Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation. Keywords Cholesterol • Late endosomes • Rab7 • NPC1 • Annexin A6 • Membrane contact sites Abbreviations A431 Human epidermoid carcinoma cells ACAT Acyl-CoA:cholesterol acyltransferase AnxA6 Annexin A6 CHO Chinese hamster ovary CHO M12 NPC1 mutant CHO cell line CMA Chaperone-mediated autophagy ER Endoplasmic reticulum FYCO1 FYVE and coiled-coil domain containing 1 GST Glutathione S-transferase LE/Lys Late endosome/lysosome (endolysosomes) LPDS Lipoprotein-deficient serum MCS Membrane contact sites MEF Mouse embryonic fibroblasts MOSPD2 Motile sperm domain containing 2 NPC1 Niemann-Pick type C1 ORP1L Oxysterol-related protein 1L OSBP Oxysterol-binding protein PFO Perfringolysin O RILP Rab interacting lysosomal protein SREBP Sterol regulatory element binding protein StARD3 StAR-related lipid transfer domain-3 TBC1D15 TBC1 domain family member 15 WT Wild type VAP-A Vesicle-associated membrane protein-associated protein A Vps13 Vacuolar protein sorting-associated protein 13 Cellular and Molecular Life Sciences Elsa Meneses-Salas and Ana García-Melero contributed equally to this work.

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Skin aging is characterized by structural and functional changes that contribute to age-associated frailty. This probably depends on synergy between alterations in the local niche and stem cell-intrinsic changes, underscored by proinflammatory microenvironments that drive pleotropic changes. The nature of these age-associated inflammatory cues, or how they affect tissue aging, is unknown. Based on single-cell RNA sequencing of the dermal compartment of mouse skin, we show a skew towards an IL-17-expressing phenotype of T helper cells, γδ T cells and innate lymphoid cells in aged skin. Importantly, in vivo blockade of IL-17 signaling during aging reduces the proinflammatory state of the skin, delaying the appearance of age-related traits. Mechanistically, aberrant IL-17 signals through NF-κB in epidermal cells to impair homeostatic functions while promoting an inflammatory state. Our results indicate that aged skin shows signs of chronic inflammation and that increased IL-17 signaling could be targeted to prevent age-associated skin ailments.