Lef1 and broad microRNA expression was reduced in aged SCs
To induce premature SC aging, first, we addressed the common phenotypes of SC aging and attempted to elucidate its central molecular machinery. Primary MSCs (CD45-, CD31-, TER119-, Pdgfra+, and Sca-1+) and HSCs (Lineage-, c-Kit+, and Sca-1+), including progenitor cells, can be isolated by the cell sorter based on cell surface markers, thus they are suitable for comparing the characteristics of the same cell fraction. Therefore, we focused on them as the first targets for exploring common changes in gene expression with aging. The global expression profiles of genes and miRNAs were compared between MSCs and HSCs derived from young (3 months) and old (24 months) mouse bone marrows using microarray and miRNA quantitative polymerase chain reaction (qPCR) array analyses. Since transcription factors predominantly determine cellular properties, we focused here on the changes in the expression of transcription factors. Among the transcription factors altered in common between MSCs and HSCs, Id1 and Cebpa were the most upregulated, and Lef1 was the most downregulated with aging, compared to those in young SCs (Fig. 1a, b; and Extended Data Table 1). Both Id1 and Cebpa have been reported to promote SC aging 12–14. In contrast, Lef1 is required for the proper functioning of TSCs 15, 16, but little is known about its association with SC aging. We next examined whether the altered expression of these three transcription factors is common in the aging of various TSCs using GREIN (GEO RNA-seq Experiments Interactive Navigator), a web-based application for analysis of public RNA-seq datasets. As a result, we found that many, but not all, datasets show decreased expression of Lef1 and increased expression of Cebpa in aged various TSCs both in mice and humans. However, the expression of Id1 was variable (Extended Data Table 2). We also found that the expression of broad miRNAs was repressed in old SCs, especially in old MSCs (Fig. 1c). The expression levels of Drosha, Dgcr8, Xpo5, Ddx5 (p68), and Ddx17 (p72), which are major regulators of miRNA biogenesis, did not vary between young and old MSCs. However, levels of age-related factors, including Cdkn1a (p21), phospho-Chk1 (p-Chk1), and γ-H2AX, were elevated (Fig. 1d). It indicates that the cause of the broad miRNA reduction in aged SCs is not age-related loss of miRNA biosynthetic activity, but rather involves the dysregulation of a specific regulatory mechanism. Similar broad miRNA downregulation was observed in aged ISCs (Extended Data Fig. 1). These results indicate that altered expression of Cebpa, Lef1, and broad miRNAs are common phenotypes in SC aging.
Lef1/DDR-miR axis was essential for proper DDR capacity
Altered expression of the two transcription factors and broad miRNAs, identified as the common phenotypes of SC aging, was examined in detail using MSCs. Consistent with the miRNA qPCR array results (Fig. 1c), a reduction of broad miRNA levels was also observed in old MSCs using miRNA microarray analysis (Fig. 2a). The expression of many miRNAs, including that of the miR-17 family, was induced by DDR after being subjected to X-ray irradiation in young MSCs, as previously reported 17, 18. Notably, their expression was abolished in old or Lef1-knockdown MSCs (Fig. 2a, b; and Extended Data Fig. 2a). Lef1 is an essential mediator of the Wnt signaling pathway with Tcf family members and β-catenin in various biological processes, and targets multiple miRNAs 19. However, Lef1 knockdown reduces the expression levels of almost all miRNAs; therefore, it cannot be conclusively established whether it directly regulates individual miRNA expression. Moreover, currently, the molecular machinery for the Wnt/miRNA biogenesis axis involves only the transcriptional activation of Dicer by β-catenin under chronic social defeat stress 20. Based on our microarray and western blot analyses, we did not observe any changes in the expression levels of miRNA biosynthesis regulators between young and old MSCs (Fig. 1a, d). These results suggest that the Lef1-mediated unknown regulatory system responsible for the expression of broad miRNAs, including the ones essential for DDR, was impaired in old SCs. To test this hypothesis, a conditional knockout model of Lef1 (Lef1cKO) was generated using the Cre/loxP system and crossed with the Cre-activatable CAG-CAT-EGFP reporter mice (Fig. 2c, d; and Extended Data Fig. 2b) 21. In young MSCs lacking Lef1, the number of cells expressing the DDR marker 53BP1 was reduced after irradiation (Fig. 2e).
We also sought to identify the miRNAs that are essential to DDR. First, as shown in Fig. 3a, 25 miRNAs were selected out of the 86 DDR-miRs, whose expression increased more than 1.5-fold through irradiation and decreased less than 0.5-fold with aging, and that were conserved in humans (Fig. 3a and Extended Data Table 3). The results of miRNA array analysis showed that Lef1 knockdown was more potent than aging, and the miRNAs repressed by Lef1 knockdown included all the miRNAs downregulated in aged MSCs. Therefore, candidate miRNAs were selected based on the criterion of reduced expression with aging. Next, the loss of DDR function analysis was performed by assessing the abrogation of the focus-forming of phosphorylated ATM (pATM), a key regulator of DDR, after irradiation in human embryonic kidney 293T cells, in which each candidate, including miR-106b, which is functionally similar to miR-17, was knocked out using genome editing. We observed that although some miRNAs had low knockout efficiency and limited effects (Extended Data Fig. 3a), all the 25 miRNAs selected were necessary for proper DDR foci formation (Fig. 3b), consistent with findings that associate many of them with DDR 17, 22–29. Although some reports suggest that the overexpression of a specific miRNA, such as miR-211, miR-326, miR-328-3p, and miR-128, in tumor cells could restore DDR capacity, including cytotoxic chemosensitivity 23, 25, 27, 28, the overexpression of any single miRNA was insufficient in restoring DDR capacity in this study, except for miR-128-2, which partially improved DDR capacity (Extended Data Fig. 3b). These results indicate that the Lef1/DDR-miR axis was essential for optimum DDR capacity and was impaired by SC aging.
Dysregulation of DDR was induced in aged TSCs in vivo
To confirm whether the decreased DDR capacity observed in the in vitro experiments occurs in aged TSCs in vivo, we examined the DDR capacity of TSCs in old mice. Consistent with our previous study using Lgr5-EGFP-IRES-creERT2 knock-in mice, which express EGFP specifically in ISCs, immunohistochemical staining for phosphorylated ATR (pATR), a key regulator of DDR, revealed decreased DDR capacity in aged Lgr5 + ISCs of the irradiated small and large intestine (Fig. 4a) 8. A similar trend was also observed in skin epidermal stem cells (EdSCs) (Fig. 4b). These indicate the decrease in DDR capacity with aging in the TSCs in vivo.
Lef1 deficiency induced premature SC aging
Since Lef1 was identified as a crucial factor for DDR capacity and its downregulation was a common phenotype in SC aging, we next attempted to induce premature SC aging to investigate its influence on organismal aging. However, to date, no critical insights have been obtained into the key organs and hierarchical relationships among tissues during the organismal aging process. We selected ISCs as the first target TSC for the induction of premature aging based on previous reports on the tissue-specific frequency of mutations 30, the incidence of tumors based on the number of stem cell divisions 31, 32 and in vivo imaging of cellular senescence marker p16Ink4a 33. The Lef1cKO mice were crossed with the Lgr5-EGFP-IRES-creERT2 and CAG-CAT-EGFP reporter mice, denoted henceforth as Lgr5-Lef1cKO mice. Continuous Cre-mediated Lef1 deficiency in ISCs was induced via the administration of tamoxifen in drinking water (Extended Data Fig. 4). However, tamoxifen induces DNA damage 34; therefore, we used the Lgr5-EGFP-IRES-creERT2 and CAG-CAT-EGFP mice administered with tamoxifen as controls. Like the findings in the old intestinal crypts, we observed the loss of DDR foci in EGFP and Msi1, a marker of ISCs including progenitor cells 35, double-positive, Lef1-deficient ISCs (Fig. 5a). To confirm the commonality of premature aging in EdSCs, Lef1cKO mice were crossed with K5-Cre mice, denoted henceforth as K5-Lef1cKO mice, that express Cre in the epidermal basal layer 36. It was observed that the K5-Lef1cKO mice lost most of their body hair, like the old mice among the previously reported Lef1-deficient mice. However, we observed abundant subcutaneous fat in the K5-Lef1cKO mice, although suppression of adipocytes differentiation was reported in N-terminally deleted Lef1 mutant mice (Fig. 5b, c) 15, 37, 38. Trichrome staining, which stains collagen fibers blue, showed that the dermal collagen levels in the skin of the K5-Lef1cKO mice were reduced compared to those in the skin of old mice (Fig. 5c). Using these mice, we examined the DDR capacity of the EdSCs. We found that DDR dysfunction occurred from a young age in the K5-Lef1cKO mice (Fig. 5d).
Lef1 deficiency induced chronic inflammation
Inflammation was also examined as it is a pro-aging factor. The Lef1 deficiency in ISCs induced the production of interleukin (IL)-6, a representative inflammatory cytokine, mainly in the intestinal epithelium, and the infiltration of F4/80-positive inflammatory macrophages in the stroma of the Lgr5-Lef1cKO mice (Fig. 6a, b). To quantitatively assess chronic inflammation, plasma concentrations of the representative inflammatory factors, IL-6 and tumor necrosis factor (TNF)α, were measured in the Lgr5-Lef1cKO mice. The results showed that the concentration of inflammatory factors was significantly elevated compared to those in young mice but not to the extent observed in old mice (Fig. 6c). These results suggest that Lef1 deficiency in ISCs is at least partially responsible for age-related dysfunctions due to chronic inflammation and is capable of inducing premature SC aging.
Induced Premature Sc Aging Affected Remote Organs
Since Lef1 deficiency in ISCs caused premature intestinal dysfunction with chronic inflammation, we further examined its effects on remote organs and systemic aging using Lgr5-Lef1cKO mice. Therefore, the number of IL-6-positive cells, especially in NeuN-positive neurons, increased considerably in the cerebral neocortex and hippocampus of the Lgr5-Lef1cKO mice (Fig. 7a, b). Lgr5 expression has been reported in some neonatal cerebellum and olfactory bulb neurons 39, 40. To show that Lef1 deficiency does not directly induce IL-6 in the neurons, we administered tamoxifen to the Lgr5-Lef1cKO mice after 12 weeks of age and confirmed that the GFP-expressing cells did not appear in the brain (Extended Data Fig. 5a). Expression levels of IL-6 are elevated in the neurons, astrocytes, and microglia of an aged brain. They are known to be associated with Alzheimer's disease and cognitive impairment 41–43. Notably, the number of p16Ink4a-positive cells, a representative marker of cellular senescence, was also markedly increased in the neocortex and the hippocampal CA3 region (Fig. 7c). However, although p16Ink4a expression was observed in the nuclei of non-neuronal cells, some of which expressed microglia (Iba1) or astrocytes (Acsbg1 and GFAP) markers, in the brains of healthy old mice, p16Ink4a was also expressed in the cytoplasm of neurons, in addition to the non-neuronal brain cells of the Lgr5-Lef1cKO mice (Fig. 7d). To reveal the influence of induction of premature ISC aging on other remote organs, known aging pathologies of the skin and kidneys were also examined. Using trichrome staining, we observed that the reduced dermal collagen production (Fig. 5c, 7e and Extended Data Fig. 5b), fibrosis of the renal glomerular mesangium, and the expansion of Bowman’s space (Fig. 7f; Extended Data Fig. 5c) were incident in the Lgr5-Lef1cKO mice. These results indicate that dysfunctions caused by SC aging impair other remote organs and contribute to the progression of organismal aging.