阿米替林对亚历山大病GFAP突变星形胶质细胞内质网应激的保护性作用

Alexander disease is the disease caused by dominant gain-of-function mutations in the glial fibrillary acidic protein (GFAP) gene, which leads to the the excessive GFAP accumulation in the astrocytes. The prognosis of Alexander disease is so poor, and there is no effective treatment. Previous literatures and previous studies of our research group confirmed that the unfolded protein response after endoplasmic reticulum stress induced by excessive GFAP aggregation is an important pathogenesis of Alexander disease. We have proved that amitriptyline can reduce GFAP expression in astrocytes and play a protective role. In this project, we are going to explore the protective effect of amitriptyline through inhibiting the unfolded protein response after endoplasmic reticulum stress on GFAP mutant astrocytes differentiated from the neural stem cells by using iPS cells from Alexander disease patients. We try to give amitriptyline to the GFAP mutant astrocytes and wild-type astrocytes respectively, and observe the shape of cells, length and numbers of the processes, GFAP and δGFAP expression, cell apoptosis of the astrocytes, detect the molecular markers in the pathways of the unfolded protein reaction, such as PERK、ATF4、CHOP、IRE1α、sXBP1、ATF6、GRP78 to confirm that amitriptyline could have a protective effect on GFAP mutant astrocytes by inhibiting the unfolded protein response after endoplasmic reticulum stress, and thus may provide intervention drugs for the pathogenesis of Alexander's disease. Amitriptyline, already on the market, can be further applied in clinical trials of the Alexander disease.

亚历山大病是胶原纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）基因突变致其在星形胶质细胞中过度聚集的遗传性脑白质病，预后差，无有效治疗。前期研究表明GFAP过度聚集诱导的内质网应激后未折叠蛋白反应是其重要发病机制之一，阿米替林可降低星形胶质细胞GFAP表达。本研究利用亚历山大病患儿iPS细胞诱导的神经干细胞及野生型神经干细胞，体外诱导分化为星形胶质细胞，给予阿米替林干预，观察并比较星形胶质细胞胞体大小、突起数量、长度以及细胞凋亡，GFAP、δGFAP表达，检测未折叠蛋白反应通路分子指标PERK、ATF4、CHOP、IRE1α、sXBP1、ATF6、GRP78水平，证实阿米替林可抑制内质网应激后未折叠蛋白反应，对GFAP突变星形胶质细胞产生保护性作用及其分子学机制，为亚历山大病提供针对发病机制的干预药物，且阿米替林已上市，可进一步应用临床。

亚历山大病是一种致死性的中枢神经系统退行性疾病，目前尚无有效治疗。目前本研究已完成亚历山大病患儿iPS细胞和野生型对照iPS细胞诱导的神经干细胞的体外诱导，诱导其向星形胶质细胞分化，并观察其细胞形态、数量，对GFAP、δGFAP表达与分布进行比较。利用免疫荧光法检测不同细胞分化阶段的细胞形态学，已对星形前体细胞Nestin、CD44以及星形胶质细胞GFAP和S100关键标志物进行检测及比较，证实亚历山大病患儿iPS细胞和野生型对照iPS细胞诱导分化的星形胶质细胞之间存在一定差异，亚历山大病患儿iPS细胞诱导分化的星形胶质细胞较野生型对照iPS细胞诱导分化的星形胶质细胞而言，其δGFAP表达显著增多，且GFAP、δGFAP表达可见团块状浓缩改变。在星形胶质细胞上进行了阿米替林（浓度为20uM）的干预，通过Western-blot技术、免疫荧光技术已初步证实阿米替林干预后24、48、72小时，AxD患者iPS细胞诱导分化的星形胶质细胞GFAP、δGFAP表达较干预前有所减少，细胞突起增多，团块状GFAP减少，分布更加均匀。阿米替林干预后未折叠蛋白反应相关通路分子指标PERK、XBP1的表达较干预前有所减少，初步证实阿米替林20uM干预可降低AxD患者iPS细胞诱导分化的星形胶质细胞GFAP、δGFAP表达，可以降低内质网应激相关通路标志物PERK、XBP1的表达，并可增加细胞突起数量，减少异常聚集的团块状GFAP，初步证实阿米替林可以抑制GFAP突变星形胶质细胞内质网应激后未折叠蛋白反应，对星形胶质细胞起到一定保护作用。阿米替林是国家食品药品监督总局已批准上市的中枢神经系统药品，目前在国内可获得，后期也可尝试应用于亚历山大病患儿，对疾病尝试一定干预，为亚历山大病患儿带来治疗上的新希望。

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