2480 - Spatio-Temporal Transcriptomics Reveals PANoptosis-Driven Neuronal Loss in Radiation-Induced Brain Injury and Extracellular Vesicle Intervention Strategy

Purpose/Objective(s):

Radiation-induced brain injury (RIBI) is a severe long-term complication following radiotherapy for head and neck tumors. This study aimed to decipher the key death program underlying early neuronal loss in RIBI and explore a clinically translatable therapeutic strategy using targeted extracellular vesicles (EVs).

Materials/Methods:

A mouse model of whole-brain irradiation with 30Gy was established, and the pathological progression of RIBI was systematically evaluated through neurological function scoring, brain water content measurement, and Nissl staining. Hippocampal tissues were collected from control mice and irradiated mice at 3 and 7 days post-irradiation. Combined with single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics (ST-RNA-seq), scWGCNA, PROGENy pathway scoring, and cell2location deconvolution analyses were performed to clarify changes in cell composition, injury-related gene modules, and spatial distribution characteristics. EVs derived from brain tissues of adult and neonatal mice were isolated and identified, and their molecular characteristics were defined by proteomics. Furthermore, rabies virus glycoprotein-derived peptide (RVG)-modified targeted EVs were constructed, and their impacts on magnetic resonance imaging (MRI)-measured white matter volume (WMV), Morris water maze behavioral performance, and 42-day peripheral organ toxicity (HE staining, ALT/AST detection) in mice were evaluated via in vivo delivery.

Results:

The results showed that irradiated mice exhibited significant cerebral edema, reduced Nissl bodies in the cortex/hippocampus, and deteriorated neurological function. Consistent with snRNA-seq and ST-RNA-seq data, the proportion of neurons significantly decreased 3 , 7 days post-irradiation, and DNA damage (marked by ?-H2AX) and NF-?B pathway activation showed brain region-specific distribution. The PANoptosis score of neurons increased, the co-labeling rate of GSDMD/MLKL/Casp3 with NeuN was elevated, and the cell communication network shifted from pro-regenerative signals to pro-death signals. In vitro and organoid experiments confirmed that they reduced ?-H2AX accumulation and PANoptosis-related pathway activation by inhibiting the cGAS–STING/IRF3/NF-?B axis. Post-treatment snRNA-seq showed that the proportion of neurons recovered by approximately 6%, and PANoptosis and NF-?B signals were significantly downregulated without obvious peripheral toxicity.

Conclusion:

This study is the first to confirm that PANoptosis is the core driving mechanism of early neuronal loss in RIBI, identifies the cGAS–STING/NF-?B axis as a key regulatory pathway.