Lipid Droplet-Associated Hsp70 Palmitoylation Drives Lysosomal Dysfunction in Ischemic Stroke

Lipid droplet (LDs) accumulate in glial cells across neurodegenerative and ischemic brain pathologies. LDs modulate cellular stress via organelle crosstalk, particularly with lysosomes, whose dysfunction and cathepsin leakage drive glial activation after cerebral ischemia. However, the molecular link between accumulated LDs and lysosomes in pathological glial activation remains incompletely understood. Using a mouse middle cerebral artery occlusion ischemia/reperfusion (I/R) model and oxygen-glucose deprivation/reoxygenation (OGD/Re) cell models, we found that both LD signal intensity and lysosomal cathepsin B leakage progressively increased from day 1 to day 14 after I/R. Etomoxir, an inhibitor of carnitine palmitoyltransferase 1 A (CPT1A), the rate-limiting enzyme for fatty acid β-oxidation, aggravated I/R-induced LD accumulation and lysosomal damage in glial cells. Exogenous LDs derived from OGD/Re-treated glial cells aggravated lysosomal damage and promoted the proliferation of astrocytes and microglia. LD accumulation was associated with Hsp70 palmitoylation and redistribution of Hsp70 from lysosomes to the extracellular space, leading to lysosomal damage and reactive glial activation. Mechanistically, using multiple genetic and pharmacological approaches, including Plin3 knockdown, an Hsp70 palmitoylation‑site mutant, recombinant human Hsp70 (rhHsp70), as well as a pan-palmitoylation inhibitor and TLR2/4 antagonist, we consistently validated that Plin3, an LD surface protein, interacts with lysosomal Hsp70, causing Hsp70 palmitoylation and redistribution, and lysosomal damage. Extracellular Hsp70 activates TLR2/4 on glial cells, promoting cell proliferation and pro-inflammatory cytokine release. Collectively, these findings indicate that pathological LDs interacting with lysosomes drive Hsp70 palmitoylation and redistribution, causing lysosomal impairment and astrogliosis in ischemic stroke. We uncover Hsp70 palmitoylation as a potential drug target for ischemic stroke therapy.