过表达A53T突变型α-synuclein细胞毒性及对损伤性刺激的影响

Cytotoxicity of A53T mutant α-synuclein over-expression and the effects on injury stimulus

目的通过比较过表达A53T(Ala53Thr)突变型α-突触核蛋白(α-synuclein)的人神经母细胞瘤SH-SY5Y细胞与正常细胞的特性及对鱼藤酮引起细胞损伤的影响,探索α-synuclein在帕金森病发病机制中的作用,为寻找治疗药物潜在靶点提供依据。方法通过比较过表达A53T突变型α-synuclein的SH-SY5Y细胞与正常细胞的细胞形态及生长曲线观察突变型α-synuclein对细胞毒性的影响;用呼吸链抑制剂鱼藤酮刺激细胞,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法观察细胞的存活率,免疫印迹法观察自噬相关蛋白的变化。结果正常细胞形态上接近于梭形,过表达A53Tα-synuclein的细胞胞体变的肿胀,小的突起增多,甚至出现多种变异的形态;过表达A53Tα-synuclein的细胞生长较正常细胞缓慢,培养相同时间达到的细胞总数相对较少;4μM鱼藤酮处理两种细胞,过表达A53Tα-synuclein细胞损伤较正常细胞大,正常细胞随鱼藤酮处理时间的延长自噬相关蛋微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)的水平出现先下降后上升的趋势(P<0.05),A53T细胞出现先上升后下降的趋势。结论过表达A53T突变型α-synuclein具有细胞毒性,且与正常细胞相比更容易受到损伤,可能与自噬敏感性增高,加速自噬耗竭相关。因此增加突变型α-synuclein的清除或增加自噬合成的相关蛋白诱导自噬可能会成为PD潜在的治疗靶点。

Objective The characteristics of A53T （Ala53Thr） mutant α-synuclein over-expression SH-SYSY neuroblastoma cells and the normal cells were compared and its impact on rotenone caused cell damage was discussed, which might reveal the possible pathogenesis of Parkinson disease （PD） and provide the novel targets for drug research of PD therapy. Methods Cytotoxicity of A53T mutant α-synuclein over-expression was observed through the cell morphology and growth curve, and then cell viability was detected by 3-（ 4, 5-dimethyl-2-thiazolyl ）- 2, 5- diphenyl-2-H-tetrazolium bromide （ MTT） method, and the autophagy-related protein was observed by immunoblotting with respiratory chain inhibitor rotenone stimulation. Results In morphology, normal cell was close to spindle, while A53T cell body became swollen with increased small protrusions, or even a variety of morphological variation. In cell growth, normal cells grew faster and had relatively less time to reach the same total number of cells; MTT results revealed that A53T mutant α-synuclein cells were more vulnerable to rotenone stimulation. In normal cells, autophagy-related protein microtubule-associated protein 1 light chain 3 （LC3-11 ） protein level was decreased firstly, and then increased, while A53T mutant cells demonstrated a different change with LC3-Ⅱ protein, which increased firstly, and then decreased （P 〈 0.05 ）. Conclusion A53T mutant α-synuclein have cytotoxicity and make cells more susceptible to injury induced by rotenone, which may related to increased autophagy sensitivity and accelerated the depletion of autophagy. Therefore, increasing mutant α- synuclein clearance or inducing autophagy may become novel targets for drug research of PD therapy.