Clearing tissues of senescent cells could represent a new therapeutic approach to delaying the onset or progression of age-related disorders and prolonging healthy human lifespan, researchers claim. Mayo Clinic College of Medicine investigators developed a transgenic mouse model in which cells displaying a specific marker of senescence can be removed on the administration of a drug. They then crossed these mice with a strain of animal that displays early age-related deficits and evaluated the effects of clearing tissues of senescent cells.
The results, reported in Nature, showed that drug-induced clearance of senescent cells from an early age delayed the onset of tell-tale age-related problems such as muscle wastage, specifically in those tissues in which the senescent cells normally accumulate. Moreover, claim Jan M. van Deursen, M.D., and colleagues, starting treatment later in life helped slow the progression of already-established age-related disorders.
The team says its findings indicate that acquisition of the senescence-associated secretory phenotype (SASP), which enables cells to secrete a variety of growth factors, cytokines, and proteases, contributes to age-related tissue dysfunction. Their published paper is titled “Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders."
Cellular senescence prevents the proliferation of damaged or dysfunctional cells that might cause cancer but also occurs in cells as a result of ageing, leading to an accumulation of senescent cells in tissues and organs, the authors explain. It has been hypothesized that factors secreted by these cells can damage surrounding tissue structure and function, but whether they are a causal factor in age-related diseases and whether their removal has any health benefits is unknown.
To look at this more closely, the Mayo Clinic team and their collaborators designed a transgenic approach to clearing senescent cells in mice, based on the drug-induced elimination of cells expressing the senescence marker p16Ink4a. The technique was a modified version of a mouse model known as FAT-ATTAC, which selectively killed adipocytes on the administration of AP20197, a drug that induces dimerization of a membrane-bound myristoylated FK506-binding protein-caspase 8 (FKBP-Casp8) fusion protein, in this case expressed in adipocytes.
To modify the approach for specifically killing senescent cells, the researchers replaced the promoter of the trangene used in FAT-ATTAC, with a 2,617-bp fragment of the p16Ink4a gene promoter that is transcriptionally active specifically in senescent cells. The transgene was also modified to express enhanced green fluorescence protein (EGP), which meant senescent cells could be detected and collected.
Nine transgenic INK-ATTAC founder lines were generated by injecting the transgene construct into fertilized eggs. Each of these founders was then bred into BubR1 hypomorphic (BubR1H/H) mice, a model that exhibits a significantly shortened lifespan and a range of age-related phenotypes.
BubR1 encodes a key member of the mitotic checkpoint, and because levels of the protein decline significantly with age it has been suggested that it represents a determinant of natural aging. BubR1H/H mice also selectively accumulate p16Ink4a -positive cells in tissues in which age-related pathologies develop, and research has shown that inactivating p16Ink4a in these mice holds back the onset of age-related phenotypes selectively in the same tissues, the authors note.
Analysis of tissues taken from the cross-bred BubR1H/H;INK-ATTAC mice at five months of age demonstrated that INK-ATTAC and GFP transcript levels were significantly raised in adipose tissue, skeletal muscle, and eye, but not in tissues in which endogenous p16Ink4a isn’t normally induced, such as the liver and heart.
The researchers next confirmed that transgenic INK-ATTAC and endogenous p16Ink4a were under the same, BubR1-independent transcriptional control mechanism in both wild-type INK-ATTAC animals and in BubR1H/H;INK-ATTAC mice, and then tested whether INK-ATTAC is expressed in senescent cells in BubR1 hypomorphic tissue. Both staining and qRT-PCR analysis indicated that INK-ATTAC expression correlated with the expression of senescence markers in inguinal adipose tissue (IAT) in the BubR1H/H;INK-ATTAC animals.
To generate further evidence for selective expression of INK-ATTAC in senescent cells, the team harvested IAT from aged BubR1H/H;INK-ATTAC animals and separated GFP+ and GFP– cell populations from single cell suspension using florescence activated cell sorting. qRT-PCR analysis 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.
The researchers needed to evaluate whether INK-ATTAC could also eliminate senescent cells. They cultured bone marrow cells taken from WT;INK-ATTAC transgenic animals in the presence of rosiglitazone to induce senescence and then monitored cell survival after activating the FKBP–Casp8 fusion protein by AP20187 treatment.
The results indicated that the vast majority of cells from the transgenic lines were either dead or in the process of dying within 48 hours of adding AP20187. In contrast, cultures that weren’t treated with AP20187 consisted almost entirely of viable cells expressing the senescence-associated beta-galactosidase (SA-β-Gal). “These data show that FKBP-Casp8 activation efficiently eliminates p16Ink4a-positive senescent cells in vitro,” the team remarks.
To see whether eliminating senescent, p16Ink4a-expressing cells had any effects on the onset of age-related phenotypes in the BubR1H/H animals, the researchers generated populations of BubR1H/H;INK-ATTAC animals that were either treated using AP20187 every third day from three weeks of age or were left untreated. All the animals were monitored to detect the development of age-related conditions known to be associated with p16Ink4a induction, including sarcopenia, cataracts, and loss of adipose tissue.
The results were remarkable, they note. Compared with untreated animals, those treated with AP20187 to clear senescent cells demonstrated significantly delayed onset of lordokyphosis (a measure of sarcopenia onset) and cataracts and exhibited preserved muscle diameter and function. Dual-energy X-ray absorptiometry (DEXA) scans of BubR1H/H;INK-ATTAC mice also showed that AP20187 treatment prevented loss of adipose tissue.
Conversely, AP20187 treatment had no effects on the development of age-related phenotypes such as cardiac arrhythmias and arterial wall stiffening, which are p16Ink4a -independent. Encouragingly, AP20187 therapy had no evident detrimental effects on mice treated continuously until eight months of age.
It was important to check whether the delayed onset of age-related pathologies associated with AP20187 treatment coincided with a reduction in the number of senescent cells in the affected tissues. Analysis of IAT from AP20187-treated BubR1H/H;INK-ATTAC mice showed a marked decrease in SA-β-Gal staining compared with IAT of untreated counterparts.
The tissue from treated animals in addition exhibited lower levels of other senescence-associated markers, along with expected reductions in INK-ATTAC and GFP. Skeletal muscle and eye tissue displayed similar reductions in senescence indicators.
In vivo studies had thus far involved treating BubR1H/H;INK-ATTAC animals from a young age. To investigate the effects of senescent cell clearance later in life and once age-related phenotypes would normally already be apparent in BubR1H/H animals, the investigators delayed the start of AP20187 treatment until five months of age.
Encouragingly, at 10 months of age, late-life treated animals demonstrated increased mean muscle fibre diameters, showed improved performance in treadmill exercise tests, and carried larger fat depots, adipocyte cell size, and subdermal adipose layer thickness than untreated controls. Moreover, senescence markers were dramatically lower in both the fat and skeletal muscle of AP20187-treated animals.
A comparison of five-month-old untreated animals with 10 month-old treated animals indicated that the improvements associated with AP20187 therapy were due to a slowed progression of age-related deficits rather than an actual reversal of aging.
The authors conclude that their results indicate that cellular senescence is causally implicated in generating age-related phenotypes. “Our proof-of-principle experiments demonstrate that therapeutic interventions to clear senescent cells or block their effects may represent an avenue for treating or delaying age-related diseases and improving healthy human lifespan.”