至健康对照水平。矛盾的是，NRF2通路激活后的转录组分析显示：与经L-SUL处理的健康对照细胞相比，经L-SUL处理的患者来源hONS细胞中，NRF2通路内差异表达的mRNA数量反而增多，尽管其代谢活性已恢复至正常水平。我们还发现磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号通路的差异表达仅存在于治疗后。
结论：本研究证实NRF2可作为帕金森病的潜在治疗靶点，并首次证明，在遗传背景各不相同的供者来源的患者细胞中，NRF2的功能可被诱导激活。然而，本研究同时发现，帕金森病患者来源细胞的应答模式与健康对照细胞并不一致。这在开发新型治疗药物时可能是一个关键考量因素。