Chaperone-Mediated Autophagy Ensures Hematopoietic Stem Cell Maintenance

Abstract

Hematopoietic stem cells (HSC) harbor extensive self-renewal capabilities along with multilineage differentiation plasticity to sustain blood production over a lifetime. Maintenance of a fully functional proteome that can rapidly change to adjust to the status of HSC activation is facilitated in part by two major intracellular proteolytic pathways, the ubiquitin proteasome system and the autophagy/lysosomal system. Alterations in HSC proteostasis have been associated with a number of degenerative and malignant diseases, underscoring the importance of elucidating the precise contribution of components of the cellular proteostasis network to HSC maintenance. In this work, we have focused in a highly selective type of autophagy, known as chaperone-mediated autophagy (CMA), whereby individual proteins bearing a unique pentapeptide motif (KFERQ-like) are targeted for degradation in lysosomes upon binding to the heat shock cognate protein of 70 kDa (HSC70). Substrate proteins are directly translocated into the lysosomal lumen through a dedicated multiprotein translocation complex. The main component of this complex is the lysosome-associated membrane protein type 2A (LAMP-2A) that also serves as substrate receptor and is the limiting component of CMA. Using novel mouse models that allow for CMA tracking (KFERQ-Dendra mice) and for selective depletion of LAMP-2A in hematopoietic cells (Vav-iCre:LAMP2Af/f mice), we have investigated the physiological role of CMA in HSCs during steady-state and upon activation and gained novel insights on the consequences of CMA failure in these cells in aging. Our work revealed that the basal CMA activity detected in quiescent HSC under steady-state conditions is significantly stimulated upon HSC activation following 5-fluorouracil (5-FU) in vivo exposure. This upregulation of CMA is necessary to ensure HSCs persistence during activation because, upon serial 5-FU injections, CMA-deficient HSCs had a significantly reduced multilineage reconstitution ability with premature bone marrow failure. We found reduced long-term colony formation of CMA-deficient HSCs in serial colony formation assays and demonstrated that these cells had a significant and progressive disadvantage of repopulating lethally-irradiated congenic recipient mice upon serial bone marrow transplantation. We also found that CMA becomes increasingly important for the maintenance of functional HSCs in aging, since as mice age, CMA-deficient HSCs showed an even greater functional defect compared to age-mated control-derived HSCs. Using comparative transcriptomics and metabolomics on HSCs from control and LAMP-2A-deficient mice, we found evidence for metabolic alterations and dysfunctional redox signaling. We confirmed that CMA-deficient HSCs have reduced rates of glycolysis, lower ATP production and higher reactive oxygen species levels than control cells. Deficient cellular energetics and increased oxidative stress are important consequences of CMA failure in HSC, since supplementation with pyruvate or treatment with anti-oxidant agents (i.e. N-acetyl-cysteine) was sufficient to restore CMA-deficient HSC function, as measured by serial colony formation. Proteomic analysis of CMA-deficient Lin-Scal+c-Kit+ (LSK) cells revealed an overall increase of acetylated and oxidized proteins, including key metabolic enzymes and proteins required for the cellular response to oxidative stress. We propose that, upon CMA failure, the inability of HSC cells to timely turning over these regulatory proteins, favors accumulation of unwanted post-translational modifications that interfere with their normal functioning. Together, our findings suggest that CMA upregulation during HSCs activation is required for the proteome remodeling that facilitates transition from quiescent to activated cells. By assuring timely turnover of selected proteins, CMA sustains the metabolic adaptation required to meet these cells' energetic needs and assures an efficient cellular response to stress. Disclosures No relevant conflicts of interest to declare.