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Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

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Abstract

Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage. Changes to DNA methylation patterns over time form the basis of an 'ageing clock', but whether old individuals retain information to reset the clock and, if so, whether this would improve tissue function is not known. Of all the tissues in the body, the central nervous system (CNS) is one of the first to lose regenerative capacity. Using the eye as a model tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal old mice. This process, which we call recovery of information via epigenetic reprogramming or REVIVER, requires the DNA demethylases Tet1 and Tet2, indicating that DNA methylation patterns don't just indicate age, they participate in ageing. Thus, old tissues retain a faithful record of youthful epigenetic information that can be accessed for functional age reversal.
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Reversal of ageing- and injury-induced vision loss by Tet-dependent
epigenetic reprogramming
Yuancheng Lu1,2, Anitha Krishnan3,9, Benedikt Brommer4,9, Xiao Tian1,2,9, Margarita Meer5,
Daniel L. Vera1,2, Chen Wang4, Qiurui Zeng1,2, Doudou Yu1,2, Michael S. Bonkowski1,2, Jae-
Hyun Yang1,2, Emma M. Hoffmann3, Songlin Zhou4, Ekaterina Korobkina3, Noah Davidsohn2,6,
Michael B. Schultz1,2, Karolina Chwalek1,2, Luis A. Rajman1,2, George M. Church2,6, Konrad
Hochedlinger7, Vadim N. Gladyshev5, Steve Horvath8, Meredith S. Gregory-Ksander3*, Bruce R.
Ksander3*, Zhigang He4* and David A. Sinclair1,2*#
1. Paul F. Glenn Center for Biology of Aging Research at Harvard Medical School;
2. Blavatnik Institute, Department of Genetics, Harvard Medical School;
3. Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School;
4. Boston Children's Hospital, Department of Neurology, Harvard Medical School;
5. Division of Genetics, Dept. of Medicine, Brigham and Women’s Hospital, Harvard Medical
School;
6. Wyss Institute for Biologically Inspired Engineering, Harvard University;
7. Department of Molecular Biology, Cancer Center and Center for Regenerative Medicine,
Massachusetts General Hospital;
8. Human Genetics, David Geffen School of Medicine, UCLA;
9. These authors contributed equally: A. K., B. B., X. T.;
* Senior authors;
# Correspondence: david_sinclair@hms.harvard.edu
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Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause
of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression
patterns that are required for cells to function optimally and recover from damage1-3.
Changes to DNA methylation patterns over time form the basis of an 'ageing clock'4,5, but
whether old individuals retain information to reset the clock and, if so, whether this would
improve tissue function is not known. Of all the tissues in the body, the central nervous
system (CNS) is one of the first to lose regenerative capacity6,7. Using the eye as a model
tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful
gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes
axon regeneration after optic nerve crush injury, and restores vision in a mouse model of
glaucoma and in normal old mice. This process, which we call recovery of information via
epigenetic reprogramming or REVIVER, requires the DNA demethylases Tet1 and Tet2,
indicating that DNA methylation patterns don't just indicate age, they participate in
ageing. Thus, old tissues retain a faithful record of youthful epigenetic information that can
be accessed for functional age reversal.
The metaphor of the epigenetic landscape, first invoked by Waddington to explain
embryonic development8,9, is increasingly seen as relevant to the other end of life9. Evidence
from yeast and mammals supports an Information Theory of Ageing, in which the loss of
epigenetic information disrupts youthful gene expression patterns1-3, leading to cellular
dysfunction and senescence.10
In mammals, progressive DNA methylation changes serve as an epigenetic clock, but
whether they are merely an effect or a driver of ageing is not known4,5. In cell culture, the ectopic
expression of the four Yamanaka transcription factors, namely Oct4, Sox2, Klf4, and c-Myc
(OSKM)11, can reprogram somatic cells to become pluripotent stem cells, a process that erases
most DNA methylation marks and leads to the loss of cellular identity4,12. In vivo, ectopic,
transgene-mediated expression of these four genes alleviates progeroid symptoms in a mouse
model of Hutchison-Guilford Syndrome, indicating that OSKM might counteract normal
ageing13. Continual expression of all four factors, however, induces teratomas14 or causes death
within days13, ostensibly due to tissue dysplasia15.
Ageing is generally considered a unidirectional process akin an increase in entropy, but
living systems are open, not closed, and in some cases can fully reset biological age, examples
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being "immortal" cnidarians and the cloning of animals by nuclear transfer16. Having previously
found evidence for epigenetic noise as an underlying cause of ageing2,3, we wondered whether
mammalian cells might retain a faithful copy of epigenetic information from earlier in life,
analogous to Shannon's "observer" system in Information Theory, essentially a back-up copy of
the original signal to allow for its reconstitution at the receiving end if information is lost or
noise is introduced during transmission17.
To test this hypothesis, we introduced the expression of three-gene OSK combination in
fibroblasts from old mice and measured its effect on RNA levels of genes known to be altered
with age, including H2A, H2B, LaminB1, and Chaf1b. We excluded the c-Myc gene from these
experiments because it is an oncogene that reduces lifespan18. OSK-treated old fibroblasts
promoted youthful gene expression patterns, with no apparent loss of cellular identity or the
induction of Nanog, an early embryonic transcription factor that can induce teratomas (Extended
Data Fig.1a-c).
Next, we tested if a similar restoration was possible in mice. To deliver and control OSK
expression in vivo, we engineered a tightly regulated adeno-associated viral (AAV) vector under
the control of tetracycline response element (TRE) promoter (Fig.1a)19,20. The TRE promoter can
be activated either by rtTA in the presence of DOX (the "Tet-ON" system) or by tTA in the
absence of DOX ("Tet-OFF"). Extraneous AAV sequences were removed so the vector could
accommodate OSK as a poly-cistron. To test if ectopic OSK expression was toxic in vivo, we
infected 5 month-old C57BL/6J mice with AAV9-rtTA and AAV9-TRE-OSK delivered
intravenously (1.0 x 1012 gene copies total), then induced OSK expression by providing
doxycycline in the drinking water (Extended Data Fig. 2a). Surprisingly, continuous induction of
OSK for over a year had no discernable negative effect on the mice (Fig.1b and Extended Data
Fig. 2b)20, ostensibly because we avoided high-level expression in the intestine (Extended Data
Fig.2c-e), thus avoiding the dysplasia and weight loss seen in other studies15.
Almost all species experience a decline in regenerative potential during ageing. In
mammals, one of the first systems to lose its regenerative potential is the CNS.6 Retinal ganglion
cells (RGCs) are part of the CNS that project axons away from the retina towards the brain,
forming the optic nerve. During embryogenesis and in neonates, RGCs can regenerate if
damaged, but this capacity is lost within days after birth7. Then, throughout adulthood, the
function of these cells continues to decline21. To date, attempts to reverse acute or chronic
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damage to the CNS have been moderately successful, and no treatments have successfully
restored eyesight.
To test whether it is possible to provide adult RGCs with the regenerative capacity, we
induced OSK in an optic nerve crush injury model. The Tet-Off system carrying OSK, either in
separate AAVs or in the same AAV, was injected into the vitreous body, resulting in efficient,
selective, and doxycycline-responsive gene expression in RGCs (Fig.1c). As a negative control, a
group of mice were also continuously treated with doxycycline to repress OSK expression
(Extended Data Fig. 3a). Two weeks after AAV delivery, we performed optic nerve crush. Axon
length and optic nerve density were determined 16 days later (Fig.1d).
The greatest extent of axon regeneration and RGC survival, independent of RGC
proliferation (Extended Data Fig.4a), occurred when all three genes were delivered via the same
AAV as a polycistron (Fig. 1e-g). Indeed, when polycistronic OSK was induced for 12-16
weeks, regenerating RGC axon fibers extended over 5 mm into the chiasm, where optic nerve
connects to brain (Extended Data Fig.4b, c). When genes were co-delivered by separate AAVs,
no effect on axon regeneration was observed, ostensibly due to the lower frequency of co-
infection (Extended Data Fig.3c, d). When delivered singly, OCT4 and SOX2 alone increased
RGC survival slightly, but none of the single factors alone had any effect on regenerative
capacity (Fig. 1e, f). Because Klf4 has been reported to repress rather than promote axonal
growth22,23, we also tested a dual-cistron of just Oct4 and Sox2, but observed no regenerative
effect even in the absence of Klf4 (Fig. 1e, f).
Utilizing the Tet-On AAV system for its rapid on-rate (Extended Data Fig. 3b and 5a, b),
we tested the effect of inducing OSK expression before or after damage. Significant
improvement in axon regeneration only occurred when OSK expression was induced after injury;
the longer the duration of OSK induction post-injury, the greater the distance the axons extended,
with no increase in the total number of RGCs (Fig. 2b-d). By comparing infected RGCs numbers
pre- and post-injury, survival rate of OSK infected RGC was ~2.5-3.0 times that of uninfected or
GFP-infected RGCs (52 vs. 17%-20%) (Extended Data Fig. 5c, d), indicating OSK's protective
and regenerative effects are largely cell-intrinsic. The PTEN-mTOR-S6K pathway, previously
shown to improve RGC survival and axon regeneration in vivo24, was not activated in OSK-
infected cells post-injury (Extended Data Fig. 6a, b), indicating a new pathway might be
involved.
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Given the necessity of post-injury OSK expression and the known role of Yamanaka
factors to reverse DNA methylation (DNAme) age during partial or fully reprogramming in
vitro4,12,25, we wondered whether neuronal injury advanced epigenomic age and whether OSK's
benefits were due to the preservation of a younger epigenome. Genomic DNA from FACS-
isolated RGCs was obtained from retinas that are intact or 4-days after axonal injury in the
presence or absence of OSK induction and subjected reduced-representation bisulfite sequencing
(RRBS). A newly published rDNAme clock26 provided the best site coverage (70/72 CpG sites)
relative to other available mouse clocks27,28, and its age estimate remained highly correlated with
chronological age of RGCs (Extended Data Fig. 7a and Methods). Consistent with the
hypothesis, in the absence of global methylation changes, injured RGCs experienced an
acceleration of the epigenetic clock and OSK expression counteracted this effect (Fig. 2e and
Extended Data Fig. 7b).
Ten-Eleven-Translocation (TET) dioxygenases are known for their ability to remove
DNA demethylation at CpG sites. Because Yamanaka factors promote in vitro reprogramming
by upregulating Tet1 and Tet2, but have no effect on Tet329,30, we tested whether Tet1 and Tet2
were required for the beneficial effects of OSK on RGCs. We utilized previously well
characterized AAVs expressing short- hairpin RNAs against Tet1 and Tet2 (sh-Tet1 and sh-
Tet2)31-33 and validated their high co-transduction rate (near 70%) with OSK AAV in the RGCs
(Extended Data Fig. 6c, d). Knockdown of either Tet1 or Tet2 blocked the ability of OSK to
promote RGC survival and regeneration (Fig. 2f and Extended Data Fig. 6e).
To explore if neuronal reprogramming might be applicable to humans, we performed
axon regeneration assays on differentiated SH-SY5Y human neuronal cultures (Fig. 2g), with
and without OSK induction (Extended Data Fig. 8a, b). Similar to results of mouse RGCs in vivo
(Extended Data Fig. 4a), OSK did not induce human neuron cell proliferation (Extended Data
Fig. 8c, d). Axon degeneration was then induced by a 24 hr treatment with vincristine (VCS), a
chemotherapeutic agent, and cells were then allowed to recover for 9 days. Again, we measured
the DNA methylation age of AAV-transduced neurons using a clock for in vitro studies34.
Paralleling the RGCs, DNA methylation age was significantly increased after VCS-induced
damage of human neurons (Extended Data Fig. 8e), and OSK expression not only prevented this
increase but also restored a younger DNA methylation age without a global reduction of DNA
methylation (Fig.2h and Extended Data Fig. 7c). At Day 9 post-damage, the neurite area was 15-
fold greater in the rejuvenated OSK-transduced cells than controls (Extended Data Fig. 8f, g).
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The recovery from damage was completely blocked by validated Tet2 knockdown (Fig. 2i, j,
Extended Data Fig. 8h) even in presence of high OSK expression (Extended Data Fig. 8i), but
was not dependent on mTOR-S6K pathway (Extended Data Fig. 8j, k), again paralleling mouse
RGCs. Thus, the ability of OSK to reprogram neurons and promote axon growth is a conserved,
DNA demethylation-dependent cell intrinsic process. We refer to this process as the recovery of
information via epigenetic reprogramming, or "REVIVER" for short.
Glaucoma, a progressive loss of RGCs and their axons that often coincides with increased
intraocular pressure, is a leading cause of age-related blindness worldwide. Although some
treatments can slow down disease progression35, it is currently not possible to restore vision once
it has been lost. Given the ability of OSK to regenerate axons after acute nerve damage, we
tested whether REVIVER could restore the function of RGCs in a chronic setting like glaucoma
(Fig. 3a). Elevated intraocular pressure (IOP) was induced unilaterally for 4-21 days by injection
of microbeads into the anterior chamber (Fig. 3b)36. AAVs or PBS were then injected
intravitreally and expressed at a time point when glaucomatous damage was established, with a
significant decrease in RGCs and axonal density (Fig. 3a, Extended Data Fig.9a, b). Four weeks
after AAV injection, OSK-treated mice presented with a significant increase in axon density
compared to mice that received either PBS or AAVs with no OSK induction (-OSK). The
increased axon density was equivalent to the axon density in the saline-only, non-glaucomatous
mice (Fig. 3c, d) and was not associated with proliferation of RGCs (Extended Data Fig.9c).
To determine whether the increased axon density in OSK-treated mice coincided with
increased vision, we tracked the visual acuity of each mouse by measuring their optomotor
response (OMR) (Fig. 3e). Compared to mice that received either PBS or -OSK AAV, those that
received OSK induction experienced a significant increase in visual acuity relative to the pre-
treatment baseline measurement, restoring about half of vision (Fig. 3f). A readout of electrical
waves generated by RGCs in response to a reversing contrast checkerboard pattern, known as
pattern electroretinogram (PERG) analysis, showed that OSK treatment significantly improved
RGC function relative to the pre-treatment baseline measurements and PBS or -OSK AAV
treatments (Fig. 3g, h). To our knowledge, REVIVER is the first treatment to reverse vision loss
in a glaucoma model.
Many treatments known to work well in young individuals often fail in older ones. For
example, an approach to regenerate retinal rod photoreceptors was effective in 1 month-old mice
but not in 7 month-olds37. Given the ability of REVIVER to regenerate axons and to restore
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vision after glaucomatous damage in young mice, we wondered if REVIVER might be effective
in aged mice, too. Optic nerve crush injury was performed on 12 month-old mice using the same
protocol as in Fig.1d according to the experimental design in Fig 4a. In aged mice, OSK AAV
treatment for two weeks post-injury doubled RGC survival, similar to that observed in 1 and 3
month-old mice (Extended Data Fig. 10a). Though the axon regeneration was slightly less than
young mice two weeks after injury (Fig. 4b, Extended Data Fig. 10b), OSK AAV treatment in
aged mice for five weeks was similar to that observed in young mice (Fig. 4b, c). These data
indicate that ageing does not significantly diminish the effectiveness of OSK AAV treatment in
inducing axon regeneration following an optic nerve crush injury.
To test whether REVIVER could reverse vision loss associated with physiological
ageing, 4 and 12 month-old mice received intravitreal injections of -OSK or +OSK AAV (Fig
4a). Compared to the 4 month-olds, there was a significant reduction in visual acuity and RGC
function at one year of age, as measured by OMR and PERG. Strikingly, this loss was
completely restored by 4-weeks of OSK expression (Fig. 4d, Extended Data Fig.10c). We did not
see a restorative effect in 18 month-old mice (Extended Data Fig. 10c, d), likely due to
spontaneous corneal opacity that develops at that age38.
Considering there was no obvious increase in RGCs and axon density in the 12m-old-
mice (Extended Data Fig. 10e, f), we suspected the increased vision was due to a functional
improvement, one that could be revealed by analyzing the transcriptome. FACS-purified RGCs
from 12-m-old mice, either untreated or treated with -OSK or +OSK AAV, were analyzed by
RNA-seq. Compared to RGCs from 5-m-old young mice, we identified 464 genes that were
differentially-expressed during ageing and also not affected by empty AAV infection (Extended
Data Fig. 11a and Supplemental Table 1-4). Remarkably, the vast majority (90%, 418) of the 464
age-deregulated genes were restored to youthful levels after 4 weeks of OSK expression (Fig. 4e,
f). Of the 268 age-downregulated genes, 44 were genes involved in sensory perception (Fig.4f),
suggesting a decline in signaling receptors or sensory function during ageing, one that can be
restored by REVIVER (Extended Data Fig. 11b, c). Interestingly, 116 of these genes are yet to
be characterized. Another 196 genes that were up-regulated during ageing are known or
predicted to be involved in ion transport (Extended Data Fig.11d).
Consistent with the axon regeneration data, the knockdown of Tet1 or Tet2 completely
blocked the ability of REVIVER to restore vision in 12 month-old mice (Fig. 4g, h). To
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determine if the DNA methylation clock was affected, we measured the rDNA methylation age
of FACS-sorted RGCs from 12 month-old mice. Four weeks of OSK AAV expression
significantly decreased DNA methylation age, and this was Tet1- and Tet2-dependent (Fig. 4i).
Together, these results demonstrate that Tet-dependent in vivo reprogramming can restore
youthful gene expression patterns, reverse the DNA methylation clock, and restore the function
and regenerative capacity of a tissue as complex as the retina.
Post-mitotic neurons in the CNS are some of the first cells in the body to lose their ability to
regenerate. In this study, we show that in vivo reprogramming of aged neurons can reverse DNA
methylation age and allow them to regenerate and function as though they were young again.
The requirement of the DNA demethylases Tet1 and Tet2 for this process indicates that altered
DNA methylation patterns may not just a measure of age but participants in ageing. These data
lead us to conclude that mammalian cells retain a set of original epigenetic information, in the
same way Shannon's observer stores information to ensure the recovery of lost information17.
How cells are able to mark and retain youthful DNA methylation patterns, then in late adulthood
OSK can instruct the removal of deleterious marks is unknown. Youthful epigenetic
modifications may be resistant to removal by the Tets by the presence of a specific protein or
DNA modification that inhibits the reprogramming machinery. Even in the absence of this
knowledge, these data indicate that the reversal of DNA methylation age and the restoration of a
youthful epigenome could be an effective strategy, not just to restore vision, but to give complex
tissues the ability to recover from injury and resist age-related decline.
Acknowledgements
We greatly thank Amy Wagers, Raul Mostoslavsky, Yang Shi, Abhirup Das, Alice Kane,
Margarete Karg, Bohan Zhang, Phillip Dmitriev, Keith Booher, Emily Chen, and Shurong Hou
for advice and assistance. The work was supported by the Harvard Medical School Epigenetics
Seed Grant Program and Development Grant Program, The Paul F. Glenn Foundation for
Medical Research, a kind gift from Edward Schulak, and NIH awards R01AG019719 (to D.A.S),
R01EY026939 and R01EY021526 (to Z.H.), and R01GM065204 (to V.N.G.). We thank Boston
Children's Hospital Viral Core, which is supported by NIH5P30EY012196, and Schepen Eye
Institute Core facilities, supported by NEI-P30EY003790. X.T. was supported by NASA
Postdoctoral Fellowship 80NSSC19K0439; D.V. by NIH training grant T32AG023480; J.-H.Y.
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was partially supported by National Research Foundation of Korea (2012R1A6A3A03040476);
B.R.K. partially by the St Vincent de Paul Foundation; and M.G.K. by NEI award
R21EY030276. This paper is dedicated to Honghua Lu, grandfather to Y.L., who passed away
bravely fighting ageing during preparation of this manuscript.
Contributions
Y.L., X.T. and D.A.S. wrote the manuscript with input from coauthors. Y.L. and D.A.S
conceived of the project with help from M.S.B.. Y.L. performed or involved in all experiments
and analysis. X.T. conducted human neuron experiments. B.B., C.W., Q.Z., D.Y., S.Z., and Z.H.
contributed to the optic nerve crush studies and imaging. A.K., Q.Z., D.Y., E.M.H., E.K.,
M.G.K., and B.R.K., contributed to the glaucoma and ageing studies. X.T., J.-H.Y., and K.H.
helped with transgenic mice work. M.S.B., M.B.S., L.R., helped with systemic AAV9
experiment. M.M. and V.G. conducted mouse RGC methylation clock analysis. D.V. performed
the RNA-seq analysis. N.D., and G.C. helped with plasmid constructs and AAV9 production.
S.H. conducted human methylation clock analysis. K.C. helped with grant applications and
project management. M.G.K., B.R.K., Z.H. and D.A.S. jointly supervised this work.
Conflict of interest
D.A.S is a consultant to, inventor of patents licensed to, and in some cases board member and
investor of MetroBiotech, Cohbar, Life Biosciences and affiliates, InsideTracker, Vium, Zymo,
EdenRoc Sciences and affiliates, Immetas, Segterra, Galilei Biosciences, and Iduna
Therapeutics. He is also an inventor on patent applications licensed to Bayer Crops, Merck
KGaA, and Elysium Health. For details see https://genetics.med.harvard.edu/sinclair. Y.L. is an
equity owner of Iduna. D.V. is an advisor to Liberty Biosecurity. M.S.B. is a shareholder in
Animal Biosciences and MetroBiotech. K.C. is an equity owner and advisor to Life Biosciences
and affiliates. N.D. and G.C are co-founder of Rejuvenate Bio. G.M.C’s disclosures see
http://arep.med.harvard.edu/gmc/tech.html. The other authors declare no competing interests.
Y.L., N.D. and D.A.S. are inventors on patent applications arising from this work.
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Methods
Mouse Lines
C57BL6/J wild type mice were purchased from Jackson Laboratory (000664) for optic nerve
crush and glaucoma model experiments. For ageing experiments, females from NIA Aged
Rodent Colonies (https://www.nia.nih.gov/research/dab/aged- rodent-colonies-handbook) were
used. Col1a1-tetOP-OKS-mCherry/ Rosa26-M2rtTA alleles were a gift from the Hochedlinger
lab (Harvard).39 All animal work was approved by Harvard Medical School, Boston Children’s
Hospital, and Mass Eye and Ear Institutional animal care and use committees.
Surgery
Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg;
Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (9 mg/kg; TranquiVed;
Vedco, Inc., St. Joseph, MO) supplemented by topical application of proparacaine to the ocular
surface (0.5%; Bausch & Lomb, Tampa, FL). All animal procedures were approved by the
IACUC of the respective institutions and according to appropriate animal welfare regulations.
Production of Adeno associated viruses (AAVs)
Vectors of AAV-TRE-OSK were made by cloning mouse Oct4, Sox2 and Klf4 cDNA into an
AAV plasmid consisting of the Tet Response Element (TRE3G promoter) and SV40 element.
The other vectors were using similar strategy or directly chemically synthesized. All pAAVs, as
listed (Supplemental Table 5), were then packaged into AAVs of serotype 2/2 or 2/9 (titers: >
5´1012 genome copies/ml). AAVs were produced by Boston Children's Hospital Viral Core.
Systemic delivery of AAV9 to internal organs
Expression in internal organs was achieved through retro-orbital injection of AAV9 (3´1011
TRE-OSK plus 7´1011 UBC-rtTA). To induce OSK expression, doxycycline (1 mg/ml; MP
biochemicals) was given in drinking water continuously, 3 weeks post-AAV injection.
Cell culture and differentiation
Ear fibroblasts (EFs) were isolated from Reprogramming 4F (Jackson Laboratory 011011) or 3F
(Hochedlinger lab, Harvard) mice and cultured at 37ºC in DMEM (Invitrogen) containing Gluta-
MAX, non-essential amino acids, and 10% fetal bovine serum (FBS). EFs of transgenic OSKM
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and OSK mice were passaged to P3 and treated with doxycycline (2 mg/ml) for the indicated
time periods in the culture medium. SH-SY5Y neuroblastoma cells were obtained from the
American Tissue Culture Collection (ATCC, CRL-2266) and maintained according to ATCC
recommendations. Cells were cultured in a 1:1 mixture of Eagle’s Minimum Essential Medium
(EMEM, ATCC, 30-2003) and F12 medium (ThermoFisher Scientific, 11765054) supplemented
with 10% fetal bovine serum (FBS, Sigma, F0926) and 1X penicillin/streptomycin
(ThermoFisher Scientific, 15140122). Cells were cultured at 37°C with 5% CO2 and 3% O2.
Cells were passaged at ~80% confluency. SH-SY5Y cells were differentiated into neurons as
previously described40 with modifications. Briefly, 1 day after plating, cell differentiation was
induced for 3 days using EMEM/F12 medium (1:1) containing 2.5% FBS, 1×
penicillin/streptomycin, and 10 µM all-trans retinoic acid (ATRA, Stemcell Technologies,
72264) (Differentiation Medium 1), followed by a 3 day incubation in EMEM/F12 (1:1)
containing 1% FBS, 1 × penicillin/streptomycin, and 10 µM ATRA (Differentiation Medium 2).
Cells were then split into 35 mm cell culture plates coated with poly-D-lysine (ThermoFisher
Scientific, A3890401). A day after splitting, neurons were matured in serum-free neurobasal/B27
plus culture medium (ThermoFisher Scientific, A3653401) containing 1 × Glutamax
(ThermoFisher Scientific, 35050061), 1 × penicillin/streptomycin, and 50 ng/ml BDNF
(Alomone labs) (Differentiation Medium 3) for at least 5 days.
Neurite regeneration assay
Differentiated SH-SY5Y cells were transduced with AAV.DJ vectors at 106 genome copies/cell.
Five days after transduction, vincristine (100 nM; Sigma, V8879) was added for 24 hrs to induce
neurite degeneration. Neurons were then washed twice in PBS and fresh Differentiation medium
3 was added back to the plates. Neurite outgrowth was monitored for 2-3 weeks by taking phase-
contrast images at 100x magnification every 3-4 days. Neurite area was quantified using Image J.
Cell cycle analysis
Cells were harvested and fixed with 70% cold ethanol for 16 hrs at 4°C. After fixation, cells
were washed twice with PBS and incubated with PBS containing 50 µg/mL propidium iodide
(Biotium, 40017) and 100 µg/mL RNase A (Omega) for 1 hr at room temperature. PI-stained
samples were analyzed on a BD LSR II analyzer and only single cells were gated for analysis.
Cell cycle profiles were analyzed using FCS Express 6 (De Novo Software).
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Human neuron methylation and epigenetic clock analyses
DNA was extracted using the Zymo Quick DNA mini-prep plus kit (D4069) and DNA
methylation levels were measured on Illumina 850 EPIC arrays. The Illumina BeadChip (EPIC)
measured bisulfite-conversion-based, single-CpG resolution DNAm levels at different CpG sites
in the human genome. Data were generated via the standard protocol of Illumina methylation
assays, which quantifies methylation levels by the β value using the ratio of intensities between
methylated and un-methylated alleles. Specifically, the β value was calculated from the intensity
of the methylated (M corresponding to signal A) and un-methylated (U corresponding to signal
B) alleles, as the ratio of fluorescent signals β = Max(M,0)/(Max(M,0)+ Max(U,0)+100). Thus, β
values ranged from 0 (completely un-methylated) to 1 (completely methylated). "Noob"
normalization was implemented using the "minfi" R package41,42. The mathematical algorithm
and available software underlying the skin & blood clock for in vitro studies (based on 391
CpGs) was previously published34.
AAV2 Virus Intravitreal Injection
Adult animals were anesthetized with ketamine/xylazine (100/10 mg/kg) and then AAV (1-3 µl)
was injected intravitreally, just posterior to the limbus with a fine glass pipette attached to the
Hamilton syringe using plastic tubing. In elevated IOP model, mice received a 1µl intravitreal
injection between 3-4 weeks following microbead injection. The injected volume of AAV-sh-
RNA is 1/5th the volume of other AAVs.
Optic Nerve Crush
Two weeks after intravitreal AAV injection, the optic nerve was accessed intraorbitally and
crushed in anesthetized animals using a pair of Dumont #5 forceps (FST). Alexa-conjugated
cholera toxin beta subunit (CTB-555, 1 mg/ml; 1-2 µl) injection was performed 2-3 days before
euthanasia to trace regenerating RGC axons. More detailed surgical methods were described
previously24.
In Vivo Doxycycline Induction or suppression
Induction of Tet-On or suppression of Tet-Off AAV2 systems in the retina was performed by
administration of doxycycline (2 mg/ml) (Sigma) in the drinking water. Induction of Tet-On
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AAV9 system systemically was performed by administration of doxycycline (1 mg/ml) (USP
grade, MP Biomedicals 0219895505) in the drinking water.
Axon Regeneration Quantification
The number of regenerating axons in the optic nerve was estimated by counting the number of
CTB-labeled axons at different distances from the crush site as described previously24.
Whole-Mount Optic Nerve Preparation
Optic nerves and the connecting chiasm were dehydrated in methanol for 5 min, then incubated
overnight with Visikol® HISTO-1™. Next day nerves were transferred to Visikol® HISTO-2™
and then incubated for 3 hrs. Finally, optic nerves and connecting chiasm were mounted with
Visikol® HISTO-2™.
Immunofluorescence
Whole-mount retinas were blocked with horse serum 4°C overnight then incubated at 4°C for 3
days with primary antibodies: Mouse anti-Oct4 (1:100, BD bioscience, 611203), Rabbit anti-
Sox2 (1:100, Cell signaling, 14962), Goat anti-Klf4 (1:100, R&D system, AF3158), Rabbit anti-
phosphorylated S6 Ser235/236 (1:100, Cell Signaling 4857), Rabbit anti-Brn3a (1:200, EMD
Millipore, MAB1585) and Guinea pig anti-RBPMS (1:400, Raygene custom order A008712 to
peptide GGKAEKENTPSEANLQEEEVRC) diluted in PBS, BSA (3%) Triton X-100 (0.5%).
Then, tissues were incubated at 4°C overnight with appropriate Alexa Fluor-conjugated
secondary antibodies (Alexa 405, 488, 567, 674; Invitrogen) diluted with the same blocking
solution as the primary antibodies, generally used at 1:400 final dilution. Frozen sections were
stained overnight with primary antibodies at 4°C and then secondary antibodies at room
temperature for 2 h. Sections or whole-mount retinas were mounted with Vectashield Antifade
Mounting Medium.
Western Blot
SDS-PAGE and western blot analysis was performed according to standard procedures and
detected with an ECL detection kit. Antibodies used: Rabbit anti-Sox2 (1:100, EMD Millipore,
AB5603), Mouse anti-Klf4 (1:1000, ReproCell, 09-0021), Rabbit anti-p-S6 (S240/244) (1:1000,
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CST, 2215), Mouse anti-S6 (1:1000, CST, 2317), Mouse anti-β-Tubulin (1:1000, Sigma-Aldrich,
05-661), Mouse anti-β-Actin−Peroxidase antibody (1:20,000, Sigma-Aldrich, A3854).
RGC Survival and Phospho-S6 Signal
RBPMS-positive cells in the ganglion layer were stained with an anti-RBPMs antibody (1:400,
Raygene custom order A008712 to peptide GGKAEKENTPSEANLQEEEVRC) and a total of
four 10X fields per retina, one in each quadrant, were enumerated. The average number per field
was determined and the percentages of viable RGCs were obtained by comparing the values
determined from the uninjured contralateral retinas. Phospho-S6 (1:100, Cell Signaling 4857)
staining was performed under the same conditions and the densities of phopsho-S6-positive
RGCs were obtained by comparing the value to uninjured contralateral retinas.
Microbead-induced mouse model of elevated IOP
Elevation of IOP was induced unilaterally by injection of polystyrene microbeads (FluoSpheres;
Invitrogen, Carlsbad, CA; 15-μm diameter) to the anterior chamber of the right eye of each
animal under a surgical microscope, as previously reported36. Briefly, microbeads were prepared
at a concentration of 5.0 × 106 beads/mL in sterile physiologic saline. A 2 μL volume was
injected into the anterior chamber through a trans-corneal incision using a sharp glass
micropipette connected to a Hamilton syringe (World Precision Instruments Inc., Sarasota, FL)
followed by an air bubble to prevent leakage. Any mice that developed signs of inflammation
(clouding or an edematous cornea) were excluded.
IOP (Intraocular pressure) measurements
IOPs were measured with a rebound TonoLab tonometer (Colonial Medical Supply, Espoo,
Finland), as previously described36,43. Mice were anesthetized by 3% isoflurane in 100% oxygen
(induction) followed by 1.5% isoflurane in 100% oxygen (maintenance) delivered with a
precision vaporizer. IOP measurement was initiated within 2-3 min after the loss of a toe or tail
pinch reflex. Anesthetized mice were placed on a platform and the tip of the pressure sensor was
placed approximately 1/8 inch from the central cornea. Average IOP was displayed
automatically after 6 measurements after elimination of the highest and lowest values. The
machine-generated mean was considered as one reading, and six readings were obtained for each
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eye. All IOPs were taken at the same time of day (between 10:00 and 12:00 hrs) due to the
variation of IOP throughout the day.
Optomotor Response
Visual acuity of mice was measured using an optomotor reflex-based spatial frequency threshold
test44,45. Mice were able to freely move and were placed on a pedestal located in the center of an
area formed by four computer monitors arranged in a quadrangle. The monitors displayed a
moving vertical black and white sinusoidal grating pattern. A blinded observer, unable to see the
direction of the moving bars, monitored the tracking behavior of the mouse. Tracking was
considered positive when there was a movement of the head (motor response) to the direction of
the bars or rotation of the body in the direction concordant with the stimulus. Each eye would be
tested separately depending on the direction of rotation of the grating. The staircase method was
used to determine the spatial frequency start from 0.15 to 0.40 cycles/deg, with intervals of 0.05
cycles/deg. Rotation speed (12°/s) and contrast (100%) were kept constant. Responses were
measured before and after treatment by individuals blinded to the group of the animal and the
treatment.
Pattern Electroretinogram (PERG)
Mice were anesthetized with ketamine/xylazine (100mg/kg and 20mg/kg) and the pupils dilated
with one drop of 1% tropicamide ophthalmic solution. The mice kept under dim red light
throughout the procedure on a built-in warming plate (Celeris, Full-Field and Pattern Stimulation
for the rodent model) to maintain body temperature at 37˚C. A black and white reversing
checkerboard pattern with a check size of 1° was displayed on light guide electrode-stimulators
placed directly on the ocular surface of both eyes and centered with the pupil. The visual stimuli
were presented at 98% contrast and constant mean luminance of 50 cd/m2, spatial frequency:
0.05 cyc/deg; temporal frequency: 1Hz. A total of 300 complete contrast reversals of PERG were
repeated twice in each eye and the 600 cycles were segmented and averaged and recorded. The
averaged PERGs were analyzed to evaluate the peak to trough N1 to P1 (positive wave)
amplitude.
Quantification of optic nerve axons
For quantification of axons, optic nerves were dissected and fixed overnight in Karnovsky’s
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reagent (50% in phosphate buffer). Semi-thin cross-sections of the nerve were taken at 1.0 mm
posterior to the globe and stained with 1% p-phenylenediamine (PPD) for evaluation by light
microscopy. Optic nerve cross sections were imaged at 60x magnification using a Nikon
microscope (Eclipse E800, Nikon, Japan) with the DPController software (Olympus, Japan) and
6-8 non-overlapping photomicrographs were taken to cover the entire area of each optic nerve
cross-section. Using ImageJ (Version 2.0.0-rc-65/1.51u), a 100 μM x 100 μM square was placed
on each 60x image and all axons within the square (0.01mm2) were counted using the threshold
and analyze particles function in image J as previously described36,43,44. Damaged axons stain
darkly with PPD and are not counted. The average axon counts in the 6-8 images were used to
calculate the axon density/mm2 of optic nerve. Individuals performing axon counts were blinded
to the experimental groups.
Quantification of retinal ganglion cells in glaucoma model
For ganglion cell counting, images of whole mount retinas were acquired using a 63x oil
immersion objective of the Leica TCS SP5 confocal microscope (Leica Microsystems). The
retinal whole mount was divided into four quadrants and two to four images (248.53µm by
248.53µm in size) were taken from the midperipheral and peripheral regions of each quadrant,
for a total of twelve to sixteen images per retina. The images were obtained as z-stacks (0.5µm)
and all Brn3a positive cells in the ganglion cell layer of each image were counted manually as
previously described44. Briefly, RGCs were counted using the “Cell Counter” plugin
(http://fiji.sc/Cell_Counter) in Fiji is Just ImageJ software (ImageJ Fiji, version 2.0.0-rc-
69/1.52n). Each image was loaded into Fiji and a color counter type was chosen to mark all
Brn3a stained RGCs within each image (0.025mm 2). The average number of RGCs in the 12 to
sixteen images were used to calculate the RGC density per square millimeter of retina. Two
individuals blinded to the experimental groups performed all RGC counts.
RGC Enrichment
Retinas were fresh dissected and dissociated in AMES media using papain, then triturated
carefully and stained with Thy1.2-PE-Cy7 anti-body (Invitrogen 25-0902-81) and Calcine Blue
live-dead cell stain, then flow sorted on a BD FACS Aria using an 130µm nozzle to collect over
10,000 Thy1.2+ and Calcine blue+ cells (1-2% of total events). Frozen cells were sent to
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GENEWIZ, LLC (South Plainfield, NJ, USA) for RNA extraction and ultra-low input RNA-seq,
or to Zymo research (Irving, CA) for DNA extraction and genome-wide reduced representation
bisulfite sequencing (RRBS).
Classic RRBS Library preparation
DNA was extracted using Quick-DNA Plus Kit Microprep Kit. 2-10 ng of starting input genomic
DNA was digested with 30 units of MspI (NEB). Fragments were ligated to pre-annealed
adapters containing 5’-methyl-cytosine instead of cytosine according to Illumina’s specified
guidelines. Adaptor-ligated fragments ≥50 bp in size were recovered using the DNA Clean &
ConcentratorTM-5 (Cat#: D4003). The fragments were then bisulfite-treated using the EZ DNA
Methylation-LightningTM Kit (Cat#: D5030). Preparative-scale PCR products were purified
with DNA Clean & ConcentratorTM-5 (Cat#: D4003) for sequencing on an Illumina HiSeq
using 2x125 bp PE.
DNA methylation age analysis of mouse RGC
Reads were filtered using trim galore v0.4.1 and mapped to the mouse genome GRCm38 using
Bismark v0.15.0. Methylation counts on both positions of each CpG site were combined. Only
CpG sites covered in all samples were considered for analysis. This resulted in total of 708156
sites. For the rDNA methylation clock reads were mapped to BK000964 and the coordinates
were adjusted accordingly26. 70/72 sites were covered for rDNA clock, compared to 102/435
sites of whole lifespan multi-tissue clock27, or 248/582 and 77,342/ 193,651 sites (ridge) of two
entire lifespan multi-tissue clocks28.
Total RNA extraction and Sample QC
Total RNA was extracted following the Trizol Reagent User Guide (Thermo Fisher Scientific). 1
µl of 10 mg/ml Glycogen was added to the supernatant to increase RNA recovery. RNA was
quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA
integrity was determined using TapeStation (Agilent Technologies, Palo Alto, CA, USA).
Ultra-low input RNA library preparation and multiplexing
RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA,
USA) and RNA integrity was ascertained using a 2100 TapeStation (Agilent Technologies, Palo
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Alto, CA, USA). RNA library preparations, sequencing reactions, and initial bioinformatics
analysis were conducted at Genewiz (South Plainfield, NJ, USA). A SMART-Seq v4 Ultra Low
Input Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech,
Mountain View, CA), and Illumina Nextera XT library was used for sequencing library
preparation. Briefly, cDNA was fragmented and adaptors were added using Transposase,
followed by limited-cycle PCR to enrich and add an index to the cDNA fragments. The final
library was assessed by a Qubit 2.0 Fluorometer and Agilent TapeStation.
Sequencing 2x150bp PE
The sequencing libraries were multiplexed and clustered on two lanes of a flowcell. After
clustering, the flowcell were loaded on the Illumina HiSeq instrument according to
manufacturer’s instructions. Samples were sequenced using a 2x150 Paired End (PE)
configuration. Image analysis and base calling were conducted by the HiSeq Control Software
(HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from Illumina HiSeq
was converted into fastq files and de-multiplexed using Illumina bcl2fastq v2.17 program. One
mis-match was allowed for index sequence identification.
RNA-seq analysis
Paired-end reads were aligned with hisat2 v2.1.046 to the Ensembl GRCm38 primary assembly
using splice junctions from the Ensembl release 84 annotation. Paired read counts were
quantified using featureCounts v1.6.447 using reads with a MAPQ >=20. Differentially-expressed
genes for each pairwise comparison were identified with edgeR v3.2648, testing only genes with
at least 0.1 counts-per-million (CPM) in at least three samples. Gene ontology analysis of
differentially-expressed genes was performed with AmiGO v2.5.1249-51. Age-associated sensory
perception genes were extracted from the mouse Sensory Perception (GO:0007600) category the
Gene Ontology database, including genes that were differentially expressed (q<=0.05) in 12
versus 5 month-old mice, excluding genes that were induced by the Control virus alone (q<=0.1).
Data availability
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
cd
e
g
f
4-wk-old
optic nerve crush
+2wks
perfusion
+2wks +2d
tTAandTRE
AAV2 CTB injection
Klf4
RBPMs
Klf4
RBPMs
Gene of
interest
Cross section
Ganglion
cells
Amacrine
cells
Bipolar
cells
Horizontal
cells
Cones Rods
Pigment
Epithelium
AAV2
Optic nerve
retina at-mount
0 4 8 12 16 20 24 28
0
10
85
90
95
100
105
110
Days
Body weight (%)
-DOX
+DOX
-DOX
+DOX
+DOX
-DOX
Tet -On OS K AAV9
Tet -On O SK Tra n sg e ne
WT
OSK Off
OSK
*
*
*
*
*
*
200 500 1000 1500
0
500
1000
1500
Distance from injury (µm)
#Axonspernerve
****
****
Oct4
Sox2
Klf4
Oct4 Sox2
Oct4 + Sox2 + Klf4
Oct4 Sox2 Klf4
GFP
Oct4 Sox2 Klf4 +DOX
Tet -Of f
OSK
OSK Off
0
400
800
1200
Survival of RGCs /mm2
****
*** *** *** ***
ns ns
d2EGFP
Oct4
Sox2
Klf4
Oct4-Sox2
O+S+K
TRE OSK
TRE OSK
Tet -On
Dox
rtTA
rtTA
tTA
Tet -Of f
tTA
Dox
TRE OSK
Dox (Tet)
*OSK: Oct4-2A-Sox2-2A-Klf4
TRE OSK
Dox (Tet)
Figure 1
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a
c
b
d
h
e
4-wk-old +2wks +2or4wpc
rtTA
TRE-OSK
+2d
optic nerve crush perfusionCTB injection
Pre-injury On Post-injury On DOX water
i
Or
g
*
*
*
*
*
*
Post-inj OSK O
Post-inj OSK On
+OSK (sh-Tet2)
+OSK (sh-Scr)
-OSK (sh-Scr)
-OSK (sh-Tet2)
?
AAV infection
Human neurons
VCS damage (24h)
5 days 9 days
sh-Scr sh-Tet2 sh-Scr sh-Tet2
0
100
200
300
400
500
**** ****
-OSK +OSK
Off
Pre-inj On
Post-inj On
Off
Post-inj On
0
500
1000
1500
2000
Survival of RGCs /mm2
**
***
ns *
2wpc
4wpc
200 500 1000 1500 2000
0
500
1000
1500
2000
Distance from injury (µm)
#Axonspernerve
Pre-inj On
****
Post-inj On
Off
Off
Post-inj On
**
****
****
2wpc
4wpc
Tet -On
j
- 1 9
0.0
0.2
0.4
0.5
0.6
0.7
0.8
0.9
DNAm Age (y)
Neurons (+OSK)
Days post VCS
linear regression (p=0.008)
*
Neurite area (103m2
f
200 500 1000 1500
0
500
1000
Distance from injury (µm)
#Axonspernerve
**** sh-Scr
sh-Tet1
sh-Tet2
**
+OSK
Figure 2
-GFPGFPOSK
0.0
0.2
0.4
0.6
DNAme Age
Injury
*** nsns
Intact
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The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
cd e
fg
8wkold +3-4wks
AAV2 injection
pERG & OMR
baseline
Elevated IOP +4wks
AAV expression
pERG & OMR
Microbead injection
cornea
lens
iris
900180 270 360
Time (ms)
0.1 cyc/deg
0.4 cyc/deg
Acuity
Stripped pattern rotation
Left eye response Right eye response
h
Baseline 4 wks Post AAV
0.00
0.15
0.30
0.45
Optomotor acuity
(cyc/deg)
***
*
ns
ns *
Baseline
0
0
-3
-6
3
6
9
12
0
-3
-6
3
6
9
12
5
10
15
Amplitude (µV)
Amplitude (µV) Amplitude (µV)
****
**
*
***
Saline
Beads (PBS)
Beads (-OSK)
Beads (+OSK)
Saline
Beads (PBS)
Beads (-OSK)
Beads (+OSK)
N1
P1
N2
N1
P1
N2
Baseline (Beads)
-OSK
N1
P1
N2
N1
P1
N2
Baseline (Beads)
+OSK
4 wks Post AAV
Saline Beads (PBS)
Beads (+OSK)
Beads (-OSK)
0
10
20
30
40
50
60
Axon density (104)/mm
2
*
*
*
*
0 7 14 21 28
0
10
20
30
40
IOP (mmHg)
Days post microbead injection
Saline
Microbeads
****
Saline PBS -OSK +OSK
Beads
Figure 3
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
d e f
gh i
200 500 1000 1500 2000
0
500
1000
1500
Distance from injury (µm)
#Axonspernerve
****
**** d2EGFP
OSK 5wks
d2EGFP
OSK 2wks
+2w +2or5w
AAV2
Aged
mice
+4wks
Regeneration?
Vision?
nerve crush
*
*
c
d2EGFPOSK
12 month; 5 wpc
4m 12 m
0.00
0.15
0.30
0.45
Age (month)
****
Optomotor acuity
(cyc/deg)
ns
*
ns
-OSK
+OSK
You ng Old Old(-OSK) Old(+OSK)
log2(1+CPM)
0
0.5
1
1.5
−6 −4 −2 0 2 4 6
−2−2−4 0 2 4 6 8
down
in aging in aging
down
up
in OSK
in OSK
162
34
256
12 up
Sig. in aging
Sig. in aging&OSK
Sig. in aging&OSK(sensory)
log2 (+OSK/-OSK)
log2 (Old/Young)
464 genes
-OSK +OSK sh-Scr sh-Tet1 sh-Tet2
0.00
0.15
0.30
0.45
Optomotor acuity
(cyc/deg)
**
**
+OSK
-OSK +OSK sh-Scr sh-Tet1 sh-Tet2
0
2
4
6
8
Amplitude (µV)
**
*** *
+OSK
Figure 4
-OSK sh-Scr sh-Tet1 sh-Tet2
0.0
0.2
0.4
0.6
0.8
DNAme Age
+OSK
*
ns
ns
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The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a
b
c
Oct4
Sox2
Klf4
c-Myc
Nanog
0
2
4
50
100
150
200
Relative mRNA expression
**
***
**
*
*
** ns
*
**
ns
H2A
H2B
H3
H4
LaminB1
Chaf1a
Chaf1b
Apob
p16
0.0
0.4
0.8
1.2
1.6
4
6
Relative mRNA expression
2m
12m
2m, OSKM (3d)
12m, OSKM (3d)
**
*
*** ***
**** * nsns
H2A
H2B
H3
H4
LaminB1
Chaf1a
Chaf1b
0.0
0.5
1.0
1.5
2.0
Relative mRNA expression
3m
15m
3m, OSK (5d)
15m, OSK (5d)
** *** ***0.06
**
Oct4
Sox2
Klf4
c-Myc
Nanog
0
2
4
100
200
300
Relative mRNA expression
***
***
ns ns
ns
ns
**
*
***
***
Ear broblasts from
young or aged transgenic mice
P3
Dox medium
3d or 5d
Cell Harvest for
gene expression
TRE OSKM
Col1a1
rtTA
Rosa26
TRE OSK
Col1a1
rtTA
Rosa26
Extended Data Figure 1
All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
TRE Luc
rtTA
UBC
+DOX-DOX
a b
c
d
e
Ey Br Pi He Th Lu Ki
Sp
Pa Te MuAd
StInCe
SC
Ey Br Pi He Th Lu Li Ki
Sp
Pa Te MuAd
StInCe
Liver
Sox2
Actin
|-Dox |+Dox | +Dox |
Tet-OnOSK
TGAAV9
Extended Data Figure 2
591317212529333741454953
0
10
50
60
70
80
90
100
110
120
130
140
150
Body weight (%)
-DOX
+DOX
-DOX
+DOX Tet -On OS K AAV9
WT
Weeks
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c d
bTet-Onsystem
a Tet-Osystem
Klf4
Klf4
RBPMs
RBPMs
+DOX
Klf4
Klf4
RBPMs
RBPMs
+DOX
Oct4
TRE Sox2 Klf4
tTA +
Oct4
+
tTA
TRE
TRE Sox2 TRE Klf4
Sox2
Oct4
RBPMS
Klf4
Merge
Sox2Oct4
RBPMS
Klf4
Merge
Extended Data Figure 3
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
*
*
*
16 wpc (OSK)
*
*
*
b
c
a
Klf4
DAPI
Ki67
DAPI
Ki67
DAPI
OSK-infected RGCs post crush injury proliferating 293T cells
*
*
*
12 wpc (OSK) 12 wpc
Extended Data Figure 4
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Tet -OF F sys tem t urn o n
tTA
tTA
TRE d2GFP
Dox
TRE d2GFP
Dox
removal
Tet -ON s yst em tu rn on
Dox
rtTA
rtTA
Dox
addition
TRE d2GFP
TRE d2GFP
a
b
Uninfected d2EGFP OSK
0
20
40
60
80
100
RGCs survival rate (%)
****
****
cd
RBPMs
d2EGFP
On DOX NeverDOX5d DOX removal 8d DOX removal
2d on DOXNo DOX 5d on DOX
RBPMs
d2EGFP
RBPMs
d2EGFP
d2EGFP
OSK
Intact Crushed
RBPMS
d2EGFP
RBPMS
d2EGFP
RBPMS
Klf4
RBPMS
Klf4
Extended Data Figure 5
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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sh-Scr sh-Tet1 sh-Tet2
0
20
40
60
80
100
shRNA transduction rate (%)
+OSK
RBPMS
pS6
OSK d2EGFP
Intact Crushed
RBPMS
pS6
RBPMS
pS6
RBPMS
pS6
e
ba
cKlf4; sh-Tet2-YFP
Klf4; sh-Tet1-YFP
Klf4; sh-Scr-YFP
sh-Scr sh-Tet1 sh-Tet2
0
500
1000
1500
Survival of RGCs /mm2
****
****
+OSK
d
Intact Crush
0
2
4
6
8
10
pS6 +%
Control
OSK
ns
ns
****
Extended Data Figure 6
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a c
b
- 1 9 - 1 9
0
20
40
50
52
54
56
58
60
Average Methylation (%)
-OSK
+OSK
Days post VCS
Extended data Figure 7
1m
6m
12 m
18 m
GFP
GFPinjured
OSKinjured
-OSK
OSKsh-Scr
OSKsh-Tet1
OSKsh-Tet2
0
10
20
30
40
Methylation level (%)
1m 12 m
0 5 10 15 20
Chronological age (m)
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
rDNA methylation age
y=0.007x+0.18
r=0.56
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The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
d e
f
c
-OSK
Day 3 Day 7 Day 9
+OSK
h
i j
+OSK
-OSK
Klf4 Tuj 1
PI-A (x 1000)
Count
050 100 150 200
0
75
150
225
300 SH-SY5Y.fcs
Fit
Diploid G1
Diploid S
Diploid G2
SH-SY5Y (Undiff.)
PI-A (x 1000)
Count
050 100 150 200
0
75
150
225
300 tTA.fcs
Fit
Diploid G1
Diploid S
Diploid G2
-OSK
PI-A (x 1000)
Count
050 100 150 200
0
75
150
225
300 OSK.fcs
Fit
Diploid G1
Diploid S
Diploid G2
+OSK
+OSK
Klf4
+Rap
+Rap
+DMSO
+DMSO
S6
p-S6
(S240/244)
β-tubulin
-OSK
Oct4 Sox2 Klf4
0
1
2
10
20
30
Relative mRNA expression
*****
**** -OSK
+OSK
Undiff. -OSK +OSK
0
10
20
30
40
Population in S phase (%)
****
NS
****
- 1 9
0.0
0.2
0.4
0.6
0.8
1.0
DNAm Age (y)
Days post VCS
Neurons (-OSK)
linear regression (p=0.55)
*
g
DMSO Rap DMSO Rap
0
100
200
300
400
Neurite area (103m2
**** ns
-OSK +OSK
Day 3 Day 7 Day 9
0
100
200
300
400
Neurite area (103m2
-OSK
+OSK
****
k
sh-Scr sh-Tet2 sh-Scr sh-Tet2
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Relative Tet2 mRNA level
*******
***
-OSK +OSK
sh-Scr sh-Tet2 sh-Scr sh-Tet2
0
20
40
60
80
Relative Oct4 expression
****
****
-OSK +OSK
Extended Data Figure 8
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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a b
c
Saline
Beads
Saline Beads
0
10
20
30
40
50
60
Axon density (104)/mm
2
**
4wkspostmicrobeads
Saline Beads
0
500
1000
2000
2500
3000
3500
4000
RGC cell density /mm2
***
4wkspostmicrobeads Brn3a
Saline
Beads
DAPI
Saline Beads (PBS)
Beads (-OSK) Beads (+OSK)
Brn3a DAPI
Saline PBS -OSK +OSK
0
500
1000
2000
2500
3000
3500
4000
RGC cell density /mm2
Beads
*
*
**
Extended Data Figure 9
All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
c d
200 500 1000 1500
0
500
1000
1500
Distance from injury (µm)
#Axonspernerve
1m
****
****
3m
12m
****
**
****
1m
3m
12 m
***
OSK
d2EGFP
e
1m 3m 12 m
0
500
1000
1500
2000
Survival of RGCs /mm2
***********
d2EGFP
OSK
18 m
0.00
0.15
0.30
0.45 ns
Optomotor acuity
(cyc/deg)
4m 12 m 18 m
0
2
4
6
8
10
12
Amplitude (µV)
**
ns
ns
ns
** -OSK
+OSK
f
4m 12 m
0
10
20
30
35
45
55
65 ns
Axon density (104)/mm
2
ns
4m 12 m
0
1000
2000
3000
4000
ns
RGC Cell density /mm2
ns
Extended Data Figure 10
All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/710210doi: bioRxiv preprint first posted online Jul. 31, 2019;
a b
c d
−6 −4 −2 0 2 4 6
−2
−4 0 2 4 6 8
down
in aging in aging
down
up
inOSK
in OSK
up
Sig. in aging
Sig. in aging&OSK
Sig. in aging&OSK(sensory)
log2 (+OSK/-OSK)
log2 (Old/Young)
464 genes
162
34
256
12
0610040J01Rik
1810053B23Rik
2900045O20Rik
4833417C18Rik
4921522P10Rik
4930453C13Rik
4930488N15Rik
4930555B11Rik
4932442E05Rik
A430010J10Rik
Abcb5
AC166779.3
Acot10
Acot12
Adamts17
Adig
AI504432
Asb15
Gm8043AU018091
C130093G08Rik
Cand2
Cd14
Cfh
Col26a1
Cracr2b
Crls1
Cryaa
CT573017.2
Cybrd1
Cyp26a1
Cyp27a1
D930007P13Rik
Ddo
DgkgDlk2
Dlx3
Dnaja1ps
Dsel
Ecscr
Eid2
Ephx1
Eps8l1
Fam20a
Fancf
Fbxw21
Fezf1
Ffar4
Flt4
Foxp4
Fzd7
Ggt1
Gja5
Gm10416
Gm10513
Gm10635
Gm10638
Gm11368
Gm11693
Gm12793
Gm13050
Gm13066
Gm13339
Gm13346
Gm13857
Gm14387
Gm14770
Gm16072
Gm16161
Gm16181
Gm17634
Gm18025
Gm19719
Gm2093
Gm22933
Gm24000
Gm24119
Gm25394
Gm28530
Gm3081
Gm32051
Gm32352
Gm33056
Gm33172
Gm34280
Gm3551
Gm35853
Gm36298
Gm36356
Gm36660
Gm37052
Gm37535
Gm37569
Gm38058
Gm38069
Gm42303
Gm42743
Gm42895
Gm42994
Gm43151
Gm44044
Gm44081
Gm44187
Gm44280
Gm44535
Gm44545
Gm44722
Gm45516
Gm45532
Gm45644
Gm46555
Gm4742
Gm47982
Gm47989
Gm48225
Gm48314
Gm9369Gm48383 Gm48398
Gm48593
Gm48804Gm29844
Gm48958
Gm49089
Gm49331
Gm49760
Gm7669
H2al2a
Htr3a
Ido2
Igfbp1
Il1rap
Inka1
Kbtbd12
Kdelc2
Kif7
Lilra5
Lrfn2
Ltc4s
Mansc1
Mc5r
Mir344c
Myoc
Neurod6 Olfr1372ps1
Otop3
P2rx5
P2ry12
Perp
Pkp1
Qsox1
Rapgef4os2
Rcn3
Sav1
Serpinh1
Siah3
Siglech
Slc10a5
Slc2a5
Smagp
Smoc2
Sstr3
St3gal3
Stc2
Tlr8
Tmem132d
Tomm20l
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... Using a transgenic mouse system for inducible creation of DSBs, he revealed that loss of epigenetic structures, an accumulation of epigenetic noise and increased predicted DNA methylation changes increase with age and DNA damage [42,43]. Importantly, the introduction of an engineered vector expressing Yamanaka transcription factors, excluding c-Myc, regenerated axons after optic nerve crush injury and restored vision in old mice [44]. The effect was dependent on the DNA demethylases Tet1, Tet2 and TDG and was accompanied by a reversal of methylation patterns and resetting the DNA methylation clock. ...
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Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA. © 2020. Mkrtchyan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. All Rights Reserved.
... Special nuclear structures known as DNA-SCARS sustain the cell growth arrest due to DNA damage [63]. Following Sinclair and colleagues [64], these changes cause a redistribution of chromatin remodeling factors and eventually facilitate the generation of "epigenetic noise", which alters the gene expression patterns that are necessary for the optimal cell function and recovery after damage. ...
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... Остановку клеточного роста, вызванную повреждением ДНК, поддерживают особые ядерные структуры -ДНКшрамы [63]. Как предполагает Дэвид Синклер, эти изменения служат причиной перераспределения факторов реорганизации хроматина и, в конечном итоге, способствуют появлению "эпигенетического шума", который нарушает паттерны экспрессии генов, необходимых для оптимального функционирования клеток и восстановления после повреждения [64]. ...
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... It has been recently reported that an adeno-associated viral (AAV) vector expressing the OSK genes under a regulatable Tet system showed regenerative and rejuvenating effects in vivo in mouse retinal ganglion cells (RGCs) and in the human neuronal cell line SH-SY5Y [35]. The human cells were injured by exposing them to vincristine (VCS), a chemotherapeutic agent, and then transduced with th OSK AAV. ...
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