Abstract
The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2,3,4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
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Data availability
There are no restrictions on data availability in this manuscript. All the information is included in the manuscript. Source data for all main and Extended Data Figures are available online as separate Excel files for each figure. Transcriptomic data have been deposited in GEO with accession number GSE142632. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD022196 (http://www.ebi.ac.uk/pride/archive/projects/PXD022196). Access to the STRING database is through https://string-db.org/. Source data are provided with this paper.
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Acknowledgements
We thank J. Zhang at the Cell Sorting Core Facility, D. Sun at the Stem Cell Isolation and Xenotransplantation facility and experts at the Analytical Image Facilities at Einstein (NCI P30CA013330) for technical assistance; M. Bourdenx for assistance with computational analysis and A. Verma and R. Elkind at Montefiore Medical Center for human hematopoietic stem-cell specimens. This work was supported by grants from the National Institutes of Health: AG021904, AG038072 and DK098408 (to A.M.C.), AG031782 (to A.M.C. and E.G.), DK105134 and CA230756 (to B.W.), R01HL146442, R01HL149714, R01HL148151 and R21HL150032 (to A.D’A.) and by the generous support of the Rainwaters Foundation (to A.M.C. and E.G.), the JPB foundation and Robert and Renée Belfer. Y.R.K. is the recipient of a T32 Training in Aging research grant (PI: Barzilai, AG023475).
Author information
Authors and Affiliations
Contributions
S.D. designed and performed biochemical, image-based and FACS-based experiments, analysed and interpreted the data, and prepared the first draft of the manuscript; Q.W. assisted with experimental design and FACS-based experiments; Y.R.K. assisted with the LTC-IC assays and analysis; A.D. performed the proteolysis assays and in vivo mouse treatments; I.T. generated and characterized the hL2AOE mice and assisted with experiments on those mice; S.K. assisted with Seahorse assays, EM and morphometric analysis and manuscript editing; V.T. and A.Z. assisted with mice handling and dissection; E.N. performed protein post-translational analysis by proteomics; M.D. performed proteomic analysis; J.A.R. performed metabolomic studies; E.G. designed the CA molecules and led the medicinal chemistry efforts for their in vivo use.; A.D’A. coordinated the proteomic and metabolomic analysis and contributed to manuscript editing; B.W. and A.M.C. coordinated the study and contributed to the design and interpretation of the experiments and to the writing and final editing of the manuscript.
Corresponding authors
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Competing interests
A.M.C. and E.G. are cofounders of Selphagy Therapeutics (Boston, MA, USA). The CA compound is under the following patent: Retinoic acid receptor antagonists as chaperone-mediated autophagy modulators and uses thereof. Gavathiotis, E., Cuervo. A. M., Xin, Q., Das, B. C. United States patent US9512092, granted 16 December 2016. All other authors declare no competing interests.
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Peer review information Nature thanks Danica Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 CMA changes in HSC with age.
a, Scheme of the KFERQ-Dendra2 fluorescent CMA reporter. b, Percentage of HSC positive for Dendra puncta at the indicated ages. n = 4 mice per group, individual points are fields. CMA+ cells = cells with >2 Dendra+ puncta. c, Dendra fluorescence in GMP cells from young and old KFERQ-Dendra mice. Left: Representative images and magnified cells. Right: Puncta per cell. n = 8 fields from 3 mice per group, individual points show average puncta/field. d, Dendra fluorescence in HSC from young and old L2AKO-KFERQ-Dendra mice. Left: Representative images and magnified cells. Right: Puncta per cell. n = 8 fields from 4 mice per group. e, Immunostaining for LAMP1 and LAMP2A in HSC from young and old mice. Top: Representative images and magnified cells. Bottom: Fluorescence intensity of each protein. n = 8 (LAMP1), 11 (LAMP2A) fields from 3 mice per group, individual points shows average intensity/field. f, Proteins bearing KFERQ-like motifs in those at higher (O>Y) and lower (O<Y) abundance in old human BM HSC compared to young ones (data set in ref. 31). Motif frequency in the total proteome is shown as reference. n = 59 human subjects (45 male and 14 female) with age range 20-60 years (with median of 33.2 years). Only proteins displaying statistically significant changes with age (P < 0.01) were included. g, STRING analysis of the proteins bearing CMA-targeting motifs that accumulate in old human BM HSC. Data shows individual values and mean ± s.e.m. One-way (b) or two-way (f) ANOVA with Tukey’s post hoc test and unpaired two-tailed t-test (c, d, e) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 20 μm (full field) and 5 μm (insets) (c-e).
Extended Data Fig. 2 CMA upregulation during HSC activation.
a, Left: Full fields of the images shown at higher magnification in Fig. 1b. Right: HSC positive for Dendra puncta. n = 4 mice per group. CMA+ cells = cells with >2 Dendra+ puncta. b, Lysosomal proteolysis of long-lived proteins in HSC untreated (Ctrl) and 8 days post 5FU injection. n = 3 independent experiments. c, Immunostaining for LAMP2A, LAMP1 and LysoTracker in HSC from mice before and 1, 3 and 8 days after single 5FU injection. Top: Representative images. Bottom: Fluorescence intensity. n = 16, 11, 7, 19 fields (LAMP2A), 9, 10, 7, 16 fields (LAMP1) and 10, 10, 6, 7 fields (LysoTracker) from 3 mice per group, individual points show average intensity/field. d, e, Dendra fluorescence in HSC from KFERQ-Dendra mice 48h post PolyI:C or vehicle injection (Basal). Representative images and magnified cells (d) and puncta per cell (e), n = 3 mice. Individual points show average puncta/field. f, g, Full fields of the images shown at higher magnification in Fig. 1b (f) and number of Dendra+ puncta per cell (g) in myeloid progenitors from KFERQ-Dendra mice before and at the indicated days post 5FU injection. n = 3 mice. h, i, Dendra fluorescence in myeloid progenitors from KFERQ-Dendra mice 48h post PolyI:C or vehicle injection (Basal). Representative images and magnified cells (h) and puncta per cell (i), n = 3 mice. Data shows individual values and mean ± s.e.m. Unpaired two tailed t-tests (b, e, i) and one-way ANOVA with Tukey’s post hoc test (a, c, g) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 10 μm (a, f), 5 μm (c), and 10 μm (full field) and 5 μm (insets) (d, h).
Extended Data Fig. 3 Effect of CMA blockage on HSC and myeloid progenitor cells.
a, Quantitative PCR for the three lamp2 spliced variants in BM from Vav-iCreL2Af/f (L2AKO) and VaviCre (Ctrl) mice. n = 3 independent experiments. b, Number of total BM cells of 2 femurs and tibias from Ctrl and L2AKO mice in basal and at day 8 post 5FU injection. n = 5 mice. c, Percentage of T cells (CD3ε+), B cells (B220+), granulocytes (CD11b+ Gr1high) and monocytes (CD11b+ Gr1middle) in Ctrl and L2AKO mice BM in basal conditions. n = 5 mice. d, Frequency of Lin- cells of 2 femurs and tibias from Ctrl and L2AKO mice in basal and day 8 post 5FU injection. n = 6 (basal) and 4 (5FU) mice. e, f, Number of LSK cells (e) and HSC cells (f) in BM of Ctrl and L2AKO mice in basal and 5FU activated conditions. n = 6 mice. g, h, Donor chimaerism in peripheral blood (g) and donor derived Lin- population (h) in BM of recipients at the indicated weeks after the primary competitive transplantation using BM from 4m Ctrl and L2AKO mice. n = 7 mice (g), n = 5 mice (h). i-k, Donor lineage distribution in peripheral blood of recipients in the secondary competitive transplantation of Ctrl and L2AKO BM. Representative FACS plots (left) and quantification (right) are shown. n = 7 mice. l, LTC-IC frequency for both Ctrl and L2AKO cells in the LTC-IC assay of Lin−Sca-1+c-Kit+ (LSK) cells from Ctrl and L2AKO mice. n = 3 mice. Data shows individual values and mean ± s.e.m. Two way ANOVA with Sidak’s post hoc test (b-d, g), unpaired two-tailed t-tests (a, e, f, h-k) and Chi-square test (l) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance.
Extended Data Fig. 4 Impact of CMA blockage in the cell cycle of HSC and myeloid progenitor cells.
a-c, Representative FACS plots of Hoechst and Ki67 staining (a) or Hoechst and BrdU staining (c) in HSC from control (Ctrl) and L2AKO mice untreated (basal) or 8 days post single 5FU injection. Quantification of FACS in a is shown in Fig. 2a and quantification of FACS in c is shown in b. n = 4 mice. d, e, Representative FACS plots of Ki67 and Hoechst staining (e) and quantification (d) of cycling HSC from Ctrl and L2AKO mice untreated (basal) or 16 days post single 5FU injection. n = 4 mice. f, g, Percentage of cycling cells (f) and representative flow cytometry plots (g) of Ki67 and Hoechst stained myeloid progenitors from Ctrl and L2AKO mice in basal conditions (top) and 8 days after a single 5FU injection (bottom). n = 7 mice per condition. h, i, ATP (h) and ROS levels (i) in myeloid progenitors from Ctrl and L2AKO mice in basal or after single 5FU injection. n = 4 (basal) and 3 (5FU) mice for h, n = 5 mice for i. Data shows individual values and mean ± s.e.m. Two-way ANOVA with Sidak’s post hoc test (b, f, h, i) and unpaired two tailed t-test (d) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance.
Extended Data Fig. 5 Characterization of macroautophagy in CMA-deficient HSC cells.
a, Immunostaining for endogenous LC3 in HSC from control (Ctr) and L2AKO mice in basal and cytokine starved conditions in presence or not of lysosomal inhibitors NH4Cl and leupeptin (N/L). Representative images of full fields and higher magnification cells (left) and LC3 flux (right) calculated as the increase in LC3+ puncta upon addition of N/L. n = 9 fields from 3 mice, individual points show average puncta/field. b, Expression of genes in the TFEB-regulated transcriptional program in Ctrl and L2AKO LSK cells. Genes are grouped as: components of the macroautophagy machinery (top left), regulators of macroautophagy initiation (right) and genes involved in lysosome biogenesis (bottom left). c, Representative electron microscopy images of HSC (from 3 independent repeats). Top: whole fields and boxed areas at higher magnification to show examples of autophagosomes (APG, yellow arrows) and autolysosomes (AUT, red arrows). Bottom: representative images of ultrastructure of the endoplasmic reticulum (green arrows) in HSC under basal and 5FU (day8) activated conditions. d-f, Average number of APG, AUT (d) and mitochondrial number per cell (e) as well as cytoplasm and total cell area (f) from electron microscopy images in c. n = 15 (Ctrl APG/AUT), 13 (L2AKO APG) and 12 (L2AKO AUT) fields (d), 11 fields (e) and 6 fields (f) from 3 independent samples. g, Average ER diameter in HSC under basal and 5FU (day8) activated conditions. n = 11 (basal ctrl), 8 (basal L2AKO), 13 (5FU ctrl) and 10 (5FU L2AKO) fields. Data shows individual values and mean ± s.e.m. Two-way ANOVA with Tukey’s multiple post hoc test (a) and unpaired two-tailed t-test (d-g) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 20 μm (full field) and 5 μm (insets) (a), 1 μm (full field) and 0.5 μm (insets) (c).
Extended Data Fig. 6 Metabolism-related changes in the transcriptome and proteome of CMA-deficient HSC.
a, Categories of metabolism-related top transcriptional changes in L2AKO HSC cells in basal conditions. b, STRING analysis of proteins more abundant in L2AKO LSK in basal conditions. c, Top categories of proteins related with overall metabolism (left) and break down of the proteins under enzyme category (right) found in the overlapped proteins in Fig. 2h. d, Scheme of glycolysis-related substrates and metabolites to highlight those significantly increased (red) or decreased (green) in L2AKO LSK. e, f, ECAR (e) and basal glycolysis, glycolytic capacity and glycolytic reserve (f) in myeloid progenitors. n = 3 independent experiments. g, Full field images of HSC straining for OxyICC or Proteostat shown in Fig. 2q, r. Bottom panel shows full field and higher magnification inset of HSC 4-HNE staining. h, Intensity of 4-HNE staining shown in g. n = 8 (Ctrl) and 11 (L2AKO) fields from 3 mice, individual points represent average intensity per field. i, Ingenuity pathway analysis of the top proteins with higher percentage of oxidation in activated L2AKO LSK cells compared with control. j, k, STRING analysis of proteins with higher percentage of oxidation in basal (j) and 5FU activated conditions (k). l, GAPDH and PK activity in 5FU-activated Ctrl and L2AKO LSK cells. n = 3 independent experiments. Data shows individual values and mean ± s.e.m. Multiple t-test by time segment (e) and unpaired two-tailed t-tests (f, h, l) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 20 μm (full field) and 5 μm (insets) (g).
Extended Data Fig. 7 Regulation of lipid metabolism by CMA during HSC activation.
a, Metabolic pathways different between Ctrl and L2AKO LSK 8 days post-5FU injection. n = 9 mice in 3 independent experiments. b, c, Immunostaining for FADS2 in LSK cells. Representative images (b) and average of intensity per cell (c). n = 16 (basal Ctrl), 21 (basal L2AKO), 17 (5FU Ctrl) and 23 (5FU L2AKO) fields from 3 independent experiments, d, In silico analysis of proteins bearing phosphorylation- or acetylation-generated KFERQ-like motifs in the total proteome, and in the group of proteins less/more abundant in L2AKO LSK cells post-5FU. Left: Percentage of proteins with KFERQ motif. Right: STRING analysis of proteins bearing acetylation-generated KFERQ motif in the L2AKO > Ctrl group. n = 3 mice in 3 independent experiments. e, Mass spectrometry analysis of acetylation-generated KFERQ motifs in proteins with lower and higher acetylation in L2AKO LSK post-5FU. Left: Percentage of proteins with or without acetylation generated KFERQ motifs. Right: STRING analysis of proteins with acetylation-generated motifs in the group of proteins with higher acetylation in L2AKO. n = 9 mice in 3 independent experiments. f, Venn diagram of proteins more abundant in L2AKO LSK than Ctrl and in old than young LSK. n = 9 mice in 3 independent experiments. Bottom: STRING analysis of proteins that accumulate in both L2AKO and old mice LSK. g-i, Metabolomic analysis in young (4m) and old (>25m) mice LSK in basal conditions. Unsupervised principal component analysis (g). Comparison of metabolites in glycolysis (h) and in the FADS2-catalysed step in linoleic acid metabolism (i). Values are integrated peak areas (arbitrary units) for each metabolite. n = 9 mice in 3 independent experiments. Data shows individual values and mean ± s.e.m. Two-way ANOVA with Tukey’s (d, e) or Sidak’s (c) post hoc test and unpaired two-tailed t-tests (h, i) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 20 μm (b).
Extended Data Fig. 8 CMA activity in haematopoietic stem cells from old mice.
a, HSC numbers in control (Ctrl) and L2AKO mice with age, n = 10,10,11 (Ctrl) and 10,11,9 (L2AKO) mice per age. b, Donor chimaerism in recipients’ peripheral blood after old Ctrl and L2AKO mice BM competitive transplantation. n = 6,5,5,6,6 (Ctrl) and 5,5,6,5,5 (L2AKO) mice per time point. c, Donor lineage distribution in peripheral blood after Ctrl and L2AKO mice BM second competitive transplantation, n = 6 (Ctrl) and 5 (L2AKO) mice. d, HSC ROS levels (MFI: median fluorescence intensity). n = 5 (young) and 6 (old) mice. e, Fluorescence puncta in HSC from KFERQ-Dendra mice. Each individual point represents a cell. n = 45 (4m), 52 (12m) and 53 (30m) cells from 4 mice per group. Red points: cells with <5 puncta. f, Representative full-field and higher magnification images (from 3 independent experiments) of dendra immunostained HSC from 30m old mice. Exposure times are indicated. Arrows: high (yellow) and low (white) cytosolic Dendra intensity. g, Flow cytometry analysis of direct Dendra fluorescence of HSC from 30m old mice. Representative plot from n = 4 mice. h, Representative full-field and higher magnification images (from 3 independent experiments) of dendra immunostained LSK cells from 30m old mice incubated or not with leupeptin. Exposure time is shown. i, CMA flux (increase in puncta number after leupeptin), n = 9 fields from 4 mice per group. j, k, Oxidized protein staining in 30m old mice HSC FACS sorted according to their cytosolic Dendra fluorescence intensity. Representative full-field and higher magnification images (j) and intensity of oxidized proteins (k), n = 26 (high intensity) and 28 (low intensity) fields from 4 mice. l, m, Cellular ROS levels (l) and GAPDH activity (m) in the same FACS sorted HSC populations as in j. n = 5 fields from 3 mice (l) and n = 3 mice (m). Data shows individual values and mean ± s.e.m. Multiple two tailed t-test (a-c), two-way ANOVA with Sidak’s post hoc test (d), one-way ANOVA with Bonferroni post hoc test (e, i) and unpaired two-tailed t-tests (k-m) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 20 μm (full field) and 5 μm (insets) (f, h, j).
Extended Data Fig. 9 Characterization of HSC with genetic or pharmacological activation of CMA.
a, b, Immunostaining images for human and mouse L2A in HSC from Ctrl and hL2AOE mice (a) and average fluorescence intensity per cell relative to Ctrl (b), n = 9 fields from 3 mice. c, Human and mouse L2A mRNA in HSC expressed as fold mL2A mRNA in Ctrl cells, n = 3 independent experiments. d, Images of Dendra immunostained HSC from 22-25m old KFERQ-Dendra-hL2AOE mice incubated or not with leupeptin, n = 3 independent experiments. e-g, CMA flux (increase in fluorescent puncta upon leupeptin) in HSC from old Ctrl (from Extended Data Fig. 8h) and KFERQ-Dendra-hL2AOE mice averaged (e) or separated by Dendra fluorescence intensity (f); fraction of cells with high cytosolic KFERQ-Dendra staining (g), n = 12 fields from 4 mice. h, Flow cytometry plots (from 3 independent experiments) of HSC sorted by their cytosolic KFERQ-Dendra fluorescence intensity. i-l, HSC frequency (i), LTC-IC frequency (j), levels of intracellular ROS (k) and PK activity (l) in HSC from 7m hL2AOE mice with upregulated L2A expression since 4m of age. n = 3 (i, j, l) and 6 (k) mice/group. m, Direct fluorescence images (from 3 independent experiments) of HSC from 22-25 months old KFERQ-Dendra mice ex vivo treated with vehicle or CMA activator (10 μM CA) for 4 weeks (left) and fluorescent puncta per cell (right). n = 9 fields from 3 mice. n, Oxidized protein staining in HSC cells from 22-25 months old mice after 7 days of culture in presence of vehicle or CA (10 μM). Nuclei are stained with DAPI. Quantification is shown in Fig. 4i. o, Viability of cells recovered at the end of LTC-IC from LSK cells of 25m mice treated ex vivo with vehicle or CA. Data shows individual values and mean ± s.e.m. Two-way ANOVA with Sidak’s (b, o) or Tukey’s (c) post hoc test, unpaired two-tailed t-test (e, g, i-m) and one-way ANOVA with Tukey’s post hoc test (f) were used. P values are shown and statistical analysis is in data source. ns: no statistical significance. Scale bars, 5 μm (a), 20 μm (full field) and 5 μm (insets) (d, m, n).
Extended Data Fig. 10 The effect of CMA on polyunsaturated fatty acid metabolism in HSC is the basis for the role of CMA in HSC function.
a, Polyunsaturated fatty acids that are generated by FADS2 activity decrease with age in human blood from healthy donor volunteers (n = 250)40. Top metabolic changes positively and negatively correlating (Spearman’s correlation) with ageing were determined (left) and placement of 3 products of FADS2 activity in the graph is marked by the blue circles as (1) for DGLA (Dihomo-γ-linolenic acid), (2) for AA (arachidonic acid) and (3) for DCA (Docosatetraenoic acid). Right shows levels of each of the three metabolites in 250 healthy individuals with age range 20-90 years. Pearson correlation was used for statistics. b, Scheme of the role of CMA in HSC function. Left: under basal conditions, functional CMA is required in HSC for protein quality control including that of enzymes involved in glucose metabolism. Failure of CMA leads to persistence of damaged enzymes and reduced glycolytic activity. Right: during HSC activation, CMA is required for increasing FADS2 activity the limiting enzyme in linoleic and α-linolenic metabolism, to activate this pathway and thus facilitate the metabolic switch from glucose to lipid metabolism. CMA changes the active/inactive enzyme ratio by selective removal of the inactive forms of FADS2. Acetylation of inactive forms of FADS2 during HSC activation completes a KFERQ-like motif in FADS2 that allows its recognition by hsc70 and subsequent targeting to lysosomes for degradation.
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This file contains Supplementary Discussion and references, Supplementary Figures, SI Fig. 1. Uncropped immunoblots from main Fig. 3 and SI Fig. 2. Gating strategies used in main and Extended Data figures.
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Dong, S., Wang, Q., Kao, YR. et al. Chaperone-mediated autophagy sustains haematopoietic stem-cell function. Nature 591, 117–123 (2021). https://doi.org/10.1038/s41586-020-03129-z
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DOI: https://doi.org/10.1038/s41586-020-03129-z
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