青光眼和 ALS 相关的 OPTN 突变体根据 Tbk1 活性、自噬和内质网应激诱导神经元细胞死亡,The FEBS Journal

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青光眼和 ALS 相关的 OPTN 突变体根据 Tbk1 活性、自噬和内质网应激诱导神经元细胞死亡
The FEBS Journal ( IF 5.5 ) Pub Date : 2021-02-06 , DOI: 10.1111/febs.15752
Swetha Medchalmi ₁ , Priyanka Tare ₁ , Zuberwasim Sayyad ₁ , Ghanshyam Swarup ₁

Affiliation

OPTN 的突变与青光眼（一种眼病）以及肌萎缩侧索硬化（ALS）（一种运动神经元疾病）有关。OPTN 中的2 bp 插入(691_692insAG 或 2bpIns-OPTN) 与青光眼和 ALS 相关。这种突变导致 127 个氨基酸后移码，产生具有 C 端异常序列的蛋白质。我们已经在运动神经元细胞系 NSC-34 和视网膜细胞系 661W 中探索了该突变体诱导细胞死亡的机制。与野生型 OPTN 相比，这种突变体在 NSC-34 和 661W 细胞中诱导了更多的细胞死亡。该突变体主要位于细胞核中，而正常的 OPTN 位于细胞质中。2bpIns-OPTN 的缺失分析表明异常序列对于细胞死亡诱导不是必需的。该突变体与 TANK 结合激酶 1 (Tbk1) 相互作用，但不与 OPTN 相互作用并激活 Tbk1。从 C/EBP 同源蛋白 (CHOP) 和一些其他基因的诱导可以看出，这种突变体在 NSC-34 细胞中诱导了内质网应激。该突变体对 CHOP、自噬体蛋白 LC3-II 和细胞死亡的诱导被 Tbk1 敲低以及抑制 ER 应激的 4-苯基丁酸废除。2bpIns-OPTN 对 CHOP 和细胞死亡的诱导是自噬依赖性的，如 Atg5 敲低的效果所示。该突变体导致 LC3 阳性聚集体的形成增加。用自噬诱导剂雷帕霉素处理细胞减少了 2bpIns-OPTN 诱导的 LC3 阳性聚集体、CHOP 和细胞死亡。这些结果表明 2bpIns-OPTN 对 Tbk1 的组成性激活导致自噬受损，从而导致内质网应激和细胞死亡。2bpIns-OPTN 对 CHOP 和细胞死亡的诱导是自噬依赖性的，如 Atg5 敲低的效果所示。该突变体导致 LC3 阳性聚集体的形成增加。用自噬诱导剂雷帕霉素处理细胞减少了 2bpIns-OPTN 诱导的 LC3 阳性聚集体、CHOP 和细胞死亡。这些结果表明 2bpIns-OPTN 对 Tbk1 的组成性激活导致自噬受损，从而导致内质网应激和细胞死亡。2bpIns-OPTN 对 CHOP 和细胞死亡的诱导是自噬依赖性的，如 Atg5 敲低的效果所示。该突变体导致 LC3 阳性聚集体的形成增加。用自噬诱导剂雷帕霉素处理细胞减少了 2bpIns-OPTN 诱导的 LC3 阳性聚集体、CHOP 和细胞死亡。这些结果表明 2bpIns-OPTN 对 Tbk1 的组成性激活导致自噬受损，从而导致内质网应激和细胞死亡。

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A glaucoma- and ALS-associated mutant of OPTN induces neuronal cell death dependent on Tbk1 activity, autophagy and ER stress

Mutations in OPTN are associated with glaucoma, an eye disease, and also with amyotrophic lateral sclerosis (ALS), a motor neuron disease. A 2-bp insertion in OPTN (691_692insAG or 2bpIns-OPTN) is associated with both glaucoma and ALS. This mutation results in frame shift after 127 amino acids, giving rise to a protein with C-terminal aberrant sequence. We have explored the mechanism of induction of cell death by this mutant in a motor neuron cell line, NSC-34, and also in a retinal cell line, 661W. Compared to wild-type OPTN, this mutant induced more cell death in NSC-34 and 661W cells. This mutant localizes predominantly in the nucleus whereas normal OPTN localizes in the cytoplasm. Deletion analysis of 2bpIns-OPTN showed that the aberrant sequence was not essential for cell death induction. This mutant interacts with TANK-binding kinase 1 (Tbk1) but not with OPTN and activates Tbk1. This mutant induced ER stress in NSC-34 cells as seen by induction of C/EBP homologous protein (CHOP) and some other genes. Induction of CHOP, autophagosomal protein LC3-II and cell death by this mutant were abrogated by Tbk1 knockdown and also by 4-phenylbutyric acid, that inhibits ER stress. Induction of CHOP and cell death by 2bpIns-OPTN was autophagy dependent as shown by the effect of Atg5 knockdown. This mutant caused increased formation of LC3-positive aggregates. Treatment of cells with autophagy inducer rapamycin reduced LC3-positive aggregates, CHOP and cell death induced by 2bpIns-OPTN. These results suggest that constitutive activation of Tbk1 by 2bpIns-OPTN leads to impaired autophagy that results in ER stress and cell death.

更新日期：2021-02-06

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