Integrating single-cell transcriptomics, molecular dynamics, and functional assays to characterize the STING-P4HA1 immunometabolic axis in Parkinson's disease

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) stabilizes the collagen triple helix and is increasingly implicated in non-canonical metabolic processes, though the structural basis of these roles remains poorly understood. We combined single-cell RNA sequencing (scRNA-seq) with high-dimensional weighted gene co-expression network analysis (hdWGCNA) to identify P4HA1 in microglia with high STING activity. Functional validation was performed in MPTP mice and MPP⁺-stimulated BV2 microglia using AAV-shRNA-mediated knockdown, Seahorse metabolic flux analysis, and assays for oxidative stress and polarization. Druggability was evaluated by virtual screening of an FDA-approved drug library, and the top complex underwent 100 ns molecular dynamics (MD) simulations assessing RMSD, Rg, SASA, and free energy landscape (FEL). P4HA1 was upregulated in Parkinson’s disease (PD) models, promoting a glycolytic shift—evidenced by increased ECAR and suppressed mitochondrial OCR—that drove ROS production, mitochondrial depolarization, and M1 microglial polarization. P4HA1 knockdown attenuated dopaminergic neuron loss and improved motor and cognitive deficits in MPTP mice. Virtual screening identified Exatecan as a high-affinity P4HA1 inhibitor (−12.2 kcal/mol). MD simulations confirmed complex stability and revealed anisotropic compaction toward a global free energy minimum. Our findings establish a structural and dynamic basis for the non-canonical immunometabolic function of P4HA1, supporting its potential as a therapeutic target in PD.