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

Abstract

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann–Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.

Lack of Annexin A6 Exacerbates Liver Dysfunction and Reduces Lifespan of Niemann-Pick Type C Protein–Deficient Mice

“…16,17 Yet, cholesterol accumulation in NP-C disease impairs the proper functioning of its endolysosomes, possibly also compromising Kupffer cell J o u r n a l P r e -p r o o f behavior. 18 Strikingly, we previously showed that restoration of hepatic NPC1 expression in Npc1 -/mice reduced hepatomegaly and hepatic cholesterol amounts, as well as bile salts, bilirubin, and transaminase levels in serum, without ameliorating the onset and progression of neurodegeneration. 19,20 In addition, several other endolysosomal proteins in the vicinity of NPC1 appear to influence NPC1-dependent activities and may contribute to the complex NP-C pathology.…”

“…[28][29][30] Interestingly, multiple ANXA6 interactions likely relevant for proper hepatic functioning are often linked to NPC1, such as autophagocytosis, cholesterol homeostasis, lysosomal function or gluconeogenesis. 30,31 Indeed, upregulated ANXA6 levels contributed to the compromised cholesterol transport in NPC1-deficient cells 21 and cholesterol-sensitive lysosomal degradation, 18 while hepatic ANXA6 deficiency hindered glucose homeostasis and survival during liver regeneration. 31 In this study, analysis of double-ko mice lacking NPC1 and ANXA6 (Npc1 -/-/Anxa6 -/-) revealed a significant reduction of lifespan compared to Npc1 -/mice, which correlated with a slight amelioration of some neurological functions, but overall worsening in motor coordination abilities.…”

Diverse Roles of Annexin A6 in Triple-Negative Breast Cancer Diagnosis, Prognosis and EGFR-Targeted Therapies

“…Most strikingly, other cell-based studies revealed Ca 2+ -insensitive, but cholesterol-dependent interaction of cytosolic AnxA6 with late endosomal membranes [ 38 ]. Interestingly, a recent study in Niemann–Pick-type C1 mutant cells identified a feedback loop involving selective lysosomal degradation of AnxA6 in the regulation of AnxA6 and cholesterol levels in this subcellular site [ 39 ]. On the other hand, upregulation of AnxA6 levels in A431, Chinese hamster ovary and other cell models triggered cholesterol accumulation in the late endosomal compartment [ 21 , 27 , 28 ].…”

“…Despite its Ca 2+ -dependent membrane-binding activity, and the implication of AnxA6 in several membrane-associated events, direct evidence for the involvement of AnxA6 in EV biogenesis and secretion is still lacking. Exosomes are generated from intraluminal membranes (ILVs) in the late endosomal compartment and recent studies identified substantial amounts of AnxA6 to be associated with ILVs [ 39 ]. Hence, one could envisage a direct physical association of AnxA6 with the final steps of exosome release at the cell surface, which needs further investigation.…”

Special Issue “Recent Developments in Annexin Biology”

“…They employed RNA sequencing to examine how the classical autophagy pathway in the host cell is altered by the influenza A virus infection, and discovered the importance of AnxA1 in enhancing autophagy in infected cells. The nexus between annexins, autophagy and human health was further elucidated by Meneses-Salas et al [ 13 ], focusing on the involvement of autophagy in the degradation of AnxA6 and its impact on the devastating childhood disorder Nieman Pick Disease. Finally, two of the studies report on the involvement of annexins in cancer.…”