Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1,2,3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5,6,7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo.
Subscription info for Chinese customers
We have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
RRBS data for DNA methylation analysis and RNA sequencing data are available in the BioSample database (NCBI) and under BioProject PRJNA655981. Illumina Human Methylation EPIC array data are available in the Gene Expression Omnibus (GEO) database (NCBI) and under GSE147436. All other relevant data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
The code for determining methylation ageing signatures is provided in the Supplementary Information.
Sinclair, D. A., Mills, K. & Guarente, L. Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. Science 277, 1313–1316 (1997).
Imai, S. & Kitano, H. Heterochromatin islands and their dynamic reorganization: a hypothesis for three distinctive features of cellular aging. Exp. Gerontol. 33, 555–570 (1998).
Oberdoerffer, P. et al. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell 135, 907–918 (2008).
Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, 3156 (2013).
Kennard, M. A. Relation of age to motor impairment in man and in subhuman primates. Arch. Neurol. Psychiatry 44, 377–397 (1940).
Goldberg, J. L., Klassen, M. P., Hua, Y. & Barres, B. A. Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296, 1860–1864 (2002).
Yun, M. H. Changes in regenerative capacity through lifespan. Int. J. Mol. Sci. 16, 25392–25432 (2015).
Waddington, C. H. & Kacser, H. The Strategy of the Genes: a Discussion of Some Aspects of Theoretical Biology (Allen & Unwin, 1957).
Sen, P., Shah, P. P., Nativio, R. & Berger, S. L. Epigenetic mechanisms of longevity and aging. Cell 166, 822–839 (2016).
Sinclair, D. A. & LaPlante, M. D. Lifespan: Why We Age—and Why We Don’t Have To 13–23, 158–175 (Simon & Schuster, 2019).
Shannon, C. E. A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423 (1948).
López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013).
Yang, J.-H. et al. Erosion of the epigenetic landscape and loss of cellular identity as a cause of aging in mammals. Preprint at https://doi.org/10.1101/808642 (2019).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Petkovich, D. A. et al. Using DNA methylation profiling to evaluate biological age and longevity interventions. Cell Metab. 25, 954–960.e6 (2017).
Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e12 (2016).
Ohnishi, K. et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 156, 663–677 (2014).
Abad, M. et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340–345 (2013).
Senís, E. et al. AAV vector-mediated in vivo reprogramming into pluripotency. Nat. Commun. 9, 2651 (2018).
Hofmann, J. W. et al. Reduced expression of MYC increases longevity and enhances healthspan. Cell 160, 477–488 (2015).
Rand, T. A. et al. MYC releases early reprogrammed human cells from proliferation pause via retinoblastoma protein inhibition. Cell Rep. 23, 361–375 (2018).
Laha, B., Stafford, B. K. & Huberman, A. D. Regenerating optic pathways from the eye to the brain. Science 356, 1031–1034 (2017).
Roska, B. & Sahel, J. A. Restoring vision. Nature 557, 359–367 (2018).
Moore, D. L. et al. KLF family members regulate intrinsic axon regeneration ability. Science 326, 298–301 (2009).
Geoffroy, C. G., Hilton, B. J., Tetzlaff, W. & Zheng, B. Evidence for an age-dependent decline in axon regeneration in the adult mammalian central nervous system. Cell Rep. 15, 238–246 (2016).
Yao, K. et al. Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas. Nature 560, 484–488 (2018).
Zhang, Y. et al. Elevating growth factor responsiveness and axon regeneration by modulating presynaptic inputs. Neuron 103, 39–51.e5 (2019).
Luo, X. et al. Enhanced transcriptional activity and mitochondrial localization of STAT3 co-induce axon regrowth in the adult central nervous system. Cell Rep. 15, 398–410 (2016).
Park, K. K. et al. Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322, 963–966 (2008).
Sun, F. et al. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature 480, 372–375 (2011).
Olova, N., Simpson, D. J., Marioni, R. E. & Chandra, T. Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging Cell 18, e12877 (2019).
Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun. 11, 1545 (2020).
Wang, M. & Lemos, B. Ribosomal DNA harbors an evolutionarily conserved clock of biological aging. Genome Res. 29, 325–333 (2019).
Wu, X. & Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 18, 517–534 (2017).
Koh, K. P. et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213 (2011).
Gao, Y. et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell 12, 453–469 (2013).
Yu, H. et al. Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair. Nat. Neurosci. 18, 836–843 (2015).
Weng, Y.-L. et al. An intrinsic epigenetic barrier for functional axon regeneration. Neuron 94, 337–346.e6 (2017).
Guo, J. U., Su, Y., Zhong, C., Ming, G. L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).
Krishnan, A. et al. Overexpression of soluble Fas ligand following adeno-associated virus gene therapy prevents retinal ganglion cell death in chronic and acute murine models of glaucoma. J. Immunol. 197, 4626–4638 (2016).
Almasieh, M. & Levin, L. A. Neuroprotection in glaucoma: animal models and clinical trials. Ann. Rev. Vis. Sci. 3, 1–30 (2016).
Levin, L. A. et al. Neuroprotection for glaucoma: requirements for clinical translation. Exp. Eye Res. 157, 34–37 (2017).
McClellan, A. J. et al. Ocular surface disease and dacryoadenitis in aging C57BL/6 mice. Am. J. Pathol. 184, 631–643 (2014).
Li, H. et al. Single-cell transcriptomes reveal diverse regulatory strategies for olfactory receptor expression and axon targeting. Curr. Biol. 30, 1189–1198.e5 (2020).
Mackay, D. S., Bennett, T. M. & Shiels, A. Exome sequencing identifies a missense variant in EFEMP1 co-segregating in a family with autosomal dominant primary open-angle glaucoma. PLoS ONE 10, e0132529 (2015).
Marmorstein, L. Y. et al. Aberrant accumulation of EFEMP1 underlies drusen formation in Malattia Leventinese and age-related macular degeneration. Proc. Natl Acad. Sci. USA 99, 13067–13072 (2002).
Wu, X., Li, G. & Xie, R. Decoding the role of TET family dioxygenases in lineage specification. Epigenetics Chromatin 11, 58 (2018).
Neri, F. et al. Genome-wide analysis identifies a functional association of Tet1 and Polycomb repressive complex 2 in mouse embryonic stem cells. Genome Biol. 14, R91 (2013).
Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).
Mozhui, K. & Pandey, A. K. Conserved effect of aging on DNA methylation and association with EZH2 polycomb protein in mice and humans. Mech. Ageing Dev. 162, 27–37 (2017).
Bar-Nur, O. et al. Small molecules facilitate rapid and synchronous iPSC generation. Nat. Methods 11, 1170–1176 (2014).
Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).
Mertens, J. et al. Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects. Cell Stem Cell 17, 705–718 (2015).
Shipley, M. M., Mangold, C. A. & Szpara, M. L. Differentiation of the SH-SY5Y human neuroblastoma cell line. J. Vis. Exp. 108, e53193 (2016).
Triche, T. J., Jr, Weisenberger, D. J., Van Den Berg, D., Laird, P. W. & Siegmund, K. D. Low-level processing of Illumina Infinium DNA Methylation BeadArrays. Nucleic Acids Res. 41, e90 (2013).
Fortin, J. P., Triche, T. J., Jr & Hansen, K. D. Preprocessing, normalization and integration of the Illumina HumanMethylationEPIC array with minfi. Bioinformatics 33, 558–560 (2017).
Horvath, S. et al. Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY) 10, 1758–1775 (2018).
Sun, D., Moore, S. & Jakobs, T. C. Optic nerve astrocyte reactivity protects function in experimental glaucoma and other nerve injuries. J. Exp. Med. 214, 1411-1430 (2017).
Krishnan, A., Kocab, A. J., Zacks, D. N., Marshak-Rothstein, A. & Gregory-Ksander, M. A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma. J. Neuroinflammation 16, 184 (2019).
Dordea, A. C. et al. An open-source computational tool to automatically quantify immunolabeled retinal ganglion cells. Exp. Eye Res. 147, 1218–1235 (2013).
Meer, M. V., Podolskiy, D. I., Tyshkovskiy, A. & Gladyshev, V. N. A whole lifespan mouse multi-tissue DNA methylation clock. eLife 7, e40675 (2018).
Thompson, M. J. et al. A multi-tissue full lifespan epigenetic clock for mice. Aging (Albany NY) 10, 2832–2854 (2018).
Horvath, S. et al. The cerebellum ages slowly according to the epigenetic clock. Aging (Albany NY) 7, 294–306 (2015).
Hoshino, A., Horvath, S., Sridhar, A., Chitsazan, A. & Reh, T. A. Synchrony and asynchrony between an epigenetic clock and developmental timing. Sci. Rep. 9, 3770 (2019).
Levine, M. et al. A rat epigenetic clock recapitulates phenotypic aging and co-localizes with heterochromatin. eLife 9, e59201 (2020).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Carbon, S. et al. AmiGO: online access to ontology and annotation data. Bioinformatics 25, 288–289 (2009).
Ashburner, M. et al. Gene Ontology: tool for the unification of biology. Nat. Genet. 25, 25–29 (2000).
The Gene Ontology Consortium. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 47, D330–D338 (2019).
We thank A. Wagers, R. Mostoslavsky, Y. Shi, A. Das, A. Pogoutse, C. Petty, A. Coffey, B. Zhang, P. Dmitriev, K. Booher, E. Chen, J. Wang, D. Vogel, M. Thompson, A. Jacobi and S. Hou for advice and assistance; and Y. Weng, H. Song and F. Wang for reagents and mice. The work was supported by the Harvard Medical School Epigenetics Seed Grant and Development Grant; The Paul F. Glenn Foundation for Medical Research; a gift from E. Schulak; NIH awards R01AG019719 and R37AG028730 (to D.A.S.), R01EY026939 and R01EY021526 (to Z.H.), R01AG067782 and R01GM065204 (to V.N.G.) and R01AG065403 (to M.E.L. and V.N.G.). We thank Boston Children’s Hospital Viral Core, which is supported by NIH5P30EY012196; and Schepens Eye Institute Core facilities, supported by NEI-P30EY003790. X.T. was supported by NIH award K99AG068303 and by NASA Postdoctoral Fellowship 80NSSC19K0439; D.L.V. was supported by NIH training grant T32AG023480; J.-H.Y. was partially supported by National Research Foundation of Korea (2012R1A6A3A03040476); B.R.K. was partially supported by the St Vincent de Paul Foundation and by NEI awards R24EY028767 and R01EY025794; and M.S.G.-K. by NEI award R21EY030276. We thank P. F. Glenn for his mentorship and support of ageing research.
D.A.S. is a consultant to, inventor of patents licensed to, board member of and equity owner of Iduna Therapeutics, a Life Biosciences company developing epigenetic reprograming therapies. D.A.S. is an advisor to Zymo Research, an epigenetics tools company. Additional disclosures are at https://genetics.med.harvard.edu/sinclair/people/sinclair-other.php. Y.L., L.A.R. and S.H. are equity owners of Iduna Therapeutics, a Life Biosciences company. D.L.V. is an advisor to Liberty Biosecurity. M.S.B. is a shareholder in MetroBiotech. K.C. is an equity owner in Life Biosciences and affiliates. N.D. and G.M.C. are co-founders of Rejuvenate Bio. Disclosures for G.M.C. can be found at http://arep.med.harvard.edu/gmc/tech.html. M.E.L. is a bioinformatics advisor to Elysium Health. Y.L., N.D. and D.A.S. are inventors on patents arising from this work (WO/2020/069373 and WO/2020/069339), filed by the President and Fellows of Harvard College. The other authors declare no competing interests.
Peer review information Nature thanks Andrew Huberman, Hongjun Song, Yasuhiro Yamada and the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Experimental outline for testing the effects of OSKM and OSK on gene expression in fibroblasts from young and old transgenic (TG) mice. b, c, Expression of OSKM (b, R26rtTA; Col1a1OSKM, n = 3 biological replicates each condition) and OSK (c, R26rtTA; Col1a1OKS-mCherry, n = 3 and 8 biological replicates) rescue age-associated transcriptional changes without inducing Nanog mRNA. mo, month(s). qPCR primers are listed in Supplementary Table 7. d, AAV-ubiquitinC (UbC)-rtTA and AAV-TRE-Luc vectors for measuring tissue distribution. e, Luciferase imaging of WT mice 2 months after intravenous injection (retro-orbital) of AAV9-UbC-rtTA;TRE-Luc (1.0 × 1012 gene copies total). DOX was delivered in drinking water (1 mg ml−1) for 7 days to +DOX mice. f, Luciferase imaging of the eye (Ey), brain (Br), pituitary gland (Pi), heart (He), thymus (Th), lung (Lu), liver (Li), kidney (Ki), spleen (Sp), pancreas (Pa), testis (Te), adipose (Ad), muscle (Mu), spinal cord (SC), stomach (St), small intestine (In) and caecum (Ce) 2 months after retro-orbital injection of AAV9-UbC-rtTA;TRE-Luc followed by treatment with DOX for 7 days. The luciferase signal was primarily in the liver. Imaging the same tissues with a longer exposure time (right) with the liver removed revealed a strong signal in the pancreas. g, Toxicity and safety studies in young and old mice after in vivo delivery of OSK-expressing AAVs. In h, i, at the age of 5 months, mice were intravenously injected with AAV9-rtTA;TRE-OSK (3 and 7 × 1011 gene copies of AAV per mouse). After 1 month, mice remained untreated (−DOX) or were treated with DOX (+DOX) for 18 months. WT mice were not injected with AAV. In j–n, at the age of 21 months, mice were injected intravenously with 5 × 1011 gene copies of AAV9-rtTA and 7 × 1011 of either AAV9-TRE-GFP (GFP) or TRE-OSK (OSK) per mouse. After 1 month, GFP, OSK, and non-injected WT mice were treated with DOX for 10 months. h, SOX2 expression in the liver of WT mice 2 months after intravenous delivery of OSK-expressing AAV9s with or without a month of DOX induction, and in the liver of OSK transgenic mice, 129S1/C57BL/6J mixed background. Uncropped scans in are shown in Supplementary Fig. 1. i, Body weight of WT mice, OSK transgenic mice, and AAV-mediated OSK-expressing mice with or without DOX in the first 4 weeks (left; n = 5, 3, 6, 4, 4 and 3 mice) and after 17 months (right, n = 5, 3, 6 and 4 mice). j, Examples of liver sections from WT or GFP mice showing the infection of AAV9. Scale bar, 100 μm. k, KLF4 and GFP protein levels in the livers of WT, GFP and OSK mice at 32 months of age. * indicates high OSK expression, + indicates induced protein expression levels in livers of OSK transgenic mice. Uncropped scans are shown in Supplementary Fig. 1. l, Tumour incidence in WT, GFP, and OSK mice at age 32 months after 10 months of DOX induction. m, n, Liver tumour scores (m) and white blood cell counts (n) for WT, GFP and OSK groups at age 32 months after 10 months of DOX induction. OSK mice were defined as either high expression (indicated by * in k) or low expression (WT, n = 11 mice; GFP, n = 10 mice; OSK high, n = 7 mice; OSK low, n = 8 mice). For m, n, there was no difference between the groups using one-way ANOVA. All data are presented as mean ± s.e.m.
Extended Data Fig. 2 Normal architecture and absence of tumours in the retina after long-term OSK expression mediated by AAV2 delivery.
a, b, Representative wholemount retina display of RBPMS (a RGC marker) and Klf4 immunofluorescence showing (a) expression from the AAV2 Tet-Off system can be turned off by DOX in drinking water (2 mg ml−1, 3 days), and (b) expression from the AAV2 Tet-On system can be turned on by DOX (2 mg ml−1, 2 days). Scale bars, 1 mm; n = 4 retinas for each condition. c, Corresponding retinal wholemount images stained for RBPMS and Klf4 are shown for each group tested: top, no injection, n = 6; middle, −OSK (intravitreal injection of AAV2-rtTA;TRE-OSK without DOX induction), 10 months post-injection, n = 3; bottom, +OSK (intravitreal injection of AAV2-tTA;TRE-OSK with DOX induction), 15 months post-injection, n = 6. All retinas are from 16-month-old mice, showing similar expression within the group. Scale bars, 100 μm. d, Volume intensity projection of en-face OCT (optical coherence tomography) retinal image with a white line indicating the location of e. e, Representative retinal cross-section B-scan images. White box indicates the location of the high magnification scans in f (retinal layers: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer and choroid). Videos of complete retinal cross-section B-scan images of the entire globe are provided as Supplementary Videos 1–3. g, Low- and high-power representative images of haemotoxylin and eosin (H&E)-stained cross-sections of corresponding eyes, verifying retinal layers. h, Quantitative measurements of retinal thickness, there was no difference between the groups at any location using two-way ANOVA with Bonferroni correction (n = 6, 3 and 6, respectively). i, Immunosuppressed NOD scid gamma mice received a subretinal injection of approximately 10,000 human retinoblastoma tumour cells. The OCT image shows a small retinal tumour and increased retinal thickness 14 days post-injection, demonstrating the ability of the OCT scan to detect tumours. *Rb indicates retinoblastoma.
Extended Data Fig. 3 Polycistronic OSK induces long-distance axon regeneration post-injury without RGC proliferation.
a, Proliferating cells in the optic nerve (for example, glial cells) in BrdU-injected mice as a positive control (n = 2 nerves). BrdU staining co-localized with Ki67, a proliferation marker. b, Representative retina wholemount staining shows OSK-expressing RGCs do not stain for BrdU in the first or second week after crush injury; n = 4 retinas. Scale bars, 100 μm. c, Imaging of optic nerves showing regenerating and sprouting axons with or without OSK AAV treatment, 12 weeks post-crush (wpc); n = 2 nerves. Scale bars, 200 μm. d, Whole-nerve imaging showing CTB-labelled regenerative axons at 16 wpc in WT mice with intravitreal injection of AAV2-tTA;TRE-OSK (n = 2 nerves). Scale bars, 200 μm. e, Survival of RBPMS-positive cells in the RGC layer transduced with different AAV2s, 16 dpc (n = 6, 4, 4, 4, 4, 4, 8 and 4 eyes). All data are mean ± s.e.m. f, g, Representative immunofluorescence (f) and sub-population proportion (g) of wholemount retinas transduced with a polycistronic AAV vector expressing Oct4, Sox2 and Klf4 in the same cell. White arrows designate triple-positive cells. n = 3 retinas. Scale bars, 100 μm. h, i, Immunofluorescence (h) and sub-population proportion (i) of wholemount retinas transduced with AAVs separately encoding Oct4, Sox2 and Klf4. Red, blue, and green arrows designate single-positive cells, with a white arrow marking a triple-positive cell, and other arrows marking double-positive cells. n = 3 retinas. Scale bar, 100 μm. One-way ANOVA with Bonferroni correction in e, with comparisons to d2EGFP shown.
Extended Data Fig. 4 Regenerative and pro-survival effects of OSK are RGC-specific and cell-autonomous.
a, Effect of OSK expression on RGC survival in young (1-month-old, n = 8), adult (3-month-old, n = 5), and old (12-month-old, n = 8) mice after optic-nerve crush-injury compared to expression of d2EGFP as a negative control (n = 6, 5 and 6, respectively). b, Axon regeneration after OSK expression compared to d2EGFP controls in young (1-month-old, n = 5, 6), adult (3-month-old, n = 6), and old (12-month-old, n = 4, 5) mice, 2 wpc. c, Number of RGCs in the intact, 2 wpc or 5 wpc retinas of 12-month-old mice expressing GFP (AAV2-tTA;TRE-d2EGFP, n = 7, 6 and 6, respectively) or OSK (AAV2-tTA;TRE-OSK, n = 5, 8 and 6, respectively). d, Axon regeneration in 12-month-old mice with OSK AAV or control AAV (d2EGFP) treatment, 5 wpc (n = 5 nerves). e, Schematic of retinal structure showing Vglut2-Cre mice selectively expressing Cre in excitatory neurons such as RGCs, whereas Vgat-Cre mice selectively express Cre in inhibitory amacrine and horizontal cells. f, Schematic of the FLEx (flip-excision) Cre-switch system. AAV2-FLEx-tTA is inverted by Cre to express tTA and therefore induces OSK only in Cre-positive cells. g, Confocal image stack demonstrating delivery of AAV2-FLEx-tTA;TRE-OSK to intact Vglut2-Cre transgenic retinas, resulting in RGC-specific OSK expression (top) and robust axon regeneration in the optic nerve (bottom). White arrows indicate RBPMS+ (AP2-)-labelled RGCs that express Klf4 (green). n = 4 independent replicates. h, Confocal image stack demonstrating delivery of AAV2-FLEx-tTA;TRE-OSK to intact Vgat-Cre transgenic retinas, resulting in amacrine-specific OSK expression (top) and poor axon regeneration in the optic nerve (bottom). White arrows indicate AP2+ (RBPMS-)-labelled amacrine cells that express Klf4 (green). n = 4 independent replicates. i, Representative image of AAV-expressing or non-expressing RGCs in intact and crushed retinas 2 wpc with AAVs expressing d2EGFP or OSK. d2EGFP: AAV2-tTA;TRE-d2EGFP, n = 6 retinas; OSK: AAV2-tTA;TRE-OSK, n = 8 retinas. j, RGC survival rate (crushed/intact) of d2EGFP- (n = 6 eyes) or Klf4-expressing cells (n = 8 eyes) and their surrounding non-expressing cells indicating a cell-autonomous pro-survival effect of OSK-expressing RGCs after crush, 2 wpc. k, Frequency of d2EGFP- or Klf4-positive RGCs pre- or 2 weeks post-injury (n = 4, 6, 6 and 8 eyes). Two-way ANOVA with Bonferroni correction in a–d, j; one-way ANOVA with Bonferroni correction in k. Scale bars (g–i), 100 μm. All data are mean ± s.e.m.
a, Representative images of retinal wholemounts transduced with AAV2-tTA (−OSK) or AAV2-tTA;TRE-OSK (+OSK) in the presence or absence of crush injury after 3 days. Retinal wholemounts immunostained for pStat3, Klf4 and RBPMS. n = 2 retinas each condition. b, Representative images of retinal wholemounts transduced with d2EGFP- or OSK-encoding AAV2 in the presence or absence of a crush injury. Retinal wholemounts immunostained for RBPMS and mTOR activation marker phosphorylated S6 (pS6). n = 4 retinas for each condition. c, Percentage of pS6-positive RGCs in intact and crushed samples (n = 4 retinas for each condition). d, Representative images of d2EGFP in retina expressed from the Tet-On AAV system. No GFP expression was observed in the absence of DOX. GFP expression reached peak levels 2 days after DOX induction and remained at a similar level at day 5 after induction. n = 2 retinas each condition. e, Representative images of retinal d2EGFP expression using the Tet-Off AAV system with various durations of DOX treatments (2 mg ml−1). Once pre-treated with DOX to suppress expression (on DOX), GFP was sparse even on day 8 after DOX withdrawal, lower than peak expression (Never DOX). n = 2 retinas each condition. f, Axon regeneration at 2 or 4 wpc in response to OSK induction either pre- or post-injury (n = 4, 5, 5, 4 and 4 eyes, respectively). g, Correlation between ribosomal DNA methylation (DNAm) age and chronological age of sorted mouse RGCs (1 month, n = 6; 12 months, n = 2; 30 months, n = 5), with the light blue region representing the confidence interval. P value of the linear regression is calculated by two-sided F-test of overall significance. In agreement with previous studies, DNA methylation age estimates of neurons tend to be lower than their chronological age but remain correlated (see Methods). h, Average DNA methylation levels across the mouse genome in RGCs from different ages and treatments, based on 703,583 shared CpG sites from RRBS of all samples (combined strands), n = 6, 8, 2, 8, 6, 4, 8, 8, 6, 5, 6, 4 and 5, respectively. i, Correlation of DNA methylation at each CpG site versus age (x-axis; 1 month, 12 months, 30 months) and versus injury (y-axis; intact, injured GFP). The heat map represents the number of sites located in each block of value coordinates. Pearson’s correlation coefficient, r = 0.34, P < 1e−200. j, Hierarchical clustered heat map of methylation levels of 4,106 CpGs that significantly changed in RGCs after crush injury (intact vs injured GFP, q < 0.05) and the effect of OSK. k, Top biological processes associated with the 698 CpGs that were significantly altered by both injury and OSK. Two-way ANOVA with Bonferroni correction in c, f. Scale bars (a, b, d, e), 100 μm. All data are mean ± s.e.m.
a, Mouse Tet1, Tet2 and Tet3 mRNA levels with or without OSK expression in RGCs (n = 6 biological replicates each condition). The P value indicated with an asterisk was calculated using an unpaired one-tailed t-test. b, Representative images of retinal wholemounts transduced with AAV2-tTA;TRE-OSK in combination with a AAV2-shRNA-YFP (yellow fluorescent protein) having either a scrambled sequence (sh-Scr) or a hairpin sequence to knockdown Tet1 (sh-Tet1) or Tet2 (sh-Tet2) expression, at titre ratio 5:5:1. Retinal wholemounts immunostained for Klf4. n = 3 retinas for each condition. c, Quantification of shRNA-YFP AAV transduction in OSK-expressing RGCs (n = 3 retinas for each condition). d, Mouse Tet1, Tet2 and Tet3 mRNA levels with sh-Scr (n = 5), sh-Tet1 (n = 4) or sh-Tet2 (n = 5) YFP AAV2 in RGCs in the presence of OSK expression. e, f, Quantification of axon regeneration (e, n = 4 eyes each condition) and RGC survival (f, n = 10, 7 and 9 eyes) at 2 wpc in retinas co-transduced with AAV2- tTA;TRE-OSK;shRNA. g, Mouse Stat3 mRNA levels after knockdown using sh-Scr (n = 5), sh-Tet1 (n = 4) or sh-Tet2 (n = 5) in RGCs in the presence of OSK expression. h, Cre-dependent Tomato expression in RGCs after intravitreal AAV2-Cre injection of Tomato reporter mice (Rosa-CAG-lox-STOP-lox-Tomato), and the co-expressed frequency of Cre and Klf4 (n = 3 eyes). i, j, RGC survival (i) and representative longitudinal sections of regenerating axons in longitudinal sections (j) in response to OSK expression (AAV2-tTA;TRE-OSK, n = 5 for each condition) compared to no expression (saline, n = 3 and 4), 16 days after crush injury in Tet2flox/flox mice injected with saline (Tet2 WT) or AAV2-Cre (Tet2 cKO). Scale bars (b, h and j), 100 μm. Two-way ANOVA in a, d, i; unpaired two-tailed Student’s t-test in g; one-way ANOVA in e, f. All data are mean ± s.e.m.
Extended Data Fig. 7 OSK-induced axon regeneration and survival require non-global active DNA demethylation through thymine DNA glycosylase.
a, Representative images of retinal wholemounts transduced with sh-Scr-H2B-GFP or sh-TDG-H2B-GFP AAV2 s for 4 weeks, demonstrating that knockdown of thymine DNA glycosylase (TDG) increased levels of 5-hydroxymethylcytosine (5-hmC). n = 4 retinas for each condition. b, Representative retinal wholemount images and images of longitudinal sections through the optic nerve showing CTB-labelled regenerative axons in WT mice, 16 dpc after an intravitreal injection of AAV2-tTA ;TRE-OSK in combination with AAV2-sh-Scr (sh-Scr) or AAV2-sh-TDG (sh-TDG) at titre ratio 5:5:1. n = 4 retinas for each condition. c, d, Quantification of regenerating axons (c) and RGC survival (d) in OSK-treated mice 16 dpc with AAVs carrying sh-Scr or sh-TDG (n = 4 nerves for each condition). e, Representative image of retinal wholemounts transduced with AAV2-tTA;TRE-OSK. Retinal wholemounts were immunostained for 5-methylcytosine (5-mC) and Klf4, showing a lack of global demethylation in OSK expressing RGCs. n = 3 retinas. f, Representative images of retinal wholemounts transduced with AAV2 vectors encoding the HA-TET1 catalytic domain (TET1-CD) or its catalytic mutant (TET1-mCD) for 4 weeks, demonstrating that overexpression of TET1-CD decreases global 5-mC levels. n = 3 retinas for each condition. g, h, Quantification of axon regeneration (g) and RGC survival (h) at 2 wpc in retinas transduced without or with AAV2 vectors encoding HA-TET1 CD mutant or HA-TET1 CD (n = 3, 4 and 3 eyes). i, A schematic diagram illustrating passive demethylation and TDG-dependent active DNA demethylation. Scale bars (a, b, e, f), 100 μm. One-way ANOVA with Bonferroni’s multiple comparison test in c, g, h; unpaired two-tailed Student’s t-test in d. There was no difference between the groups in g and h using two-way ANOVA and one-way ANOVA, respectively. All data are mean ± s.e.m.
Extended Data Fig. 8 OSK induces axon regeneration and reversal of DNA methylation age in human neurons.
a, mRNA levels of mouse Oct4, Sox2 and Klf4 in human neurons transduced with vectors packaged by AAV-DJ, a recombinogenic hybrid capsid that is efficient for in vitro transduction. −OSK: AAV-DJ-tTA (n = 3); +OSK: AAV-DJ-tTA;TRE-OSK (n = 3). b, Percentage of cells in S phase, as measured by propidium iodide (PI)-staining (n = 4). c, FACS profiles of G1, S and G2 phases in undifferentiated SH-SY5Y cells and differentiated cells transduced with −OSK and +OSK vectors. d, Experimental outline for testing axon regeneration in human neurons after vincristine (VCS) damage. e, f, DNA methylation (DNAm) age of human neurons without damage (intact), and 1 or 9 days after VCS damage in the absence (e) or presence (f) of OSK expression, measured using the skin and blood clock suited to in vitro studies (see Methods). The linear regression P value in e (P = 0.55) indicates nonlinear DNA methylation age changes, and in f (P = 0.008) indicates a continuous decrease in DNA methylation age (n = 3, 3 and 6). g, Average DNA methylation levels among 850,000 probes from the EPIC array in human neurons without damage (intact), and 1 or 9 days after VCS damage in the absence or presence of OSK expression (n = 3, 3 and 6). h, i, Representative images (h, similar results were confirmed in two series of experiments) and quantification (i) of neurite area at different time points after VCS damage (n = 6, 7, 5, 5, 7 and 5; 2 independent experiments). Cells were not passaged after damage to avoid cell–cell contact for quantifying maximum axon regeneration. j–l, Human TET1, TET2 and TET3 mRNA level with scrambled shRNA (sh-Scr) or sh-Tet2 AAV in human neurons in the presence or absence of OSK expression (n = 4; 2 independent experiments). m, Representative images of human neurons in each AAV treated group, 9 days after VCS damage. Similar results were confirmed in three series of experiments. n–p, Neurite area (n), axon number (o) and axon length (p) in each AAV-treated group 9 days after VCS damage (n = 20, 21, 24 and 23; 3 independent experiments). q, Mouse Oct4 mRNA levels (from OSK AAV) in human neurons with sh-Scr or sh-Tet2 AAV and in presence or absence of OSK AAV (n = 4). r, The effect of mTOR inhibition by rapamycin (Rap, 10 nM) on axon regeneration of differentiated neurons with or without OSK (n = 18, 19, 13 and 11; 2 independent experiments). s, S6 phosphorylation levels in human neurons 5 days after treatment with rapamycin (Rap, 10 nM). Similar results were seen in two independent experiments. Uncropped scans are shown in Supplementary Fig. 1. t, Neurite area of neurons expressing TET1 catalytic domain (TET1-CD) or its catalytic mutant (TET1-mCD) 9 days after VCS damage (n = 24, 28 and 21; 2 independent experiments). One-way ANOVA with Bonferroni’s multiple comparison test in b, e–g and t; two-way ANOVA with Bonferroni’s multiple comparison test in a, i, j–l and n–r. All bar graphs are mean ± s.e.m.
Extended Data Fig. 9 Vision restoration and regenerative effect of OSK rely on functional improvement of existing RGCs.
a, Axon density and representative photomicrographs of PPD-stained optic nerve cross-sections, 4 weeks after microbead or saline injection (baseline, n = 5 eyes each condition). Scale bars, 25 μm. b, Quantification of RGCs and representative confocal microscopic images from retinal flat-mounts stained with anti-Brn3a (red), an RGC-specific marker, and DAPI (4′,6-diamidino-2-phenylindole, blue), a nuclear stain, 4 weeks after microbead or saline injection (baseline, n = 5 eyes each condition). Scale bar, 100 μm. c, Axon density and representative micrographs from PPD-stained optic nerve cross-sections, 4 weeks after AAV2 or PBS injection (treated, n = 9, 7, 6 and 8 eyes). Scale, 50 μm. d, Quantification of RGCs and representative confocal microscopic images 4 weeks after PBS or AAV injection (treated, n = 7, 5, 6 and 5 eyes). Scale bar, 100 μm. e, PERG measurement at different ages 4 weeks after −OSK (n = 16, 14 and 11 eyes) or +OSK treatment (n = 20, 12 and 14 eyes). Similar results from 2 independent experiments are combined. f, Visual acuity in 18-month-old mice treated with −OSK (n = 11 eyes) or +OSK (n = 14 eyes) AAV for 4 weeks. g, h, Axon (g; n = 4, 6, 10 and 9 nerves) and RGC (h; n = 5, 4, 10 and 8 retinas) density in 4- and 12-month-old-mice, 4 weeks after −OSK or +OSK AAV injection. i, Scatter plot of OSK-induced changes and age-associated changes in mRNA levels in RGCs, with differentially expressed genes labelled. Gene selection criteria are in Methods. j, Hierarchical clustered heat map showing relative mRNA levels of age-associated sensory perception genes in FACS-sorted RGCs from untreated young (5-month-old) or old (12-month-old) mice or old mice treated with either −OSK or +OSK AAV. Sensory genes were extracted from the mouse Sensory Perception (GO:0007600) category of the Gene Ontology database. Gene selection criteria are in Methods. −OSK: AAV2-rtTA;TRE-OSK for c–h, AAV2-TRE-OSK for i, j; +OSK: AAV2-tTA;TRE-OSK for c–j. Unpaired two-tailed Student’s t-test in a, b, f; one-way ANOVA with Bonferroni’s multiple comparison test in c, d; two-way ANOVA with Bonferroni correction in e, g, h. All data are mean ± s.e.m.
a, b, Top biological processes based on transcriptome data that were either lower expressed in old compared to young RGCs and reversed by OSK (a), or higher expressed in old RGCs compared to young and reversed by OSK (b). c, Heat map showing relative mRNA levels of genes involved in the negative regulation of neural projection development, among the 464 differentially expressed genes during ageing. The accumulation of the gene Efemp1 during ageing is suspected to have a role in diseases of the retina. d, RGC Efemp1 mRNA levels measured by qPCR (relative to GAPDH) compared between young mice, old mice, and old mice treated with −OSK or +OSK AAV. Old RGCs with sh-Scr, sh-Tet1 or sh-Tet2 knockdown combined with +OSK AAV are included for comparison (n = 7, 6, 5, 4, 5, 5 and 6 eyes). e, Principal component 1 value of 1 month, 12 month and 30 month RGC training samples in the PCA analysis. Values are standardized to have a mean = 0 and s.d. = 1 (n = 6, 2 and 6). f, DNA methylation ageing signatures of 6-week-old RGCs isolated from axon-intact retinas infected with GFP-expressing AAV, or from axon-injured retinas infected with GFP- or OSK- expressing AAV at 4 dpc (n = 4, 4 and 8 eyes). g, Top biological processes associated with the 1,226 signature CpG sites. h, i, Transcription factor (TF) binding (h) and histone modifications (i) specifically enriched at the 1,226 signature CpG sites, compared to five sets of randomly selected CpGs. j, Correlation of Tet1 and Tet2 knockdown-induced changes in methylation (5-mC and 5-hmC together) at the selected CpGs. r = 0.4, P = 2.53e−45. k, Delta value of ribosomal DNA methylation age (months) of 12-month-old RGCs infected for 4 weeks with +OSK (n = 5 retinas). Values are relative to the average of RGCs infected with −OSK AAV. l, Ribosomal DNA methylation age (months) of 12-month-old OSK-treated RGCs infected for 4 weeks with sh-Tet1 or sh-Tet2 (n = 4, 5 retinas). Values are relative to the average of RGCs infected with sh-Scr. m, PERG amplitudes in old (12-month-old) mice treated with −OSK, +OSK or +OSK together with either sh-Scr or sh-Tet1/sh-Tet2-mediated knockdown for 4 weeks (n = 8, 7, 5, 6 and 6 eyes). n, Working model. The loss of youthful epigenetic information during ageing and injury (including genome-wide changes to DNA methylation, acceleration of the DNA methylation clock, and disruption of youthful gene expression patterns) causes a decline in tissue function and regenerative capacity. OSK-mediated reprogramming recovers youthful epigenetic information, reverses the DNA methylation clock, restores youthful gene expression patterns, and improves tissue function and regenerative capacity, a process that requires active DNA demethylation by TET1/TET2 and TDG. The PRC2 complex may serve to recruit TET1 and TET2 to specific sites in the genome, and DNA methylation by DNA methyltransferases (DNMTs) may be important as well. One-way ANOVA with Bonferroni’s multiple comparison test in d–f, m. All data are mean ± s.e.m.
This file contains Supplementary Figures 1-2, Supplementary Discussion, Supplementary Code and Supplementary Tables 1-7.
DNAm.csv; DNAm values used for developing and analyzing the DNA methylation ageing signatures.
Complete cross section B-scan images for an entire globe of a 16-month-old mouse retina, without intravitreal injection of AAV2.
Complete cross section B-scan images for an entire globe of a 16-month-old mouse retina, 10 months post intravitreal injection of AAV2-rtTA;TRE-OSK (-OSK, no OSK expression).
Complete cross section B-scan images for an entire globe of a 16-month-old mouse retina, 15 months post intravitreal injection of AAV2-tTA;TRE-OSK (+OSK, continuous OSK expression).
About this article
Cite this article
Lu, Y., Brommer, B., Tian, X. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). https://doi.org/10.1038/s41586-020-2975-4
Clinical Epigenetics (2021)
In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche
Nature Communications (2021)
Nature Reviews Genetics (2021)
Nature Reviews Neuroscience (2021)
Nature Aging (2021)