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Th-Ink-ing about Aging - Clearance of p16(Ink4a)-Positive Senescent Cells Delays Ageing-associated Disorders


From Nature
By Stuart P. Atkinson

Cellular senescence is a mechanism by which dysfunctional or aging cells are growth arrested and plays an important role in controlling tumourigenesis. Senescent cells also accumulate in various tissues and organs with ageing (Campisi, 2005) but whether this is causally implicated in age-related tissue dysfunction and whether their removal is potentially beneficial remains unknown. To better understand this concept, researchers from the laboratory of Jan M. van Deursen at the Mayo Clinic College of Medicine, Minnesota, USA designed a transgenic strategy based on previous work (Pajvani et al), through which senescent cells can be selectively killed by apoptosis upon the administration of AP20187. AP20187 a synthetic drug that induces dimerisation of a membrane-bound myristoylated FK506-binding-protein–Caspase 8 (FKBPCasp8) fusion protein expressed specifically via a 2,617-bp fragment of the p16Ink4a gene promoter that is transcriptionally active in senescent, but not non-senescent cells (Baker et al). Most senescent cells express p16Ink4a, a cyclin-dependent kinase inhibitor and tumour suppressor that enforces growth arrest by activating the Retinoblastoma (Rb) protein (Rodier and Campisi) and additionally, p16Ink4a expression of is known to increase with ageing in several rodent and human tissues (Krishnamurthy et al). The construct also contained an internal ribosome entry site (IRES) followed by an open reading frame (ORF) coding for enhanced green fluorescence protein (EGFP) to allow for detection of p16Ink4a-positive senescent cells.   The construct was injected into fertilized eggs yielding transgenic mouse founder lines, designated INK-ATTAC (INK-linked apoptosis through targeted activation of Caspase).

Lines generated which contained the construct where then bred onto a BubR1 hypomorphic (BubR1H/H) genetic background to give BubR1H/H-INK-ATTAC strains. BubR1H/H mice have a markedly short lifespan and exhibit a variety of age-related phenotypes (Baker et al 2004, Hartman et al and Matsumoto et al) and selectively accumulate p16Ink4a-positive cells in certain tissues in which age-associated pathologies develop (Baker et al 2008). Screening of inguinal adipose tissue (IAT) from nine BubR1H/H-INK-ATTAC strains at 5 months of age found GFP fluorescence in two strains (-3 and -5) and further QPCR analysis in these two strains found high GFP expression in adipose tissue, skeletal muscle and eye, but not in tissues in which endogenous p16Ink4a is not induced, such as the liver and heart. Further analysis found that fatty tissues of aged BubR1H/H-INK-ATTAC stained strongly for senescence-associated-β-galactosidase (SA-β-Gal), and that INK-ATTAC expression correlated with expression of senescence markers in IAT by QPCR. Single cell sorting of these cells on their GFP expression demonstrated that GFP+ cells not only expressed much higher levels of p16Ink4a than GFP– cells but also had elevated levels of other key senescence markers. Finally, it was demonstrated that p16Ink4a induction in wild type-INK-ATTAC MEFs through ectopic expression of oncogenic Ras and serial passaging lead to the production of GFP-positive SA-β-Gal-positive INK-ATTAC cells.

The ability of INK-ATTAC to eliminate senescent cells was then demonstrated by AP20187 treatment of bone marrow cells (from WT-INK-ATTAC-3 and -5 strains) that had been treated with rosiglitazone to induce senescence; 48 hours after AP20187 treatment the vast majority of the cells were dead or dying. Next, the effect of senescent cell clearance was investigated in BubR1H/H-INK-ATTAC-3 and -5 mice and, remarkably, AP20187-treatment led to a delayed onset of age-related disease compared to untreated mice.   Sarcopenia (the degenerative loss of skeletal muscle mass and strength associated with aging) onset and cataract formation were delayed, muscles fibres of treated cells were larger in treated mice with treadmill exercise tests indicated preservation of muscle function and adipose tissue loss was diminished, with individual adipocytes larger in size.   However, the overall survival of treated vs. untreated mice was similar, perhaps due to the lack of induction of INK-ATTAC in the heart and aorta, coupled with the fact that heart failure is presumed to be the major cause of death in BubR1H/H mice. Both sets of mice showed cardiac arrhythmias and arterial wall stiffening, which arose in a p16Ink4a-independent fashion. The delayed onset of age-related pathologies was next shown to be correlated to the reduction in senescent cells as the IAT of treated mice had less SA-β-Gal staining compared to untreated, while skeletal muscle and eye tissues had a similar reduction in senescence indicators. Further, BrdU incorporation was higher in IAT and muscle tissue of treated cells, suggesting that cellular replicative senescence is decreased upon treatment.

The above studies were conducted at weaning age, and so to study the effect of the clearance of senescent cells in older mice, AP20187 treatment of BubR1H/H-INK-ATTAC mice was started at 5 months and analysed at 10 months. Fully matured cataracts apparent at the time of treatment remained unchanged, however late-life treated animals had increased mean muscle fibre diameters and showed improved performance in treadmill exercise tests in comparison to untreated mice, while most fat depots of the treated mice became enlarged and adipocyte cell size and subdermal adipose layer thickness were significantly increased. This was again correlated to a reduction in senescence markers in fat and skeletal muscle.

Taken together, these results indicate that the selective expression of INK-ATTAC in p16Ink4a-positive senescent cells can be appropriately utilised for mediating cell death, that the clearance of senescent cells by AP20187-treatment in BubR1H/H-INK-ATTAC mice delays age-related disease and that late-life clearance of p16Ink4a-positive senescent cells attenuates the progression of age-related decline in BubR1 hypomorphic mice. This is an exciting piece of work showing a link between the loss of functional cells in a tissue and tissue disease/dysfunction with age, and may allow us to answer further pertinent questions in the future. Does clearance of senescent cells in wild type mice (as opposed to the BubR1H/H mice) delay symptoms associated with normal aging, does this affect lifespan, and, partially explored within this paper, does long term clearance of senescent cells affect normal growth and development?



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