Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism
Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism
复制标题
DOI：
10.1074/jbc.m117.797092
复制
发表时间：
2017-11-24
影响因子：
4.8
通讯作者：
Sucic, Sonja
中科院分区：
文献类型：
--
作者：
Asjad, H. M. Mazhar;Kasture, Ameya;Sucic, Sonja
编码人多巴胺转运蛋白（hDAT，SLC 6A 3）的基因的点突变会导致婴儿/青少年肌张力障碍和帕金森综合征。为了解开这些疾病的分子机制，并研究可能的药物治疗，在这里，我们检查了13种致病DAT突变体，这些突变体在HEK 293细胞中异源表达时保留在内质网中。在这些突变体中的三个，即hDAT-V158 F、hDAT-G327 R和hDAT-L368 Q中，用药物伴侣诺核糖核苷或热休克蛋白70（HSP 70）抑制剂匹非林补救折叠缺陷，使得这些DAT突变体的内质网输出和放射性配体结合以及底物摄取恢复。在果蝇中，DAT缺乏导致睡眠减少。因此，我们利用突变hDAT在果蝇中的靶向转基因表达的能力，以探索这些hDAT突变体是否也可以在完整的生物体中被拯救。降冰片烯或匹非林处理支持hDAT递送至多巴胺能神经元的突触前末梢，并在表达hDAT-V158 F或hDAT-G327 R的DAT缺陷（fumin）果蝇系中将睡眠恢复至正常长度。相比之下，hDAT-L368 Q在果蝇DAT突变体背景中的表达引起发育致死，表明药物伴侣不能补救的毒性作用。我们的观察确定了这些突变最有可能适用于受影响儿童的药物救援。此外，我们的研究结果还强调了将细胞培养中的药物伴侣作用转化为临床情况的挑战。由于果蝇和人类之间多巴胺能神经传递的进化保守性，D.黑腹菌也许能让我们弥合这一鸿沟。
Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum when heterologously expressed in HEK293 cells. In three of these mutants, i.e. hDAT-V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit was remedied with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin- such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or pifithrin- treatment supported hDAT delivery to the presynaptic terminals of dopaminergic neurons and restored sleep to normal length in DAT-deficient (fumin) Drosophila lines expressing hDAT-V158F or hDAT-G327R. In contrast, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, indicating a toxic action not remedied by pharmacochaperoning. Our observations identified those mutations most likely amenable to pharmacological rescue in the affected children. In addition, our findings also highlight the challenges of translating insights from pharmacochaperoning in cell culture to the clinical situation. Because of the evolutionary conservation in dopaminergic neurotransmission between Drosophila and people, pharmacochaperoning of DAT in D. melanogaster may allow us to bridge that gap.