Loss of DJ-1 Alleviates Microglia-Mediated Neuroinflammation in Parkinson's Disease via Autophagy-Lysosomal Degradation of NLRP3

Qianqian Miao^1,2,3*Tiantian Wang^3,4Haoran Wang^5,6Yanxia Yu⁷Xing Jin^3,7*

• ¹Department of Pharmacy, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
• ²Suzhou Municipal Hospital, Suzhou, China
• ³Gusu School, Nanjing Medical University, Suzhou, China
• ⁴The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
• ⁵Nanjing Medical University, Nanjing, China
• ⁶Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
• ⁷Department of Clinical Pharmacy, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China

Objective: This study aimed to investigate the role and underlying mechanism of DJ-1 in regulating NLRP3 inflammasome-mediated neuroinflammation during Parkinson's disease. Methods: We used LPS to stimulate primary microglia in vitro and performed stereotactic injection of LPS into the substantia nigra of mice in vivo 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. In vivo, 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. Results: 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. In vivo, 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. Conclusion: 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.