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NNadir

(33,368 posts)
Wed Dec 16, 2020, 11:25 PM Dec 2020

Reversing Aging: Reprogramming to recover youthful epigenetic information and restore vision

The paper I'll discuss in this post is this one: Reprogramming to recover youthful epigenetic information and restore vision (Lu, Y., Brommer, B., Tian, X., Sinclair, D. et al. Nature 588, 124–129 (2020))

Somatic DNA is the DNA which actually exists in living tissues; it need not and mostly is not, be transmitted to progeny. Many diseases, notably many cancers, are derived from somatic cell mutations.

Many people are familiar with the 4 nucleotide bases associated with DNA. During lifetime, these bases can undergo modifications that affect cell function; this paper describes aging as being a function of methylation. (The proteins which surround and wrap DNA, the histones, also have an effect on nucleic acid function. Some years ago I worked on a project to determine acetylation sites in histones to show the mechanism of certain kinds of blood cancers.)

The authors in this paper have replaced aged nucleic acids with unaffected DNA to regenerate sight in nearly blind mice.

The abstract, which is open sourced, has a lot of information.

From the introductory text:

The metaphor of the epigenetic landscape, which was first invoked to explain embryonic development8, is increasingly being seen as relevant to the later stages of life9. Evidence from yeast and mammals supports an information theory of ageing10,11, in which the loss of epigenetic information disrupts youthful gene expression patterns1,2,3, leading to cellular dysfunction and senescence12,13.

DNA methylation patterns are laid down during embryonic development to establish cell type and function. During ageing, for reasons that are currently unclear, these patterns change in ways that can be used to calculate DNA methylation age—a representation of biological age that can predict future health and lifespan4. In cell culture, the ectopic expression of the four Yamanaka transcription factors OCT4, SOX2, KLF4 and MYC (OSKM) can reprogram cultured somatic cells to become pluripotent stem cells14—a process that erases cellular identity and resets DNA methylation age4,15. In a premature-ageing mouse model of Hutchinson–Gilford progeria syndrome, cyclic transgene-mediated expression of the four genes encoding these transcription factors alleviates symptoms and extends lifespan, raising the possibility that OSKM might also counteract normal ageing16. The continuous expression of all four factors in mice, however, often induces teratomas17,18,19 or is fatal within days16, ostensibly due to tissue dysplasia18.

Ageing is generally considered to be a unidirectional process, akin to an increase in entropy. However, living systems are open, not closed, and in some cases biological age can be fully reset, for example in ‘immortal’ jellyfish and through the cloning of animals by nuclear transfer. Having previously found evidence for epigenetic noise as an underlying cause of ageing3,13, we wondered whether mammalian cells might retain a faithful copy of epigenetic information from earlier in life that could serve as instructions to reverse ageing10...


A virus which is frequently utilized to exchange DNA is the AAV (adeno-associated virus) virus. The viral DNA is stripped out of the virus and the desired DNA is placed into it, and the viral machinery is utilized to inject the "new" DNA into the cell.

Among the 4 Yamanaka transcription factors, MYC, and oncogene, was deleted in the insertion into cells, and thus OSKM becomes OSK, three transcription factors.

Pictures and captions from the text show what's going on:

Fig. 1: AAV2-delivered polycistronic OSK promotes axon regeneration and RGC survival after optic nerve injury.



The caption:

a, Schematic of the Tet-On and Tet-Off dual AAV vectors. OSK denotes Oct4-P2A-Sox2-T2A-Klf4. b, Schematic (top) and representative retinal wholemount (bottom left) and cross-sections (bottom right) (n = 10), two weeks after intravitreal injection of AAV2-tTA;TRE-OSK, showing Klf4 (green) expression in the RGCs (blue, visualized by RBPMS as marker). Scale bars, 1 mm (left); 100 ?m (right). c, Experimental outline of the optic-nerve crush-injury study using the Tet-Off system. 555-CTB was used for anterograde axonal tracing. d, The number of axons 16 days after crush injury, at distances distal to the lesion, in mice treated with AAV2 encoding: destabilized enhanced green fluorescent protein (d2EGFP); Oct4; Sox2; Klf4; Oct4 and Sox2; all three OSK genes on three monocistronic AAV2s (indicated as separated by + signs); or all three OSK genes on a single polycistronic AAV2 (n = 5, 4, 4, 4, 4, 4 and 7 eyes, respectively). In d, e, suppression of polycistronic OSK expression by DOX (n = 5 eyes) is shown as OSK off. e, Representative images of longitudinal sections through the optic nerves of 4-week-old (young) mice after receiving intravitreal injections of AAV2-tTA;TRE-OSK in the presence (n = 5) or absence (n = 7) of DOX. CTB-labelled axons are shown 16 days post-crush. Asterisks indicate the optic-nerve crush site. f, Representative confocal images of longitudinal sections through the optic nerve showing CTB-labelled axons in 12-month-old (old) mice, 5 weeks post-crush, with AAV2-mediated expression of either GFP or OSK. Representative results from n = 5 eyes. Scale bars (e, f), 200 ?m. g, The number of axons 16 days post-crush at multiple distances distal to the lesion site in transgenic Cre lines that selectively express OSK either in RGCs (Vglut2-Cre) or in amacrine cells (Vgat-Cre) after intravitreal injection of AAV2-FLEx-tTA;TRE-OSK (n = 4 eyes for each condition). Further details are given in Extended Data Fig. 4e–h. One-way ANOVA with Bonferroni correction (d, g), with comparisons to d2EGFP shown in f. All data are presented as mean ± s.e.m.


Some more text from the paper:

Almost all species experience a decline in regenerative potential during ageing. In mammals, one of the first systems to lose regenerative potential is the CNS5,6,7. Retinal ganglion cells (RGCs) of the CNS project axons away from the retina to form the optic nerve. If damaged, RGCs of embryos and neonates can regenerate axons, but this capacity is lost within days after birth, probably due to a molecular program that is intrinsic to the RGCs6,22. Attempts to reverse optic-nerve damage have been only moderately successful, and there are currently no treatments that can restore eyesight either in late-stage glaucoma or in old age23.


The authors utilized their mouse model to see if they could reverse this aging process. How they did it is suggested in this figure and its caption:

Fig. 2: DNA demethylation is required for OSK-induced axon regeneration after injury.



The caption:

a, Strategies for induction of OSK pre- and post-injury. b, Survival of RGCs (number of RGCs per mm2) at 2 and 4 weeks post-crush (wpc) in response to OSK induction either pre- or post-injury (inj.) (n = 4 eyes, except for the post-injury ‘on’ group at 2 weeks post-crush, for which n = 7 eyes). c, Representative longitudinal sections through the optic nerve showing CTB-labelled axons at 4 weeks post-crush, without (top) or with (bottom) the induction of OSK after injury. Asterisks indicate the optic-nerve crush site. Results are representative from n = 4 eyes. Scale bars, 200 ?m. d, Ribosomal DNA methylation age (in months) of 6-week-old RGCs isolated from axon-intact retinas infected with or without GFP-expressing AAV2 (intact), or from axon-injured retinas infected with GFP- or OSK-expressing AAV2 at 4 days post-crush (injured) (n = 6, 4, 8 and 8 eyes, respectively). DNA methylation age estimates of neurons tend to be lower than their chronological ages but remain correlated (see Extended Data Fig. 5g and Methods). e, Hierarchical clustered heat map of DNA methylation levels of CpGs that significantly changed in RGCs after crush injury (intact GFP vs injured GFP, q < 0.05) and the effect of OSK at 4 days post-crush (injured OSK). f, Axon regeneration in response to OSK expression (AAV2-tTA; TRE-OSK), 16 days post-crush in Tet2flox/flox mice infected with saline (Tet2 wild type (WT); n = 3 for each condition) or AAV2-Cre (Tet2 cKO; n = 4 for each condition). One-way ANOVA with Bonferroni’s multiple comparison test (b, d, f). For all bar graphs, data are mean ± s.e.m.


In order to restore function, the demethylation of DNA is necessary, but not sufficient to restore the function of the axons concerned with vision, as gene knockdown work showed. The OSK transcription factors are also necessary.

Vision can be restored in mice in which glaucoma damage has been induced:

Fig. 3: Four weeks of OSK expression reverses vision loss after glaucomatous damage has already occurred.



The caption:

a, Experimental outline of the induced glaucoma studies using 8-week-old mice. b, Intraocular pressure (IOP) measured weekly by rebound tonometry for the first 4 weeks after the injection of microbeads (n = 29 mice) or saline (n = 10 mice). c, High-contrast visual stimulation to measure optomotor response. Reflexive head movements were tracked in response to the rotation of a moving striped pattern that increased in spatial frequency. d, Optomotor response of each mouse before treatment (baseline) and 4 weeks after intravitreal injection of AAVs (treated; see f for key) (n = 10, 7, 12 and 12 mice for each condition; similar results from 3 independent experiments combined). e, Representative PERG waveforms in response to a reversing contrast checkerboard pattern, recorded from the same eye both at the pre-injection baseline and at 4 weeks after ?OSK (top) or +OSK (bottom) AAV injection. f, Mean PERG amplitudes of recordings from each mouse in d at the baseline before treatment and at 4 weeks after intravitreal AAV injection (n = 10, 7, 12 and 12 mice for each condition; similar results from 3 independent experiments combined). ?OSK (not induced), AAV2-rtTA;TRE-OSK; +OSK (induced), AAV2-tTA;TRE-OSK. For b, a two-way ANOVA with Bonferroni correction between groups was used; for d, f, a two-way mixed ANOVA with Bonferroni correction between groups was used for the overall effect of time and treatment, and before and after treatments were analysed using a paired two-tailed Student’s t-test. Data in b, d and f are presented as mean ± s.e.m.


Fig. 4: Restoration of youthful vision, transcriptome and DNA methylation ageing signature in old mice.



The caption:

a, Experimental outline for testing the effect of reprogramming in old mice. b, Visual acuity in young (4-month-old) and old (12-month-old) mice after 4 weeks of ?OSK or +OSK treatment (n = 16, 20, 14 and 12 eyes, respectively; similar results from 2 independent experiments combined). c, d, Hierarchical clustered heat map (c) and scatter plot (d) showing mRNA levels of 464 genes that were differentially expressed between young and old RGCs and the effect of OSK. RGCs were sorted from the retinas of untreated young (5-month-old, n = 5), old (12-month-old, n = 6) or treated old (12-month-old; ?OSK, n = 5; +OSK, n = 4) mice. The gene selection criteria for c, d are described in Methods. e, DNA methylation ageing signatures of RGCs from 12-month-old mice infected for 4 weeks with ?OSK or +OSK (n = 4 and 3 retinas). f, Visual acuity in old (12-month-old) mice treated for 4 weeks with ?OSK, +OSK, or +OSK together with short hairpin scramble RNA (shScr), shTet1 or shTet2 (n = 8, 7, 5, 6 and 6 eyes, respectively). ?OSK: AAV2-rtTA;TRE-OSK (b); AAV2-TRE-OSK (c, d); AAV2-tTA;TRE-d2EGFP (e); +OSK: AAV2-tTA;TRE-OSK (b–f). Two-way ANOVA with Bonferroni correction (b); Kruskal–Wallis test (e); one-way ANOVA with Bonferroni correction, with comparisons to the ?OSK group (f). For all bar graphs, data are mean ± s.e.m.


The basis experiments were, for information, repeated in cultured human neuronal cells.

Paralleling the mouse RGC data, the expression of OSK in differentiated human neurons effectively counteracted axonal loss and the advancement of DNA methylation age induced by vincristine—a chemotherapeutic drug that induces axon injury—in the absence of cell proliferation or global DNA demethylation (Extended Data Fig. 8a–g). Nine days after damage, the neurite area was 15-fold greater in the rejuvenated OSK-transduced cells compared with control cells (Extended Data Fig. 8h, i).


Some concluding remarks from the discussion:

Here we show that it is possible to safely reverse the age of a complex tissue and restore its biological function in vivo. Using the eye as a model system, we present evidence that the ectopic expression of OSK transcription factors safely induces in vivo epigenetic restoration of aged CNS neurons, without causing a loss of cell identity or pluripotency. Instead, OSK promotes a youthful epigenetic signature and gene-expression pattern that causes the neurons to function as though they were young again. The requirement for active demethylation in this process supports the idea that changes in DNA methylation patterns are involved in the ageing process and its functional reversal (Extended Data Fig. 10n). However, we do not wish to imply that DNA methylation is the only epigenetic mark involved in this process...

...Perhaps the most interesting question raised by these data is how cells encode and store youthful epigenetic information. Possibilities for information storage include covalent DNA modifications, DNA-binding proteins, RNA-guided chromatin modifying factors, and RNA–DNA hybrids that are established early in life. The role of these youth marks would be akin to the ‘observer’ in information theory, which preserves an original backup copy of information in case it is lost or obscured by noise11. We suggest that epigenetic reprogramming, either by gene therapy or other means, could promote tissue repair and the reversal of age-related decline in humans.


Cool, I think.

Have a nice day tomorrow. For those of us in the US Northeast, be safe in the storm. If possible work from home or stay at home.


9 replies = new reply since forum marked as read
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Reversing Aging: Reprogramming to recover youthful epigenetic information and restore vision (Original Post) NNadir Dec 2020 OP
What I wouldn't give to have young eyes again! MLAA Dec 2020 #1
This is an interesting paper. Some rare kinds of blindness have successfully been treated... NNadir Dec 2020 #2
Would sure be great if my vision could be restored as in my youth captain queeg Dec 2020 #3
KNR and bookmarking. niyad Dec 2020 #4
,could this technology repair a pancreas? I_UndergroundPanther Dec 2020 #5
It may not be exactly the same type of approach but yes, there's a lot of gene therapy work on... NNadir Dec 2020 #9
This message was self-deleted by its author CatLady78 Dec 2020 #6
Less dense article here... masmdu Dec 2020 #7
This message was self-deleted by its author CatLady78 Dec 2020 #8

NNadir

(33,368 posts)
2. This is an interesting paper. Some rare kinds of blindness have successfully been treated...
Wed Dec 16, 2020, 11:47 PM
Dec 2020

...with gene therapy.

This work refers to a specific type of eye malfunction, that associated with glaucoma, but the implications of this work seem to me to be much broader.

It could become a real treatment, I would imagine an expensive treatment, but a real one.

captain queeg

(10,036 posts)
3. Would sure be great if my vision could be restored as in my youth
Wed Dec 16, 2020, 11:54 PM
Dec 2020

Really, my vision was great well into my 40s. later 50s I started using reading glasses sometimes. Now I can't read without them. Distance is still pretty good.

I_UndergroundPanther

(12,452 posts)
5. ,could this technology repair a pancreas?
Thu Dec 17, 2020, 12:30 AM
Dec 2020

To actually regrow the inslet cells? Or turn the clock back to when you had a functioning pancreas?

NNadir

(33,368 posts)
9. It may not be exactly the same type of approach but yes, there's a lot of gene therapy work on...
Thu Dec 17, 2020, 08:20 AM
Dec 2020

...diabetes. Here's a relatively recent review of what's being done:

Type 1 and 2 diabetes mellitus: A review on current treatment approach and gene therapy as potential intervention (Tan et al., Diabetes & Metabolic Syndrome: Clinical Research & Reviews Volume 13, Issue 1, January–February 2019, Pages 364-372)

It is well understood that diabetes has a genetic component.

We are in the early stages of gene therapy. A "one shot" genetic therapy has been approved for curing a certain rare form of blindness.

The economics of one shot medications depends on the prevalence of the disease. As diabetes is common throughout the world, assuming that a "one size fits all" gene modification works, and no designer genetic manipulation is necessary, it might actually fall into the category of "expensive but often affordable."

I have not personally studied gene therapy approaches to diabetes, but it seems that a great deal has been written about it.

Response to NNadir (Original post)

Response to masmdu (Reply #7)

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