Hepatic retromer is essential for systemic cholesterol homeostasis by regulating lysosomal cholesterol handling.

Disturbed hepatic cholesterol homeostasis is associated with atherosclerotic cardiovascular disease (ASCVD) and metabolic dysfunction-associated steatotic liver disease (MASLD). The endo-lysosomal system plays a central role in cholesterol uptake and intracellular trafficking, yet the molecular mechanisms governing these processes remain poorly understood. Here, we identify hepatic vacuolar protein sorting 35 (VPS35), a core component of the retromer complex, as a critical regulator of hepatocellular and systemic cholesterol homeostasis. Liver-specific VPS35 ablation in mice elevated plasma and hepatic cholesterol levels due to impaired hepatic uptake of atherogenic lipoproteins, defective cholesterol trafficking through the endo-lysosomal system, and increased de novo cholesterol biosynthesis. The decreased hepatic uptake of atherogenic lipoproteins was associated with altered subcellular distribution of the low-density lipoprotein receptor (LDLR) and reduced levels of LDLR-related protein 1 (LRP1). The increased hepatic cholesterol content was associated with altered lysosomal ultrastructure and pH and decreased levels of a wide range of lysosomal proteins, including key proteins for lysosomal cholesterol egress, such as lysosomal acid lipase (LAL), scavenger receptor class B member 2 (SCARB2), Niemann-Pick type C1 (NPC1), and NPC2. Collectively, these findings reveal a previously unrecognized function of the hepatic retromer complex in systemic cholesterol homeostasis. Beyond its established role in endosomal cargo transport, VPS35 is essential for lysosomal cholesterol handling by preserving key proteins involved in lysosomal cholesterol metabolism.