Tale of Telomerase: Lessons and Limits in a Late-Life Launch
Recent studies show the potential -- and the limits -- of inducing telomerase expression in aging mice. We place these results in context and explore their implications for new treatments targeting age-related degeneration in humans.
The connection between telomeres, telomerase, and cellular and organismal "aging" was a matter of significant scientific interest but little public awareness until the early 1990s, when Dr. Michael West founded Geron Corporation. In the process of launching that venture, and in the following years, West succeeded in embedding a controversial thesis deeply into the public imagination: that the (re)activation of telomerase in somatic cells could retard or even reverse the degenerative aging process. There were always problems with this thesis, and with public (mis)understandings of it, but its sheer simplicity and public prominence has in direct and indirect ways advanced scientific research that has answered many of the questions that thesis forced upon the scientific community, and opened up important new avenues for research in telomre biology and in biomedical gerontology.
The most direct and important fruits of that expansion of research into telomerase have been studies on the pharmacological and transgenic activation of telomerase in the tissues of aging mice. Several such reports have appeared over the years, each hailed prematurely as evidence of the life- and health-extending power of the enzyme. The most important of these studies have been a series of experiments by María Blasco, PhD, SENS Foundation Research Advisory Board member and Director of the Molecular Oncology Programme at Spain's National Cancer Research Centre (CNIO). A tantalizing new report in this series has just appeared(9) -- but to understand it in context, we will first review those that led up to it.
"Healthy" Animals Riddled with Cancer
In the first of these studies,(1,2) Dr. Blasco's group targeted transgenic overexpression of the catalytic subunit of murine telomerase (mTERT) to the stem and proliferative cell pools of a wide range of epithelial tissues, by placing their construct under the control of the bovine keratin 5 promoter. Despite the fact that these "K5-mTERT" transgenic mice had mean telomere lengths similar to those of wild-type mice, the transgenic mTERT caused a more vigorous epidermal tissue proliferative response -- resulting in "Antagonistic effects of telomerase on cancer and aging". On the one hand, K5-mTERT mice had improved wound healing and reduced rates of inflammatory and degenerative renal lesions and testicular atrophy. On the other hand, this more ready tissue renewal came coupled with "a higher incidence of both induced and spontaneous tumors... [and] some hyperproliferative lesions, such as mucosal hyperplasia of the stomach or the intestine, as well as acinar hyperplasia in the mammary glands... These results suggested that TERT overexpression facilitates cell proliferation and cooperates with other mutations in tumor development in the absence of significant changes in telomere length."(3)
Similar findings were independently reported in another model of consitutive mTERT expression across multiple tissues, which led to a substantial increase in precancerous mammary lesions and invasive mammary carcinomas in female mice;(5) and again on a more tissue-specific in their own later report that constitutive trangenic expression of TERT in thymocytes and peripheral T-cells led to higher incidence and wider tissue distribution of T-cell lymphoma in mice.(6)
Some researchers might have ended their work with these animals on this unpromising note. Fortunately, Dr. Blasco was able to continue following these animals to the end of their lives.
During their first year of life, the higher incidence of cancer and other diseases of excessive cellular proliferation predictably caused mTERT-transgenic mice to suffer substantially higher mortality than did their wild-type controls. Yet as the surviving transgenic and control animals entered into their second year alive, the survival curves that had previously been progressively diverging began to converge once again. And by the end of their lives in the laboratory, the authors reported that in spite of their high rates of early cancer and premature mortality, "K5-mTERT mice show an extension of the maximum lifespan from 1.5 to 3 months, depending on the transgenic line, which represents up to a 10% increase in the mean lifespan compared to wild-type littermates."(3)
In fact, the reported increase in lifespan in treated mice was only internally valid: while longer-lived than their own founder strain in the CNIO lab, the transgenic mice's lifespans were well within the historical norm of the background strain, making the apparent extension of life uncertain -- a problem which plagues many studies of potential interventions in aging, whether genetic(13,17) or pharmacological.(13) Still, even a normal lifespan was somewhat surprising (and to some, provocative), granted these animals' multiple proliferative lesions and subtantial early mortality -- particularly in light of the importance of cancer in limiting survival in even wild-type mice.
You Break It, You Fix It
To overcome the increased risk of cancer in mTERT-transgenic animals, Blasco's group crossed their K5-mTERT mice with animals harboring transgenic "overdoses" tumor-suppressor genes (p53, alone and in combination with p16, and p19ARF), thereby combining the improved tissue renewal of the K5-mTERT mice with the robust cancer defense of highly vigilant growth-arrest machinery.(4) Mice whose senescence machinery had been thus supplemented had previously been shown to suffer far less cancer than their wild-type cousins, but to live no longer because of a range of diseases of failed tissue renewal. Combining strong cancer resistance with aggressive activation of proliferation in the remaining cells seemed a way to have the best of both worlds.
In some ways, it did. Relative to their enhanced 53- and p53/p16/p19ARF controls, the animals with added K5-mTERT transgenes "showed a better preservation of both the thickness of the epidermis and of the subcutaneous fat layer" and "a better preservation of the GI tract epithelia" with age, along with greater resistance to dextran sodium sulfate-induced intestinal ulcers, and delayed "onset of a large variety of age-related pathologies, mostly atrophies and inflammatory processes".(4) Additionally, these animals exhibited functional improvements in neuromuscular coordination, based on superior tightrope test performance and glucose tolerance as measured in young adulthood. And the strong cancer resistance of Sp53 and Sp53/TgTert mice was preserved when transgenic mTERT was added to their genetic toolkit.
But surprisingly, it is not at all clear that these cancer-resistant, tissue-renewal-enhanced animals were ultimately healthier than unmodified laboratory mice. True, the crossbreeds exhibited a greater median lifespan relative to the experiment's internal controls -- but they still failed to live any longer than historical controls of the C57BL6-DBA/2 background strain. And the effect on maximum lifespan was not even decisive in comparison to internal control mice, let alone in comparison to historical controls.(4)
Never Too Late -- and Maybe Too Early?
Then in 2011, long-time telomerase researcher Ronald DePinho (then at the Dana-Farber Cancer Institute) and colleagues published a report suggesting the possibility that telomerase induction could not only delay age-related phenotypes similar to those that had been postponed by mTERT overexpression in these studies, but could potentially reverse some of them if the onset of treatment postdated the appearance of degenerative changes. To test the effects of telomerase induction in an animal model with substantial telomere erosion and tissue denudation, DePinho's group generated TERT knock-in mice, in whom the native TERT was replaced by an inducible gene construct that would remain transcriptionally inactive absent the necessary pharmacological activator. Similar to mice with disabling mutations in mTERT or mTERC (the RNA template subunit of the telomerase holoenzyme), these animals developed an increasingly-severe "premature aging" phenotype with each generation of breeding, as the telomeres of the germ cells are progressively eroded rather than being preserved in the gonads and lengthened upon fertilization. These animals thus exhibited progeroid phenotypes including testicular and splenic atrophy, intestinal crypt cell apotosis and depopulation, widespread DNA damage response signaling, and a severely shortened median lifespan.(7)
As DePinho's group reported to widespread attention, activation of these animals' previously-untranscribable mTERT reversed numerous aspects of their degenerative phenotype. Testicular and splenic volumes were replenished; expression of senescence-associated genes was reduced; fecundity was increased; germ line and intestinal crypt cell apoptosis was became less frequent; and subventricular zone neurogenic proliferation increased, restoring in the process the animals' imipaired olfactory function. And similar to the "natural" course of the survival Blasco's K5-mTERT mice, reactivation of their decommissioned TERT quckly decelerated the downward trajectory of their survival curves.(7) However artificial their "aging," their apparent "rejuvenation" was striking and clearly caught the public imagination.
In the same year, Blasco's group also reported results suggesting the potential of late-life telomerase induction to ameliorate the degenerative phenotype of aging, even in wild-type mice. Pharmacological activation of mTERT expression in several tissues in 1- and 2-year-old mice led to a reduction in the numbers of critially-short telomeres in peripheral blood leukocytes, and a decelerated course of several aging phenotypes. Treated animals had reduced hepatic steatosis, and less age-related loss of bone mineral density and subcutaneous fat, along with a better-preserved capacity for wound healing and hair regrowth. While the incidence of lymphomas and hepatomas were nominally elevated in treated mice, no statistically-significant effects on tumorigenesis were observed in individual organs or in the organisms as a whole relative to untreated animals. And there was no deleterious effect on lifespan.(8)
Combined, these results suggested an intriguing possibility. Up until DePinho's report(7), all of Blasco's transgenic mTERT studies, and a similar report by others,(5) had been carried out in animals whose overexpression of mTERT had begun during embryonic development and continued throughout the lifespan. Most of excess of proliferative pathology and mortality in these animals had manifested in the early and middle years of the rodents' lifespans, during and shortly after the mitotic explosion of embryonic and postnatal development. By contrast, the benefits of mTERT overexpression on health and mortality were most evident later in life, long after the achievement of mature body size, when mitotic activity had slowed.(3) And while DePinho's study involved the correction of an artificial "aging" phenotype that was imposed on the mice by the experimenters themselves rather than the intrinsic metabolic forces of "normal" aging, the observed "rejuvenation" effects in his study(7) and inBlasco's own telomerase induction study(8) clearly supported the contention that telomerase could confer benefits to stem cell function long after telomere erosion had already begun to exact a heavy burden on tissue function. And neither study reported a significant concomitant increase in risk of malignant disease.
All of this posed a provocative question. Could it be that much of the carcinogenic risk associated with telomerase induction across the lifespan came from accelerating the intense mitotic pressure (and therefore, mutational risk) of the first year of life? And if so, could much of the risk associated with the therapy be avoided by delaying mTERT expression until middle age and beyond, when embryonic and early postnatal development are already completed, and "normally"-aging tissues are beginning to suffer denudation from stem and progenitor cell pool senescence?
The Danger On The Rocks Is Surely Past(?)
In a new report, Blasco and colleagues have now reported the effects of somatic mTERT gene therapy initiated in early middle age (1 y) and late adulthood (2 y) in wild-type mice.(9) Using an adenoviral vector of a serotype (AAV9) with multi-tissue tropism, mice were transduced with an mTERT gene therapy construct (AAV9-mTERT mice) that increased protein levels in multiple tissues, achieving fold increases in some cases equivalent to those previously reported(4) in embryonic transgenic animals.
Similarly to some previous reports, animals undergoing aging after receiving AAV9-mTERT were characterized by better-maintained subcutaneous fat layers, lower insulin levels, and a reduction in bone mineral density loss as compared with untreated animals.(9) More surprisingly, AAV9-mTERT mice showed signs of reduced deterioration of primarily postmitotic tissues such as muscle and nerve. Thus, animals with telomerase therapy introduced at the 2 y mark suffered less age-related impairment of balance and coordination on the rotarod test in the months after treatment. Similarly, the substantial loss of neuromuscular function that occurred in untreated animals between the first and second year of life mice -- as exhibited in declining performance on a tightrope test -- was abolished in animals treated at 1 y and evaluated 11 mo later. And there was also a trend toward improved memory on the object recognition test.(9) While small and not statistically significant, this trend may yet suggest that a real potential for telomerase therapy for memory function exists, because of the relatively modest level of mTERT gene transfer to the brain.
That overexpression of mTERT would decelerate the age-related decline in the function of postmitotic tissues is not immediately intuitive, but it is consistent with superior maintenance of the replicative capacity of tese tissues' stem and progenitor cell pools, and also with the possible roles of the catalytic subunit of telomerase in mitochondrial structure and function.(10-12) Of note, mice treated with mTERT gene therapy at 2 y exhibited reduced age-related decline in expression of genes involved in mitochondrial and metabolic function, such as PGC-1α, ATP synthase, and estrogen-related receptor-α.(9) This potential support for the existence such non-canonical roles for TERT should stimulate further investigation into this emerging area of research.
Bringing together the global effects of AAV-mTERT on the organisms' health and vitality, there was an internally significant, although ultimately inconclusive, suggestion of an increase in lifespan in treated mice:
Figure: Survivorship in Late-Life Somatic mTERC Gene Therapy Mice. Comparisons are to vector bearing green fluorescent protein alone (AAV9-eGFP) or to untreated wild-type control animals. Reproduced from (9).
Notably, these (relative) increases in life expectancy appeared to correlate with at least one measure of telomerase activity. A subgroup analysis suggested that those animals in whom the percentage of peripheral blood lymphocytes with short (<50% of mean) telomere lengths was either maintained or reduced over the course of therapy, may have survivee longer than those animals in whom this percentage continued to increase with age. Also, an additional experimental group was treated with somatic gene therapy with a catalytically-inactive mutant mTERT; these animals lived no longer than internal controls, and while only a subset of the full panel of tissues were evaluated, these animals did not exhibit the superior maintenance of tissue structure and function that acrued to animals treated with the active subunit.(9)
Limitations: Experimental and Therapeutic
These results of this new study are exciting, but several caveats must be noted about the results themselves, and their implications for medical therapies against the degenerative aging process in humans. As in the previous studies, the apparent increases in survival in this new report were, in fact, ambiguous. The study was substantially underpowered to detect a true increase in maximal lifespan; and even taking the results at face value, the reported survival data -- even for treated animals -- were, once again, well within the range typical for well-husbanded, untreated control mice reported in other studies. As others have emphasized in similar cases,(13,17) whether the same treatment would extend life in animals who would otherwise had led normally-longevious lives cannot be determined from such a result.
Additionally, there was relatively little effort invested in ruling out a possible effect of Calorie restriction (CR) in this study. A reduction in energy intake, over and above that required to avoid obesity, and in combination with adequate nutrition, remains the most robust non-germline intervention to increase health and longevity in mammals, and it is therefore important to rule out any therapy-associated CR in testing interventions aimed at intervention in the degenerative aging process. The authors of the current study did report that they "did not find significant differences in body fat content or in body weight of the different mouse cohorts,"(9) but this was apparently based on a single measurement of these parameters before and sometime after treatment. Such measurements are far from sufficient to rule out CR in murine lifespan study. As exhaustively documented in a widely-overlooked review of the history of such studies by the highly experienced and painstaking Dr. Stephen Spindler, seemingly minor and temporary changes in energy intake can exert significant effects on health and longevity outcomes. We highlighted this invaluable paper in a previous post because demonstrates how critical it is to the design of robust murine lifespan studies to incorporate robust and regular measures of food intake. The lack of such measures leaves the proper interpretation of nearly all reports of decelerated degenerative aging and expansion of the bounds of the lifespan in doubt -- including this one.
Presuming, however, that the life- and healthspan benefits reported in this study should be taken at face value, the ultimate question is their human translatability -- and there are reasons to be skeptical that a similar therapy could be safely used to retard the degenerative aging process in humans. Some would note first that safe and effective gene therapy is not yet available for our species -- but that, like many other matters in biomedical gerontology, is only a matter of time and investment. Of greater concern is the safety of telomerase therapy, granted the very different body plans of humans as compared to mice. Even untreated, wild-type mice express telomerase in many tissues, and in these tissues the enzyme is under a quite relaxed regulatory regime. Moreover, the C57Bl/6 background strain used in this report(9) has exceptionally long telomeres, even for a laboratory mouse.
Yet despite their long telomeres at birth and relatively promiscuous telomerase activity, laboratory mice still undergo net telomere erosion and widespread cellular senescence with aging, showing that they have limits to replicative capacity that additional, therapeutic increases in telomerase expression may fail to overcome -- limitations that do not exist in our own species. Indeed, while primary mouse embryonic fibroblasts derived from TERT-transgenic mice still undergo replicative senescence in culture,(5), stable telomerase expression in transfected human cells confers indefinite replicative potential.(18)
Under a different set of selective pressures, Homo sapiens have evolved extremely tight control over telomerase, principally because of the greater risk of cancer intrinsic to a body composed of a higher number of cells. Development and maintenance of a larger body necessitates additional rounds of cell division, each of which entails DNA replication and the attendant risk of mutation. Moreover, even if each such cell is initially generated with perfect genetic fidelity, every additional cell in the body constitutes an additional node of risk of later mutation, and the progression of such a mutation-bearing cell into the nidus of neoplastic disease requires the acquisition of a telomere-lengthening mechanism. At minimum, long-term overexpression of telomerase provided by somatic gene therapy provides additional time for a replicating mutant cell to acquire such a mechanism on its own, and may also provide a gene that is itself available for exploitation.
While telomerase overexpression initiated late in life may not have significantly increased the burden of cancer in Blasco's AAV-mTERT mice, there were worrisome suggestions in several organs. Similarly, even transient induction of telomerase in DePinho's late-generation, progeroid mice caused an increase in chromosomal instability, albeit without any observed increase in carcinogenesis.(7) With bodies composed of far more cells, and with a far greater post-treatment life expectancy, a small increase in chromal instability or authentic tendency toward malignant transformation that occurrs in the cells of telomerase-treated rodents would be expected to pose a greater risk of progressing into life-threatening disease in aging, TERT-treated humans.
But supposing that Blasco's latest observations in late-life telomerase (9) could be directly and safely translated into a therapy for aging humans -- what then? Despite the apparent "rejuvenation" of the structure and function of some tissues reported by DePinho in late-generation telomerase-knockout mice upon telomerase induction,(8) extending flagging telomeres across a wide range of tissues in this study of wild-type mice undergoing "normal" degenerative aging did not reverese aging phenotypes, but was only able to decelerate the age-related slide into frailty and dysfunction, even in the subset of tissues with strong AAV9-mTERT expression. By contrast, the rejuvenation biotechnologies whose development SENS Foundation advocates and works to catalyze with key research investments will remove or repair the cellular and molecular damage that underlies age-related pathology, leading to the arrest and regression of degenerative aging changes.
And dependence on telomerase expression to maintain aging cells in better health would place place recipients of telomerase therapy into a terrible dilemma. Irrespective of what transgenic telomerase therapy would in itself do to recipients' risk of cancer, it is incompatible with the sole foreseeable means of putting an irreversible end to cancer as a cause of age-related death and suffering. As explained in detail elsewhere,(14-16) the relentless evolutionary ingenuity of cancer as a disease leaves each of us at perpetual risk of cancer -- and will leave us at progressively greater risk of cancer as a cause of death as other drivers of age-related mortality are eliminated by emerging rejuvenation biotechnologies -- until all telomere-lengthening machinery is ablated from human cells. Any therapy that relies upon, or introduces, telomerase activity in human cells in situ is therefore incompatible with the goal of building an impregnable wall against neoplastic disease, whatever the merits of telomerase therapy as a standalone intervention.
Building on a decade of prior work, each study following logically from the last, this new report from Blasco's group at CNIO reveals telomerase therapy as a surprisingly wide-ranging intervention against the age-related slide into frailty and, perhaps, mortality. Much has been learned in the process, even if it is never turned into a human therapy. And more broadly, it demonstrates once again -- as with other legacy "anti-aging" therapies based on modulation of metabolic pathways -- that this degeneration is a modifiable target of biomedical intervention. Our bodies become plagued with ill health with age because of the progressive accumulation of cellular and molecular damage in our tissues. Retard that damage's accumulation (as in this study), and the body's slide into disability, dysfunction, and disease is retarded. Repair the damage of aging directly -- restore the structural integrity of aging cells and tissues to the molecular fidelity of youth -- and the health and vigor that characterizes youth will be restored, and maintained into an indefinite tomorrow.
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