Hypothalamic stem cells control ageing speed partly through exosomal miRNAs

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Abstract

It has been proposed that the hypothalamus helps to control ageing, but the mechanisms responsible remain unclear. Here we develop several mouse models in which hypothalamic stem/progenitor cells that co-express Sox2 and Bmi1 are ablated, as we observed that ageing in mice started with a substantial loss of these hypothalamic cells. Each mouse model consistently displayed acceleration of ageing-like physiological changes or a shortened lifespan. Conversely, ageing retardation and lifespan extension were achieved in mid-aged mice that were locally implanted with healthy hypothalamic stem/progenitor cells that had been genetically engineered to survive in the ageing-related hypothalamic inflammatory microenvironment. Mechanistically, hypothalamic stem/progenitor cells contributed greatly to exosomal microRNAs (miRNAs) in the cerebrospinal fluid, and these exosomal miRNAs declined during ageing, whereas central treatment with healthy hypothalamic stem/progenitor cell-secreted exosomes led to the slowing of ageing. In conclusion, ageing speed is substantially controlled by hypothalamic stem cells, partially through the release of exosomal miRNAs.

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Figure 1: Ageing-associated htNSC loss and its impact on ageing speed.
Figure 2: Ageing acceleration and lifespan shortening due to htNSC loss.
Figure 3: Slowdown of ageing by hypothalamic implantation of IκBα-htNSCs.
Figure 4: Exosomes and exosomal miRNAs secreted by htNSCs.
Figure 5: Contribution of htNSCs to exosomal miRNAs in the CSF.
Figure 6: Slowdown of ageing by treatment of htNSC-derived exosomes.

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  • 27 June 2018

    The microarray data generated and analysed in this Article have been uploaded to the Gene Expression Omnibus (GEO) under accession number GSE113383. Accordingly, the statement in the 'Data availability' section of the Article has been rephrased online.

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Acknowledgements

This study was supported by NIH R01 DK078750, AG031774, HL113180 and DK099136 (D.C.).

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Contributions

Y.Z., M.S.K., J.Y. and C.H. performed hypothalamic injections and cell implantation; Y.Z. performed immunostaining, cloning, virus production and CSF sampling; M.S.K. performed behavioural experiments and exosome treatment; B.J. performed cell culture, exosome and miRNA characterization; J.Y. performed lifespan follow-up and initial behavioural and miRNA analysis; J.P.Z.-H. performed cell culture and imaging; D.C. conceived the hypothesis, designed and organized the study and wrote the paper.

Corresponding author

Correspondence to Dongsheng Cai.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks K. Jin, T. Wyss-Coray and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Ageing-related physiology and histology in C57BL/6 mice.

Male C57BL/6 mice at indicated ages (months, M) were maintained in standard housing conditions and under standard chow-feeding conditions without any experimental treatment, except for analysis of ageing-related physiological parameters and tissue histology as indicated. n = 18 mice per group (behaviours) and n = 5 mice per group (histology). Data are mean ± s.e.m. Source data

Extended Data Figure 2 Viral injection and additional information on TK1/GCV model.

a, Lentiviruses of CMV-promoter-driven GFP were injected into the hypothalamic third ventricle (3V) of C57BL/6 mice via a pre-implanted cannula. One week after injection, brain sections were made and examined for GFP immunostaining. Scale bars, 50 μm. Images represent four independent experiments. b, c, AgRP-Cre mice and POMC-Cre mice received an injection in the hypothalamic third ventricle of Hsv-TK1 lentivirus followed by GCV treatment and were examined three months later for the number of AgRP and POMC neurons in the ARC through Cre immunostaining. d, AgRP-Cre mice and POMC-Cre mice were injected with rAAV2-FLEX-rev-ChR2:tdTomato virus or vehicle into the ARC, followed by injection of Sox2-promoter-driven Hsv-TK1 lentivirus (TK) or control lentivirus (Con) into the hypothalamic third ventricle. GCV was administrated into the third ventricle twice per week for three weeks. Subsequently these mice were subjected to an optogenetic stimulation-induced feeding response as described in the Methods. Food intake before and after optogenetic stimulation were also measured. e, MWM training information for Fig. 1f. **P < 0.01, ***P < 0.001; two-tailed Student’s t-test (b, c), one-way ANOVA with Tukey’s post hoc test (d, e); n = 4 mice per group (b, c), n = 5 mice per group (d) and n = 8 mice per group (e). Data are mean ± s.e.m. Source data

Extended Data Figure 3 Ablation of htNSCs in hypothalamic third ventricular wall by DTR/diphtheria toxin.

Mid-aged male C57BL/6 mice (15 months old) were injected in the hypothalamic third ventricle with Sox2-promoter-directed DTR lentivirus (DTR) or control lentivirus, followed by four-week (twice per week) intraperitoneal injection of diphtheria toxin (DT) or vehicle. a, Diagram of lentiviral DTR. b, Evaluation of Sox2-promoter-driven DTR lentiviruses in cultured htNSCs by immunostaining of DTR and Sox2. Scale bars, 50 μm. Images represent 3 independent experiments. ce, Immunostaining of hypothalamic sections (c, d) and physiological analyses (e) of these mice at three months after viral injection. Control values in c and d represent similar observations in DTR/DT+ and DTR+/DT groups. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test (c, d), one-way ANOVA with Tukey’s post hoc test (e); n = 5 mice per group (c, d) and n = 8 mice per group (e). Data are mean ± s.e.m. Source data

Extended Data Figure 4 Assessing exosomes secreted by htNSCs.

a, Immunostaining of CD81 in cultured htNSCs and astrocytes. Scale bars, 10 μm. b, Electron microscopic images of htNSCs. Right, high magnification of the outlined area on the left. Black arrows indicate the presence of multivescular bodies. c, Flow cytometry analysis of CD81 of htNSC-derived secreted exosomes. Grey area indicates the appropriate isotype control. d, Purified exosomes secreted from cultured htNSCs were profiled using nanoparticle analysis. Data represent three independent experiments.

Extended Data Figure 5 Additional assessments on secreted exosomes from htNSCs.

a, Secreted exosomes isolated from htNSCs were subjected to fractioning by density-gradient ultracentrifugation, different fractions were analysed by immunoblotting with anti-TSG101 antibody. b, Secreted exosomes isolated from htNSCs were subjected to pull-down by anti-CD81 antibody or appropriate isotype control and then analysed for levels of candidate miRNAs. n = 4 independent biological samples per group (a) and n = 5 independent biological samples per group (b). Data are mean ± s.e.m. Source data

Extended Data Figure 6 Small RNA bio-analyser assay of secreted exosomes.

a, Representative traces of exosomal small RNA and miRNA secreted by htNSCs, IκBα-htNSCs, MSCs and hypothalamic neuronal GT1-7 cells. b, Quantification of exosomal miRNAs secreted by indicated cells according to the results from small RNA/miRNA bio-analysis. n = 5 independent biological samples per group. Data are mean ± s.e.m. Source data

Extended Data Figure 7 Growth factors and cytokines secreted by htNSCs.

Indicated cells were cultured in medium without EGF and bFGF for 2 days, and the medium was collected and analysed using the Mouse Growth Factor Array for indicated growth factors and cytokines. Array images contain three independent biological samples per cell model (blots on the top and the design of array on the bottom).

Extended Data Figure 8 Effects on htNSCs and animal physiology by Rab27a shRNA.

a, b, Cultured htNSCs were infected with Sox2-promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA or control scramble shRNA lentivirus (a) and examined for Ki67 by immunostaining (b) at 2–3 days after viral infection. Scale bars, 60 μm. c, C57BL/6 mice (12-month-old males) were injected in the hypothalamic third ventricle with Sox2-promoter-driven Cre lentivirus and Cre-dependent Rab27a shRNA or control scramble shRNA lentivirus or vehicle. Ageing-related physiology was analysed in mice at six weeks after viral injection. *P < 0.05, **P < 0.01; one-way ANOVA with Tukey’s post hoc test (c); n = 8 mice for vehicle, n = 9 mice for control shRNA, n = 10 mice for Rab27a shRNA (c). Data are mean ± s.e.m. Source data

Extended Data Figure 9 Electron microscopic examination of htNSC-secreted exosomes.

Exosomes secreted from cultured htNSCs were obtained and purified using differential ultra-centrifugation and examined for the purity and size distribution by electron microscopy using the protocol detailed in the Methods. Scale bar, 250 nm.

Extended Data Figure 10 Additional information for the anti-ageing models used in this study.

a, C57BL/6 mice (16-month-old males) were treated via hypothalamic third-ventricle cannula with exosomes (100 ng protein, purified from htNSCs) or vehicle, three times per week for four months, and the hypothalamic tissues were dissected and examined for indicated mRNAs. Expression levels of mRNAs are presented in arbitrary units (au), and the value of each species in the control group was normalized to 1. b, The training session information for MWM in Fig. 6c. c, The training session information for MWM in Fig. 6d. *P < 0.05 (c) or as indicated (a); two-tailed Student’s t-test (a) or one-way ANOVA with Tukey’s post hoc test (c); n = 5 mice per group (a), n = 8 mice for Con/GCV with vehicle, n = 7 mice for TK1/GCV with vehicle and TK1/GCV with exosome (b), and n = 7 mice per group (c). Data are mean ± s.e.m. Source data

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Zhang, Y., Kim, M., Jia, B. et al. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs. Nature 548, 52–57 (2017). https://doi.org/10.1038/nature23282

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