TFEB Probably Involved in Midazolam-Disturbed Lysosomal Homeostasis and Its Induced β-Amyloid Accumulation
TFEB Probably Involved in Midazolam-Disturbed Lysosomal Homeostasis and Its Induced β-Amyloid Accumulation
复制标题
TFEB 可能参与咪达唑仑扰乱的溶酶体稳态及其诱导的 β-淀粉样蛋白积累
DOI：
10.3389/fnhum.2019.00108
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发表时间：
2019-05-21
影响因子：
2.9
通讯作者：
Liu, Xuesheng
中科院分区：
文献类型：
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作者：
Cheng, Dan;Tan, Qilian;Liu, Xuesheng
阿尔茨海默病（Alzheimer's disease，AD）是最常见的神经退行性疾病之一，β-淀粉样蛋白（beta-amyloid，A β）在AD的发病机制中起主导作用。转录因子EB（TFEB）是自噬和溶酶体生物合成的主要调节因子，通过调节自噬-溶酶体途径参与AD的发病过程。迄今为止，在神经退行性疾病患者手术期间麻醉剂的选择以及对这些患者的影响和潜在机制的评价很少报道。在本研究中，使用过表达APP的HEK 293-APP细胞和Hela细胞。用不同浓度的咪达唑仑处理细胞不同时间，用溶酶体示踪剂染色，荧光显微镜下观察溶酶体形态。使用ImageJ软件分析溶酶体的数量和大小。通过核分离和Western Blot检测细胞核中TFEB和APP切割的细胞内蛋白的水平。最后，采用ELISA检测药物处理后细胞中A β 40和A β 42的水平。我们发现，30 μ M咪达唑仑减少溶酶体的数量和增加其在HEK 293和HeLa细胞的大小。然而，15 μ M咪达唑仑在24小时短暂扰乱溶酶体内稳态，并在36小时恢复。值得注意的是，在24 h时，不同浓度咪达唑仑处理组之间溶酶体体内平衡受到干扰的程度无显著差异。此外，30 μ M咪达唑仑阻止TFEB在正常或饥饿细胞中转运至细胞核。最后，在30 μ M咪达唑仑处理的HKE 293-APP细胞中，细胞内C-末端片段β（CTF β、CTF α、A β 40和A β 42）水平均显著升高。总的来说，TFEB转运至细胞核的抑制可能涉及咪达唑仑干扰的溶酶体稳态及其诱导的体外A β蓄积。这些结果提示了咪达唑仑加速AD发病的风险，并提示TFEB可能是降低咪达唑仑依赖性神经毒性的候选靶点。
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and beta-amyloid (A beta) plays a leading role in the pathogenesis of AD. The transcription factor EB (TFEB), a main regulating factor of autophagy and lysosome biosynthesis, is involved in the pathogenesis of AD by regulating autophagy-lysosomal pathways. To date, the choice of anesthetics during surgery in patients with neurodegenerative diseases and evaluation of the effects and underlying mechanisms in these patients have rarely been reported. In this study, the HEK293-APP cells overexpressing APP and Hela cells were used. The cells were treated with midazolam at different concentrations and at different times, then lysosomes were stained by lysotracker and their morphology was observed under a fluorescence microscope. The number and size of lysosomes were analyzed using the ImageJ software. The levels of TFEB in the nucleus and APP-cleaved intracellular proteins were detected by nuclear separation and Western Blot. Finally, ELISA was used to detect the levels of A beta 40 and A beta 42 in the cells after drug treatment. We found that 30 mu M midazolam decreased the number of lysosomes and increased its size in HEK293 and HeLa cells. However, 15 mu M midazolam transiently disturbed lysosomal homeostasis at 24 h and recovered it at 36 h. Notably, there was no significant difference in the extent to which lysosomal homeostasis was disturbed between treatments of different concentrations of midazolam at 24 h. In addition, 30 mu M midazolam prevents the transport of TFEB to the nucleus in either normal or starved cells. Finally, the intracellular C-terminal fragment beta(CTF beta, CTF alpha, A beta 40 and A beta 42 levels were all significantly elevated in 30 mu M midazolam-treated HKE293-APP cells. Collectively, the inhibition of TFEB transport to the nucleus may be involved in midazolam -disturbed lysosomal homeostasis and its induced A beta accumulation in vitro. The results indicated the risk of accelerating the pathogenesis of AD by midazolam and suggested that TFEB might be a candidate target for reduction of midazolam-dependent neurotoxicity.