Lipoic acid rejuvenates aged intestinal stem cells by preventing age‐associated endosome reduction
2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês
10.15252/embr.201949583
ISSN1469-3178
AutoresGang Du, Yicheng Qiao, Zhangpeng Zhuo, Jiaqi Zhou, Xiaorong Li, Zhiming Liu, Li Yang, Haiyang Chen,
Tópico(s)Biochemical Acid Research Studies
ResumoArticle9 July 2020Open Access Source DataTransparent process Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction Gang Du Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Yicheng Qiao Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhangpeng Zhuo Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jiaqi Zhou Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Xiaorong Li Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhiming Liu Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Yang Li Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Haiyang Chen Corresponding Author [email protected] orcid.org/0000-0001-8305-4060 Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Gang Du Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Yicheng Qiao Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhangpeng Zhuo Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jiaqi Zhou Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Xiaorong Li Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhiming Liu Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Yang Li Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Haiyang Chen Corresponding Author [email protected] orcid.org/0000-0001-8305-4060 Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Author Information Gang Du1,2, Yicheng Qiao2, Zhangpeng Zhuo2, Jiaqi Zhou2, Xiaorong Li2, Zhiming Liu1, Yang Li2 and Haiyang Chen *,1,2 1Laboratory for Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China 2Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China *Corresponding author. Tel: +86 028 85164205; E-mail: [email protected] EMBO Rep (2020)21:e49583https://doi.org/10.15252/embr.201949583 See also: P Zhang and BA Edgar (August 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The age-associated decline of adult stem cell function is closely related to the decline in tissue function and age-related diseases. However, the underlying mechanisms that ultimately lead to the observed functional decline of stem cells still remain largely unexplored. This study investigated Drosophila midguts and found a continuous downregulation of lipoic acid synthase, which encodes the key enzyme for the endogenous synthesis of alpha-lipoic acid (ALA), upon aging. Importantly, orally administration of ALA significantly reversed the age-associated hyperproliferation of intestinal stem cells (ISCs) and the observed decline of intestinal function, thus extending the lifespan of Drosophila. This study reports that ALA reverses age-associated ISC dysfunction by promoting the activation of the endocytosis–autophagy network, which decreases in aged ISCs. Moreover, this study suggests that ALA may be used as a safe and effective anti-aging compound for the treatment of ISC-dysfunction-related diseases and for the promotion of healthy aging in humans. Synopsis Supplementation of alpha-lipoic acid (ALA) reverses the age-associated hyperproliferation of intestinal stem cells (ISCs) and the observed decline of intestinal function by promoting the activation of the endocytosis-autophagy network in aged Drosophila. The expression of ALA synthase (LAS) continuously decreases during aging, which leads to a significant reduction of endogenous ALA in aged Drosophila midguts. ALA regulates the abundance of endosomes in the ISCs of Drosophila. ALA counteracts the endocytosis-autophagy-mediated EGFR-activity in Drosophila ISCs. Introduction Organismal aging is characterized by a continuous decrease of the functional abilities of tissues and organs. In many vertebrate organs, resident adult stem cells, which possess high proliferative and differentiate capacities that compensate for cell loss, are responsible for both tissue homeostasis and organ functionality throughout the lifespan of the organism. This is particularly important in tissues with a high turnover rate, such as the intestinal epithelium (Guo et al, 2016). Although stem cells can be regarded as immortal, they are also subject to an age-associated decline of their self-renewal and differentiation properties (Schultz & Sinclair, 2016). Previous studies reported that damage accumulation in stem cells is closely linked to both organismal aging and age-related diseases such as cancer, inflammatory diseases, type 2 diabetes, and degenerative diseases (Rando, 2006; Apidianakis & Rahme, 2011; Li & Jasper, 2016; Holmberg et al, 2017). However, the detailed mechanisms that ultimately lead to this decline in stem cell function in response to aging still remain largely unknown. Identifying small molecular compounds that enhance the regenerative capacity of adult stem cells in model animals could not only yield drugs that promote healthy aging but could also provide additional insight into uncovering the mechanisms of this age-associated functional decline in stem cells. Alpha-lipoic acid (ALA; 1,2-dithiolane-3-pentanoic acid, also known as thioctic acid) is an organosulfur-containing compound, which is present in all eukaryotic cells (Solmonson & DeBerardinis, 2018). In humans, ALA can be endogenously synthesized using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine, and iron–sulfur clusters (Cronan, 2016; Shaygannia et al, 2018). In addition, as a vitamin-like substance, ALA can also be absorbed by digestion. As an essential cofactor for the energetic metabolism, multiple mitochondrial dehydrogenase complexes require ALA for catalysis, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase (Solmonson & DeBerardinis, 2018). ALA has been reported to play critical roles in diverse biological processes, including the stabilization and regulation of mitochondrial multi-enzyme complexes, the elimination of reactive oxygen species (ROS), the oxidation of carbohydrates and amino acids, and the coordination of energetic metabolism (Park et al, 2014). Although healthy young humans can synthesize sufficient ALA to meet the body's needs, the level of ALA significantly declines with age, which is assumed to be linked to age-associated organic dysfunction (Hagen et al, 2002; Park et al, 2014). As a safe and natural ingredient, ALA has been widely administrated as a nutritional supplement to treat diverse age-associated diseases, including diabetes, obesity, diabetic polyneuropathy and retinopathy, atherosclerosis, hypertension, and Alzheimer's disease (Shay et al, 2009; Park et al, 2014; Salehi et al, 2019). In addition, it has been reported that ALA administration can reverse memory impairment and protect retinal pigment epithelial cells in aged mice (Voloboueva et al, 2005; Farr et al, 2012). Although the mechanism of how ALA benefits aged tissues and organs remains largely unknown, these data indicate that ALA supplementation in elders may promote healthy aging. Interestingly, recent studies have shown that ALA may also participate in the regulation of stem cell functions. ALA administration has been reported to reduce the loss of hematopoietic stem cells in G protein-coupled receptor kinase-depleted mice (Le et al, 2016). ALA can significantly increase the cardiac differentiation efficiency of embryonic stem cells (Shen et al, 2014). Moreover, ALA supplementation enhanced the therapeutic effect of mesenchymal stem cells in rats with cardiac injury (Abd El-Fattah et al, 2019). However, despite these clues, the role of ALA in the context of stem cell aging still remains unexplored. Due to its simple organizational structure, straightforward genetic manipulation, and well-defined stem cell lineage, the Drosophila midgut has emerged as a suitable model system for the study of mechanisms underlying the age-related decline in stem cell function. Consequently, the Drosophila midgut can be used to identify potential strategies that enhance the regenerative capacity of adult stem cells. Drosophila intestinal stem cells (ISCs) specifically express Notch ligand Delta (Dl) and the transcription factor escargot (Esg), which reside in the basement membrane of the midgut epithelium. Here, ISCs proliferate to self-renew and produce progenitor cells (either enteroblasts [EBs] or enteroendocrine mother cells [EMCs], depending on the Notch activity). EBs further differentiate into absorptive enterocytes (ECs), and EMCs produce secretory enteroendocrine cells (EEs; Fig EV1A). The number of ISCs and progenitor cells is relatively small and remains stable in young and healthy midguts, while it increases several folds in response to aging (Biteau et al, 2008; Choi et al, 2008; Schultz & Sinclair, 2016). Furthermore, the differentiation capacity of ISCs and EBs showed a continuous decrease with aging (Cui et al, 2019). This leads to the accumulation of Esg and Dl expressing cells in the aged midgut (Biteau et al, 2008; Choi et al, 2008; Park et al, 2009). Multiple signaling pathways, such as c-Jun N-terminal kinase (JNK) signaling (Biteau et al, 2008), insulin signaling (Biteau et al, 2010; Cheng et al, 2014; Kao et al, 2015), mTOR signaling (Johnson et al, 2013), p38-MAPK signaling (Park et al, 2009), DGF/VEGF signaling (Choi et al, 2008), and ROS signaling (Hochmuth et al, 2011; Chen et al, 2017), have been reported to regulate ISC aging. Combined with notable transcriptional alteration, these signalings regulate the changes of biologic behaviors of ISCs during aging. This further disrupts the intestinal barrier and the acid–base balance of the digestive tract (Li et al, 2016). Interestingly, decreasing the number of Esg-positive (Esg+) cells in the midgut (either by genetic manipulation or drug administration) significantly increased the lifespan of Drosophila (Gervais & Bardin, 2017). Therefore, the Drosophila midgut is an ideal model to investigate the function and the underlying mechanism of ALA in the regulation of the behaviors of stem cells upon aging. Click here to expand this figure. Figure EV1. Alpha-lipoic acid (ALA) synthesis reduces in aged Drosophila midguts, and orally administered ALA rejuvenates aged intestinal stem cells (ISCs; related to Fig 1) Model of Drosophila intestinal stem cell (ISC) lineages. One ISC (Dl+ and Esg+) produces a new ISC and differentiates into a diploid precursor enteroblast (EB; Esg+ and Su(H)GBE+) with high Notch or a diploid precursor enteroendocrine mother cell (EMC). The EMC divides once to produce a pair of diploid enteroendocrine cells (EEs; Pros+). The post-mitotic EB further differentiates into pre-enterocyte (pre-EC; Esg+ and Pdm1+), which continues to differentiate into an octoploid mature enterocyte (ECs; Pdm1+). Quantification of luciferase activity after administration of endogenous chemicals. Error bars show the SD of six independent experiments. Immunofluorescence images of pH3 staining with the midgut section from the R4 region in 40-day flies and 40-day flies with ALA administration started at 26th day after fly eclosion. pH3 (red) staining was used to visualize the mitosis of ISCs. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region in 40-day flies and 40-day flies with ALA administration after fly eclosion (lifelong administration). esg-GFP (green) indicates ISCs and their differentiating cells. Dl (red) staining was used to visualize ISCs. A cartoon illustrating the sorting of esg-GFP+ cells using FACS for RT–qPCR analysis (see Materials and Methods Details). Data information: DAPI-stained nuclei are shown in blue. Scale bars represent 25 μm (C) and 10 μm (D). Error bars represent SDs. Student's t-tests, *P < 0.05, **P < 0.01, and non-significant (NS) represents P > 0.05. Source data are available online for this figure. Download figure Download PowerPoint Using Drosophila midgut as a model system enabled the disclosure of the role of ALA in the prevention of the functional decline of ISCs and the extension of the lifespan of Drosophila. This study reports that ALA increases Drosophila lifespan, regulates age-associated acid–base homeostasis, and prevents the age-associated hyperproliferation of ISCs through an endocytosis-mediated mechanism. Furthermore, this study suggests that ALA can be used as an effective and safe anti-aging compound to promote healthy aging in humans. Results Orally administered ALA rejuvenates aged ISCs When Drosophila age, the ISCs in their midguts undergo a malignant increase of their proliferation rate and a decrease of differentiation efficiency (Biteau et al, 2008; Choi et al, 2008; Cui et al, 2019). This leads to the continuous accumulation of Esg+ cells (ISCs and their differentiating progenies) in the midguts of aged flies (Biteau et al, 2008; Choi et al, 2008; Cui et al, 2019). To disclose the regulation of the age-associated functional decline of ISCs by endogenous small molecules, the repressive effect on age-related Esg+ cell accumulation of 10 selected endogenously and de novo synthesized chemicals in Drosophila midguts was tested using an "esg-luciferase" reporter system (Figs 1A and B, and EV1B). This system is based on the luciferase reporter expression by esg-GAL4-driven UAS-luciferase in Esg+ cells, which allows the tracing and quantification of real-time changes of ISCs and their differentiating progenies in aging Drosophila midguts. Among these tested endogenous chemicals, ALA administration started at an intermediate age (26 days) and showed a most remarkable repressive effect of Esg+ cell accumulation in aged (40 days) Drosophila midguts (Figs 1B and EV1B). We tested three concentrations (0.01, 0.05, and 0.5 mM) of ALA administration and found 0.5 mM ALA administration showed the best effect of preventing Esg+ cell accumulation in aged midguts (Fig 1B). Moreover, the luciferase activity of aged flies (40 days) in response to ALA administration started at day 26 after fly eclosion and was even less than that of 26-day flies (intermediate-age flies). Since it has been reported that the ALA level significantly decreases with age in humans (Park et al, 2014), this study focused on exploring the role of ALA in preventing stem cell aging. Figure 1. Alpha-lipoic acid (ALA) synthesis reduces in aged Drosophila midguts, and orally administered ALA rejuvenates aged intestinal stem cells (ISCs) A. A model illustrating the Drosophila "esg>luciferase" reporter system of chemical administration. The chemicals were fed to Drosophila with esg-GAL4-driven luciferase expression on the 26th day after eclosion. After administration for 14 days, Drosophila were dissected, and the activity of luciferase in their midguts was measured. B. Quantification of the luciferase activity of flies with indicated ages and ALA administration. Error bars show the standard deviation (SD) of six independent experiments. C–F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. G. Quantification of the number of esg-GFP+ cells and Dl+ cells in experiments (C–F). n is indicated. The numbers of quantified guts from left to right are 15, 17, 16, 16, 15, 17, 16, and 16. H. Quantification of the number of pH3+ cells in experiments (C-F). n is indicated. I. Relative mRNA fold change of Las in sorted esg-GFP+ cells of wild-type (esg-GFP/CyO) Drosophila during aging. The Las expressions of Drosophila with different ages are plotted relative to 14-day Drosophila, which was set to 1. Error bars indicate the SD of three independent experiments. J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin. K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments. L. Quantification of the content of ALA in midguts of 14-day, 26-day, and 40-day flies using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. M–O. LC-ESI-MS/MS chromatograms of ALA in midguts of 14-day flies (M), 26-day flies (N), and 40-day flies (O). Arrows indicate the peak of ALA. The area of ALA peak indicates the content of ALA. cps, counts per second; MRM, Multiple Reaction Monitoring. When the estimated peak of ALA is undetected in the chromatogram, the content of ALA is considered as 0. Data information: DAPI-stained nuclei are shown in blue. Scale bars represent 10 μm (C–F). Error bars represent SDs. Student's t-tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and non-significant (NS) represents P > 0.05. See also Fig EV1. Source data are available online for this figure. Source Data for Figure 1 [embr201949583-sup-0007-SDataFig1.xlsx] Download figure Download PowerPoint To further analyze the anti-ISC-aging effect of ALA, ISCs and their differentiating progenies were visualized in aged midguts using an esg-GFP reporter line. Simultaneously, the number of ISCs was analyzed, which were identified by the Dl antibody staining and the proliferation rate of ISCs, as indicated by the phosphorylated histone 3 (pH3+; a mark of mitosis) antibody staining. Consistent with previous studies (Biteau et al, 2008; Choi et al, 2008), the numbers of esg-GFP+ cells, Dl+ cells, and pH3+ cells continuously increased with age in the Drosophila midguts (Fig 1C–E, G and H). However, 40-day flies (aged flies) that were fed with 0.5 mM ALA for 14 days (starting at the 26th day after eclosion) showed significantly less accumulation of esg-GFP+ cells, Dl+ cells, and pH3+ cells compared with 40-day flies without ALA feeding (Figs 1E–H and EV1C). This indicates that ALA can alleviate ISC aging in old flies. More importantly, esg-GFP+ cells, Dl+ cells, and pH3+ cells of 40-day flies, that received ALA administration starting at the 26th day, were even less than the cells of 26-day flies (intermediate-age flies; Fig 1D and F–H). This was consistent with the result obtained from the "esg-luciferase" reporter system (Fig 1B). This suggested that ALA could not only reduce but also reverse ISC aging based on the change of ISC proliferation rate. In addition, 40-day flies with lifelong ALA administration (starting after fly eclosion) showed less ISC accumulation in midguts. This was found by comparing the numbers of esg-GFP+ cells, Dl+ cells, and pH3+ cells with those of 40-day flies with ALA administration from an intermediate age (26th day after eclosion; Figs EV1D and 1F). These results show that ALA administration could rejuvenate aged ISCs in Drosophila. Reduced ALA synthesis in aged flies may cause the functional decline in ISCs In animals, ALA is de novo synthesized from octanoic acid in mitochondria. ALA synthase (LAS; an iron–sulfur cluster mitochondrial enzyme that catalyzes the final step in the de novo pathway of ALA biosynthesis) controls the rate of ALA production in the body (Cronan, 2016; Shaygannia et al, 2018; Solmonson & DeBerardinis, 2018). There is only one Las homologous gene in Homo sapiens and Drosophila melanogaster, and the protein sequence of LAS shares 93% similarity and 67% identity. Previous studies have reported a strong inverse correlation between LAS reduction and the status of a number of diseases, including diabetes, atherosclerosis, and neonatal-onset epilepsy (Mayr et al, 2011; Padmalayam, 2012; Yi et al, 2012; Xu et al, 2016). However, whether LAS expression is also decreased during aging remains unknown. To analyze the expression pattern of Las in Drosophila ISCs during aging, real-time quantitative PCR (RT–qPCR) analyses were performed using sorted esg-GFP+ cells (Fig EV1E). These data showed a significant decrease of Las transcription in ISCs and their differentiating progenies during aging (Fig 1I). Moreover, Western blotting analysis showed a remarkable decrease of LAS protein in aged midguts of Drosophila (Fig 1J–K). To further demonstrate that the abundance of ALA decreases in guts of aged flies, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analyses were performed. These data indicated that the abundance of ALA indeed dramatically decreased in aged midguts of Drosophila (Figs 1L–O and EV2A–E). Thus, both the mRNA and protein levels of Las in Drosophila ISCs undergo a significant reduction in response to aging, which in turn causes a reduction of ALA in midguts of aged flies. Click here to expand this figure. Figure EV2. ALA synthesis reduces in aged flies and regulates lifespan of Drosophila (related to Figs 1, 2, 3) LC-ESI-MS/MS chromatogram of blank. LC-ESI-MS/MS chromatogram of ALA standard. Mass spectra of ALA. LC-ESI-MS/MS chromatogram of 8-aminooctanoic acid (internal standard). Mass spectra of 8-aminooctanoic acid (internal standard). Las transcript levels were reduced in Las RNAi (v22037) and Las RNAi (TH02737.N) flies, relative to their levels in control (UAS-GFP) flies. Error bars show the SD of three independent experiments. Food intake measured using the CAFE assay of Drosophila at 26th day with and without ALA administration as indicated. Error bars show the SD of three independent experiments. Survival (percentage) of female W1118 Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 200 (W1118 + 0 mM ALA) and 200 (W1118 + 0.5 mM ALA). The other two independent experiments related to Fig 3G. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 200 (Canton-S + 0 mM ALA) and 200 (Canton-S + 0.5 mM ALA). The other two independent experiments related to Fig 3I. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA at 26-day as indicated. The numbers of quantified flies: 200 (Canton-S + 0 mM ALA) and 200 (Canton-S + 0.5 mM ALA). The other two independent experiments related to Fig 3J. Data information: Error bars represent SDs. P-values for lifespan curves (H, I, and J) were calculated by the log-rank test. The statistical tests used in other panels were Student's t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and non-significant (NS) represents P > 0.05. Source data are available online for this figure. Download figure Download PowerPoint To test whether the reduction of LAS expression in aged ISCs contributes to the age-associated ISC hyperproliferation of old midguts, the expression of LAS in ISCs was knocked down using esgts-GAL4-mediated RNA interference (RNAi; Fig EV2F). LC-ESI-MS/MS analyses indicated that knockdown of LAS led to a significant reduction of ALA synthesis in midguts (Fig 2A–C). Moreover, reduction of LAS expression in young flies indeed caused ISC accumulation (Figs 2D, E and G and H), which was observed in old wild-type Drosophila (Fig 1E). More importantly, ALA administration fully reversed the ISC accumulation phenotype caused by LAS depletion in young flies (Fig 2E–H). To further demonstrate that LAS reduction could lead to age-associated ISC hyperproliferation, the lineage of LAS-depleted ISCs was traced by performing mosaic analysis with repressible cell marker (MARCM), which labels all progenies of a single activated ISC in one clone with a visible GFP marker (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006). Based on Pdm1 staining (which labels differentiated ECs), depletion of LAS did not show obvious ISC differentiation defects (Fig 2I–K). However, we found that the average size of LAS-depleted MARCM clones was obviously bigger compared to the control clones (Fig 2I, J and L). This suggested that LAS regulates the rate of ISC proliferation. These findings suggested that the reduced LAS expression in ISCs in response to aging may be a cause of age-related functional decline in ISCs. Figure 2. Reduced ALA synthesis in flies causes the functional decline in ISCs A, B. LC-ESI-MS/MS chromatograms of midguts of flies carrying Act5Cts-GAL4-driven UAS-lacZ (control, A) or Las RNAi (B). C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. D–F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. G. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (D–F). n is indicated. The numbers of quantified guts from left to right are 17, 19, 18, 17, 19, 18, 17, 19, and 18. H. Quantification of the luciferase activity of midguts with indicated genotypes and manipulation. Error bars show the SD of six independent experiments. I, J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of MARCM clone size of experiments in (I, J). n is indicated. Each dot corresponds to one clone. Data information: DAPI-stained nuclei are shown in blue. Scale bars represent 10 μm (D–F, I, J). Error bars represent SDs. Student's t-tests, ****P < 0.0001, and non-significant (NS) represents P > 0.05. See also Fig EV2. Source data are available online for this figure. Source Data for Figure 2 [embr201949583-sup-0008-SDataFig2.xlsx] Download figure Download PowerPoint ALA prevents the age-related decline of intestinal functions and extends the lifespan of Drosophila Previous studies have shown that the functional decline of ISCs during aging caused a significant decrease in digestive functions of Drosophila, including the loss of gas
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