JC paper

PPT 1 Hello everyone! Today we will discuss this ‘Naturally occurring p16Ink4a-positive cells shorten healthy lifespan’ paper from Dr.Deursen’s(yang vanderosen) Lab. Based on our previous knowledge that during senescence, cells enter an irreversibly arrested state in response to stress, and senescence can function to suppress tumors or pre-neoplasms. For years researchers have suspected that senescent cells play a role in aging; however, conclusive evidence has been lacking. Here Dr.Deursen’s team use a transgenic approach to eliminate senescent cells in mice and establish clear, causal contributions by these cells not only to life span, but also to age-related functional decline in specific organ systems. PPT 2 First, I’ll briefly introduce p16Ink4a. The INK4a/ARF locus is got involved in cell cycle control. The INK4a/ARF locus encodes 2 overlapping proteins, p16INK4a and p14ARF, by using different first exons and common second and third exons. These structurally very different proteins both act as negative regulators of the cell cycle, p16INK4a inhibits the activation of CDK4 and CDK6 by cyclin D, hence preventing subsequent phosphorylation of pRB and thus cell cycle progression. On the right-side graph, we can see the linear relationship between Log2-transformed p16INK4a expression and chronological age shows significant increase of p16INK4a expression with aging. So p16INK4a expression emerges were regarded as an general aging biomarker. PPT 3 And another thing we should know before we get into this paper is that in this new study, the Mayo researchers modified a previously published mouse model to eliminate senescent cells. This earlier model, called FAT-ATTAC (fat apoptosis through targeted activation), expresses an FK506 binding protein-caspase 8 fusion protein driven by a Fabp4 promoter selectively in adipocytes. Treatment with the synthetic compound AP20187, causes the fusion protein to dimerize and ablate the FKBP–Casp8-expressing cells. Here you can see in the b panel, if we didn’t treatment the cells with AP20187 there will be no interaction between two FKBP regions and the cells will also keep health. But after AP20187 was added into the system, the FKBP region would be dimerized and results in widespread apoptosis with loss of adipocyte membrane integrity and lipid contents. This is the modified version they used in this study. Senescent cells express high levels of a biomarker called p16Ink4a. To modify the FAT-ATTAC model to selectively ablate senescent cells, they replaced the Fabp4 promoter with a 2,617-bp fragment of the p16Ink4a gene promoter that is transcriptionally active in senescent, but not non-senescent cells. Further, they also added an internal ribosome entry site (IRES) followed downstream by an enhanced green fluorescent protein (EGFP) cassette to label the cells expressing the FKBP–Casp8 fusion protein. This novel transgenic line, called INK-ATTAC, permits ablation of senescent cells with AP20187. In this way, they can clear the senescent cell and avoid removing useful cells. PPT 4 After we know the background of this paper, let’s start discussing the Figure 1. The first part of this paper is that validate the selectivity of the model they will use in this study. The case they show us to proof that in Figure 1 is that clearance of senescent fat progenitor cells attenuates age-related lipodystrophy. First, they collected GFP+ and GFP− cell populations from inguinal white adipose tissue (iWAT) of 12-month-old ATTAC mice by FACS. In the panel b, after separate the GFP+ and GFP− cells, they find that GFP+ cells expressed much higher levels of Ink4a and FKBP-Casp8 than GFP− cells, as well as a broad panel of senescence markers. Also, as you can see in the panel c, GFP+ cells, but not GFP− cells, were also highly positive for senescence-associated-β-galactosidase. Senescence-associated beta-galactosidase (SA-β-gal or SABG) is a hypothetical hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides only in senescent cells. So, senescence-associated beta-galactosidase, along with p16Ink4A, is regarded to be a biomarker of cellular senescence. Based on the result of panel b and c, we can see that the GFP+ cells could be identified as senescence cells in this study. Then they will show the AP20187 can actually lead to cell apoptosis. As you can see in the panel d, iWAT of 18-month-old ATTAC mice treated bi-weekly with AP from 12 months onwards had eightfold less GFP+ adipocyte progenitors than vehicle-injected controls, but no significant difference in other cell types. And then they apply SA-β-Gal staining in panel e confirmed that Ink4a-positive senescent cells in iWAT increased between 12 and 18 months, and that AP eliminated most of these cells. In the Figure f, Consistent with senescence of progenitor cells, transmission electron microscopy (TEM) on SA-β-Gal-stained iWAT showed that X-Gal crystals were present in small perivascular cells rather than endothelium, white blood cells or adipocytes. Here X-Gal is a kind of chromogenic substrate to detect the SA-β-Gal. After incubating with a lysosomal hydrolase at pH6.0 will produce blue precipitate. Roughly we can regard cells contain X-Gal crystal as senescence cells. In the Figure g, X-Gal crystals were found in 0.2% and 1.6% of total iWAT cells from AP-treated and control mice, respectively. So based on the Figure defg they proofed that the majority of the senescence cells were eliminated by adding AP20187. The rest panel they will show us that Clearance of senescent fat progenitor cells attenuates age-related lipodystrophy. Age-dependent fat tissue disfunction is characterized by decreased adipogenesis and adipocyte atrophy. So, in the Figure h and I, we can see clearance of Ink4a-positive cells prevented loss of fat mass occurring between 12 and 18 months. Consistent with this, adipocyte size decreased between 12 and 18 months of age in the panel j , and the transcript levels of two key transcriptional regulators of adipogenesis Pparg and Cebpa were also decreased in the panel K. So Figure 1 tell us that the GFP + cells can be roughly regarded as senescence cells in the following part and these cells can be eliminated via add AP20187 in the system. In addition senescence contributes to age-dependent fat tissue alterations. But AP treatment of ATTAC mice could prevent these decreases. After validating their model, they start using this model to proof that p16Ink4a+ cells will shorten lifespan and healthspan. To examine the effect of p16Ink4a-positive cell clearance on health and lifespan, they sequentially established two cohorts of ATTAC transgenic mice. The initial cohort was on a C57BL/6-129Sv-FVB mixed genetic background fed a diet containing 9% fat. We note that this diet shortens lifespan compared to diets with 5% fat typically used in lifespan studies. The later cohort was on a congenic C57BL/6 background fed a standard 5% fat diet. Then mice were injected twice a week with AP or vehicle at 12 months of age until they became moribund or died of natural causes. Because at 12 months of age p16Ink4a+ cells will start to accumulate in several tissues. Mice separate from the longevity cohorts were examined for a series of age-sensitive outcomes at 18 months, an age at which relatively few mice in each of the cohorts had died. Data for both sexes combined showed that median lifespans of mixed and C57BL/6 AP-treated animals were increased by 27% and 24%, respectively. Median lifespans for each sex separately were also significantly extended in AP-treated cohorts irrespective of genetic background, with increases ranging from 17% to 35%. Also, from Figure c, we can see that the Maximum lifespan14 was significantly increased for mixed AP-treated males and females combined (P = 0.0295), but not for females and males individually. Maximum lifespan was not extended for C57BL/6 AP-treated animals, either combined or separately. Here I should emphasize that they operationally use ‘maximum lifespan’ to refer to the upper percentile of lifespan and not the observed sample maxima. In addition, AP treatment of mice lacking the ATTAC transgene did not improve lifespan. Then the Figure 3 will show us that clearance of senescent cells will prolong healthspan. As we can see in panel a and supplementary Figure, in both cohorts, AP treatment had no effect on the incidence or spectrum of macroscopically detectable tumours at autopsy. But tumour latency was increased. In the other hands, we can also judge the health of mice by appearance. In the panel b, AP-treated mice were overtly indistinguishable from vehicle-injected littermates at 18 months of age, but typically had a healthier appearance by 22 months. What mice look like at 18 months were not shown here, but we can easily distinguish the healthier mice at 22 months by their mental state. The AP positive mice seems more energetic than AP negative mice. Despite a lack of overt difference at 18 months, AP-treatment prevented age-dependent reductions in both spontaneous activity and exploratory behavior measured by open-field testing, which was independent of sex and genetic background. Then to further investigate the effect of p16Ink4a+ cells on physiological functions that change with age, they focused on kidney and heart health. Aged kidneys are characterized by the formation of sclerotic glomeruli, which affect glomerular filtration rates, impair kidney function, and lead to increased blood urea nitrogen levels. Indeed, glomerulosclerosis significantly increased between 12 and 18 months in vehicle-treated ATTAC mice. As we can see in panel a, the first row show the mice glomeruli(ge lao mai rei u lai) stained by haematoxylin(hei me taix lin) and eosin which will stain the nucleus blue and cytoplasm red. Compare to the AP positive mice, the glomeruli of AP negative mice there is an area of collagenous sclerosis(s ke le rou sis) runs across the middle of this glomeruli. And the second row show the glomeruli stained by PAS which will detect polysaccharides in the cell. In the pathological examination of glomeruli, it will highlight the neutrophil. So we can see significantly Neutrophil infiltration in the AP negative mice. And this is a marker of the glomerulosclerosis. And in the panel b, we can also see that the percent of sclerotic glomeruli of AP negative mice is significantly higher than AP positive mice and vehicle treated mice independent of sex and genetic background. And in the panel c, the age-related increases in blood urea nitrogen were also relieved by AP treatment independent of sex and genetic background. So, based on both panel a, b and c we can see that AP treatment can markedly reduce glomerulosclerosis and age-related increases in blood urea nitrogen independent of sex and genetic background. In the panel d, SA-β-Gal staining of kidney sections confirmed that AP-mediated disposal of senescent cells could reduce the accumulation of SA-β-Gal which is an enzyme only in senescent cells. By TEM shown in the panel f, they observed X-Gal crystals in 0.3% and 1.2% of renal cells of 18-month-old treated and untreated mice, respectively. In the panel e, to their surprise, SA-β-Gal+ cells were not located in the glomeruli but in proximal tubules, this phenomenon raising the question that how senescent tubular brush-border epithelial cells might promote glomerulosclerosis. Based on previous study angiotensin receptor blockers could attenuate age-related glomerulosclerosis under normotensive conditions, which has led to the idea that overactivation of the local renin-angiotensin-aldosterone system (RAAS) drives the formation of sclerotic glomeruli. Consistent with this, as we can see in panel g, they use qPCR to quantify the expression level of angiotensin receptor 1a (Agtr1a) which is a key component of RAAS system, increased between 12 and 18 months in AP negative mice, but no such increase was observed in AP-treated mice. Also in the panel h, western blotting confirmed that Agtr1a protein levels were lower in AP-treated kidney samples. In the panel I, we can see both renal tubules and glomeruli contributed to this decline, as demonstrated by immunolabelling of kidney sections for Agtr1a. These data suggest that senescent renal epithelial cells may produce senescence-associated secretory phenotype components that hyperactivate the local RAAS in kidney. After they explored how senescence cells affect kidney, they also look into how p16Ink4a+ cells contribute to cardiac ageing. As we can see in panel a, hearts of 12-month-old mice showed SA-β-Gal activity which are these light blue dot shown in the picture at the atrial and ventricular surface, the SA-β-Gal activity was increased with ageing in vehicle-treated but not AP-treated mice. They also observed SA-β-Gal+ smooth muscle cells in the aortic root wall of vehicle-treated mice. In the panel b and c, TEM revealed that ciliated epithelial cells and fibroblasts in the pericardium contained X-Gal crystals. And AP treatment can markedly reduce the percentage of visceral pericardium cells with X-Gal crystals. In the panel d, Morphometric analysis of heart sections showed that ventricular wall thickness was the same in vehicle- and AP-treated animals, regardless of sex. However, Ventricular cardiomyocytes of AP-treated mice were much smaller we can see in the panel e and f, suggesting that they had more cardiomyocytes than vehicle-treated mice. Taken together, these data suggest that p16Ink4a-positive cells are key drivers of this age-related cardiac phenotype. Besides, Cardiac ageing is also characterized by decreasing stress tolerance, which has been attributed to decreasing numbers of ATP-sensitive potassium (KATP) channels in the sarcolemma of cardiac myocytes due to age-related reductions in the expression of Sur2a, a key regulatory subunit of KATP channels34. As we can see in panel g, Cardiac Sur2a expression indeed decreased between 12 and 18 months in vehicle-treated mice, but no such decline was observed in AP-treated mice, suggesting that cardiac stress tolerance was preserved. To test this, they measured the time to death from arrhythmogenesis after injection of 580 microgram per kilogram of the β-adrenergic agonist isoproterenol. The result was shown in panel h, consistent with age-related loss of cardiac stress resistance, 18-month-old vehicle-treated mice died faster than 12-month-old mice. By contrast, the time to death was not accelerated in AP-treated mice. In the panel I, they did more physiological stress test. They conducted echocardiographic measurements of ventricular mass before and after eliciting cardiac stress over a 6-day period, by administering 10 microgram per kilogram isoproterenol twice a day. Cardiac mass significantly increased in 18-month-old vehicle-treated mice, whereas AP-treated mice were capable of handling the applied stress without such adaptive response, similar to young mice. To summarize this paper, basically they eliminated p16Ink4a+ cells from non-progeroid mice using ATTAC to begin to address how senescent cells influence health and lifespan. They make the following conclusions. First, age-related accumulation of p16Ink4a+ cells negatively affects longevity. Second, removal of p16Ink4a+ cells from midlife on delays the progression of neoplastic disease irrespective of genetic background and diet. Third, several age-dependent changes occur more slowly during a six-month period of p16Ink4a+ cell clearance starting at midlife. Finally, local p16Ink4a+ cells in fat, kidney and heart exert their effects through distinct mechanisms, involving progenitor cell dysfunction, RAAS overactivation, and Sur2a downregulation, respectively.