Afsari Farinaz, Christensen Kenneth V, Smith Garrick Paul, Hentzer Morten, Nippe Olivia M, Elliott Christopher J H, Wade Alex R
Department of Biology and.
Neuroscience Drug Discovery DK, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark.
Hum Mol Genet. 2014 Sep 1;23(17):4465-78. doi: 10.1093/hmg/ddu159. Epub 2014 Apr 9.
Our understanding of Parkinson's disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor 'BMPPB-32'. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK(-) null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.
致病基因突变的发现彻底改变了我们对帕金森病(PD)的理解。其中最常见的是LRRK2激酶基因中的G2019S突变,该突变导致激酶活性增加。然而,激酶活性增加与帕金森病之间的联系尚不清楚。此前,我们发现果蝇中人类LRRK2 - G2019S转基因的多巴胺能表达会导致老年动物出现与活动相关的视力丧失,我们推测这可能是由于幼年动物未能正确调节神经元活动所致。为了验证这一假设,我们基于频率标记的稳态视觉诱发电位,采用了一种敏感的视觉功能测量方法。光谱分析使我们能够识别果蝇视觉系统多个层面的信号,野生型视觉反应曲线在质量上与人类皮层的相似。hLRRK2 - G2019S的多巴胺能表达增加了整个视网膜网络的对比敏感度。为了测试这是否是由于激酶活性增加所致,我们给果蝇喂食针对LRRK2的激酶抑制剂。新型LRRK2激酶抑制剂“BMPPB - 32”使第1天和第14天果蝇的对比敏感度恢复正常。生化和细胞实验表明,BMPPB - 32比LRRK2 - IN - 1更具特异性。我们在体内证实了这一点,发现dLRRK(-)缺失果蝇对LRRK2 - IN - 1显示出较大的脱靶效应,而对BMPPB - 32则没有。我们的数据将G2019S - LRRK2突变增加的激酶活性与神经元功能障碍联系起来,并证明了果蝇视觉系统在分析遗传疾病和疗法的神经学效应方面的作用。