The PI4K2A-OSBPL6/ORP6-PS axis mediates lysosomal membrane repair to restore neuronal lipid homeostasis and promote neuronal survival after spinal cord injury

Dysfunction of the neuronal macroautophagy/autophagy-lysosome system is a critical contributor to neuronal death following spinal cord injury (SCI), but the underlying mechanisms remain elusive. Our study demonstrated that SCI induced impaired autophagic flux and lysosomal membrane permeabilization (LMP) in neurons. By combining <i>in vivo</i> bulk RNA sequencing with validation experiments, we observed the transient upregulation of the membrane repair factor PI4K2A, which was specifically enriched in lysosomes, after SCI. Crucially, ER-MS and IP-MS analyses revealed an interaction between PI4K2A and the endoplasmic reticulum lipid transfer protein OSBPL6/ORP6. This interaction led to the transport of phosphatidylserine (PS) to damaged lysosomal membranes, promoting LMP repair and subsequently reducing lipid droplet accumulation, which suppressed neuronal death. Furthermore, overexpression of neuronal PI4K2A <i>in vivo</i>, through an OSBPL6- and PS-dependent mechanism, reduced LMP-mediated lipid droplet accumulation and increased neuronal survival, thereby improving functional recovery after SCI. Collectively, our findings establish the PI4K2A-OSBPL6/ORP6-PS axis as a novel and essential mechanism for lysosomal membrane repair in neurons. This pathway is crucial for maintaining neuronal lipid homeostasis and represents a promising therapeutic target for reducing neuronal loss and improving functional recovery after central nervous system trauma.<b>Abbreviations</b>: AIF1/IBA1: allograft inflammatory factor 1; Baf A1: bafilomycin A₁; BMS: Basso Mouse Scale; CNS: central nervous system; co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DS5: DS55980254; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HSPA5/GRP78: heat shock protein family A (HSP70) member 5; HT22: hippocampal neuronal cell line; KEGG: Kyoto Encyclopedia of Genes and Genomes; LD: lipid droplet; LC-MS: liquid chromatography-mass spectrometry; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LGALS3/GAL3: lectin, galactoside binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanolamine; MFGE8/lactadherin: milk fat globule EGF and factor V/VIII domain containing; MS: mass spectrometry; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); NEFH/NF200: neurofilament, heavy polypeptide; OSBPL6/ORP6: oxysterol binding protein-like 6; OSBPL8/ORP8: oxysterol binding protein-like 8; PC: phosphatidylcholine; PLA2G4A/cPLA2: phospholipase A2, group IVA (cytosolic, calcium dependent); PITT: phosphoinositide-initiated membrane tethering and lipid transport; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PLS-DA: partial least squares discriminant analysis; PS: phosphatidylserine; PtdIns: phosphatidylinositol; PTDSS1: phosphatidylserine synthase 1; PUFAs: polyunsaturated fatty acids; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; ROS: reactive oxygen species; SCI: spinal cord injury; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TGs: triglycerides.