Chaperone-mediated autophagy protects against hyperglycemic stress

ABSTRACTChaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.KEYWORDS: CMAfishglucose intoleranceLamp2AmetabolismNFE2L2/NRF2 AcknowledgementsWe thanks Atika Zouine and Vincent Pitard for technical assistance at the Flow cytometry facility, Centre National de la Recherche Scientifique (CNRS) unité mixte de service (UMS) 3427, Institut National de la Santé Et de la Recherche Médicale (INSERM) US 005, University of Bordeaux, F-33000 Bordeaux, France. We thanks Christel Poujol for technical assistance at the Bordeaux Imaging Center (BIC), CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging and supported by the French National Research Agency (ANR-10-INBS-04). We thanks Alexandre Stella from the ProteoToul Services - Proteomics Facility of Toulouse for the proteomics analysis. We also want to thanks F. Terrier, and A. Lanuque for the preparation of diets and care of fish, L. Peron for manufacturing the light emitting device, and the IE ECP Ecology and Fish Population Biology Facility for the access to the widefield AXIO Imager M2 microscope. Schematic figures were created with BioRender.com tools.Disclosure statementNo potential conflict of interest was reported by the author(s).Supplementary dataSupplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2267415Data availability statementThe authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.Additional informationFundingThis project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 101030643 and the Aquaexcel3.0 grant agreement No 871108, and by the European Maritime and Fisheries Fund [PFEA470019FA1000005].