Impaired immune surveillance accelerates accumulation of senescent cells and aging methods
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Perforin deficiency promotes age-related disorders and death
The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1 -/- mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1 -/- mice showed a reduction of more than 2-fold in voluntary exercise ( P < 0.05; Fig., Supplementary Figure ), and their grip strength and coordination in a "hang-wire" test were significantly decreased, compared to old WT mice ( P = 0.044; Fig. ). Mammals also tend to lose weight during late stages of life. Old Prf1 -/- mice also suffered from progressive weight loss (Fig. ), which was comprised of both muscle loss and fat loss...
ABT-737 alleviates age-related phenotype of Prf1 -/- mice
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family -. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo -. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1 -/- mice, for two consecutive days monthly for a period of two months (Fig. ). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. ). While the activity...
ABT-737 extends the lifespan of progeroid mice
The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA +/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice ( P = 0.019; Fig. ). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig., Supplementary Figure ). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1 -/W...
Histological analysis
The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:100, #103124, Biolegend), APC anti-Gr-1 (1:100, #108412, Biolegend), APC anti-B220 (1:100, #103212, Biolegend), Alexa647 anti-NK1.1 (1:100, #108720, Biolegend), Alexa647 anti-CD3 (1:100, #100209, Biolegend). For immunostaining, tissue sections (10 µm) were mounted on slides and air-dried for 15 min, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min, and permeabilized in 0.2% Triton X-100 for 15 min. Slides were blocked in a solution of 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h, then incubated with primary antibody overnight at...
Flow cytometry and ImageStreamX analysis
For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#100209, Biolegend) mixture for 30 min in 4 °C. The dilution of these antibodies was 1:100 for the studies. After wash, DAPI was shortly introduced in order to exclude dead cells. Cells were analyzed in a SORP-LSRII instrument (BD Biosciences) and FlowJo v10 software. The cell gating strategy is shown in the Supplementary Figure.
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The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1 -/- mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1 -/- mice showed a reduction of more than 2-fold in voluntary exercise ( P < 0.05; Fig., Supplementary Figure ), and their grip strength and coordination in a "hang-wire" test were significantly decreased, compared to old WT mice ( P = 0.044; Fig. ). Mammals also tend to lose weight during late stages of life. Old Prf1 -/- mice also suffered from progressive weight loss (Fig. ), which was comprised of both muscle loss and fat loss (Supplementary Figure ). Another aspect of organism-level fitness reduction is kyphosis, an exaggerated rounding of the spine. Old Prf1 -/- mice exhibited a higher incidence of age-related kyphosis than old WT mice ( P < 0.05; Fig. ). An age-related pathology re...
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family -. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo -. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1 -/- mice, for two consecutive days monthly for a period of two months (Fig. ). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. ). While the activity of the control group decreased over the two months as expected from aged mice, the ABT-737-treated group increased their activity 2-fold at the end of the first month and this increase was retained till the end of the experiment ( P = 0.026; Fig. ). At the end of the two-month per...
The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA +/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice ( P = 0.019; Fig. ). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig., Supplementary Figure ). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1 -/- mice were reduced upon ABT-737 treatment in LMNA +/G609G mice (Fig., Fig. ). We next determined the lifespan of LMNA +/G609G mice treated with ABT-737 or with a vehicle. Strikingly, ABT-737-treatment resulted in a significant increase in median survival (377 vs. 353 days, P =...
The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:100, #103124, Biolegend), APC anti-Gr-1 (1:100, #108412, Biolegend), APC anti-B220 (1:100, #103212, Biolegend), Alexa647 anti-NK1.1 (1:100, #108720, Biolegend), Alexa647 anti-CD3 (1:100, #100209, Biolegend). For immunostaining, tissue sections (10 µm) were mounted on slides and air-dried for 15 min, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min, and permeabilized in 0.2% Triton X-100 for 15 min. Slides were blocked in a solution of 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h, then incubated with primary antibody overnight at 4 °C in the blocking solution. The following day the slides were washed twice with 0.2% Triton X-100 and once with PBS, each time for at least 30 min. In case primary antibodies were not fluorescently labeled, samples were incubated with Dylight-549-conjugated secondary antibody (1:...
For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#100209, Biolegend) mixture for 30 min in 4 °C. The dilution of these antibodies was 1:100 for the studies. After wash, DAPI was shortly introduced in order to exclude dead cells. Cells were analyzed in a SORP-LSRII instrument (BD Biosciences) and FlowJo v10 software. The cell gating strategy is shown in the Supplementary Figure.
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