Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan.
Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan.
Neurobiol Dis. 2015 Feb;74:25-31. doi: 10.1016/j.nbd.2014.11.005. Epub 2014 Nov 14.
The sigma-1 receptor (SIGMAR1) is one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated that accumulation of SIGMAR1 was common to neuronal nuclear inclusions in polyglutamine diseases including Huntington's disease. Our study also indicated that SIGMAR1 might shuttle between the cytoplasm and the nucleus. In the present study, we investigated the role of SIGMAR1 in nuclear inclusion (NI) formation, using HeLa cells transfected with N-terminal mutant huntingtin. Cell harboring the mutant huntingtin produced SIGMAR1-positive NIs. SIGMAR1 siRNA and a specific inhibitor of the proteasome (epoxomicin) caused significant accumulation of aggregates in the cytoplasm and nucleus. A specific inhibitor of exportin 1 (leptomycin B) also caused NIs. Huntingtin became insolubilized in Western blot analysis after treatments with SIGMAR1 siRNA and epoxomicin. Furthermore, proteasome activity increased chronologically along with the accumulation of mutant huntingtin, but was significantly reduced in cells transfected with SIGMAR1 siRNA. By contrast, overexpression of SIGMAR1 reduced the accumulation of NIs containing mutant huntingtin. Although the LC3-I level was decreased in cells treated with both SIGMAR1 siRNA and control siRNA, the levels of LC3-II and p62 were unchanged. SIGMAR1 agonist and antagonist had no effect on cellular viability and proteasome activity. These findings suggest that the ubiquitin-proteasome pathway is implicated in NI formation, and that SIGMAR1 degrades aberrant proteins in the nucleus via the ER-related degradation machinery. SIGMAR1 might be a promising candidate for therapy of Huntington's disease.
σ-1 受体(SIGMAR1)是内质网(ER)伴侣蛋白之一,通过与泛素-蛋白酶体途径相关的 ER 相关降解机制参与错误折叠蛋白的降解。在各种神经退行性疾病中形成包涵体的 ER 功能障碍也变得明显。最近,我们证明在包括亨廷顿病在内的多聚谷氨酰胺疾病中,神经元核内包涵体的积累是常见的。我们的研究还表明,SIGMAR1 可能在细胞质和细胞核之间穿梭。在本研究中,我们使用转染 N 端突变 huntingtin 的 HeLa 细胞研究了 SIGMAR1 在核包涵体(NI)形成中的作用。携带突变 huntingtin 的细胞产生 SIGMAR1 阳性 NI。SIGMAR1 siRNA 和蛋白酶体的特异性抑制剂(环氧酶素)导致细胞质和细胞核中聚集体的显著积累。出口蛋白 1(莱普霉素 B)的特异性抑制剂也会导致 NI。用 SIGMAR1 siRNA 和环氧酶素处理后,在 Western blot 分析中 huntingtin 变得不溶。此外,随着突变 huntingtin 的积累,蛋白酶体活性在时间上增加,但在转染 SIGMAR1 siRNA 的细胞中显著降低。相比之下,SIGMAR1 的过表达减少了含有突变 huntingtin 的 NI 的积累。虽然用 SIGMAR1 siRNA 和对照 siRNA 处理的细胞中 LC3-I 水平降低,但 LC3-II 和 p62 水平不变。SIGMAR1 激动剂和拮抗剂对细胞活力和蛋白酶体活性没有影响。这些发现表明泛素-蛋白酶体途径参与 NI 形成,并且 SIGMAR1 通过 ER 相关降解机制降解核内异常蛋白。SIGMAR1 可能是治疗亨廷顿病的有前途的候选药物。