Snapin mediates neuronal PANoptosis after mild traumatic brain injury via H2S-dependent S-sulfhydration of CTSD.

INTRODUCTION: Traumatic brain injury (TBI) induces complex secondary neuronal death, yet no specific therapeutic strategies are available to ameliorate post-traumatic neurological dysfunction. PANoptosis is a coordinated cell death pathway involving apoptosis, pyroptosis, and necroptosis; however, it remains unknown whether PANoptosis contributes to mild TBI (mTBI)-induced neuronal death and what precise mechanisms govern this process. OBJECTIVE: This study aimed to investigate whether Snapin regulates neuronal PANoptosis following mTBI and to elucidate its functional mechanism through the hydrogen sulfide (H2S)/ cathepsin D (CTSD) pathway. METHODS: We utilized a controlled cortical impact (CCI) protocol to create a model of mTBI. Neuronal Snapin was conditionally knocked down via AAV-shSnapin. PANoptosis-related proteins were evaluated using immunofluorescence staining and Western blot. Endogenous H2S levels were measured with a sulfide ion-selective electrode. The interaction between Snapin and cystathionine β-synthase (CBS) was examined by molecular docking and co-immunoprecipitation, while S-sulfhydration of CTSD was detected using a modified biotin switch assay. Therapeutic effects were assessed through behavioral tests including the wire-grip test, open field test, beam balanced walk, novel object recognition, and Morris water maze. RESULTS: Snapin was significantly upregulated in cortical and hippocampal neurons after mTBI. Knockdown of Snapin attenuated neurodegeneration, reduced PANoptosis-related proteins, and improved neurofunctional recovery. Mechanistically, Snapin bound to CBS, disrupting H2S metabolic homeostasis and reducing endogenous H2S levels. The decrease in H2S limited S-sulfhydration of pro-CTSD, promoting its maturation into active CTSD and ultimately inducing PANoptosis. Both pepstatin A and NaHS treatment conferred neuroprotection, reducing neuronal death and neuroinflammation. CONCLUSION: Snapin promotes mTBI-induced neuronal PANoptosis by disrupting the H2S/CTSD pathway through specific binding with CBS. Therapeutic strategies targeting Snapin, restoring H2S homeostasis, or inhibiting CTSD activation may significantly alleviate neurological impairment and neuroinflammation post-mTBI, highlighting novel therapeutic avenues for mTBI treatment.