Proteomics Evidence of the Role of TDMQ20 in the Cholinergic System and Synaptic Transmission in a Mouse Model of Alzheimer’s Disease

The interaction between copper ions and amyloid peptide Aβ has been reported to be involved in Alzheimer’s disease (AD) pathology. Based on copper coordination biochemistry, we designed specific copper chelators [tetradentate monoquinolines (TDMQs)] in order to regulate copper homeostasis in the AD brain and inhibit the deleterious oxidative stress catalyzed by copper-Aβ complexes. We previously reported that TDMQ20, a highly selective copper chelator selected as a drug candidate, was able to extract copper from the Cu-Aβ1–16 complex and restore cognitive and behavioral deficits in AD mouse models. For a better understanding of the mechanism of action of TDMQ20, we decided to investigate the change of profile of proteins expressed in 5xFAD mice after an oral treatment of TDMQ20 (dose = 10 mg/kg, once every two days for 3 months, in total 45 times). Clioquinol (CQ), a non-specific chelator, has been used as a comparator. Here, we report the proteomic alterations in the cortex of 5xFAD mice using iTRAQ (isobaric tags for relative and absolute quantification) proteomics technology. The results indicated that 178 differentially expressed proteins (DEPs) have been identified in the AD mouse group with respect to wild type (WT) animals (AD/WT). After treatment by TDMQ20, 35 DEPs were found common in AD/WT and TDMQ20/AD groups in an opposite change manner (up- or down-regulated, respectively). In addition, among the 35 DEPs mentioned above, 10 common target proteins have been identified in AD/WT, TDMQ20/AD, and CQ/AD groups, among which 3 target proteins were successfully validated by western blot analysis. In particular, the expression levels of ChAT and CHRM4 are significantly increased upon TDMQ20 treatment with respect to 5xFAD mice, while CQ did not significantly change the expression of these proteins. Our study suggests the involvement of the copper chelator TDMQ20 on the cholinergic system, a feature that may explain the improved cognitive and behavioral performance in AD mice upon oral treatment of TDMQ20.

铜离子与淀粉样蛋白肽Aβ的相互作用已被证实与阿尔茨海默病（Alzheimer’s disease, AD）的病理进程密切相关。基于铜配位生物化学理论，我们设计了特异性铜螯合剂——四齿单喹啉类化合物（tetradentate monoquinolines, TDMQs），以期调控AD脑内的铜稳态，并抑制铜-Aβ复合物所介导的有害氧化应激。此前我们曾报道，高选择性铜螯合剂TDMQ20作为候选药物，能够从Cu-Aβ1–16复合物中萃取出铜离子，并改善AD小鼠模型的认知与行为缺陷。为深入解析TDMQ20的作用机制，我们对5xFAD小鼠以10 mg/kg的剂量口服给药TDMQ20，每两日一次，持续3个月，总计给药45次，随后分析其体内蛋白表达谱的变化。本研究以非特异性螯合剂氯碘羟喹（clioquinol, CQ）作为对照。本研究采用iTRAQ（同量异位素标签相对与绝对定量，isobaric tags for relative and absolute quantification）蛋白质组学技术，对5xFAD小鼠大脑皮层的蛋白质组学改变进行了检测。结果显示，相较于野生型（wild type, WT）小鼠，AD模型小鼠组中共鉴定出178个差异表达蛋白（differentially expressed proteins, DEPs）。经TDMQ20处理后，在AD/WT组与TDMQ20/AD组中，共发现35个表达变化趋势相反的共同差异表达蛋白，二者分别呈现上调与下调趋势。此外，在上述35个差异表达蛋白中，在AD/WT、TDMQ20/AD及CQ/AD三组中均鉴定出10个共同靶蛋白，其中3个靶蛋白已通过蛋白质印迹（western blot）分析得到验证。尤为关键的是，相较于未处理的5xFAD小鼠，TDMQ20处理可显著上调ChAT与CHRM4的表达水平，而CQ并未对这些蛋白的表达产生显著影响。本研究表明，铜螯合剂TDMQ20可作用于胆碱能系统，这一特性或可解释口服TDMQ20治疗后AD小鼠认知与行为能力得到改善的现象。