Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Abstract

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.

Selective Degradation Permits a Feedback Loop Controlling Annexin A6 and Cholesterol Levels in Endolysosomes of NPC1 Mutant Cells

“…L. Liscum (Tufts University School of Medicine, Boston, MA, USA) and D. Ory (Washington University, St. Louis, MO, USA). The AnxA6-depleted CHO M12-A6ko cell line was generated in our laboratories using CRISPR/Cas9 editing technology as described [25]. Control (GM5659D) and NPC1 mutant human skin fibroblasts (GM03123) were from the Coriell Institute for Medical Research (Camden, NJ, USA) and cultured in DMEM, 10% FCS, L-glutamine (2 mM), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 • C, 5% CO 2 .…”

“…AnxA6-dependent Rab7 inactivation in NPC1 mutant cells was associated with a reduction of MCS between LE/Lys and the ER. Vice versa, AnxA6 depletion in NPC1 mutant cells caused elevated Rab7 activity, increased MCS formation, and significantly reduced the amount of cholesterol in LE/Lys [25].…”

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport

“…Thus, NPC1 meets all the criteria for a tether and appears to be an important regulator of MCS formation. Reduced MCS on loss of NPC1 activity was also found to be associated with reduced GTP‐bound (active) Rab7 due to recruitment of the Rab7 GAP TBC1D15 by endosomal AnnexinA6 . GTP‐Rab7 was restored on depletion of AnnexinA6, resulting in increased ER‐LE/Lys MCS and, excitingly, reversal of the cholesterol accumulation.…”

“…This explains how NPC1 transports cholesterol from the LE/Lys lumen to the cytosolic leaflet of the limiting membrane, but the next step in cholesterol egress, transport from the limiting membrane to the ER, has remained elusive. Two independent studies, one from our lab and the other from Meneses‐Salas et al, have gone some way to resolving this fundamental gap in our knowledge, uncovering an additional role for NPC1 in MCS formation . Both studies found that ER‐LE/Lys MCS were reduced on loss of functional NPC1, suggesting a regulatory role for NPC1 in MCS formation.…”

“…Although Rab7 was found to bind ORP1L independently of GTP/GDP‐binding state, both protrudin and PDZD8 specifically bind GTP‐bound Rab7 and NPC1 was recently also shown to interact with GTP‐bound Rab7 . Moreover, a recent study demonstrated reduced ER‐LE/Lys contact when GTP‐bound Rab7 was decreased, whereas increasing Rab7‐GTP, by inhibition of the Rab7 GAP TBC1D15, expanded the MCS …”