褪黑素和DAPK1抑制剂协同靶向DAPK1治疗阿尔兹海默病的机制研究

Melatonin can decrease tau hyperphosphorylation, Aβ pathology and neuron death, which are three neuropathological hallmarks of Alzheimer's disease (AD). However, the molecular mechanisms by which melatonin decreases tau and Aβ pathology are not fully understood. Large evidences showed that Death-associated protein kinase 1 (DAPK1) has key roles in AD. Moreover, inhibiting DAPK1 not only decreases tau phosphorylation, Aβ production, and neuron death, but also enhances learning and memory. However, little is known about whether and how DAPK1 expression and activity is regulated during AD. Our preliminary data show that melatonin decreased DAPK1 protein stability by proteasome pathway, thereby decreasing tau phosphorylation at an AD-relate site and increasing tau function such as neurite outgrowth. In addition, the treatment of melatonin and DAPK1 inhibitor has synergistical effects. Together, these results lead us to hypothesize that melatonin targets DAPK1 to reduce tau and Aβ pathology in AD and the combination therapy with melatonin and DAPK1 inhibitor may dramatically alleviate AD pathology. In this proposal, we will elucidate the novel molecular mechanism by which melatonin regulates DAPK1 during AD using cell models, and determine the effects of combination therapy of melatonin and DAPK1 inhibitor on in vivo models such as DAPK1-Tg, APP/PS1 and hTau mouse models, and determine the relationship among DAPK1 expression, melatonin receptors and AD pathology in different brain subregions in human AD samples. This project would provide a novel therapeutic option for AD treatments.

褪黑素可减轻阿尔兹海默病（AD）病变，如tau过度磷酸化、Aβ沉积和神经元死亡，但其分子机制不明确。大量数据表明死亡相关蛋白激酶1（DAPK1）在AD中起关键作用；而抑制DAPK1，可降低tau磷酸化水平、Aβ生成和神经元死亡，并提高小鼠的认知功能，但其上游作用机制仍未知。预实验发现褪黑素可通过泛素-蛋白酶体途径降低DAPK1的蛋白水平，从而降低AD相关位点的tau磷酸化水平，促进tau的功能如神经突触生长；且褪黑素和DAPK1抑制剂联合干预有协同效应。据此推测褪黑素通过靶向DAPK1而减轻AD病变，褪黑素和DAPK1抑制剂可协同治疗AD。我们将采用细胞模型阐明褪黑素靶向调节DAPK1的新型分子机制，利用DAPK1-Tg、APP/PS1和hTau小鼠揭示褪黑素和DAPK1抑制剂协同治疗AD的作用效果，并使用AD临床样本探讨DAPK1、褪黑素受体和AD病变的相关性，有望开发AD治疗新方法。

阿尔茨海默病（AD）是当今世界痴呆最主要的病因，中国患病人数多，经济费用高。然而目前AD发病机制尚不清楚，缺乏有效治疗方法。褪黑素可减轻AD病变，如tau过度磷酸化、Aβ沉积和神经元死亡，但其分子机制不明确。大量数据表明死亡相关蛋白激酶1（DAPK1）在AD中起关键作用；而抑制DAPK1，可降低tau磷酸化水平、Aβ生成和神经元死亡，并提高小鼠的认知功能，但其上游作用机制仍未知。我们在细胞、离体小鼠模型和人类样本中使用综合方法测试了DAPK1蛋白稳定性、tau磷酸化和功能，以研究褪黑素对DAPK1调节的影响。我们使用生物素-褪黑素进行了下拉试验，以证明褪黑素与DAPK1的直接结合。我们对hTau小鼠进行皮下注射褪黑素5个月，并利用免疫荧光分析法、Western Blotting和Thioflavin-S染色法对小鼠脑组织进行tau蛋白病变的检测。此外，我们还研究了褪黑素、DAPK1表达和Pin1磷酸化的相关性，因为通过DAPK1抑制Pin1活性会导致tau过度磷酸化。结果发现褪黑素以转录后的方式降低DAPK1蛋白水平。此外，褪黑素直接与DAPK1结合，并诱导其在蛋白酶体依赖通路中泛素化。此外，褪黑素和DAPK1抑制剂对DAPK1的抑制通过抑制Pin1活性协同降低与AD相关的多个位点的tau磷酸化，Pin1是一种已知抑制tau过度磷酸化的脯氨酸异构酶，并促进神经突生长和微管组装。我们发现褪黑素可显著降低tau蛋白的AD相关位点磷酸化水平，并可有效降低NFTs并减少神经元丢失。最后，在AD大脑中，DAPK1和褪黑素水平呈负相关。综上所述，褪黑素通过调节DAPK1从而控制tau磷酸化及其功能是一种新的机制，并为AD的治疗提供了新选择。