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Notum Produced by Paneth Cells Attenuates Regeneration of Aged Intestinal Epithelium

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Notum Produced by Paneth Cells Attenuates Regeneration of Aged Intestinal Epithelium Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Pentinmikko, Nalle et al. "Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium." Nature 571, 7765 (July 2019): 571 © 2019 The Author(s) As Published http://dx.doi.org/10.1038/s41586-019-1383-0 Publisher Springer Science and Business Media LLC Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/126506 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. 1 Paneth cell produced Notum attenuates regeneration of aged intestinal 2 epithelium 3 4 Nalle Pentinmikko1, Sharif Iqbal1*, Miyeko Mana2*, Simon Andersson1, Armand B. Cognetta III3, 5 Radu M. Suciu3, Jatin Roper4, Kalle LuopaJärvi1, Eino Markelin1, Swetha Gopalakrishnan1, Olli- 6 Pekka Smolander1, Santiago Naranjo2, Tuure Saarinen5,6, Anne Juuti6, Kirsi Pietiläinen5, Petri 7 Auvinen1, Ari Ristimäki7, Nitin Gupta8, Tuomas Tammela9, Tyler Jacks2,10, David M. Sabatini2,10,11, 8 BenJamin F. Cravatt3, Ömer H. Yilmaz2, and Pekka KataJisto1,12# 9 10 1 Institute of Biotechnology, HiLIFE, University of Helsinki, 00014, Helsinki, Finland 11 2 The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, 12 Cambridge, Massachusetts 02139, USA. 13 3 The Skaggs Institute for Chemical Biology, Department of Chemical Physiology, The Scripps 14 Research Institute, La Jolla, California 92037, USA. 15 4 Department of Medicine, Division of Gastroenterology, Duke University, Durham, North Carolina, 16 USA 17 5 Obesity Research Unit, Research Programs Unit, Diabetes and Obesity, University of Helsinki 18 00014, Helsinki, Finland 19 6 Helsinki University Hospital, Abdominal Center, Department of Gastrointestinal Surgery, Helsinki, 20 Finland 21 7 Department of Pathology, Research Programs Unit and HUSLAB, University of Helsinki and 22 Helsinki University Hospital, Helsinki, Finland 23 8 Atlanta Gastroenterology Associates; 980 Johnson Ferry Road; Suite 820; Atlanta GA 30342, 24 USA. 25 9 Cancer Biology and Genetics, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 26 New York, New York, USA 27 10 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, 28 Massachusetts, USA. 29 11 Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute, Department of 30 Biology, MIT, Cambridge, Massachusetts 02142, USA. 31 12 Department of Biosciences and Nutrition, Karolinska Institutet, 14183, Stockholm, Sweden 32 * These authors contributed equally 33 # Correspondence to: [email protected] 34 35 Word Count: 2547 36 Figures: 3 main, 9 extended data 37 Supplemental information: 1 table, 3 data 38 39 A decline in stem cell function impairs tissue regeneration during aging, but the role of the 40 stem cell supporting niche in aging is not well understood. The small intestine is maintained 41 by actively cycling intestinal stem cells (ISCs) that are regulated by the Paneth cell niche1,2. 42 Here we show that the regenerative potential of human and mouse intestinal epithelium 43 diminishes with age due to defects in both stem cells and their niche. The functional decline 44 was caused by decrease in stemness maintaining Wnt signalling due to production of an 45 extracellular Wnt-inhibitor, Notum, in aged Paneth cells. Mechanistically, high mTORC1 46 activity in old Paneth cells inhibits PPARa activity3 and lowered PPARa increased Notum 47 expression. Genetic targeting of Notum or Wnt-supplementation restored function of old 48 intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the 49 regenerative capacity of old stem cells and promoted recovery from chemotherapy induced 50 damage. Our results reveal an unappreciated role for the stem cell niche in aging and 51 demonstrate that targeting of Notum can promote regeneration of old tissues. 52 53 Tissue turnover and regenerative capacity decrease upon aging in many tissue types4-6. The 54 intestinal epithelium is one of the fastest renewing tissues in the human body and has been reported 55 to regenerate without loss of self-renewal in long term in vitro organoid culture7. However, 56 complications in the gastrointestinal system increase with age8-10, and intestines of old mice 57 regenerate slower after radiation-induced damage11, suggesting reduced stem cell activity. 58 59 To assess possible aging-induced changes in the human intestinal epithelium, we used the capacity 60 of ISC containing epithelial crypts to form clonogenic organoids7 as an in vitro assay of intestinal 61 regenerative potential. We observed a significant age-induced reduction in the organoid forming 62 capacity of colonic crypts biopsied from healthy human donors (Fig. 1a). As the heterogeneous 63 human colon material does not allow downstream analysis of stem cell intrinsic and extrinsic effects, 64 we next analysed the effects of age on mouse small intestinal epithelium. Crypts from old (>24 65 months) mice formed significantly fewer organoids than those isolated from young (3-9 months) 66 animals (Extended Data Fig. 1a). Importantly, regenerative growth of de novo crypts was also 67 diminished in the organoids formed by old crypts (Fig. 1b, Extended Data Fig. 1b,c), indicating a 68 reduction in stem cell function. Furthermore, the reduced crypt formation observed during serial 69 passage of secondary crypt domains demonstrated that the decline in epithelial regeneration was 70 due to alterations intrinsic to the epithelium (Extended Data Fig. 1d,e). 71 72 Intestinal tissue renewal is largely maintained by the Lgr5 expressing ISCs, located between Paneth 73 cells at the crypt base. ISCs divide regularly and produce transit-amplifying (TA) progenitor cells that 74 divide several additional times and gradually differentiate. Paneth cells produce antimicrobial 75 peptides and multiple signalling factors, such as EGF, Wnt3, Delta-like ligands, and cyclic ADP 76 ribose (cADPR)2,12, which regulate stemness and function of the neighbouring ISCs. To more 77 specifically address the separate roles of stem cells and their niche in age-associated intestinal 78 decline, we used the Lgr5-EGFP-IRES-creERT2 reporter mice1, which allow the identification and 79 isolation of Paneth cells, Lgr5–EGFPhi ISCs, and TA cells that can be further divided to immediate 80 EGFPmed and late EGFPlo progenitors. 81 82 The aged mouse crypts did not present gross histological alterations, and the fraction of ISCs and 83 TA cells that were EdU+ or Ki67+ was unchanged in old mouse and human samples (Extended Data 84 Fig. 1f-h). However, flow cytometry of crypts from old mice revealed a significant drop in frequency 85 of Lgr5hi ISCs (Fig. 1c), whereas Paneth cell frequency was significantly increased in old mice and 86 humans (Fig. 1c, Extended Data Fig. 1i,J). As the Lgr5-EGFP-IRES-creERT2 mouse model exhibits 87 mosaic expression of the EGFP containing construct1, the alterations in cellular frequencies were 88 also validated by immunohistochemical analyses of Olfactomedin4 and Lysozyme to enumerate 89 ISCs and Paneth cells, respectively (Extended Data Fig. 1k). Reduction in ISC number together with 90 their unchanged EdU+ frequency suggested that old crypts may have a lower output of cells possibly 91 contributing to the villus blunting and slower intestinal turnover during aging13. As Paneth cells 92 positively regulate the number and function of Lgr5hi stem cells in young animals2,12, the decoupling 93 of the Lgr5hi:Paneth cell ratio in old animals (Extended Data Fig. 1l) raised the possibility that 94 interactions between these two cell types change during aging. To address this, we interrogated the 95 organoid forming capacity of co-cultured Lgr5hi and Paneth cells isolated from young and old animals 96 (Fig. 1d). Strikingly, old Paneth and Lgr5hi cells both showed cell-type specific age-induced effects, 97 and initiated organoids with reduced efficiency. Consistent with previous work, neither cell type 98 formed organoids efficiently alone2,12, but when co-cultured with Paneth cells, Lgr5hi cells from young 99 animals formed organoids at higher rate than old Lgr5hi cells. Interestingly, the age-induced stem 100 cell defect was partially rescued by co-culturing with young Paneth cells, whereas old Paneth cells 101 failed to fully support organoid formation by young Lgr5hi cells. These data indicate that both stem 102 cell intrinsic and extrinsic epithelial factors reduce the regenerative potential during intestinal aging. 103 104 Decline in Fatty Acid Oxidation (FAO) was recently shown to intrinsically reduce the function of aged 105 intestinal stem cells14. Surprisingly, we noted that old Paneth cells extrinsically decreased clonogenic 106 growth of young Lgr5hi cells even in a long-term coculture (Extended Data Fig. 1m,n). While the 107 original Paneth cells existed at least 14 days in such cocultures (Extended Data Fig. 1o), new Paneth 108 cells are continuously produced by the stem cells, suggesting that exposure to old Paneth cells had 109 long term effects on ISCs and their progeny. To mechanistically understand how age-induced 110 changes in the niche-stem cell communication may influence stem cells, we performed RNA 111 sequencing on both cell types (Supplemental Table 1). Interestingly, old Paneth cells showed 112 particular deregulation of genes encoding secreted or plasma membrane-associated proteins 113 (Extended Data Fig. 2a,b). Among the key stemness regulating factors, we noted no alterations in 114 Wnt3 or Egf expression, whereas expression of cADPR producing Bst1 was reduced (Extended 115 Data Fig. 2c However, targeting15 of Bst1 did not mimic the effects of aging on an ad libitum diet 116 (Extended Data Fig. 2d,e). 117 118 As the aged mouse intestine was also recently reported to harbour reduced Wnt activity13, we next 119 focused on the extracellular Wnt inhibitor Notum that was significantly increased in old Paneth cells 120 (Fig.
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