2020 Jan 23
Deficient Autophagy in Epithelial Stem Cells Drives Aging in the Freshwater Cnidarian
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Deficient Autophagy in Epithelial Stem Cells Drives Aging in the Freshwater Cnidarian
Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3 months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.
Aging model system; Autophagy sensor; Epithelial stem cells; Evolution of aging; Hydra regeneration; Rapamycin; WIPI2; p62/SQSTM1.
© 2020. Published by The Company of Biologists Ltd.
Conflict of interest statement
Competing interestsThe authors declare no competing or financial interests.
Inducible aging phenotype in cold-sensitive (A) Phylogenetic position of Hydra oligactis ( Ho_CS). Hydra among metazoans. (B) Anatomy of a male H. oligactis animal. (C) Schematic view of Hydra gastric tissue. Mes, mesoglea. (D) Morphological changes observed in Ho_CS (top) and Ho_CR (bottom) animals at various time points after transfer to 10°C (day 0); arrowheads indicate testes, arrows indicate head regions. Scale bars: 500 µm. (E) Survival rates among Ho_CR and Ho_CS cohorts maintained at 10°C for 120 days. (F) Head regeneration in Ho_CR or Ho_CS animals selected for the presence or the absence of testes, bisected at mid-gastric level on day 24 post-transfer (dpt) and monitored for 24 days post-amputation (dpa). (G) Head regeneration measured in Ho_CS animals bisected at 18°C (blue, black) or at various time points after transfer to 10°C (9, 23, 30, 37 dpt) and monitored for 15 days.
Somatic interstitial loss upon gametogenesis-induced aging and pharmacological induction of aging in asexual (A,B) Modulations in haploid cell content (A) and cell cycle profiles (B) detected by flow cytometry in Ho_CS animals. Ho_CS and Ho_CR animals maintained at 18°C or at 10°C for 25, 35 or 45 days; starv., 7-day starvation. Error bars represent s.d. (C) Fraction of interstitial cells (i-cells, single and pairs) over epithelial (epith) cells counted in macerated tissues (300 cells minimum per condition). (D) Expression of foxN1 in i-cells, Kazal-1 in gland cells and prdl-b in nematoblasts in Ho_CS and Ho_CR animals before and after transfer to 10°C ( n=20 animals/condition). Scale bar: 300 µm. (E) Hydroxyurea (HU) treatment given as three successive 24 h pulses eliminates all cycling i-cells without affecting the ESCs, which cycle three to four times slower. Arrowheads indicate feeding times. (F) Fraction of i-cells over epithelial cells measured in Ho_CR and Ho_CS animals maintained at 18°C and analyzed before (day 0) or after HU treatment initiation (4, 7, 14 days). (G) HU-induced morphological changes noted in animals taken 12 or 23 days after day 0. Inset in the top row shows a magnification of the boxed area. (H) Head regeneration of Ho_CS and Ho_CR animals bisected 9 days after HU treatment initiation ( n=3×10). (I) Survival rate of HU-treated H, v Ho_CS and Ho_CR animals ( n=6×10). Error bars represent s.e.m. values in H,I. All HU treatments were performed as shown in E.
Disorganization of the epithelial epidermal layer in aging (A,B) Phalloidin staining of the epidermis in Ho_CS animals. Ho_CR and Ho_CS animals transferred to 10°C and fixed at the indicated time points. Arrows indicate disorganized regions of the epidermis, arrowheads indicate shortened myofibrils. Scale bars: 50 µm. (C) Phalloidin staining of the epidermis in control or HU-treated Ho_CS animals maintained at 18°C and fixed after 28 days. (D,E) BrdU-labeling index values measured after 96 h BrdU exposure performed at the indicated time points either after transfer to 10°C (D) or after HU treatment at 18°C as in Fig. 2E. In D, each dot corresponds to a replicate in which at least 300 cells were counted. In E, animals were maintained at 18°C. * P<0.05, **** P<0.0001 (unpaired t-test). (F) Scheme comparing the impact of i-cell loss in Hv animals, in which epithelial stem cells adapt (Wenger et al., 2016), and in Ho_CS, in which a more limited i-cell loss is lethal, suggesting a lack of epithelial adaptation.
Deficiency in the inducibility of the autophagy flux in ESCs from (A) Toluidine-stained transversal sections of gastric regions from 1- and 11-day starved animals. Lower panel show enlarged areas of the panels above. epi, epidermis; gc, gastric cavity; mg, mesoglea. (B) Detection of autophagic vacuoles (arrowhead) and digestive vacuoles (dv, pink) in epithelial cells immunostained for LC3 (green) and stained with MitoTracker (red) and DAPI (white). Scale bar: 10 µm. (C) Enlarged view of the LC3 Ho_CS animals. + structures shown in B. Arrowheads indicate circular LC3 + structures surrounding sequestered portions of cytoplasm. Scale bar: 5 µm. (D,E) Number of LC3 + vacuoles (D) or LC3 puncta (E) in ESCs of regularly fed or 17-day starved Hv, Ho_CR and Ho_CS animals maintained at 18°C. (F) Number of LC3 + vacuoles in ESCs of Ho_CS maintained at 10°C for 35 or 45 days. (G) Structure of the mCherry-eGFP-hyLC3A/B dual autophagy sensor. (H,I) Live imaging of ESCs expressing the autophagy sensor in regularly fed Hv, Ho_CR or Ho_CS animals maintained at 18°C either untreated (H) or exposed to MG132 immediately before imaging (I). Green arrowheads indicate autophagosomes, orange arrowheads autophagosomes losing GFP fluorescence and red and white arrowheads autolysosomes. (J) Live imaging of ESCs expressing the autophagy sensor in Hv or Ho_CS animals exposed before imaging to MG132 for 3 h and to BafA (100 nM) for 16 h or not . Green arrowheads indicate autophagosomes. (K) Distribution of the LC3 + vacuoles between autophagosomes (black triangles) and autolysosomes (red triangles). (L) Number of LC3 + puncta in the cells shown in J. For box and whisker plots, the box indicates the 25th to 75th percentile, the line shows the median and the whiskers indicate the smallest and the largest value. Points represent individual values. P-values calculated using unpaired t-test. *** P=0.0003.
Modulation of p62/SQSTM expression levels in (A) Structure of the human (Hu) and Ho_CS animals. Ho p62/SQSTM1 (p62) proteins (see Fig. S12). ‘Ab’ indicates the region used to raise the anti- Hydra p62 antibody. (B) p62 levels in p62(RNAi) animals exposed or not to MG132; tub: α-tubulin. (C) Epithelial cells immunostained with the anti- Hydra p62/SQSTM1 (red) and the anti-human LC3 (green) antibodies, co-stained with DAPI (white). Arrows point to p62-labeled granules associated with LC3. Scale bar: 10 µm. (D,E) p62 levels in animals treated with increasing levels of BafA (D, left), or maintained at 10°C for 35 days and exposed or not to BafA (D, right), or starved for 1 or 14 days and exposed to MG132 (E). (F-H) p62 levels and ubiquitin patterns in animals exposed to MG132 (F,G), or knocked down for p62 and exposed to MG132 (H). Except in D (right), all animals were maintained at 18°C and drug treatments given for 16 h.
Rapamycin treatment delays aging in (A,B) Aging phenotype at 58 dpt (A) and survival rate (B) in Ho_CS without enhancing the autophagy flux. Ho_CS animals exposed, or not, to rapamycin from day 3 (dpt). Error bars represent s.e.m. (C) Toluidine-stained transversal sections of gastric regions from untreated (left) or rapamycin-treated (right) Ho_CS animals taken at 35 dpt. Red boxes indicate the enlarged areas shown on the right; pink brackets indicate gastrodermis thickness; black arrowhead indicates sperm cells in the testis lumen and red arrowhead indicates sperm cells engulfed in an epithelial cell. gc, gastric cavity; te, testis. (D) BrdU-labeling index values measured after 96 h BrdU exposure in Ho_CS animals maintained at 10°C in the presence (red triangles) or absence (black circles) of rapamycin. (E) Number of LC3 + vacuoles in ESCs of Ho_CS animals exposed (red dots) or not (blue dots) to rapamycin. Error bars represent s.d.; P-values calculated using the unpaired t-test. (F,G) p62 levels assessed by western blotting (F) or proteomic analysis (G) in Ho_CR and Ho_CS animals maintained at 18°C or transferred to 10°C and exposed to rapamycin for 35 days. tub: α-tubulin. (H) Live imaging of epithelial cells transiently expressing the mCherry-eGFP-hyLC3A/B autophagy sensor in animals maintained at 18°C and exposed to rapamycin (0.8 µM) from day 0. Arrowheads indicate newly formed autophagosomes with full GFP fluorescence and limited mCherry fluorescence (green); mature autophagosomes with double fluorescence GFP/mCherry (yellow); or autolysosomes with quenched GFP fluorescence (red).
Deficient autophagy flux upon (A) Scheme showing the role of WIPI2 in autophagosome formation (adapted from Dooley et al., 2014). (B) Structure of the human and WIPI2 silencing in H. vulgaris. Hydra WIPI2 proteins and alignment of the regions involved in ATG12-5-16 complex or PI3P binding. (C) Left: RNAi procedure used to knock down WIPI2 expression in intact animals. Red arrowheads indicate feedings preceding siRNAi delivery, black arrows indicate siRNA electroporation (EP), green arrow indicates co-electroporation of the mCherry-eGFP-hyLC3 autophagy sensor on EP7 . Right: WIPI2 and CBP (CREB-binding protein) RNA levels measured by RT-PCR in animals exposed to WIPI2 or scrambled (scr) siRNAs. D, day. (D) Phenotypic analysis of WIPI2(RNAi) and scrambled(RNAi) animals post-EP6 or -EP7. Scale bar: 1 mm. Note that all images have been cut and aligned on black background for ease of comparison. (E) Mortality rates recorded after EP7 and head regeneration efficiency in animals bisected at mid-gastric level 24 h after EP6. (F) Live detection of LC3 vacuoles in epithelial cells from WIPI2(RNAi) or scr(RNAi) animals 2 days post-EP7. MG132 added just before imaging. Green arrows indicate formation of GFP + puncta in scr(RNAi) cells. (G-J) WIPI2 silencing in HU-treated animals. (G) p62 and tubulin levels in Hv animals electroporated six times with WIPI2- or scr-siRNAs. (H) Fold change (FC) in p62 levels in animals exposed or not to HU and knocked down or not for WIPI2; each triangle represents a distinct experiment. (I,J) Imaris quantification of LC3 puncta (I), and autophagosomes and autolysosomes (J) in epithelial cells of WIPI2(RNAi) or scr(RNAi) animals expressing the autophagy sensor, imaged 2 days post-EP7. Vacuoles were quantified 3 h after MG132 exposure. For box and whisker plots, the box indicates the 25th to 75th percentile, the line shows the median and the whiskers indicate the smallest and the largest value. Points represent individual values. P-values calculated using the unpaired t-test.
Comparative view of the timing of the different signs of aging recorded in The data linked to survival are shown in Fig. 1E and Ho_CS and Ho_CR sexual animals. Fig. S1, budding in Fig. S1, prey capture in Fig. 1C and Fig. S1N, regeneration in Fig. 1F,G, i-cell loss in Fig. 2C,D, epithelial cell proliferation in Fig. 3D, and Fig. S4A, animal contractility in Fig. S1M, and muscle fiber organization in Fig. 3A-C.
The autophagic flux in epithelial cells of aging and non-aging Inducibility of the autophagy flux upon starvation, MG132 or rapamycin treatments as deduced from the formation of LC3-positive vacuoles and puncta Hydra. in vivo, or the accumulation of p62/SQSTM1. Thickness of curved arrows indicate the relative extent of ATG inducibility.
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