铜过载促进β-淀粉样蛋白诱导的阿尔茨海默病中 NLRP3/Caspase-1/GSDMD 介导的细胞焦亡。

Copper Overload Promotes β-amyloid Induced NLRP3/Caspase-1/GSDMD-Mediated Pyroptosis in Alzheimer's Disease.

机构信息

Neurology Department, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 200080, Shanghai, China.

Neurology Department, Shanghai Xuhui Central Hospital, 200041 Shanghai, China.

出版信息

J Integr Neurosci. 2024 Oct 21;23(10):194. doi: 10.31083/j.jin2310194.

DOI:10.31083/j.jin2310194

PMID:39473154

Abstract

PURPOSE

Alzheimer's disease (AD) is characterized by cognitive decline and abnormal protein accumulation. Copper imbalance and pyroptosis play significant roles in the pathogenesis of AD. Recent studies have suggested that dysregulated copper homeostasis contributed to β-amyloid accumulation, which may activate the NOD-like receptor protein 3 (NLRP3)-related pyroptosis pathway, promoting neuronal damages and AD progression. Therefore, the present study aims to investigates whether copper facilitates AD through exacerbating β-amyloid (Aβ) induced activation of NLRP3/Caspase-1/Gasdermin D (GSDMD)-mediated neuronal cell pyroptosis.

METHODS

Mouse hippocampal HT-22 cells were cultured with Aβ1-42 oligomer for 24 h as AD Model group. CuCl treatment was administered to the AD cell model, and cell survivability levels were detected by Cell Counting Kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL), and other relevant kits. Mitochondrial function was evaluated using Mitochondrial membrane potential dye JC-1 and transmission electron microscopy (TEM). After intervention with the NLRP3 inhibitor MCC950, activation of the NLRP3/Caspase-1/GSDMD pathway by copper ions (Cu) was confirmed via Western Blot. Thioredoxin T (ThT) fluorescence assay was performed to observe the aggregation effect of Aβ induced by Cu overload.

RESULTS

CuCl treatment of the AD cell model resulted in up-regulation of the levels of Lactate Dehydrogenase (LDH), Interleukin-1β (IL-1β), and IL-18 expression, which indicated activation of pyroptosis. We observed a significant decrease in mitochondrial membrane potential, mitochondrial swelling, and loss of mitochondrial cristae by fluorescence microscopy and TEM. ThT fluorescence imaging showed that Cu promoted Aβ aggregation and up-regulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ACS), Caspase-1, Cleaved Caspase-1, GSDMD, and Gasdermin D N-terminal (GSDMD-NT). The NLRP3 inhibitor MCC950 partially reversed Cu-mediated pyroptosis in HT-22 cells.

CONCLUSIONS

Exposure to copper ions disrupt mitochondrial copper homeostasis, promotes Aβ aggregation, and activates NLRP3 inflammasomes, further promoting the Aβ aggregation activated pyroptosis in AD cell models.

摘要

目的

阿尔茨海默病（AD）的特征是认知能力下降和异常蛋白积累。铜失衡和细胞焦亡在 AD 的发病机制中起重要作用。最近的研究表明，铜稳态失调导致β-淀粉样蛋白（Aβ）积累，可能激活 NOD 样受体蛋白 3（NLRP3）相关的细胞焦亡途径，促进神经元损伤和 AD 进展。因此，本研究旨在探讨铜是否通过加剧β-淀粉样蛋白（Aβ）诱导的 NOD 样受体蛋白 3（NLRP3）/半胱氨酸天冬氨酸蛋白酶 1（Caspase-1）/Gasdermin D（GSDMD）介导的神经元细胞焦亡来促进 AD。

方法

将小鼠海马 HT-22 细胞用 Aβ1-42 寡聚体培养 24 h 作为 AD 模型组。用氯化铜（CuCl）处理 AD 细胞模型，用细胞计数试剂盒（CCK-8）、末端转移酶介导的 dUTP 缺口末端标记（TUNEL）和其他相关试剂盒检测细胞存活率。用线粒体膜电位染料 JC-1 和透射电子显微镜（TEM）评估线粒体功能。用 NLRP3 抑制剂 MCC950 干预后，通过 Western Blot 证实铜离子（Cu）对 NLRP3/Caspase-1/GSDMD 通路的激活作用。用硫氧还蛋白 T（ThT）荧光法观察 Cu 过载诱导的 Aβ 聚集效应。

结果

AD 细胞模型中 CuCl 处理导致乳酸脱氢酶（LDH）、白细胞介素-1β（IL-1β）和白细胞介素-18 表达上调，提示细胞焦亡激活。荧光显微镜和 TEM 观察到线粒体膜电位显著下降、线粒体肿胀和线粒体嵴丢失。ThT 荧光成像显示，Cu 促进了 Aβ 的聚集，并上调了 NLRP3、凋亡相关斑点样蛋白含有 CARD（ASC）、半胱氨酸天冬氨酸蛋白酶 1（Caspase-1）、裂解的 Caspase-1、GSDMD 和 GSDMD-N 端（GSDMD-NT）。NLRP3 抑制剂 MCC950 部分逆转了 Cu 介导的 HT-22 细胞焦亡。