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Telomerase reverses ageing process (full text; published pdf attached)
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Dramatic rejuvenation of prematurely aged mice hints at potential therapy.
"In conclusion, this unprecedented reversal of age-related decline in the central nervous system and other organs vital to adult mammalian health justify exploration of telomere rejuvenation strategies for age-associated diseases, particularly those driven by accumulating genotoxic stress."
Nature Nov 28 2010
Ewen Callaway
Premature ageing can be reversed by reactivating an enzyme that protects the tips of chromosomes, a study in mice suggests.
Mice engineered to lack the enzyme, called telomerase, become prematurely decrepit. But they bounced back to health when the enzyme was replaced. The finding, published online today in Nature1, hints that some disorders characterized by early ageing could be treated by boosting telomerase activity.
It also offers the possibility that normal human ageing could be slowed by reawakening the enzyme in cells where it has stopped working, says Ronald DePinho, a cancer geneticist at the Dana-Farber Cancer Institute and Harvard Medical School in Boston, Massachusetts, who led the new study. "This has implications for thinking about telomerase as a serious anti-ageing intervention."
Other scientists, however, point out that mice lacking telomerase are a poor stand-in for the normal ageing process. Moreover, ramping up telomerase in humans could potentially encourage the growth of tumours.
Eternal youth
After its discovery in the 1980s, telomerase gained a reputation as a fountain of youth. Chromosomes have caps of repetitive DNA called telomeres at their ends. Every time cells divide, their telomeres shorten, which eventually prompts them to stop dividing and die. Telomerase prevents this decline in some kinds of cells, including stem cells, by lengthening telomeres, and the hope was that activating the enzyme could slow cellular ageing.
Two decades on, researchers are realizing that telomerase's role in ageing is far more nuanced than first thought. Some studies have uncovered an association between short telomeres and early death, whereas others have failed to back up this link. People with rare diseases characterized by shortened telomeres or telomerase mutations seem to age prematurely, although some tissues are more affected than others.
"They are not studying normal ageing, but ageing in mice made grossly abnormal."
David Harrison
Jackson Laboratory, Bar Harbor, Maine
When mice are engineered to lack telomerase completely, their telomeres progressively shorten over several generations. These animals age much faster than normal mice - they are barely fertile and suffer from age-related conditions such as osteoporosis, diabetes and neurodegeneration. They also die young. "If you look at all those data together, you walk away with the idea that the loss of telomerase could be a very important instigator of the ageing process," says DePinho.
To find out if these dramatic effects are reversible, DePinho's team engineered mice such that the inactivated telomerase could be switched back on by feeding the mice a chemical called 4-OHT. The researchers allowed the mice to grow to adulthood without the enzyme, then reactivated it for a month. They assessed the health of the mice another month later.
"What really caught us by surprise was the dramatic reversal of the effects we saw in these animals," says DePinho. He describes the outcome as "a near 'Ponce de Leon' effect" - a reference to the Spanish explorer Juan Ponce de Leon, who went in search of the mythical Fountain of Youth. Shrivelled testes grew back to normal and the animals regained their fertility. Other organs, such as the spleen, liver and intestines, recuperated from their degenerated state.
The one-month pulse of telomerase also reversed effects of ageing in the brain. Mice with restored telomerase activity had noticeably larger brains than animals still lacking the enzyme, and neural progenitor cells, which produce new neurons and supporting brain cells, started working again.
"It gives us a sense that there's a point of return for age-associated disorders," says DePinho. Drugs that ramp up telomerase activity are worth pursuing as a potential treatment for rare disorders characterized by premature ageing, he says, and perhaps even for more common age-related conditions.
Cancer link
The downside is that telomerase is often mutated in human cancers, and seems to help existing tumours grow faster. But DePinho argues that telomerase should prevent healthy cells from becoming cancerous in the first place by preventing DNA damage.
David Sinclair, a molecular biologist at Harvard Medical School in Boston, agrees there is evidence that activating telomerase might prevent tumours. If the treatment can be made safe, he adds, "it could lead to breakthroughs in restoring organ function in the elderly and treating a variety of diseases of aging."
Other researchers are less confident that telomerase can be safely harnessed. "Telomere rejuvenation is potentially very dangerous unless you make sure that it does not stimulate cancer," says David Harrison, who researches ageing at the Jackson Laboratory in Bar Harbor, Maine.
Harrison also questions whether mice lacking telomerase are a good model for human ageing. "They are not studying normal ageing, but ageing in mice made grossly abnormal," he says. Tom Kirkwood, who directs the Institute for Ageing and Health at Newcastle University, UK, agrees, pointing out that telomere erosion "is surely not the only, or even dominant, cause" of ageing in humans.
DePinho says he recognizes that there is more to ageing than shortened telomeres, particularly late in life, but argues that telomerase therapy could one day be combined with other therapies that target the biochemical pathways of ageing. "This may be one of several things you need to do in order to extend lifespan and extend healthy living," he says.
References
1. Jaskelioff, M. et al. Nature doi:10.1038/nature09603 (2010).
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Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice
Nature | Letter
Published online: 28 November 2010
Mariela Jaskelioff, Florian L. Muller, Ji-Hye Paik, Emily Thomas, Shan Jiang, Andrew C. Adams, Ergun Sahin, Maria Kost-Alimova, Alexei Protopopov, Juan Cadinanos, James W. Horner, Eleftheria Maratos-Flier & Ronald A. DePinhoron_depinho@dfci.harvard.edu
1Belfer Institute for Applied Cancer Science and Departments of Medical Oncology, Medicine and Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Division of Endocrinology, Diabetes & Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
DOI: doi:10.1038/nature09603
ABSTRACT:
An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo1. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses1. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2+ neural progenitors, Dcx+ newborn neurons, and Olig2+ oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons2, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk1, 3 and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.
Accelerating structural and functional decline across diverse organ systems is observed in the aged1, 3, 4. The loss of genome integrity and associated DNA damage signalling and cellular checkpoint responses are well-established intrinsic instigators that drive tissue degeneration during ageing5. Of particular relevance to this study, age-progressive loss of telomere function in mice has been shown to provoke widespread p53 activation resulting in activation of cellular checkpoints of apoptosis, impaired proliferation and senescence, compromised tissue stem cell and progenitor function, marked tissue atrophy and physiological impairment in many organ systems1, 6.
Mounting evidence in humans has also provided strong association of limiting telomeres with increased risk of age-associated disease7 and with onset of tissue atrophy and organ system failure in degenerative diseases such as ataxia-telangiectasia, Werner syndrome, dyskeratosis congenita and liver cirrhosis, among others1, 3. In cell-based models of ataxia-telangiectasia and Werner syndrome, enforced TERT can restore normal cellular proliferative potential8. These findings build on seminal cell culture studies showing that enforced TERT expression can endow primary human cells with unlimited replicative potential9. Importantly, TERT overexpression in epithelial tissues of cancer-resistant mice leads to extended median lifespan10. In addition, intercrossing wild-type and late generation mTerc-/- mice with severe degenerative phenotypes results in healthy offspring11, indicating that viable late generation mTerc-/- germ cells can be restored to normal telomere function on introduction of a wild-type mTerc allele at the time of fertilization. However, to our knowledge, there are no genetic or pharmacological studies showing somatic reversal of age-related degenerative phenotypes driven by endogenous genotoxic stresses in adult mammals. Here, in telomerase-deficient mice experiencing severe tissue degeneration, we investigated whether endogenous telomerase-mediated restoration of telomere function throughout the organism would quell DNA damage signalling and either arrest, or possibly reverse, cellular checkpoint responses and associated tissue atrophy and dysfunction. Notably, the mice enlisted into this study are adults exhibiting significant progeroid phenotypes.
Construction and functional validation of the germline TERT-ER knock-in allele are detailed in Supplementary Fig. 1. In the absence of 4-OHT, ER fusion proteins remain in an inactive misfolded state12 and thus we first sought to verify whether mice homozygous for TERT-ER recapitulated the classical premature ageing phenotypes of mice null for mTerc or mTert. To that end, mice heterozygous for TERT-ER (hereafter G0TERT-ER) were intercrossed to produce first generation mice homozygous for TERT-ER (G1TERT-ER) which were then intercrossed to produce successive G2, G3 and G4TERT-ER cohorts. G1-G4TERT-ER cells have no detectable telomerase activity (Fig. 1a). Accordingly, G4TERT-ER primary splenocytes had hallmark features of short dysfunctional telomeres, including decreased telomere-specific fluorescence in situ hybridization (FISH) signal and Robertsonian fusions (Fig. 1b, e, f). Moreover, G4TERT-ER fibroblasts failed to divide after five to six passages and adopted a flat, senescence-like morphology (Fig. 1c, d). Adult G4TERT-ER mice showed widespread tissue atrophy, particularly in highly proliferative organs including extreme testicular atrophy and reduced testes size due to apoptotic elimination of germ cells, resulting in decreased fecundity (Fig. 2a, d and Supplementary Fig. 2a), marked splenic atrophy with accompanying increased 53BP1 (also known as Trp53bp1) foci consistent with DNA damage (Fig. 2b, e, h) and intestinal crypt depletion and villus atrophy in conjunction with numerous apoptotic crypt cells and increased 53BP1 foci (Fig. 2c, f, i and Supplementary Fig. 2b). Finally, median survival of G4TERT-ER mice is significantly decreased relative to that of telomere intact mice (43.5 versus 86.8 weeks, ***P < 0.0001, Supplementary Fig. 2f). Thus, G4TERT-ER mice phenocopy late generation mTert-/- and mTerc-/- animals13, 14, indicating that TERT-ER is inactive in the absence of 4-OHT.
Next, we assessed the impact of telomerase reactivation on telomere dysfunction-induced proliferative arrest. On passage of adult G4TERT-ER fibroblast cultures, cells adopted flat senescent-like morphology at approximately five population doublings (Fig. 1d, upper panel). These quiescent cultures showed prominent G0/G1 accumulation in the cell cycle by fluorescence-activated cell sorting (FACS) analysis and rare cell division events by time-lapse video microscopy (not shown). However, upon replating these cells in media containing 100 nM 4-OHT, telomerase reactivation led to elongated telomeres, prompt resumption of proliferation over greater than eight additional passages tested, and reduction in the G0/G1 phase fraction (Fig. 1c; data not shown). Coincidently, high levels of cyclin-dependent kinase inhibitor, p21CIP1 (also known as Cdkn1a), declined upon 4-OHT treatment of the G4TERT-ER cultures, allowing cell cycle re-entry (Supplementary Fig. 2e). This pattern of p21CIP1 regulation aligns with previous work documenting its role as a key mediator of cell cycle arrest induced by telomere dysfunction in mouse tissues15. Parallel G0 or G4TERT-ER fibroblasts maintained in 4-OHT at initial isolation did not undergo passage-induced senescence and instead showed sustained proliferation (>20 passages; Fig. 1c, d).
These cell-based studies prompted systemic analyses of the impact of 4-OHT-mediated telomerase reactivation in the setting of entrenched tissue degeneration. At the end of 4 weeks of continuous 4-OHT exposure, documentation of telomerase-mediated telomere restoration and function in G4TERT-ER tissues included increased telomere-FISH signal in primary splenocytes (Fig. 1b, e, f), decreased p53 activation and expression of p21CIP1 in liver (Supplementary Fig. 2d, e), and marked decrease in 53BP1 foci in splenocytes (Fig. 2b, e) and intestinal crypt cells (Fig. 2c, f). These molecular changes paralleled striking tissue rejuvenation including reduced apoptosis of testes germ cells (data not shown) and intestinal crypt cells (Supplementary Fig. 2b, i), reduced tissue atrophy with restoration in normal testes and spleen size (Fig. 2d, h and Supplementary Fig. 2a) and, most strikingly, increased fecundity (Fig. 2g). Moreover, median survival increased in G4TERT-ER mice treated with a 4-week course of 4-OHT (**P < 0.005, Supplementary Fig. 2f). Sustained 4-OHT treatment had no effect on G0TERT-ER age- and gender-matched controls which were included in all experiments. Together, these data indicate that, despite an entrenched degenerative state, endogenous telomerase reactivation results in marked extinction of DNA damage signalling, alleviation of cellular checkpoint responses and reversal of tissue atrophy in highly proliferative organ systems of the late generation TERT-ER mice.
Although the marked impact of telomerase reactivation on highly proliferative organs is encouraging, we sought to assess more intensively the potential benefits on brain health, which is a prime determinant of age-progressive declining health in humans. Along these lines, it is worth noting that the ageing mammalian brain shows accumulating DNA damage16 and a progressive restriction of neurogenesis and impaired re-myelination due to a decline in neural stem and progenitor cell proliferation and differentiation17. As neural stem/progenitor cells (hereafter NSCs) support neurogenesis, particularly in the subventricular zone (SVZ), we first examined the properties of NSCs derived from adult G0 and G4TERT-ER mice. As reported previously for late generation mTerc-/- mice6, 14, 18, vehicle-treated G4TERT-ER NSC cultures showed decreased self-renewal activity relative to G0TERT-ER controls and this defect was partially corrected with 4-OHT treatment (Fig. 3a, d). G4TERT-ER neurospheres were not only rarer but also smaller in diameter than G0TERT-ER controls, and their average diameter was restored to normal by 4-OHT treatment (Fig. 3a and Supplementary Fig. 2c). These self-renewal profiles tracked with activated p53-mediated DNA damage signalling in vehicle-treated G4TERT-ER NSC cultures, which was extinguished with 4-OHT treatment and absent in the G0TERT-ER controls (Fig. 3b, e). Examination of NSC differentiation capacity revealed significant (twofold) reduction in G4TERT-ER NSC capacity to generate neurons relative to 4-OHT-treated G4TERT-ER cultures and 4-OHT- or vehicle-treated G0TERT-ER controls (Fig. 3c, f). Consistent with previous work14, 18, there was no impact on astrocyte differentiation (data not shown).
On the basis of these cell culture observations, we examined the SVZ, a region where NSCs reside and have an active role in adult brain physiology. In adult mice, NSCs give rise to transit-amplifying progenitor cells that divide rapidly and contribute to generation of neuroblasts, astrocytes and myelinating oligodendrocytes. Consistent with previous reports of an SVZ proliferation defect in mTerc-/- mice6, 14, 18 and wild-type aged mice19, vehicle-treated G4TERT-ER mice show a profound decrease in proliferating (Ki67+) cells in the SVZ relative to G0TERT-ER controls. Notably, 4-OHT-treated G4TERT-ER mice show a striking, albeit partial, restoration of proliferation following only 4 weeks of treatment (Fig. 4, first row). This resumed SVZ proliferation mirrors well restoration of Sox2+ cells, a marker of NSCs (Fig. 4, second row), and doublecortin (Dcx)-positive cells, an early neuronal lineage marker, together demonstrating preservation of neural stem/progenitor reserves and their neurogenic capacity in vivo (Fig. 4, third row). Finally, quantitative FISH analysis shows telomere elongation in the SVZ after 4 weeks of 4-OHT treatment (Supplementary Fig. 3). Thus, the markedly constrained neural progenitor proliferation and neurogenesis profile associated with telomere dysfunction can be ameliorated by reactivation of endogenous telomerase activity.
To test the hypothesis that telomerase reactivation leads to tissue rejuvenation, we conducted detailed morphological and functional fitness analyses of different brain structures upon telomerase reactivation. First, we examined the white matter of the corpus callosum and observed that aged G4TERT-ER mice have far fewer Olig2+ mature oligodendrocytes (Fig. 4, fourth row). This cellular deficiency is associated with reduced brain weight (Fig. 5a, b) and significantly thinner myelin sheathing of neurons with g ratios (numerical ratio between the diameter of the axon proper and the outer diameter of the myelinated fibre) of 0.7756± 0.0054 for G4TERT-ER mice versus 0.7032± 0.0049 for G0TERT-ER (mean± s.e.m., ***P < 0.0001) (Fig. 5c, d). Remarkably, endogenous telomerase reactivation reinstates normal numbers of mature oligodendrocytes (Fig. 4) and reverses the hypomyelination phenotype at the level of mean myelin sheath diameters (with g ratios of 0.7058± 0.0006 and 0.7164± 0.0063 for 4-OHT-treated G4 and G0TERT-ER mice, respectively) (Fig. 5c, d). Furthermore, a 4-OHT treatment course of only 4 weeks is sufficient to cause significant partial reversion of the brain size defect, with G4TERT-ER brain weights increasing from 77.3± 3.3% of G0TERT-ER brain weights in the vehicle group to 89.7± 4.0% in the 4-OHT group (Fig. 5a, b). Importantly, telomere elongation can be detected in the corpus callosum after 4 weeks of telomerase reactivation (Supplementary Fig. 3c). Thus, endogenous telomerase reactivation exerts a swift impact on oligodendrocyte proliferation and differentiation, and promotes repopulation of white matter structures with mature oligodendrocytes and active myelin deposition.
Lastly, we investigated the physiological effect of telomere dysfunction and telomerase reactivation on olfactory function. Age-associated hyposmia, as evidenced by an increased olfactory threshold and a reduced ability in odour identification and discrimination, is a well established phenomenon in aged humans20. In rodents, ageing is associated with diminished olfactory neurogenesis and deficits in fine olfactory discrimination19, 21. Olfactory interneurons in the olfactory bulb that receive and process information from the olfactory sensory neurons in the olfactory epithelium derive from SVZ stem cells2. Rodents demonstrate avoidance responses towards predators' odorants as well as spoiled smells like aliphatic acids, aliphatic aldehydes and alkyl amines, which are processed in the olfactory bulb22. Given the marked decrease in SVZ neurogenesis of G4TERT-ER mice and the fact that the olfactory bulb retains high telomerase activity in adult wild-type mouse brains23, we sought to determine whether telomere dysfunction results in a functional deficit of these mice to detect and process odorants for elicitation of instinctive avoidance/defensive behaviours.
Pathology within the olfactory epithelium which may be considered a basis of age-related olfactory dysfunction, was ruled out by confirmation of grossly normal histology of the olfactory epithelium in both cohorts (Supplementary Fig. 4). Next, we ruled out alterations in exploration behaviour and overall locomotion by monitoring total distance travelled by the animals in the absence of odorants, which was similar for all experimental groups (Supplementary Table 1; Fig. 5e). We then performed innate avoidance tests using serially diluted 2-methylbutyric acid (2-MB), an odorant that rouses innate aversive responses in mice. Whereas G0TERT-ER mice demonstrated avoidance responses at all 2-MB concentrations tested (1.87 x 10-4 M to 1.87 x 10-6 M), G4 mice showed attraction/neutral behaviours at concentrations lower than 1.87 x 10-4 M (Fig. 5e, f). Strikingly, following only 4 weeks of 4-OHT treatment, the performance of G4TERT-ER mice was markedly improved, with avoidance behaviours being apparent at all 2-MB concentrations (Fig. 5e, g). Accordingly, the frequency of entry into the odour zone was higher for vehicle-treated G4TERT-ER mice than for the other three experimental groups (Supplementary Table 2). These findings are consistent with significant alleviation of the olfactory defect stemming from the documented wave of telomerase-mediated SVZ neurogenesis and oligodendrocyte maturation which would promote repopulation of olfactory bulbs with functional interneurons and improve olfactory neuron function via remyelination.
Here, we report the generation of a novel mouse model to explore the impact of physiological telomerase reactivation across diverse adult cell types and organ systems. In G4TERT-ER mice with advanced degenerative phenotypes, short-term telomerase reactivation restored telomere reserves, quelled DNA damage signalling, and alleviated cellular checkpoint responses in several high-turnover organ systems with significant functional impact including increased fecundity. From this, we speculate that some tissue stem/progenitor cells are retained in a quiescent and intact state yet can be enlisted to resume normal repopulating function upon elimination of genotoxic stress at telomeres. Despite chromosomal instability, the brief course of telomerase reactivation was not sufficient to promote carcinogenesis (data not shown), a finding consistent with a role for telomerase in promoting progression of established neoplasms24. However, it remains possible that more prolonged telomerase reactivation schedules or applications in later life may provoke carcinogenesis.
As noted, age-associated compromise in mammalian brain function is associated with extensive accumulation of DNA damage and progressive reduction in neurogenesis and myelination. Indeed, many aspects of this central nervous system decline are accelerated and worsened in the setting of telomere dysfunction (refs 25, 26, this study). Our data establish that telomerase reactivation in adult mice with telomere dysfunction can restore SVZ neurogenesis and, consistent with its role in sustaining new olfactory bulb neurons, can ameliorate odour detection with improved performance in innate odour avoidance tests. These results are consistent with previous studies showing that prolonged inhibition of neurogenesis in the SVZ has a negative effect on odour detection thresholds27. In conclusion, this unprecedented reversal of age-related decline in the central nervous system and other organs vital to adult mammalian health justify exploration of telomere rejuvenation strategies for age-associated diseases, particularly those driven by accumulating genotoxic stress.
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