Highlights
A modified FOXO4-p53 interfering peptide causes p53 nuclear exclusion in senescent cells
This FOXO4 peptide induces targeted apoptosis of senescent cells (TASC)
TASC neutralizes murine liver chemotoxicity from doxorubicin treatment
TASC restores fitness, hair density, and renal function in fast and naturally aged mice
Summary
The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.
Introduction
Unresolved DNA damage can impair cellular function, promote disease development, and accelerate aging (López-Otín et al., 2013). To prevent such undesired consequences, cells are equipped with a range of DNA repair mechanisms (Hoeijmakers, 2009). However, these mechanisms are not flawless. When repair falls short, tissue integrity is still at least initially maintained by independent stress-response mechanisms as apoptosis and cellular senescence (de Keizer, 2017). Senescent cells are permanently withdrawn from the cell cycle and generally develop a persistent pro-inflammatory phenotype, called the senescence-associated secretory phenotype (SASP) (Coppé et al., 2008). The SASP influences the cellular microenvironment, which can be beneficial early in life or in an acute setting of wound healing (Demaria et al., 2014, Muñoz-Espín et al., 2013). However, unlike apoptotic cells, which are permanently eliminated, senescent cells can prevail for prolonged periods of time and accumulate with age (Krishnamurthy et al., 2004). Because of their low but chronic SASP, persistent senescent cells are thought to accelerate aging and the onset of age-related diseases (de Keizer, 2017). Indeed, senescence has been associated with a plethora of (age-related) pathologies, and, conversely, genetic clearance of senescent cells can delay features of aging (Baker et al., 2016). It remains largely unclear how damaged cells avoid apoptosis in favor of senescence. We set out to address this question and to determine whether therapeutic targeting of senescent cells could not only delay but also counteract the loss of tissue homeostasis after acute damaging medical treatments, such as chemotherapy, or chronic damage caused either by accelerated or natural aging.
Results
FOXO4 Is Elevated in Senescent Cells and Maintains Their Viability
To identify potential pivots in senescent cell viability, we initiated this study by investigating whether apoptosis-related pathways are altered in senescent cells. We performed unbiased RNA sequencing on samples of genomically stable primary human IMR90 fibroblasts and IMR90 induced to senesce by ionizing radiation (IR) (Rodier et al., 2011). As senescent cells are reportedly apoptosis-resistant (Wang, 1995), we expected pro-apoptotic genes to be repressed. Surprisingly, however, senescent IMR90 showed an upregulation of prominent pro-apoptotic “initiators” PUMA and BIM while the anti-apoptotic “guardian” BCL-2 was reduced (Figures 1A and S1A). This suggested senescent IMR90 are primed to undergo apoptosis but that the execution of the death program is restrained. We reasoned such a brake could potentially be a transcriptional regulator and focused on transcription factors that have previously been linked to apoptosis, including STAT1, 2, and 4; RELB; NFκB; TP53; and FOXO4 (Figures 1B and S1B). Interference with JAK-STAT signaling is known not to affect the viability of senescent cells (Xu et al., 2015), and we have previously observed similar effects for NFκB and p53 inhibition (Freund et al., 2011, Rodier et al., 2009). Our interest was therefore directed to a factor that has not yet been studied as such, FOXO4 (Figure 1B). FOXO4 belongs to a larger mammalian family, with FOXO1 and 3 being its major siblings. FOXOs are well studied in aging and tissue homeostasis as targets of insulin/IGF signaling and as regulators of reactive oxygen species (de Keizer et al., 2011, Eijkelenboom and Burgering, 2013, Martins et al., 2016). Whereas senescence-inducing IR showed only mild effects on the expression of FOXO1 and 3, both FOXO4 mRNA and protein expression progressively increased (Figures 1C and 1D). We therefore wondered whether FOXO4 could function to balance senescence and apoptosis. We stably inhibited FOXO4 expression using lentiviral shRNA (Figure 1E). FOXO4 inhibition prior to senescence-induction resulted in a release of mitochondrial cytochrome C (Figure 1F) and BAX/BAK-dependent caspase-3 cleavage (Figure 1G). In addition, FOXO4 inhibition in cells that were already senescent, but not their control counterparts, reduced viability and cell density (Figures 1H and S1C). Together, these show that after acute damage FOXO4 favors senescence over apoptosis and maintains viability of senescent cells by repressing their apoptosis response.
FOXO4-DRI Disrupts PML/DNA-SCARS and Releases Active p53 in Senescent Cells
Interference with FOXO4 signaling could be a strategy to eliminate senescent cells and thereby potentially target senescence-related diseases. However, shRNA-mediated repression of FOXO4 would be complicated to translate to the clinic. Thus, we decided to design compounds that could structurally interfere with FOXO4 function instead. Immunofluorescence experiments showed FOXO4 to be gradually recruited to euchromatin foci after senescence-induction (Figures 2A–2C and S2A–2D ). As senescence develops, promyelocytic leukemia (PML) bodies fuse with 53BP1-containing DNA-SCARS to jointly regulate expression of the SASP (Rodier et al., 2011). High-resolution structured illumination microscopy (SIM) of nuclei of senescent cells showed FOXO4 to reside within these PML bodies, adjacent to 53BP1-containing DNA-SCARS (Figure 2D; Movies S1 andS2; Figures S2E–S2I).
p53 controls both apoptosis and senescence (Kruiswijk et al., 2015) and localizes to DNA-SCARS in senescent cells (Rodier et al., 2011). Under those conditions p53 is phosphorylated by ATM on Ser15, which blocks its MDM2-mediated degradation (Rodier et al., 2009). Consistent with the observation of FOXO4 residing in PML bodies, FOXO4 localized next to phosphorylated ATM substrates (Figure S2I) and pS15-phosphorylated p53 (Figure 2E). This raised the question whether FOXO4 could maintain senescent cell viability by binding p53 and inhibiting p53-mediated apoptosis in favor of cell-cycle arrest.
FOXOs can interact with p53, and the interaction domain has been characterized by nuclear magnetic resonance (NMR) (Wang et al., 2008). To interfere with the FOXO4-p53 interaction, we therefore designed a cell-permeable peptide comprising part of the p53-interaction domain in FOXO4 (Figures 2F and 2G). FOXO1 and FOXO3 are essential to numerous endogenous processes as development, differentiation, and tumor suppression, roles not prominently attributed to FOXO4 (Hosaka et al., 2004, Nakae et al., 2003, Paik et al., 2007, Renault et al., 2009). Another difference with FOXO1 and 3 is that FOXO4 is only marginally expressed in most tissues (Figures S2J and S2K), and FOXO4 knockout mice do not show a striking phenotype (Hosaka et al., 2004, Paik et al., 2007). We therefore chose a region in FOXO4 that is conserved in both humans and mice but differs from FOXO1 and FOXO3 (Figure S2L).
Research on peptide chemistry has shown that protein domains containing natural L-peptides can sometimes be mimicked by using D-amino acids in a retro-reversed sequence (Guichard et al., 1994). Modification of peptides to such a D-retro inverso (DRI)-isoform can render peptides new chemical properties, which may improve their potency in vitro and in vivo (Borsello et al., 2003). Several DRI-modified peptides have been shown to be well tolerated and therapeutically effective in clinical trials. These include a double-blinded, randomized, placebo-controlled Phase IIb trial (Beydoun et al., 2015, Deloche et al., 2014, Suckfuell et al., 2014) and a Phase I trial for systemic treatment of solid tumors (Warso et al., 2013), together showing there is precedence for DRI peptides in clinical therapy. This provided the rationale for designing the FOXO4 peptide in a DRI conformation, henceforth named FOXO4-DRI. We performed competition experiments by NMR to investigate whether FOXO4-DRI can inhibit the interaction between p53 and FOXO4 in vitro. Titration of a recombinant N-terminal domain of p53 (aa 1–312) to a solution containing the 1H, 15N-labeled FOXO4 Forkhead (FH) domain (aa 486–206) induced a progressive chemical shift perturbation (CSP) of 1H, 15N HSQC cross peaks, indicating specific binding of p53 to FOXO4 (Figure 2H). Stepwise addition of the FOXO4-DRI peptide to this complex caused the CSPs of FOXO4 to be reverted back to the unbound state, indicating FOXO4-DRI competes with FOXO4 for p53 binding in a dose-dependent manner and doing so with higher affinity (Figure 2I).
To facilitate cellular uptake of FOXO4-DRI, it was designed as a fusion with HIV-TAT, a basic and hydrophilic sequence that allows energy-independent cellular uptake of cargo through transient pore formation (Herce and Garcia, 2007). Using an antibody against HIV-TAT, we observed FOXO4-DRI to be taken up as soon as 2–4 hr after administration and to remain detectable for at least 72 hr (Figure 2J). Given that the affinity of antibodies is generally low, this indicates FOXO4-DRI effectively enters senescent cells at high intracellular concentrations, which remain abundant and stable over a prolonged period of time. Following its uptake, FOXO4-DRI reduced the number of senescence-induced FOXO4 foci, PML bodies, and 53BP1 DNA-SCARS while not affecting the number of small 53BP1 foci (Figure 2K).
FOXO4 can regulate expression of the p53-target p21cip1 in senescent cells (de Keizer et al., 2010), and through p21cip1, p53 can induce p16in4a-independent cell cycle arrest in senescent cells (Di Leonardo et al., 1994). Moreover, p53 can induce apoptosis either through transactivating pro-apoptosis genes or in a transcription-independent manner by translocating to the mitochondria (Mihara et al., 2003). Examination of the promoter of Cdkn1a, the gene encoding p21Cip1, showed a canonical FOXO target sequence to be flanked by two p53 binding sites (Figure 2L). We therefore investigated the effect of FOXO4-DRI on p21Cip1 and p53. FOXO4-DRI reduced senescence-associated p21Cip1 levels (Figure 2L) and promoted the accumulation and nuclear exclusion of active pSer15-p53 (Figures 2M and S2M). Together, these results show that by competing with endogenous FOXO4 for p53 binding, FOXO4-DRI disrupts senescence-associated FOXO4/PML/DNA-SCARS and causes nuclear exclusion of active p53.
FOXO4-DRI Can Selectively and Potently Target Senescent Cells for p53-Dependent Apoptosis
Given the reported pro-apoptotic role of active p53 when recruited to mitochondria, we next assessed the effects on senescent cell viability. Incubation of senescent and control IMR90 with increasing concentrations of FOXO4-DRI showed FOXO4-DRI to potently and selectively (11.73-fold difference) reduce the viability of senescent versus control IMR90 (Figure 3A) and other normal cells (Figure S3A). Real-time cell density measurements revealed the effect to occur as soon as 24–36 hr after administration (Figure 3B). Neither the same peptide in L-isoform (Figure 3C) nor an unrelated DRI-peptide based on a distinct Forkhead protein, FOXM1 (Kruiswijk et al., 2016), affected senescent cell viability (Figure 3D). These results show that FOXO4-DRI can target senescent cells, and they highlight the importance of the DRI-modification for its potency.
Two classes of anti-senescence compounds have been reported so far: Quercetin/Dasatinib, either alone or in combination (Zhu et al., 2015), and the pan-BCL inhibitors ABT-263/737 (Chang et al., 2016, Yosef et al., 2016). Quercetin and Dasatinib have been reported to be non-specific (Chang et al., 2016). We found no selectivity toward senescent IMR90 (Figure S3B), and therefore this cocktail was not explored further. ABT-263 (Chang et al., 2016) and ABT-737 (Yosef et al., 2016) target the BCL-2/W/XL family of anti-apoptotic guardians (see also Figure 1A). Indeed, ABT-737 showed selectivity for senescent IMR90 (Figure S3B). However, already at low doses, it appeared to influence control cells as well (Figure S3B). Also in a treatment regimen where both compounds were added in consecutive rounds of lower concentrations, FOXO4-DRI proved to be selective against senescence yet safe to normal cells (Figures 3E and S3C).
We next addressed the role of p53 in FOXO4-DRI-mediated clearance of senescent cells. Stable knockdown of p53 reduced the ability of FOXO4-DRI to target senescent IMR90 (Figures 3F and S3D). A similar effect was observed when the senescent cells were co-incubated with the pan-caspase inhibitors QVD-OPH or ZVAD-FMK (Figure 3G), suggesting a caspase-dependent effect. Indeed, real-time imaging in the presence of a caspase-3/7-activatable dye showed FOXO4-DRI to specifically induce caspase-3/7 activation in senescent, but not control, cells (Figure 3H; Movies S3 and S4). Together, these data show that FOXO4-DRI potently and selectively reduces the viability of senescent cells by competing with FOXO4-p53 binding, thereby triggering release of active p53 to the cytosol and inducing cell-intrinsic apoptosis through caspase-3/7. This establishes FOXO4-DRI as a genuine inducer of TASC. (targeted apoptosis of senescent cells).
FOXO4-DRI Counteracts Chemotherapy-Induced Senescence and Loss of Liver Function
Given the potency of FOXO4-DRI against senescence in vitro, we wondered whether FOXO4-DRI could be of therapeutic use against senescence-related pathologies. We therefore employed three independent in vivo senescence models, one for chemotoxicity (Figure 4), one for accelerated aging (Figures 5 and 6), and one for natural aging (Figure 7). In all of these, we made use of the recently developed senescence-detection system: p16::3MR. In this system the promoter of the major senescence gene p16ink4a drives expression of Renilla luciferase (RLUC) to allow longitudinal visualization of senescence. In addition, it expresses a thymidine kinase (TK) from the herpes aimplex virus, which induces apoptosis when cells are presented with its substrate ganciclovir (GCV) (Figure S4A; Demaria et al., 2014).
Off-target toxicity limits the maximum tolerated dose of chemotherapeutic drugs and causes long term health problems in cancer survivors, including an acceleration of aging (Henderson et al., 2014). Chemotherapy can induce senescence (Ewald et al., 2010), and we therefore determined whether therapeutic removal of senescence could influence chemotoxicity. As an example, we used the common chemotherapeutic drug doxorubicin, which can indeed induce senescence (Cahu et al., 2012, Roninson, 2003) and liver toxicity in rodents and humans (Damodar et al., 2014). In agreement with these reports, doxorubicin induced senescence in IMR90 in vitro, evident by elevated SA-β-GAL activity, expression of p16ink4a, and the early and late SASP factors IL-1α and IL-6 (Orjalo et al., 2009), respectively (Figures 4A–4C and S4B). As seen for IR-senescent cells (Figures 2 and 3), doxorubicin-induced senescent cells showed an upregulation in FOXO4 foci (Figures 4B and 4C) and FOXO4-DRI potently and selectively lowered the viability of doxorubicin-senescent versus control IMR90 (Figure 4D). In line with the IR-senescence data, low effective doses of FOXO4-DRI were well tolerated in normal IMR90 compared to ABT-737 while being very potent against doxorubicin-senescent cells at higher doses (Figure 4E). Also in this setting, the potency of FOXO4-DRI was more pronounced when applied in consecutive rounds (Figures 4F and S4C).
It could be that FOXO4-DRI merely lowers the threshold for cells to enter apoptosis after DNA damage. This would impair its potential for in vivo or clinical translation. Incubation of normal IMR90 with FOXO4-DRI, administered at various time-points prior to doxorubicin exposure, did not influence the sensitivity of cells to doxorubicin (Figure 4G). In contrast, doxorubicin-senescent cells were effectively cleared. Thus, FOXO4-DRI does not predispose healthy cells to DNA damage, but selectively targets cells that have undergone senescence as a consequence of earlier doxorubicin exposure. Together this prompted us to try a similar sequential treatment regimen of FOXO4-DRI in doxorubicin-exposed mice in vivo.
In follow-up of the in vitro data, doxorubicin progressively induced senescence in vivo as detected by p16ink4a-driven RLUC in p16::3MR mice (Figure S4D). Furthermore, as seen in patients, doxorubicin reduced total body weight (Figure 4J) and induced expression of FOXO4 foci and IL-6 in the liver (Figures 4K and 4L). Strikingly, these effects were neutralized after sequential treatment with FOXO4-DRI (Figures 4H–4L). We therefore wondered whether liver function was also affected. Doxorubicin strongly induces plasma levels of aspartate aminotransferase (AST), an established indicator of liver damage (Damodar et al., 2014) (Figure 4M). Excitingly, FOXO4-DRI potently counteracted the doxorubicin-induced increase in plasma AST (Figure 4N). To address whether these effects are mediated through clearance of senescence, we combined treatment of FOXO4-DRI with GCV to facilitate senescence clearance through the TK suicide gene of p16::3MR construct. GCV reduced doxorubicin-induced p16-RLUC expression (Figure S4E) and plasma AST levels (Figures 4M and S4F), indicating AST reduction is indeed caused by clearance of senescent cells. In both cases FOXO4-DRI did not further enhance these effects. Together, these data indicate that FOXO4-DRI is effective in reducing doxorubicin-induced senescence in vitro and in vivo and in doing so neutralizes the doxorubicin-induced loss in body weight and liver toxicity. Thus, FOXO4-DRI is effective against chemotoxicity.
FOXO4-DRI Counteracts Senescence and Features of Frailty in Fast-Aging XpdTTD/TTD Mice
We next wondered whether FOXO4-DRI could influence the healthspan of mice in which senescence and the concomitant loss of tissue homeostasis were not actively induced but were allowed to develop spontaneously as a consequence of aging. As is the case for humans (Ferrucci et al., 2005), we expected strong biological variation in senescence and the SASP in naturally aged wild-type mice. To reduce the effects of biological noise, we therefore decided to first employ fast-aging mice. We sought a model that recapitulates features of natural aging and does not suffer from age-related pathologies caused by other processes such as apoptosis (de Keizer, 2017). This we found in XpdTTD/TTD, a model based on the human premature-aging syndrome trichothiodystrophy (TTD) (de Boer et al., 2002, de Boer et al., 1998). Using the p16::3MR reporter system, we observed that already at a young age XpdTTD/TTD animals show high levels of p16-positive senescence (Figure 5A). As also seen for doxorubicin-induced senescence, FOXO4-DRI reduced this effect (Figure 5B); this result indicates that XpdTTD/TTD is a valid fast-aging model for studying the effects of FOXO4-DRI on spontaneously developed senescence in vivo.
Underscoring their aging phenotype, XpdTTD/TTD mice show accelerated loss of hair (Figure 5D; de Boer et al., 1998). While not initially focused on this phenotype, we observed a robust improvement of fur density in FOXO4-DRI-treated XpdTTD/TTDmice (Figures 5C and 5D and S5A). To address this more quantitatively, we determined the infrared-measured abdominal surface temperature of the mice. Due to the lack of fur, the abdominal temperature of XpdTTD/TTD mice was several degrees higher than wild-type counterparts, an effect reduced by FOXO4-DRI (Figure 5E). A second unexpected observation was found in the behavior of the treated mice. Whereas XpdTTD/TTD mice generally show less exploratory behavior compared to wild-type littermates, FOXO4-DRI-treated animals were noticeably more active (Figure S5B). To also investigate this more quantitatively, we scored the responsiveness of the mice to gentle physical stimuli. Despite individual variation, XpdTTD/TTD mice were on average considerably more responsive to such stimuli after FOXO4-DRI treatment (Figure 5F). Finally, as a more objective measure of activity, we tracked voluntary physical activity in a set-up in which the mice were continuously housed in cages with free access to running wheels. Despite significant individual differences, XpdTTD/TTD mice were found to run 1.37 ± 0.54 km/day on average, compared to 9.37 ± 1.1 km/day seen for wild-type mice, arguing they are indeed less mobile (Figure 5G). In line with the behavioral results, exposure of the mice to FOXO4-DRI increased running wheel activity over time in the majority of these (Figures 5H and 5I). Together, these results indicate that FOXO4-DRI can reduce cellular senescence and counteract hair loss and general frailty in fast-aging XpdTTD/TTD mice.
FOXO4-DRI Counteracts Loss of Renal Function in Fast-Aging XpdTTD/TTD Mice
The phenotypical and behavioral results described above are difficult to connect to a molecular mechanism. We therefore decided to focus on the role of senescence in aging-induced decline in function of specific tissues. Pilot measurements of various metabolites in plasma samples of XpdTTD/TTD mice suggested they suffer from decreased renal function. As injected compounds tend to accumulate in the kidney, these together argued for investigating the potential of therapeutic removal of senescence in this organ. Urea is secreted through urine but becomes detectable in the blood when glomerular filtration rates drop. Plasma urea is therefore a marker of declined renal filtering capacity (Gowda et al., 2010, Lyman, 1986). In fact, it was recently established that semigenetic clearance of senescence can delay the aging-induced increase in plasma urea, establishing senescence as a culprit for loss of renal filtering capacity during aging (Baker et al., 2016). As evident from the increase in plasma urea levels, renal function indeed declines in wild-type mice as they age (Figure 6A; 26 weeks versus 130 weeks). This was faithfully recapitulated early in life in XpdTTD/TTD mice (Figure 6A; 26 weeks WT versus 26 weeks TTD). Both naturally aged wild-type and young XpdTTD/TTD kidneys showed a strong increase in SA-β-GAL activity and IL-6 expression in the tubular regions (Figures 6B and 6C). In addition, they also showed a significant increase in tubular cells positive for FOXO4 foci (Figure 6D), together indicating that both modes show elevated senescence. Using an ex vivo system of aged kidney slices, FOXO4-DRI induced strong terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity within 3 days (Figures 6E and S6A–S6D ), indicating that FOXO4-DRI can also induce apoptosis in these cells. Altogether this provided rationale for investigating the potency of FOXO4-DRI on tubular senescence and renal function in vivo.
A limitation to the therapeutic potential of the senolytic pan-BCL inhibitors ABT-263/ABT-737 is their tendency to cause severe thrombocytopenia (Schoenwaelder et al., 2011). This is undesirable when actually aiming to restore healthspan of aged individuals. Comparing platelet levels before and 30 days after treatment showed FOXO4-DRI not to noticeably influence platelet levels (Figure 6F) or other whole blood values (Figure S6E). Neither did it cause deleterious effects on non-proliferative tissues as far as tested, e.g., the heart (Figure S6F). Encouraged by the passing of at least these initial safety concerns, we progressed to measuring the effects of FOXO4-DRI on renal senescence and functional capacity. In line with the SA-β-GAL data, tubuli of XpdTTD/TTD kidneys show severe loss of LMNB1 (Figure 6G), a robust molecular marker of senescence (Freund et al., 2012). This is paralleled with elevated IL-6 (e.g., Figure 6J), indicative of SASP, and elevated urea levels in the blood (e.g., Figure 6K). SASP factors as IL-6 may be the cause for the observed loss in renal function, and we wondered how FOXO4-DRI would function under such high-SASP conditions. In vitro experiments showed FOXO4-DRI to be more potent against senescent cells in which SASP was transiently boosted by recombinant IL1α/β or lipopolysaccharide (LPS), whereas an IL1 receptor antagonist or the general anti-inflammatory drug cortisol reduced its potency (Figures 6H and 6I). Thus, FOXO4-DRI actually is most effective against senescent cells expressing high levels of SASP and could as such be particularly effective against loss of renal function. Excitingly, while not substantially influencing total body nor kidney weight (Figure S6G), FOXO4-DRI treatment normalized the percentage of tubular cells lacking LMNB1 (Figure 6G), the tubular IL-6 elevation (Figure 6J), and the elevations in plasma urea levels (Figure 6K). To address whether this is mediated by senescence clearance, we again made use of the ability of the 3MR construct to eliminate senescent cells through GCV. As GCV is typically administered i.p., we treated a cohort of XpdTTD/TTD–p16::3MR mice i.p. with FOXO4-DRI and GCV. GVC and FOXO4-DRI induced a comparable reduction in plasma urea in both groups (Figure 6L). Thus, FOXO4-DRI targets high SASP-expressing senescent cells that have naturally developed in the kidneys of fast-aging XpdTTD/TTD mice and in doing so restores kidney homeostasis.
FOXO4-DRI Counteracts Frailty and Loss of Renal Function in Naturally Aged Mice
Encouraged by these results, we decided to challenge whether FOXO4-DRI could also target senescence and tissue homeostasis in normal mice that were allowed to age naturally. As expected, the biological variation in p16-driven senescence was substantial in aged p16::3MR, compared to young XpdTTD/TTD-p16::3MR (Figures 7A and 7C). The variation in running wheel activity was too large to perform meaningful experiments (Figure S7A). Nonetheless, while again not influencing platelet levels (Figure 7B), FOXO4-DRI significantly reduced p16-driven RLUC (Figure 7C) and could improve fur density (Figure 7D) and responsiveness (Figure 7E). Furthermore, in the kidneys of these mice, FOXO4-DRI increased the number of LMNB1 positive cells (Figure 7F), reduced IL-6 expression (Figure 7G), and restored renal filtering capacity measured by decreased plasma urea (Figure 7H). As an extra control, the plasma levels of a second metabolite indicative of reduced renal function, creatinine, were measured. This also was reduced by FOXO4-DRI, independently confirming the beneficial effect of FOXO4-DRI on the restoration of renal filtering capacity in naturally aged mice (Figure 7I). As seen for the XpdTTD/TTD–p16::3MR mice, i.p. administration of FOXO4-DRI or GCV equally reduced plasma urea and creatinine levels (Figure 7J). Thus, senescent cells are causal for the reduction in renal function in fast-aging XpdTTD/TTD and naturally aged wild-type mice, and by selective targeting of high-SASP expressing senescent cells in the tubuli, FOXO4-DRI can restore kidney homeostasis. By inducing TASC, FOXO4-DRI may thus be a potent drug to restore loss of health after natural aging and is an attractive option to explore further in the battle against those age-related diseases that are at least in part driven by senescence.
Discussion
With life expectancy projected to increase in the foreseeable future (Vaupel, 2010), it is important to develop strategies to extend and restore healthspan. Cell-penetrating peptides (CPPs) are relatively understudied in aging research. Further analysis of their use is warranted as they serve several major advantages. Counter to broad-range inhibitors, CPPs can in theory target any surface-exposed stretch of amino acids to block specific protein-protein interactions and, in doing so, they can selectively modulate very specific downstream signaling events (discussed in de Keizer (2017)). Other compounds, classified as senolytics, have been described to influence senescent cell viability. As a CPP, FOXO4-DRI differs from these by being designed around a specific amino acid sequence in a molecular target only mildly expressed in most normal tissues (see e.g., Figures S2J and S2K). Though a more thorough analysis is required, at least as far as tested here FOXO4-DRI appears to be well tolerated, which is an absolutely critical milestone to pass when aiming to treat relatively healthy aged individuals (de Keizer, 2017).
FOXO4-DRI effectively disrupts the p53-FOXO4 interaction (Figures 2H and 2I), but the importance of the FOXO4 protein itself is more complicated in DNA damage and senescence. As FOXO4-DRI causes nuclear exclusion of active p53, the levels of p21Cip1 decline (Figures 2L–2N). However, the loss of p21Cip1 alone is insufficient to induce apoptosis and was actually shown to induce a senescence-escape instead (Brown et al., 1997). Rather, the exclusion of p53 itself has been reported to induce apoptosis directly when relocated to mitochondria (Mihara et al., 2003), thereby explaining the FOXO4-DRI effects. FOXO4 shRNAs induce apoptosis in senescent IMR90 (Figures 1E–1H), arguing that full FOXO4 inhibition might also be of use against senescence. True as this may be, chronic FOXO4 reduction is not advisable as FOXOs play a role in DNA-damage repair and Foxo4−/− mice are susceptible to acute damage (Zhou et al., 2009). In contrast to loss of FOXO4, FOXO4-DRI does not sensitize healthy cells to acute DNA damage (Figure 4G). Thus, while permanent FOXO4 inhibition is inapplicable, the fact that as a CPP it can block a specific protein-protein interaction makes FOXO4-DRI selective and thereby well tolerated and effective.
Based on these positive effects, it is now possible to envision a point on the horizon where the disease indications are identified that could benefit most from FOXO4-DRI therapy. High SASP-secreting cells are likely to play a much larger role in disease development than more sterile senescent cells. Through SASP, senescent cells may permanently confer a state of stemness in neighboring cells and thereby impair tissue function and renewal, an effect that we recently described in the senescence-stem lock model for aging (de Keizer, 2017). FOXO4-DRI has a strong preference for targeting high-SASP subpopulations of senescent cells, but it is unclear what causes heterogeneity in the SASP. It will be a major achievement to unravel those mechanisms and to steer these such that therapeutic targeting is most beneficial. In that sense, identification of senescence-driven pathologies that rely on SASP may help in optimizing candidates for therapy. XpdTTD/TTD is a pleiotropic model for aging that can be effectively used as a basis for such research. It is a well-established model for osteoarthritis, especially in cohorts of older age than we used here (52 weeks) (Botter et al., 2011) and for the unhealthy loss in muscle (sarcopenia) and fat mass (Wijnhoven et al., 2005).
Last, it is relevant to note that independent of aging and age-related diseases, FOXO4-DRI may be of use against the progression, stemness, and migration of malignant cancer. Given that SASP factors influence these (Campisi, 2013), it will be particularly interesting to determine whether FOXO4-DRI affects those p53-wt cancer cells that have adopted a more migratory and stem-like state due to reprogramming by chronic SASP exposure. In any case, the here reported beneficial effects of FOXO4-DRI provide a wide range of possibilities for studying the potential of therapeutic removal of senescence against diseases for which few options are available.