Miao Qianqian, Wang Tiantian, Wang Haoran, Yu Yanxia, Jin Xing
Department of Pharmacy, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China.
Suzhou Municipal Hospital, Suzhou, China.
Front Immunol. 2025 Sep 8;16:1656729. doi: 10.3389/fimmu.2025.1656729. eCollection 2025.
This study aimed to investigate the role and underlying mechanism of DJ-1 in regulating NLRP3 inflammasome-mediated neuroinflammation during Parkinson's disease.
We used LPS to stimulate primary microglia and performed stereotactic injection of LPS into the substantia nigra of mice to investigate changes in DJ-1 expression following inflammatory stimulation. To evaluate the functional effects of DJ-1 on NLRP3 inflammasome activation, we used siRNA to knock down DJ-1 in primary microglia or BMDMs and analyzed downstream inflammatory responses as well as the specificity of this regulation. , we used microglia-specific AAV to selectively silence DJ-1 in the substantia nigra to further evaluate the anti-inflammatory effect of DJ-1 deficiency. To validate the direct interaction between DJ-1 and NLRP3, we performed co-immunoprecipitation and proximity ligation assay. We used the autophagy inhibitor 3-MA and activator rapamycin to investigate how NLRP3 is degraded upon DJ-1 deficiency in CRISPR-Cas9-engineered DJ-1 knockout HEK-293 cells.
DJ-1 were significantly upregulated following LPS or LPS plus ATP stimulation in primary microglia. Similarly, after stereotactic LPS injection into the substantia nigra, we observed a significant upregulation of DJ-1 expression. Knockdown of microglial DJ-1 markedly suppressed NLRP3 inflammasome activation, as evidenced by reduced mature caspase-1 and decreased IL-1β secretion. We confirmed this phenomenon in BMDM and found that DJ-1 knockdown specifically inhibited NLRP3 inflammasome activation, with no effect on NLRC4 or AIM2 inflammasomes. , microglia-specific DJ-1 knockdown significantly attenuated microglial NLRP3 inflammasome activation in the substantia nigra and exerted neuroprotective effects after LPS treatment. Furthermore, DJ-1 was found to directly bind NLRP3 and stabilize its conformation, thereby preventing autophagic degradation.
This study demonstrates that DJ-1 deficiency in microglia inhibits NLRP3-driven inflammation by promoting NLRP3 degradation through the autophagy-lysosome pathway. Future studies should focus on identifying the specific binding sites and structure of DJ-1 with NLRP3, as well as investigating whether inhibiting DJ-1 in microglia could serve as a potential therapeutic target for suppressing neuroinflammation in Parkinson's disease.
本研究旨在探讨DJ-1在帕金森病中调节NLRP3炎性小体介导的神经炎症中的作用及潜在机制。
我们用脂多糖（LPS）刺激原代小胶质细胞，并将LPS立体定向注射到小鼠黑质中，以研究炎症刺激后DJ-1表达的变化。为了评估DJ-1对NLRP3炎性小体激活的功能影响，我们使用小干扰RNA（siRNA）敲低原代小胶质细胞或骨髓来源的巨噬细胞（BMDM）中的DJ-1，并分析下游炎症反应以及这种调节的特异性。此外，我们使用小胶质细胞特异性腺相关病毒（AAV）选择性沉默黑质中的DJ-1，以进一步评估DJ-1缺乏的抗炎作用。为了验证DJ-1与NLRP3之间的直接相互作用，我们进行了免疫共沉淀和邻近连接分析。我们使用自噬抑制剂3-甲基腺嘌呤（3-MA）和激活剂雷帕霉素来研究在CRISPR-Cas9基因编辑的DJ-1敲除的人胚肾293（HEK-293）细胞中，DJ-1缺乏时NLRP3是如何被降解的。
在原代小胶质细胞中，LPS或LPS加三磷酸腺苷（ATP）刺激后DJ-1显著上调。同样，在将LPS立体定向注射到黑质后，我们观察到DJ-1表达显著上调。敲低小胶质细胞中的DJ-1可显著抑制NLRP3炎性小体的激活，表现为成熟的半胱天冬酶-1减少和白细胞介素-1β分泌降低。我们在BMDM中证实了这一现象，并发现敲低DJ-1特异性抑制NLRP3炎性小体的激活，对NLRC4或AIM2炎性小体无影响。此外，小胶质细胞特异性敲低DJ-1可显著减弱黑质中小胶质细胞NLRP3炎性小体的激活，并在LPS处理后发挥神经保护作用。此外，发现DJ-1直接结合NLRP3并稳定其构象，从而防止自噬降解。
本研究表明，小胶质细胞中DJ-1的缺乏通过自噬-溶酶体途径促进NLRP3降解，从而抑制NLRP3驱动的炎症。未来的研究应集中于确定DJ-1与NLRP3的特异性结合位点和结构，以及研究抑制小胶质细胞中的DJ-1是否可作为抑制帕金森病神经炎症的潜在治疗靶点。
