G2019S LRRK2 Increases Stress Susceptibility Through Inhibition o DAF-16 Nuclear Translocation in a 14-3-3 Associated-Manner in Caenorhabditis elegans

Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson's disease (PD). Oxidative stress plays a key role in the pathogenesis of PD. Mutations in LRRK2 have been shown to increase susceptibility to oxidative stress. To explore mechanisms underlying susceptibility to oxidative stress in LRRK2 mutants, we generated stable Caenorhabditis elegans (C. elegans) strains in which human LRRK2 proteins including wild type LRRK2 (WT), G2019S LRRK2 (G2019S), and G2019S-D1994A kinase-dead LRRK2 (KD) were expressed in all neurons. Human 14-3-3 beta was injected into LRRK2 transgenic worms to allow co-expression of 14-3-3 beta and LRRK2 proteins. We found that G2019S transgenic worms had increased sensitivity to stress (heat and juglone treatment) and impaired stress-induced nuclear translocation of DAF-16. In addition, G2019S inhibited ftt2 (a 14-3-3 gene homolog in C. elegans) knockdown-associated nuclear translocation of DAF-16. Comparably, overexpression of human 14-3-3 beta could attenuate G2019S-associated toxicity in response to stress and rescued G2019S-mediated inhibition of sod-3 and dod-3 expression. Taken together, our study provides evidence suggesting that 14-3-3-associated inhibition of DAF-16 nuclear translocation could be a mechanism for G2019S LRRK2-induced oxidative stress and cellular toxicity. Our findings may give a hint that the potential of 14-3-3 proteins as neuroprotective targets in PD patients carrying LRRK2 mutations.

富含亮氨酸重复激酶2（LRRK2）的突变是家族性帕金森病（PD）的常见病因。氧化应激在帕金森病的发病机制中起关键作用。已表明LRRK2的突变会增加对氧化应激的敏感性。为了探究LRRK2突变体对氧化应激敏感性的潜在机制，我们构建了稳定的秀丽隐杆线虫（C. elegans）品系，其中包括野生型LRRK2（WT）、G2019S LRRK2（G2019S）和G2019S - D1994A激酶失活的LRRK2（KD）在内的人LRRK2蛋白在所有神经元中表达。将人14 - 3 - 3β注入LRRK2转基因线虫中，以实现14 - 3 - 3β和LRRK2蛋白的共表达。我们发现G2019S转基因线虫对压力（热和胡桃醌处理）的敏感性增加，并且应激诱导的DAF - 16核转位受损。此外，G2019S抑制了ftt2（秀丽隐杆线虫中14 - 3 - 3基因的同源物）敲低相关的DAF - 16核转位。相比之下，人14 - 3 - 3β的过表达能够减轻G2019S相关的应激毒性，并挽救G2019S介导的sod - 3和dod - 3表达的抑制。总之，我们的研究提供的证据表明，14 - 3 - 3相关的DAF - 16核转位抑制可能是G2019S LRRK2诱导的氧化应激和细胞毒性的一种机制。我们的研究结果可能提示14 - 3 - 3蛋白作为携带LRRK2突变的帕金森病患者的神经保护靶点的潜力。