Midazolam Enhances Mutant Huntingtin Protein Accumulation via Impairment of Autophagic Degradation In Vitro.
Midazolam Enhances Mutant Huntingtin Protein Accumulation via Impairment of Autophagic Degradation In Vitro.
复制标题
咪达唑仑通过削弱体外自噬降解来增强突变亨廷顿蛋白的积累。
DOI：
10.1159/000491895
复制
发表时间：
2018
影响因子：
--
通讯作者：
Liu Xuesheng
中科院分区：
文献类型：
--
作者：
Zhang Jiqian;Dai Wei;Geng Pengcheng;Zhang Li;Tan Qilian;Cheng Dan;Wei Pengfei;Yang Zhilai;Zhang Lei;Gu Erwei;Xu Guanghong;Liang Chaozhao;Liu Xuesheng
背景/AimsAutophagy是一种众所周知的“清洁”错误折叠的突变huntingtin蛋白（mHtt）的途径，在多聚谷氨酰胺疾病中起着相当大的作用。迄今为止，关于多谷氨酰胺疾病患者手术中麻醉剂的选择以及这些患者中麻醉剂的作用和潜在机制的评估的研究很少。方法采用稳定表达含有74个谷氨酰胺重复单元的绿色荧光蛋白标记Htt蛋白的sgfp -Htt (Q74)-PC12细胞。用15 μM咪达唑仑和100 mM海藻糖（阳性对照）处理细胞，通过检测自噬相关蛋白和底物的变化来评估自噬诱导和自噬降解，并采用MTT法评估细胞活力。通过质粒转染过表达组织蛋白酶D来恢复咪达唑仑损伤的自噬降解。结果咪唑安定增加细胞内mHtt水平呈时间和剂量依赖性。此外，海藻糖或氯喹增强或阻断自噬通量可分别降低或增加咪达唑仑诱导的mHtt升高。咪达唑仑通过mtor依赖的信号通路诱导自噬，但自噬降解受损，p62和LC3 II水平持续升高，组织蛋白酶D水平下降。然而，组织蛋白酶D过表达逆转了咪达唑仑的作用。咪达唑仑可使GFP-Htt (Q74)-PC12细胞活力降低20%，而过表达组织蛋白酶D可使其活性降低。结论咪达唑仑可通过损害自噬降解来提高mHtt水平，降低Htt (Q74)-PC12细胞活力，过表达组织蛋白酶D可使其活性恢复。
Background/AimsAutophagy is a well-known pathway to “clean” the misfolded mutant huntingtin protein (mHtt), which plays a considerable role in polyglutamine diseases. To date, there have been few studies of the choice of anesthetic during surgery in patients with polyglutamine diseases and evaluation of the effects and underlying mechanisms of anesthetics in these patients.MethodsGFP-Htt (Q74)-PC12 cells, which stably express green fluorescent protein-tagged Htt protein containing 74 glutamine repeating units, were used throughout this study. Cells were treated with 15 μM midazolam and 100 mM trehalose (positive control), and the induction of autophagy and autophagic degradation were assessed by detecting changes in autophagy-related proteins and substrates, and cell viability was assessed using the MTT assay. Overexpression of cathepsin D by plasmid transfection was used to restore midazolam-impaired autophagic degradation.ResultsMidazolam increased intracellular mHtt levels in a time-and dose-dependent manner. Additionally, enhancing or blocking autophagic flux by trehalose or chloroquine could decrease or increase midazolam-induced mHtt elevation, respectively. Midazolam induced autophagy in the mTOR-dependent signaling pathway, but autophagic degradation was impaired, with a continuous rise in p62 and LC3 II levels and decrease in cathepsin D. However, overexpression of cathepsin D reversed the effects of midazolam. Midazolam led to a 20% decrease in GFP-Htt (Q74)-PC12 cell viability, which could be abrogated by overexpression of cathepsin D.ConclusionsMidazolam increased mHtt levels and decreased Htt (Q74)-PC12 cell viability via impairment of autophagic degradation, which could be restored by overexpression of cathepsin D.