TELOMERASE REACTIVATION REVERSES TISSUE DEGENERATION IN AGED
The enzyme telomerase allows for replacement of short bits of DNA sequences known as telomeres, which are otherwise shortened when a cell divides via mitosis. In telomerase deficient cells such as somatic cells, loss of telomere function provoke p53 (tumor suppressor protein) activation which triggers the activation of cellular checkpoints of apoptosis. When mice are engineered to lack telomerase completely, their telomeres progressively shorten over several generations. These animals were found to age much faster than normal mice, barely fertile and suffer from age-related conditions such as neurodegeneration and reduced sense of smell. They also die young. To find out if these dramatic effects are reversible, Mariela’s team engineered mice such that the inactivated telomerase could be switched back on by feeding the mice a chemical called 4-hydroxytamoxifen. The researchers allowed the mice to grow to adulthood without the enzyme, and then reactivated it for 4 weeks. Mice homozygote for the telomerase dysfunction gene sustained increase DNA damage signaling and proliferative tissue degenerative phenotypes upon successive generational mating and advancing age. Of importance is the reversed neurogeneration upon telomere reactivation because brain cells (neurons) are naturally non-renewable and thus good indicators of aging.
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Tissue degeneration and premature ageing can be reversed by reactivating an enzyme called telomerase that protects the tips of chromosomes. At the 3’ prime end of DNA are sequences called Telomere that help protect the DNA and shortens as the cell divides in somatic cells. The telomere becomes reduced to a critical length where the cell then undergoes apoptosis. However in cancer or stem stems, the length of the telomere is regenerated by the enzyme telomerase.
Normally the way that telomerase is activated in cells is by means of an "estrogen receptor" (ER), to which a form of the hormone estrogen (17β-estradiol to be precise) can bind and enable transcription of Telomerase reverse transcriptase gene. However, the Estrogen receptors protein can be modified such that it is dysfunctional in the absence of 4-hydroxytamoxifen. A special form of the Tert gene that includes this 4-OHT induced ER can be "knocked-in" to the mouse germ line. In the Knock-In procedure, TERT gene was genetically modified such that its protein product itself is conjugated to the estrogen receptor protein, to make Tert-ER. This engineered protein (TERT/estrogen receptor fusion) is dysfunctional (mis-folded) in the absence of 4-OHT and refolds only when it is bound to 4-OHT. Thus this genetically engineered Tert-ER gene was inserted into an embryonic cell which is then implanted into a surrogate mother. The resulting mice were mated with normal mice and then intercrossed to obtain a homozygote healthy but telomerase dysfunction mice. 4-OHT was effectively supplied to the TERT-ER mouse (in the form of a time-release subcutaneous pellet) to turn telomerase expression on and off at the experimenter's will.
- To assess the impact of telomerase reactivation in adult mice with severe telomere dysfunction.
- To test the hypothesis that telomerase reactivation leads to tissue rejuvenation.
- To assess potential benefits of telomerase reactivation in highly proliferative cells such as splenocytes, testes germ cells and brain cells.
- To investigate the physiological effect of telomere dysfunction and telomerase reactivation on olfactory functions in mice
MATERIALS AND METHOD
TERT-ER mice were generated with traditional knock-in methods. A knock-in targeting vector containing the ERT2-LBD domain upstream and in frame with the mTert genomic sequence (exon 1 through intron 2) and a Lox-pgk-Neo-Lox fragment was introduced into embryonic stem cells. An antibiotic, Neomycin was used to select for cells that took up the mTert gene. Neomycin-resistant clones yielded five independent lines, two of which were injected into C57BL/6 blastocysts and implanted into surrogate mothers, yielding 10 high-percentage chimeras. Germ line transmission was confirmed by crossing the chimeras to C57BL/6 females. Heterozygous TERT-ERneo animals were crossed to EIIa-Cre animals to delete the NeoR cassette and further intercrossed to homozygosity. The EIIa-Cre allele was then bred out of the line and heterozygous animals were backcrossed to C57BL\6 at least 3 times. Four generations of successive generations of telomerase deficient mice was obtained. All studies were performed on adult (30–35 week old) males, heterozygous (G0TERT-ER) or homozygous (G4TERT-ER) for this allele. 2.5mg 4-OHT time-release pellets were inserted subcutaneously to reach steady state blood levels of 1 ng/mL 4-OHT. Brains from animals perfused with 10% formalin were fixed for 24 hours and coronally sectioned using a brain matrix. Telomeric repeats amplification protocol (TRAP) was combined with real-time detection of amplification products to determine telomerase activity. 0.5 μg total protein extract was used in each reaction. End products were resolved by PAGE in a 12.5% non-denaturing gel and stained with Invitrogen. For Western blots, 40 μg of protein were loaded per lane. Antibodies used include phospho-p53, p21, Actin, and HRP-conjugated secondary antibodies. Telomere activity was also quantified using a telomere specific Fluorescent in situ hybridization (FISH). FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complement. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes
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Neurobasal media supplemented with 4-OHT were used to plate the neural stem/progenitor cells. A cytometer was used for cell quantification. For innate olfactory avoidance tests, mice were fasted for 20 hours and habituated for 20 minutes to the test cage where the responses were recorded with a mounted video camera. A filter paper scented with water and higher concentration of 2-methyl butyric acid was placed in the cage and mouse behavior recorded for 3minutes.
RESULTS AND DISCUSSIONS
G1-G4 TERT-ER cells were examined for premature ageing phenotypes and found to lack telomerase activity (Fig 1a). Primary splenocytes had hallmark feautures of short dysfunctional telomeres, including decreased telomere-specific fluoresence in situ hybridization (Fig 1b, e, f). Adult G4 TERT-ER mice showed intense tissue atrophy particularly in high proliferative organs including extreme testicular atrophy and reduced testes size due to apoptotic elimination of germ cells, resulting in decreased fecundity (Fig 2a,d). There is a marked splenic atrophy with accompanying increased 53BP1 foci indicative of DNA damage (Fig 2b,e,h). Median survival of G4 TERT-ER mice is significantly decreased relative to that of telomere intact mice (Fig. 2f). cultured G4 neural stem cells showed decreased self-renewal activity relative to the G0 controls and this result shows that the defect was partially corrected with 4-OHT treatment (Fig 3a,d). Examination of NSC differentiation capacity revealed significant reduction in G4 TERT-ER neural stem cell capacity to generate neurons relative to 4-OHT treated cultures and 4-OHT treated controls (Fig 3c,f). Notably, 4-OHT treated G4 mice show a striking restoration of proliferation following only 4 weeks of treatment (Fig 4). Cellular deficiency associated with reduced brain weight was observed in G4 (Fig 5a, b) signifying a reduced functional fitness. But 4-OHT was shown to sufficiently cause a partial reversion of the brain defect. 4-OHT treated rodents demonstrated avoidance respones towards methyl-butyric acid, a predator ordorant which are processed in the olfactory bulb (Fig 5e,g). The frequency of entry into odour zone was higher for G4 TERT-ER mice than other control groups.
One of the basic implicit assumption prior to this experiment was that telomere damage is one of the genotoxic stress factors that drive age-associated organ decline and disease risk. This asumption was supported by Sahin, et al (2009) and by our observed reversal of systemic degenerative phenotypes in TERT-ER adult mice. Short-term telomerase reactivation restored telomere reserves, reduced DNA damage signalling, and alleviated cellular chekcpoint responses in several highly proliferative tissues.
Most importantly is that markedly constrained neural progenitor proliferation and neurogenesis profile associated with telomere dysfunction can be enhanced by reactivation of endogenous telomerase activity. This is of great importance because nerve cells are known to be naturally non-renewable.
Though, Mariella, et al 2011, claimed that brief course of telomere reactivation was not sufficient to promote carcinogenesis, other researchers such as Hu and Dephino, 2012, showed that third- and fourth-generation mice with telomerase activated by 4-OHT had a median survival of 30 days and more frequent tumor infiltration to the spleen, kidney, liver, lung, bone marrow and brain than did control-treated mice, 70 percent of which lived beyond 50 days. This result suggests that further experimentation is required before clinical trials begin with humans. Also, telomere shortening is only one of the factors that cause ageing. Ageing in human is caused by many different interrelated factors. Thus the extent to which telomerase activation could reverse ageing in humans remains unclear.
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In conclusion, though human anatomy is more complex that in mice, the reversal of age-related decline in the CNS 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.
Mariela, J, Florian L.M. (2011). Telomerase reactivation reverses degenration in aged telomerase-deficient mice. Nature. 469, 102-106.
Sahin, E. and Dephinho, R.A.(2010). Linking functional decline of telomers, mitochondria and stem cells during ageing. Nature 464, 520-528.
Hu, J, Dephino, R. (2012). Blocking telomerase kills cancer cells but provokes resistance, progression. MD Anderson news release.