Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A.

Chaperone-mediated autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not fully understood but might involve response to stress conditions including oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) has an impact on CMA modulation. In this work, we identified and validated 2 NFE2L2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NFE2L2 deficiency and overexpression was linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in nfe2l2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide, or pharmacological activation of NFE2L2 with sulforaphane or dimethyl fumarate also increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA activity, by NFE2L2. ABBREVIATIONS: ACTB: actin, beta, ARE: antioxidant response element; ATG5: autophagy related 5; BACH1: BTB domain and CNC homolog 1; ChIP: chromatin immunoprecipitation; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; DMF: dimethyl fumarate; ENCODE: Encyclopedia of DNA elements at the University of California, Santa Cruz; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA: glucosylceramidase beta; GFP: green fluorescent protein; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LAMP2B: lysosomal associated membrane protein 2B; LAMP2C: lysosomal associated membrane protein 2C; LAMP1: lysosomal associated membrane protein 1; MAFF: MAF bZIP transcription factor F; MAFK: MAF bZIP transcription factor K; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PQ: paraquat; PI: protease inhibitors; qRT-PCR: quantitative real-time polymerase chain reaction; RNASE: ribonuclease A family member; SFN: sulforaphane; SQSTM1/p62: sequestosome 1; TBP: TATA-box binding protein.

Role of Choline in the Modulation of Degenerative Processes: In Vivo and In Vitro Studies.

The purpose of the present study was to examine the nutraceutical potential of choline as an added value to its well-known brain nutrient role. Several toxicity, antitoxicity, genotoxicity, antigenotoxicity, and longevity endpoints were checked in the somatic mutation and recombination test in in vivo Drosophila animal model. Cytotoxicity in human leukemia-60 cell line (HL-60) promyelocytic and NIH3T3 mouse fibroblast cells, proapoptotic DNA fragmentation, comet assay, methylation status, and macroautophagy (MA) activity were tested in in vitro assays. Choline is not only safe but it is also able to protect against the DNA damage caused by an oxidative genotoxin. Moreover, it improves the life extension in the animal model. The in vitro results show that it is able to exhibit genetic damage against leukemia HL-60 cells. Single-strand breaks in DNA are observed at the molecular level in treatments with choline, although only a significant hypermethylation on the long interspersed elements-1 and a hypomethylation on the satellite-alpha DNA repetitive DNA sequences of HL-60 cells at the lowest concentration (0.447 mM) were observed. Besides, choline decreased MA at the lower assayed concentration and the MA response to topoisomerase inhibitor (etoposide) is maintained in the presence of treatment with 0.22 mM choline. Taking into account the hopeful results obtained in the in vivo and in vitro assays, choline could be proposed as a substance with an important nutraceutical value for different purposes.

Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging.

Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.

Functional interaction between autophagy and ciliogenesis.

Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signalling motivated us to explore the possible functional interactions between this signalling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signalling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. Whereas abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signalling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie some common ciliopathies.