DAPK1调控小胶质细胞NLRP3炎症小体组装激活生成IL-1β

Our previous research reported that interleukin (IL)-1β production was decreased by inhibiting the downstream signal of Toll like receptors (TLRs) in microglia. Consequently, the neuronal death was attenuated. However, IL-1β production is also tightly controlled by NOD like receptors (NLRs), which is another important patter recognized receptor. NLRP3 is the most important member of NLRs family. After NLRP3 is activated, it recruits the ASC and pro-caspase-1, and forms the active inflammasome complex. Then, it activates caspase-1, which cleaves the pro-IL-1β and produces IL-1β. Our recent research suggested that death associated protein kinase (DAPK) 1 was required for full IL-1β production after activation of NLRP3 inflammasome in microglia. And cathepsin B (CTSB) may be the upstream molecule of DAPK1. So, we hypothesize that the release of CTSB after phagocytosis of Aβ by microglia activates DAPK1, which enhances NLRP3 inflammasome complex activation. Consequently, it cleaves po-IL-1β. To test this hypothesis, four specific aims will be completed. (1) Determine whether DAPK1 is required for full IL-1β production and activation of NLRP3 inflammasome in microglia; (2) Determine whether DAPK1 catalytic activity is required for full IL-1β production and activation of NLRP3 inflammasome in microglia; (3) Determine the binding site of DAPK1 and NLRPs; (4) Determine if CTSB is a upstream molecule of DAPK1. Our proposal will help us define the new function of microglia and provide a chance to find a new target for neurodegeneration therapy.

我们发现通过调控TLR受体下游信号可以抑制小胶质细胞IL-1β生成，减轻神经损伤。而IL-1β生成还受到NOD样受体（NLRs）调控，NLRP3是该家族重要成员。NLRP3激活后与ASC、pro-caspase-1组装成炎症小体，使pro-caspase-1活化，后者裂解IL-1β的前体使其成熟。我们发现死亡相关蛋白激酶（DAPK）1可以调控小胶质细胞NLRP3的激活，组织蛋白酶B可能是DAPK1的上游信号。故，假设小胶质细胞吞噬Aβ后释放出的组织蛋白酶B激活DAPK1，后者又使NLRP3炎症小体激活，从而调控IL-1β生成。本项目将开展以下工作：明确DAPK1及DAPK1的催化活性是否调控小胶质细胞生成IL-1β和 NLRP3炎症小体组装激活；探明DAPK1与NLRP3的结合位点；研究CTSB与DAPK1的相互作用。本项目将为深入认识小胶质细胞功能实现对神经退行性疾病的治疗提供全新依据。

背景：白介素-1β (interleukin-1β, IL-1β) 的生成受到 Toll样受体下游信号和NOD样受体（NOD like receptors, NLRs）信号双重调控。NLRP3是NLRs家族的重要成员，激活后与pro-caspase-1、ASC组装形成炎性小体，使pro-caspase-1切割活化，激活的caspase-1裂解pro-IL-1β使之成熟。NLRP3/caspase-1/ IL-1β信号通路参与神经炎症反应。死亡相关蛋白激酶(death-associated protein kinase, DAPK)1与阿尔茨海默病（Alzheimer’s disease, AD）发生发展有关，并可以调控炎症反应。然而，DAPK1是否参与调控小胶质细胞IL-1β生成过程及其相应的机制，目前尚不清楚。..研究内容：本项目包括离体实验部分和在体实验部分。离体实验部分通过基因干预技术和药物抑制剂研究DAPK1及其催化活性对小胶质细胞IL-1β生成和NLRP3炎性小体激活的影响，再明确cathepsin B是否是DAPK1激活的上游信号。在体实验部分探讨DAPK1是否参与调控小鼠脑内IL-1β生成、NLRP3炎性小体激活和认知功能改变。..结果和关键数据：离体实验表明LPS+Aβ25-35刺激可使小胶质细胞产生IL-1β，并激活NLRP3炎性小体和DAPK1；下调DAPK1表达水平或抑制其活性减少IL-1β成熟和caspase-1活化，上调DAPK1表达水平增加IL-1β成熟和caspase-1活化；DAPK1的调控作用发生在NLRP3炎性小体组装激活阶段；LPS+Aβ25-35刺激导致溶酶体cathepsin B释放入胞浆，是DAPK1激活的上游信号。在体实验表明侧脑室注射Aβ25-35可使小鼠海马区小胶质细胞、NLRP3炎性小体和DAPK1激活，并产生IL-1β；DAPK1抑制剂可减少Aβ25-35所致的IL-1β产生和NLRP3炎性小体激活，并改善小鼠认知能力。..科学意义：小胶质细胞参与的炎症反应是神经疾病发生和加重的重要因素，本研究有助于深入认识小胶质细胞功能，为神经退行性疾病的治疗提供全新的依据。