Cellular senescence as a source of chronic microinflammation that promotes the aging process

Abstract

Why and how do we age? This physiological phenomenon that we all experience remains a great mystery, largely unexplained even in this age of scientific and technological progress. Aging is a significant risk factor for numerous diseases, including cancer. However, underlying mechanisms responsible for this association remain to be elucidated. Recent findings have elucidated the significance of the accumulation of senescent cells and other inflammatory cells in organs and tissues with age, and their deleterious effects, such as the induction of inflammation in the microenvironment, as underlying factors contributing to organ dysfunction and disease development. Cellular senescence is a cellular phenomenon characterized by a permanent cessation of cell proliferation and secretion of several proinflammatory cytokines (senescence associated secretory phenotypes). Notably, the elimination of senescent cells from aging individuals has been demonstrated to alleviate age-related organ and tissue dysfunction, as well as various geriatric diseases. This review summarizes the molecular mechanisms by which senescent cells are induced and contribute to age-related diseases, as well as the technologies that ameliorate them.

1. Introduction

Aging is an inevitable phenomenon in human life, characterized by the functional decline of all organs and tissues. Historically, the aging process was regarded as a random phenomenon of life that occurs according to the second law of thermodynamics.¹⁾ However, recent studies of the aging process in other species have demonstrated that there are several organisms that rarely exhibit signs of aging, suggesting that aging is not an inevitable phenomenon of life.^2)-5) Aging is a significant etiological factor for numerous human diseases, including neurodegenerative diseases and cancer. A prevalent pathology associated with the aging process is “chronic inflammation”, which is largely responsible for the functional deterioration of organs and tissues, as well as the development of numerous diseases during the aging process^6),7) (Fig. 1). In humans, extensive chronic inflammation has been identified in various organs and tissues of the elderly, and the extent of this inflammation increases with age.⁸⁾ It is noteworthy that research has revealed a tendency among centenarians to exhibit low levels of chronic inflammation, a phenomenon that is also observed in organisms demonstrating minimal signs of aging.⁹⁾

Fig. 1

Age-related diseases and hallmarks of aging. The hallmarks of aging have been shown to regulate the process of aging and, concomitantly, to promote the development of age-related diseases. For the hallmarks of aging, we reference the report by Lopez-Otin, C. et al.¹¹⁸⁾

The mechanism by which chronic inflammation is induced during aging is not well understood; however, recent studies have implicated cellular senescence in this process.¹⁰⁾ Cellular senescence is defined as a programed response to genetic stress and epigenomic abnormalities, exhibiting numerous intracellular metabolic and phenotypic changes.¹¹⁾ A finite proliferative state was observed in cultured human normal fibroblasts and first described as cellular senescence in 1961 (replicative senescence).¹²⁾ In the context of cellular senescence, the term “senescent cells” refers to cells that have undergone permanent cell cycle arrest in response to external growth stimuli. In addition to this, senescent cells have been observed to upregulate anti-apoptotic pathways, and secrete various proinflammatory cytokines, a phenomenon referred to as a senescence-associated secretory phenotype (SASP).¹³⁾ In the 2010s, Baker et al. employed genetic engineering to develop a model that can induce apoptosis in senescent cells in a drug-dependent manner.^14),15) Their findings indicated that the artificial removal of p16^high senescent cells from old individuals suppressed the development of age-related diseases, including cancer, kidney dysfunction, and dementia, thereby extending healthy life expectancy. This demonstrated that the elimination of senescent cells (senolysis) can effectively counteract the process of aging and the onset of age-related diseases. Consequently, the development of senescent cell removal technology has emerged as a global topic of significant interest. However, the cells responsible for the development of chronic inflammation during the aging process have not yet been fully elucidated. The factors that contribute to SASP vary depending on the specific organ, tissue, and cell type in which cellular senescence occurs, the duration of senescence and the origin of the senescent stimulus. SASP may also act in an anti-inflammatory manner.¹⁶⁾ In addition, senescent cells may have some beneficial effects.^17)-21) The excessive elimination of p16-positive senescent liver endothelial cells has been observed to exacerbate liver fibrosis in certain pathological conditions.^22),23) Therefore, the impact of cellular senescence on the organism is heterogeneous and the comprehensive mechanism is not yet fully elucidated.

In recent years, the DNA methylation patterns, encompassing both the loss and gain of DNA methylation, have emerged as the most effective method to estimate the biological aging of an individual.²⁴⁾ On a genome-wide basis, age-dependent changes in DNA methylation encompass global hypomethylation and region-specific hypermethylation.²⁵⁾ The global loss of DNA methylation has been demonstrated to result in the de-repression of the expression of endogenous retroviral genes and retrotransposons.²⁶⁾ This phenomenon has been shown to induce senescence.^27),28) The age-related loss of DNA methylation has been demonstrated to cause the accumulation of senescent cells in individuals, thereby suggesting the possibility of regulating aging. However, the molecular basis underlying the age- and cell proliferation-dependent loss of DNA methylation remains to be elucidated. One hypothesis posits that impaired DNA methylation replication may be involved. To maintain the cellular phenotypes in a variety of organisms, DNA methylation must be replicated during cell proliferation through the unique ubiquitylation-dependent mechanisms.²⁹⁾ This review summarizes the molecular basis of age-related DNA methylation loss, the induction of cellular senescence, the age-related changes and pathogenesis of geriatric diseases caused by the accumulation of cellular senescence, and finally, the methods to suppress aging and age-related diseases by eliminating senescent cells.

2. Mechanisms of senescence induction

Cellular senescence is induced by a variety of genotoxic stimuli, including telomere shortening, DNA damage, mitochondrial dysfunction, and oncogene activation.³⁰⁾ It is also induced by epigenome abnormalities. The most decisive phenotype of senescent cells is a durable cessation of cell proliferation in response to various mitogenic stimuli. The p53 and pRb family pocket proteins are both essential for permanent cell cycle arrest. However, the activation of p53 and pRb alone is insufficient to induce of senescence.³¹⁾ Fluorescent ubiquitination-based cell cycle indicator-based live-cell imaging analysis revealed that all senescence-inducing stimuli induced mitosis-skipping, thereby showing the conversion of green-colored S-G2 cells to red-colored G1 cells without mitosis.³²⁾ This mitosis-skipping process is regulated by G2 phase-specific activation of p53, which results in the transcriptional activation of p21 and the repression of both Cdk1 and Cdk2. These effects, in turn, lead to the premature activation of anaphase-promoting complex/cyclosome Cdh1.³³⁾ This activation results in the degradation of various mitotic regulators even in the G2 phase. p53 in the G2 phase has also been shown to enhance pRb1 function and suppress transcription of the factors required for mitotic initiation (Fig. 2).³⁴⁾ A comparable mechanism underlying permanent cell cycle exit at S/G2 phase has recently been reported.³⁵⁾

Fig. 2

Mechanism of senescence induction. All senescence-inducing stimuli ultimately activate DNA damage responses. Activated DNA damage responses stabilize p53 in G2 in certain situations. In this particular instance, p53 transcriptionally induces p21, which in turn prematurely activates APC/C^Cdh1, resulting in the degradation of most mitotic regulators. Activated p53 in G2 also represses transcription of most mitotic regulatory genes by regulating pRb. This interplay between these two parallel pathways ensures the comprehensive loss of mitotic regulators in G2, leading to the induction of mitotic skipping. As a result, cells enter a state of senescence.

SASP is another aspect of senescence, whose induction is also regulated by p53 pathways. The activation of p53 has been shown to suppress p38 mitogen-activated protein kinase, a process that is critical for SASP induction.³⁶⁾ Consequently, following mitosis skipping, activated p53 must be downregulated. Our research has identified the SCF^Fbxo22-KDM4A complex as an E3 ubiquitin ligase that is specific for methylated p53, its activation form, during the late phase of senescence.³⁷⁾ The subsequent degradation of methylated p53 has been shown to induce p16 expression, a hallmark of senescence, and the subsequent SASP.

3. Molecular mechanisms of DNA methylation replication and its implications in age-dependent loss of DNA methylation

DNA methylation plays a pivotal role in the regulation of gene transcription, chromosomal inactivation, genomic imprinting and transposon/endogenous retrovirus silencing.³⁸⁾ Subsequent to DNA replication, methylated DNA cannot be replicated directly, resulting in the formation of hemi-methylated DNA in which only the template is methylated, but not the nascent strand.³⁹⁾ Ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF-1) specifically binds to hemi-methylated DNA and targets two Lys residues at the N-terminus of PCNA-associated factor 15 or the N-terminal tail of histone H3 for dual monoubiquitylation.^40)-42) In the euchromatic region, ubiquitinated PCNA-associated factor 15 promotes the localization of DNA methyltransferase 1 (DNMT1) at DNA methylation sites through specific interaction with the replication foci targeting sequence domain of DNMT1 and in turn activates it.⁴²⁾ In the heterochromatic region, hemimethylated DNA remaining after passage through the replication machinery is initially recognized by cell division cycle associated 7, another hemimethylated DNA binding protein,⁴³⁾ and its partner nucleosome remodeling factor lymphoid specific helicase. Mutations in these genes are associated with immunodeficiency, centromeric instability, and facial abnormality syndrome, which is characterized by DNA hypomethylation in the heterochromatin region.⁴⁴⁾ The nucleosome remodeling activity⁴⁵⁾ facilitates UHRF-1-dependent dual monoubiquitination of histone H3, thereby recruiting and activating DNMT1.⁴³⁾ These mechanisms of DNA methylation replication, particularly in heterochromatin, are likely responsible for the loss of DNA methylation during cellular proliferation. A recent report indicated that the loss of DNA methylation induced by the depletion of UHRF-1 and DNMT1 can lead to colorectal cancer cells undergoing non-canonical cellular senescence independent of p53 and p16/pRB.²⁸⁾ However, it has also been reported that reduced DNA methylation can promote cell proliferation in AML cells.⁴⁶⁾

Loss of DNA methylation in the heterochromatin region is likely to reactivate endogenous retroviral genes, which has been reported to induce cellular senescence (Fig. 3).^27),28) The reactivation of these retroviral genes has been detected in multiple organs of aged primates, mice, and even in human tissues and serum.²⁷⁾ Surprisingly, certain endogenous retroviruses are capable of being unlocked, thus facilitating the transcription of viral genes and the formation of retrovirus-like particles. These particles act as a transmissible factor, inducing senescence in surrounding cells. Indeed, senescent cells exhibit derepression of long interspersed nuclear element-1 retrotransposable elements, which in turn activates a interferon 1.⁴⁷⁾ Interferon 1 activation contributes to the maintenance of SASP in senescent cells. Taken together, captured age-related epigenetic variation in intrinsic aspects of the aging process may serve as a surrogate marker of multiple cellular and genomic processes. This includes, but is not limited to, the potential deterioration of mechanisms involved in maintaining the epigenome. However, clear experimental results showing that impaired DNA methylation replication mainly contributes to the reactivation of endogenous retroviruses have not yet emerged.

Fig. 3

The low fidelity of DNA methylation replication at heterochromatin regions leads to senescence induction through the reactivation of endogenous retroviral genes. Genomic information is maintained but DNA methylation information is lost during cell proliferation. The loss of DNA methylation at heterochromatin regions is likely to reactivate the transcription of retrotransposons and endogenous retroviral genes. These activations, in turn, induce cellular senescence.

4. Senolysis, a promising strategy for preventing aging and age-related diseases

Targeting of senescent cells as a strategy to ameliorate age-related dysfunction has been established from observations using various models to eliminate senescent cells in vivo. These models include INK-ATTAC mice, in which FK506-binding-protein-caspase 8 was inserted under control of the p16 gene promoter,^{14),15),48)-51)} 16-3MR mice in which the p16 promoter in the BAC cassette drives herpes simplex virus 1 thymidine kinase, and knock-in mouse models in which the exon of the endogenous p16 locus was replaced with a Cre^ERT2 cassette.^52)-54) However, a recent preprint revealed that the luminescence of p16-3MR mice appeared to be non-specific in some senescent models,⁵⁵⁾ suggesting a future re-evaluation of this system.

Senolytic drugs (senolytics) were first identified based on the observation that senescent cells are highly resistant to apoptosis.⁵⁶⁾ The anti-apoptotic cascade, also known as senescent cell anti-apoptotic pathways, was upregulated in senescent cells, and 46 molecules targeting senescent cell anti-apoptotic pathways were identified as potential senolytic candidates.⁵⁶⁾ Subsequently, dasatinib, a multi-receptor tyrosine kinase inhibitor; quercetin, a flavonoid polyphenol; and fisetin, a flavonoid analogous to quercetin, were identified as first-generation senolytics.⁵⁷⁾ Dasatinib + quercetin treatment has garnered significant scientific validation, with studies demonstrating its efficacy in addressing various health concerns, including frailty,^56),58) osteoporosis,⁵⁹⁾ hepatic stenosis,⁶⁰⁾ insulin resistance,⁶¹⁾ neurodegenerative diseases,⁶²⁾ exercise capacity,⁶³⁾ pulmonary fibrosis,⁶⁴⁾ and chronic kidney disease.⁶⁵⁾ Moreover, this treatment has been associated with an increase in life expectancy.^56),58)

p53 is a key regulator of apoptosis. The expression of forkhead box protein O4 (FOXO4) is increased in senescent cells and prevents apoptosis by inhibiting the nuclear translocation of p53.⁶⁵⁾ A FOXO4-DRI peptide contains a p53-binding domain of FOXO4 with a D-amino acid substitution that interferes with the interaction between endogenous FOXO4 and p53 and has senolytic activity.⁶⁶⁾

BCL-2 family proteins (BCL-2, BCL-XL, and BCL-W) are upregulated in senescent cells and contribute to apoptosis resistance. Consequently, BCL-2 family inhibitors, such as ABT-263 (Navitoclax), ABT-737, A1331852, and A1155463, have been identified as senolytic agents.⁶⁷⁾ Despite the wide range of applications, ABT-263 may cause severe thrombocytopenia as a side effect, which prevents its clinical use for senolysis.⁶⁸⁾ Proteolysis-targeting chimeras (PROTACs) technology has been developed to overcome the adverse effects of senolytics.^69),70) PZ15227, a PROTAC drug, has been engineered by tethering ABT-263, which targets BCL-XL to cereblon E3 ligase for degradation. PZ15227 has been shown to effectively clear senescent cells and rejuvenate tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia.⁷¹⁾ ARV825, a PROTAC drug for bromodomain family protein degrader, degrades BRD4 and shows senolytic effects.⁷²⁾ In addition to the aforementioned agents, the development of senolytic drugs is underway, with a focus on targeting diverse biological pathways.

In senescent cells, proteostasis dysfunction produces large amounts of the aggregated proteins.⁷³⁾ The excess of protein aggregates is likely to damage lysosomal membranes, leading to intracellular acidification. Intracellular acidification rapidly leads to BNIP3-mPTP-mediated apoptosis.^74),75) To avert this form of cell death, senescent cells upregulate glutaminase 1 (GLS1) expression, which produces ammonia. Excess ammonia can neutralize cytoplasmic acidosis, thereby enabling the survival of senescent cells⁷³⁾ (Fig. 4). Intriguingly, lysosomal membrane damage has been demonstrated to induce inflammatory secretory phenotypes.⁷⁶⁾ Consequently, GLS1 inhibitors may possess the capacity to eliminate not only senescent cells but also other cells that induce inflammation. Consistent with this, the administration of BPTES, a GLS1 inhibitor, to aged or diseased mice has been reported to improve several age-related dysfunctions and diseases.⁷³⁾

Fig. 4

Mechanism underlying the role of GLS1 in senescence survival. Even in conditions that do not cause stress, a significant proportion of nascent proteins are misfolded. Furthermore, senescent cells exhibit increased metabolic active, resulting in the production of greater quantities of nascent proteins compared with their normal counterparts. Thus, the surplus of nascent proteins is predisposed to generate additional misfolded proteins, which amass within lysosomes as protein aggregates. The subsequent accumulation of these aggregates within lysosomes engenders a state of intracellular acidosis. Intracellular acidosis stabilizes GLS1, which, in turn, produces excess ammonia and serves to neutralize the intracellular acidosis. This regulatory process is instrumental in preventing the lethality of senescent cells. In contrast, normal counterparts do not exhibit the same degree of accumulation of protein aggregates in lysosomes or the development of intracellular acidosis. Taken together, these data suggest that a transient inhibition of GLS1 selectively induces senescent cell death.

5. Immune surveillance of senescent cells

In recent years, a considerable body of research in animal models has demonstrated the close communication between senescent cells and the immune system.^77),78) It is widely accepted that the elimination of senescent cells is primarily orchestrated by the host’s innate and adaptive immune surveillance systems, comprising macrophages, natural killer (NK) cells, and cytotoxic T cells.^79),80) Therefore, compromised immune surveillance systems result in the accumulation of senescent cells and accelerate the aging process. SASP factors are generally associated with immunogenic phenotypes that promote self-elimination by host immune surveillance.^81)-83)

Immune surveillance of senescent cells is conducted by different types of immune cells, depending on the context of the tissue microenvironment.^82)-86) Senescent cells generate distinct ligands for corresponding immune cells, thereby facilitating their elimination, contingent upon the microenvironment. Senescent hepatic stellate cells in CCl₄-induced hepatitis exhibit an upregulation in the expression of major histocompatibility complex class I-related chains A and UL-16 binding protein 2, which in turn activate NK group 2, member D in NK cells.⁷⁹⁾ These cells then are targeted and eliminated by NK cells. The elimination of senescent premalignant hepatocytes, induced by oncogene activation, is orchestrated by a CD4-positive T cell-mediated immune response, which is activated by the secretion of SASP factors.⁸⁰⁾ In addition, recent findings revealed that senescent fibroblasts present in aged human skin are targeted by cytotoxic CD4-positive T cells in the maintenance of youthful skin.⁸⁷⁾

We recently found that naturally occurring senescent cells in various organs, including the liver, lungs, and kidneys are eliminated by cytotoxic CD8-positive T cells.⁸⁸⁾ However, we have also be aware that a subset of senescent cells exhibits a high degree of resistance to host immune surveillance. We and others found that senescent cells exhibit heterogeneous expression of the immune checkpoint protein PD-L1.^88),89) This heterogeneous expression of PD-L1 appears to be due, at least in part, to impaired protein degradation systems.^88),90),91) Another immune checkpoint molecule, PD-L2, has also been reported to be upregulated in several types of senescent cancer cells following chemotherapy.⁹²⁾ The expression of PD-L2 in senescent tumor cells has been shown to result in resistant tumor immunity, because PD-L2-deficient senescent tumor cells fail to recruit immune suppressor cells, and the tumor undergoes regression by CD8^＋ T cells. Taken together, the expression of immune checkpoint molecules in senescent cells has been demonstrated to result in the evasion of these cells from the host immune surveillance system. Consistent with these observations, we found that the administration of an anti-PD-1 antibody promoted immune surveillance of senescent cells and improved several age-related organ dysfunctions.⁸⁸⁾ A recent finding has indicated that lipopolysaccharide-induced p16-positive alveolar macrophages exhibit elevated levels of PD-L1.⁹¹⁾ The engagement of Fcγ receptors by an anti-PD-L1 antibody, but not an anti-PD-1 antibody, has been shown to reduce the proportion of short-term lipopolysaccharide-induced p16-positive alveolar macrophages. In this regard, it should be noted that anti-PD-1 and anti-PD-L1 antibodies exhibited distinct Fcγ receptor dependency, and that anti-PD-L1 antibody engagement of Fcγ receptors led to a reduction in a specific type of macrophages within the tumor microenvironment.⁹³⁾ In addition, an ongoing debate exists concerning the classification of p16-positive macrophages as senescent or activated macrophages.⁹⁴⁾ Taken together, the effects of these two antibodies on p16-positive cells may vary depending on the duration of administration and the specific target cell types.

6. Senescent cells and cancer

Japan is currently experiencing an increase in the number of cancer patients, many of whom are treated with anti-cancer drugs. Chemotherapy activates the senescence program in cancer cells, leading to the induction of tumor senescent cells. The elimination of these tumor senescent cells is a crucial aspect of the host immune system during malignant transformation, as evidenced by a mouse carcinoma model.⁹⁵⁾ The secretion of SASP factors by tumor senescent cells induced by anti-cancer therapy or radiation therapy has been demonstrated to stimulate the proliferation of surrounding cancer cells and promote tumor progression. However, they also recruit various immunocompetent cells into tumors and enhance tumor immunity. In this regard, tumor senescent cells possess a high degree of immunogenicity due to alterations in the cell surface proteome status of tumor cells. These alterations, in turn, activate IFN signaling and major histocompatibility complex-I antigen presentation, thereby activating cytotoxic CD8-positive T cells.^96),97) Therefore, tumor senescent cells are likely to function as a part of anti-tumor immunity.

Recently, the impact of senescent cancer stromal cells in the microenvironment, particularly cancer-associated fibroblasts (CAFs), has garnered significant attention. CAFs represent a significant stromal component within solid tumors. A subpopulation of these cells may promote cancer progression.⁹⁸⁾ Our research revealed that senescent CAFs accumulate in the mouse bladder with age⁹⁹⁾ and create a tumor-permissive niche by secreting CXC motif chemokine ligand 12.¹⁰⁰⁾ Notably, elimination of senescent cancer stromal cells has been shown to impede the proliferation of bladder tumors. Thus, the targeting of senescent CAFs emerges as a potentially efficacious strategy to impede bladder tumor growth. Furthermore, an attempt was made to identify the transcriptomic signature of human senescent CAFs in the bladder to predict the prognostic value for patients with bladder cancer. Then, an investigation was conducted to ascertain the association between the human senescent CAF signature identified in mouse senescent CAFs in the bladder and relevant clinical parameters. The findings, which were based on data from TCGA, revealed a significant association between the signature and various factors, including age, overall survival, metastasis, recurrence, the pathological T stage, and grade of cancer cells in human bladder cancer. The hazard ratio of the signature was equivalent to that of the pathological T stage, suggesting that the signature may possess clinical relevance for prognostic prediction.¹⁰⁰⁾

The significance of senescent CAFs in cancer progression has been documented in other organs as well. In the context of pancreatic ductal adenocarcinoma (PDAC), senescent CAFs have been observed to promote tumor fibrosis, immunosuppressive function of tumor-associated macrophages, and T cell dysfunction, thereby enhancing PDAC progression.¹⁰¹⁾ Senescent CAFs in PDAC also impede the activation of CD8-positive T cells, thereby diminishing the effectiveness of immunotherapy.¹⁰²⁾ In the context of breast cancer, senescent CAFs limit the functionality of NK cell activity, therefore promoting cancer progression. The presence of senescent CAFs correlates with cancer recurrence and poor prognosis in human patients.¹⁰³⁾ Senescent CAFs can facilitate the accumulation of tumor-associated neutrophils and impede T-cell infiltration in lung adenocarcinoma.¹⁰⁴⁾ Although the population of senescent CAFs is generally a minor within the tumor microenvironment, these cells appear to promote the progression of various types of cancer. Consequently, stromal senescent CAFs may also serve as a target for cancer therapies (Fig. 5).

Fig. 5

Senotherapeutics may be effective in suppressing cancer growth. The impact of senescent cancer stromal cells on cancer growth is a multifaceted phenomenon, exhibiting both positive and negative effects. Senotherapeutics targeting senescent stromal cells may be a promising strategy for anticancer therapy.

7. Impaired protein quality control in neurodegenerative diseases

A plethora of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), have been identified.¹⁰⁵⁾ These diseases are characterized by the accumulation of misfolded proteins, which can be triggered by specific microenvironmental stresses within the brain.^106),107) The misfolded proteins are likely to form protein aggregates in neurons that are partially resistant to known proteolytic pathways and form inclusion bodies and extracellular plaques.¹⁰⁸⁾ Neurons exhibit a heightened vulnerability to these aggregates in comparison to dividing cells, as the presence of toxic aggregates within neurons hinders the capacity for dilution through cell division.¹⁰⁹⁾ Furthermore, neurons possess extended neurites, which necessitate the long-distance transportation of misfolded proteins to lysosomes. Given the post-mitotic nature of neuron survival and their capacity for prolonged survival, it is reasonable to hypothesize the presence of specialized protective mechanisms that are designed to detect and remove toxic aggregates. Thus, we postulated that senescent cells, which have been shown to contain protein aggregates as described above, might also employ systems analogous to those of neurons.

An investigation into differentially expressed genes between normal human fibroblasts before and after the induction of senescence as a post-mitotic model yielded the identification of LONRF2 as a gene that is upregulated in parallel with the mitotic to post-mitotic transition.¹¹⁰⁾ Experiments with artificially induced protein aggregates have demonstrated that LONRF2 selectively ubiquitylates aggregated but not natural proteins. The LONRF2 protein is notable for its possession of two distinct domains, the RING finger and the LON substrate binding domains. Both of these domains are indispensable for the binding and ubiquitylation of misfolded proteins. The latter domain was initially identified as a misfolded protein binding domain in bacterial LON protease, which selectively degrades misfolded proteins.^111),112) The LONRF family of proteins consists of LONRF1, 2 and 3. Notably, although LONRF1 and LONRF3 are ubiquitously expressed in various organs, LONRF2 is predominantly expressed in mature neurons. The product of Lonrf2 has the capacity to bind selectively to and ubiquitylate misfolded TDP43 and hnRNP M1 proteins. These proteins have been observed to aggregate in neurons in conditions associated with ALS and frontotemporal lobar degeneration.¹⁰⁾

LONRF2-knockout mice exhibited age-dependent motor neuron degeneration and cerebellar ataxia phenotypes, such as reduced muscle strength and motor function deficits. These phenomena presumably result from reduced spinal motor neuron viability, Purkinje cell viability, and granular and molecular layer thickness in the cerebellum.¹¹⁰⁾ These phenotypes are substantiated by the neuropathological evidence of an increased number of neurons with aggregates of ataxin 2, phospho- TAR DNA binding protein 43, and G3BP1 in the spinal cord, cerebellum, and cortex. Furthermore, the loss of LONRF2 function in motor neurons derived from induced pluripotent stem (iPS) cells resulted in reduced cell survival. Thus, LONRF2-knockout mice exhibit characteristics analogous to those observed in ALS.¹¹³⁾ These findings provide in vivo evidence that LONRF2 plays a key role in protein quality control in neurons and protects against neurodegenerative diseases. Most intriguingly, ectopic expression of LONRF2 may restore the abnormalities seen in motor neurons derived from human iPS cells established from ALS patients.¹¹⁰⁾ The analysis of motor neurons derived from ALS-hiPS cells revealed that those from patients with ALS exhibited reduced maximum neurite lengths. However, these neurites were maintained at greater lengths in Lonrf2-expressing cells. Taken together, LONRF2 in conjunction with LONRF1 and LONRF3, emerges as a potentially efficacious therapeutic agent for the treatment of neurodegenerative diseases, including ALS.

8. Conclusions

A plethora of evidence suggests that the process of aging is accompanied by systemic chronic inflammation.¹¹⁴⁾ The factors that regulate the fundamental mechanisms of aging have been proposed as the “hallmarks of aging”.¹¹⁵⁾ These include genomic instability, progenitor cell depletion/dysfunction, telomere and epigenetic changes, deregulated protein homeostasis, altered nutrient sensing, mitochondrial dysfunction, altered cell-cell communication, chronic low-grade inflammation, fibrosis, deregulated microbiota, and cellular senescence. As indicated by previous research, the factors of aging, including, but not limited to, cellular senescence, tend to progress in concert and encompass the pathophysiology of multiple diseases. These include, but are not limited to, age-related dysfunction (including frailty, immobility, sarcopenia/muscle loss, mild cognitive impairment, urinary incontinence, and other geriatric syndromes), and a decline in resilience (a reduced ability to recover from stress such as injury, surgery, chemotherapy, infection, or a reduced antibody response to vaccination).¹¹⁶⁾ The concept of “explaining aging with a unified theory” posits that interventions targeting a single fundamental mechanism may also prove effective in addressing other mechanisms. For instance, interventions that target cellular senescence (senolysis and reinforcement of immune surveillance) may result in a reduction of inflammation, a decrease in progenitor exhaustion, a decline in fibrosis, a reduction in mitochondrial dysfunction, and a partial restoration of the microbiota in experimental animal models of aging and chronic disease. In addition, the use of senomorphics, which function by curtailing SASP to induce senostasis, has emerged as a promising strategy to suppress chronic tissue microinflammation and ameliorate various age-related dysfunctions.¹¹⁷⁾

Although senolysis is applicable to senescent cells in most organs, it is not well suited for non-dividing terminally differentiated cells such as neurons. In this context, the targeting of protein aggregation emerged as a promising therapeutic approach for the treatment of neurodegenerative diseases. As the accumulation of misfolded protein aggregates is a hallmark of aging cells, including senescent cells and aged neurons, the targeting of misfolded proteins, such as selective degradation and suppression of their accumulation, emerges as a promising alternative approach to prevent aging. Finally, even if preclinical data are promising, senolytic drugs, senescent cell-associated inflammation-inhibiting technologies and misfolded protein degradation technologies should not be recommended for the prevention or treatment of disease in the marketplace or in clinical practice unless or until the safety and efficacy of these technologies have been thoroughly substantiated through meticulously designed and rigorous clinical trials. It has been documented that excessive elimination of senescent cells can result in detrimental effects in the long term or in specific pathological contexts.^12)-23) In other words, it is imperative to optimize the administration of senolytics, taking into account variables such as dosage, duration, and the specific types of senescent cells targeted. Concurrently, experimental findings employing animal models and preclinical studies are yielding valuable data and insights into the role of cellular senescence as a therapeutic target for age-related diseases. These findings have the potential to facilitate the clinical application of these technologies in the near future.

Acknowledgements

We thank Dr. Yoshikazu Johmura and Dr. Teh-Wei Wang for critical discussions. This study was supported by the Japan Agency for Medical Research and Development (AMED) under grant nos. JP23zf0127003h, JP23gm1410013h, JP20gm5010001s, and JP20ck010655h; MEXT/Japan Society for the Promotion of Science KAKENHI under grant nos. JP20H00514, JP20K21497, and JP19H05740; and the Princess Takamatsu Cancer Research Fund.

Conflict of interest

M.N. is a scientific advisor and shareholder at reverSASP Therapeutics.

Notes

Edited by Shigekazu NAGATA, M.J.A.

Correspondence should be addressed to: M. Nakanishi, Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan (e-mail: [e-mail]).

References

• 1)  Alzeer, J. (2022) Halalopathy: Role of entropy in the aging process. Am. J. Biomed. Sci. Res. 16, 147-154.
• 2)  Jones, O.R., Scheuerlein, A., Salguero-Gomez, R., Camarda, C.G., Schaible, R., Casper, B.B. et al. (2014) Diversity of ageing across the tree of life. Nature 505, 169-173.
• 3)  Ruby, J.G., Smith, M. and Buffenstein, R. (2018) Naked mole-rat mortality rates defy Gompertzian laws by not increasing with age. Elife 7, e31157.
• 4)  Reinke, B.A., Cayuela, H., Janzen, F.J., Lemaitre, J-F., Gaillard, J-M, Lawing A.M. et al. (2022) Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity. Science 376, 1459-1466.
• 5)  Da Silva, R., Conde, D.A., Baudisch, A. and Colchero F. (2022) Slow and negligible senescence among testudines challenges evolutionary theories of senescence. Science 376, 1466-1470.
• 6)  Walford, R.L. (1969) The immunologic theory of aging. Gerontologist 4, 195-197.
• 7)  Teissier, T., Boulanger, E. and Cox, L.S. (2022) Interconnections between Inflammageing and Immunosenescence during ageing. Cells 11, 359.
• 8)  Baechle, J.J., Chen, N., Makhijani, P., Winer, S., Furman, D. and Winer D.A. (2023) Chronic inflammation and the hallmarks of aging. Mol. Metab. 74, 101755.
• 9)  Arai, Y., Martin-Ruiz, C.M., Takayama, M., Abe, Y., Takebayashi, T., Koyasu, S. et al. (2015) Inflammation, but not telomere length, predicts successful ageing at extreme old age: a longitudinal study of semi-supercentenarians. eBioMedicine 2, 1549-1558.
• 10)  Saito, Y., Yamamoto, S. and Chikenji, T.S. (2024) Role of cellular senescence in inflammation and regeneration. Inflamm. Regen. 44, 28.
• 11)  DiMicco, R., Krizhanovsky, V., Baker D. and d'Adda di Fagagna, F. (2021) Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat. Rev. Mol. Cell Biol. 22, 75-95.
• 12)  Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell. Res. 25, 585-621.
• 13)  Coppe, J-P., Desprez, P-Y., Krtolica, A. and Campisi, J. (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99-118.
• 14)  Baker, D.J., Wijshake, T., Tchkonia, T., LeBrasseur, N.K., Child, B.G., van de Sluis, B. et al. (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232-236.
• 15)  Baker D.J., Childs, B.G., Durik, M., Wijers, M.E., Sieben, C.J., Zhong, J. et al. (2016) Naturally occurring p16^Ink4a-positive cells shorten healthy lifespan. Nature 530, 184-189.
• 16)  Shive, C. and Pandiyan, P. (2022) Inflammation, immune senescence, and dysregulated immune regulation in the elderly. Front. Aging 3, 840827.
• 17)  Born, E., Lipskaia, L., Breau, M., Houssaini, A., Beaulieu, D., Marcos, E. et al. (2023) Eliminating senescent cells can promote pulmonary hypertension development and progression Circulation 147, 650-666.
• 18)  Reyes, N.S., Krasilnikov, M., Allen, N.C., Lee, J.Y., Hyams, B., Zhou, M. et al. (2022) Sentinel p16^Ink4a＋ cells in the basement membrane form a reparative niche in the lung. Science 378, 192-201.
• 19)  Wang, J., Lu, X., Sakk, V., Klein, C.A. and Rudolph, K.L. (2014) Senescence and apoptosis block hematopoietic activation of quiescent hematopoietic stem cells with short telomeres. Blood 124, 3237-3240.
• 20)  Chiche, A., Le Roux, I., von Joest, M., Sakai, H., Aguin, S.B., Cazin, C. et al. (2017) Injury-induced senescence enables in vivo reprogramming in skeletal muscle. Cell Stem Cell 20, 407-414.
• 21)  Helman, A., Klochendler, A., Azazmeh, N., Gabai, Y., Horwitz, E., Anzi, S. et al. (2016) p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion. Nat. Med. 22, 412-420.
• 22)  Grosse, L., Wagner, N., Emelyanov, A., Molina, C., Lacas-Gervais, S., Wagner, K-D. et al. (2020) Defined p16^High senescent cell types are indispensable for mouse healthspan. Cell Metab. 32, 87-99.e6.
• 23)  Zhao, H., Liu, Z., Chen, H., Han, M., Zhang, M., Liu, K. et al. (2024) Identifying specific functional roles for senescence across cell types. Cell 187, 7314-7334.
• 24)  Bell, C.G., Lowe, R., Adams, P.D., Baccarelli, A.A., Beck, S., Bell, J.T. et al. (2019) DNA methylation aging clocks: challenges and recommendations. Genome Biol. 20, 249.
• 25)  Xiao, F-H., Wang, H-T. and Kong, Q-P. (2019) Dynamic DNA methylation during aging: “Prophet” of age-related outcomes. Front. Genet. 10, 107.
• 26)  Groh, S. and Schotta, G. (2017) Silencing of endogenous retroviruses by heterochromain. Cell Mol. Life Sci. 74, 2055-2065.
• 27)  Liu, X., Liu, Z., Wu, Z., Ren, J., Fan, Y., Sun, L. et al. (2023) Resurrection of endogenous retroviruses during aging reinforces senescence. Cell 186, 287-304.
• 28)  Chen, X., Yamaguchi, K., Rodgers, B., Goerhig, D., Vindrieux, D., Lahaye, X. et al. (2024) DNA methylation protects cancer cells against non-canonical senescence. bioRxiv: 2024.08.23.609297.
• 29)  Nishiyama, A. and Nakanishi, M. (2021) Navigating the DNA methylation landscape of cancer. Trends Genet. 11, 1012-1027.
• 30)  Campisi, J. and d'Adda di Fagagna, F. (2007) Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell Biol. 8, 729-740.
• 31)  Shay, J.W., Pereira-Smith, O.M. and Wright, W.E. (1991) A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell. Res. 196, 33-39.
• 32)  Johmura, Y., Shimada, M., Misaki, T., Naiki-Ito, A., Miyoshi, H., Motoyama, N. et al. (2014) Necessary and sufficient role for a mitosis skip in senescence induction. Mol. Cell 55, 73-84.
• 33)  Kernan, J., Bonacci, T. and Emanuele, M. (2018) Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle. BBA-Mol. Cell Res. 1865, 1924-1933.
• 34)  Li, M. and Zhang, P. (2009) The function of APC/C^Cdh1 in cell cycle and beyond. Cell Div 4, 2.
• 35)  Zeng, J., Hills, S.A., Ozono, E. and Diffley, J.F.X. (2023) Cyclin E-induced replicative stress drives p53-dependent whole-genome duplication. Cell 186, 528-542.
• 36)  Freund, A., Patil, C.K. and Campisi, J. (2011) p38 MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. EMBO J. 30, 1536-1548.
• 37)  Johmura, Y., Sun, J. Kitagawa, K., Nakanishi, K., Kuno, T., Naiki-Ito, A. et al. (2016) SCF^Fbxo22-KDM4A targets methylated p53 for degradation and regulates senescence. Nat. Commun. 7, 10574.
• 38)  Greenberg, M.V.C. and Bouc'his, D. (2019) The diverse roles of DNA methylation in mammalian development and disease. Nat. Rev. Mol. Cell Biol. 20, 590-607.
• 39)  Petryk, N., Bultmann, S., Bartke, T. and Defossez, P-A. (2021) Staying true to yourself. Mechanisms of DNA methylation maintenance in mammals. Nucleic Acid Res. 49, 3020-3032.
• 40)  Nishiyama, A., Yamaguchi, L., Sharif, J., Johmura, Y., Kawamura, T., Nakanishi, K. et al. (2013) Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 502, 249-253.
• 41)  Ishiyama, S., Nishiyama, A., Saeki, Y., Moritsugu, K., Morimoto, D., Yamaguchi, L. et al. (2017) Structure of the Dnmt1 reader module complexed with a unique two-mono-ubiquitin mark on histone H3 reveals the basis for DNA methylation maintenance. Mol. Cell 68, 350-360.
• 42)  Nishiyama, A., Mulholland, C.B., Bultmann, S., Kori, S., Endo, A., Saeki, Y. et al. (2020) Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation. Nat. Commun. 11, 1222.
• 43)  Wassing, I.E., Nishiyama, A., Shikimachi, R., Jia, Q., Kikuchi, A., Hiruta, M. et al. (2024) CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes. Sci. Adv. 10, eadp5753.
• 44)  Unoki, M., Funabiki, H., Velasco, G., Francastel, C. and Sasaki, H. (2019) CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome. J. Clin. Invest. 129, 78-92.
• 45)  Jenness, C., Giunta, S., Muller, M.M., Kimura, H., Muir, T.W. and Funabiki, H. (2018) HELLS and CDCA7 comprise a bipartite nucleosome remodeling complex defective in ICF syndrome. Proc. Natl. Acad. Sci. U.S.A. 115, E876-E885.
• 46)  Yang, L., Rau, R. and Goodell, M.A. (2015) DNMT3A in haematological malignancies. Nat. Rev. Cancer 15, 152-165.
• 47)  De Cecco, M., Ito, T., Petrashen, A.P., Elias, A.E., Skvir, N.J., Criscione, S.W. et al. (2019) L1 drives IFN in senescent cells and promotes age-associated inflammation. Nature 556, 73-78.
• 48)  Childs, B.G., Baker, D.J., Wijshake, T., Conover, C.A., Campisi, J. and Van Deursen, J.M. (2016) Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 354, 6311.
• 49)  Sturmlechner, I., Durik, M., Sieben, C., Baker, D. and Van Deursen, J.M. (2017) Cellular senescence in renal ageing and disease. Nat. Rev. Neph. 13, 77-89.
• 50)  Bussian, T.J., Aziz, A., Meyer, C.F., Swenson, B.L., van Deursen, J.M. and Baker, D.J. (2018) Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature 562, 578-582.
• 51)  Childs, B.G., Zhang, C., Shuja, F., Sturmlechner, I., Trewartha, S., Velasco, R.F. et al. (2021) Senescent cells suppress innate smooth muscle cell repair functions in atherosclerosis. Nat. Aging 1, 698-714.
• 52)  Jeon, O.K., Kim, C., Laberge, R-M., Demaria, M., Rathod, S., Vasserot, A.P. et al. (2017) Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med. 23, 775-781.
• 53)  Demaria, M., O'Leary, M.N., Chang, J., Shao, L., Liu, S., Alimirah, F. et al. (2017) Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7, 165-176.
• 54)  Moiseeva, V., Cisneros, A., Sica, V., Deryagin, O., Lai, Y., Jung, S. et al. (2023) Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration. Nature 613, 169-178.
• 55)  Hori, N., Kawamoto, S., Uemura, K., Okumura, Y., Tanaka, K., Park, J.H. et al. (2024) Shaving back fur uncovers hidden issues in p16-3MR mice. bioRxiv: 2024.06.24.600181.
• 56)  Zhu, Y., Tchkonia, T., Pirtskhalava, T., Gower, A.C., Ding, H., Giorgadze, N. et al. (2015) The Achilles' heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 14, 644-658.
• 57)  Zhu, Y., Doornebal, E.J., Pirtskhalava, T., Giorgadze, N., Wentworth, M., Fuhrmann-Stroissnigg, H. et al. (2017) New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463. Aging 9, 955-963.
• 58)  Xu, M., Pirtskhalava, T., Farr, J.N., Weigand, B.M., Palmer, A.K., Weivoda, M.M. et al. (2018) Senolytics improve physical function and increase lifespan in old age. Nat. Med. 24, 1246-1256.
• 59)  Farr, J.N., Xu, M., Welvoda, M.M., Monroe, D.G., Fraser D.G., Onken, J.N. et al. (2017) Targeting cellular senescence prevents age-related bone loss in mice. Nat. Med. 23, 1072-1079.
• 60)  Ogrodnik, M., Miwa, S., Tchkonia, T., Tinlakos, D., Wilson, C.L., Lahat, A. et al. (2017) Cellular senescence drives age-dependent hepatic steatosis. Nat. Commun. 8, 15691.
• 61)  Palmer, A.K., Xu, M., Zhu, Y., Pirtskhalava, T., Weivoda, M.M., Hachfeld, C.M. et al. (2019) Targeting senescent cells alleviates obesity-induced metabolic dysfunction. Aging Cell 18, e12950.
• 62)  Zhang, P., Kishimoto, Y., Grammatikakis, I., Gottimukkala, K., Cutler, R.G., Zhang, S. et al. (2019) Senolytic therapy alleviates Ab-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model. Nat. Neurosci. 22, 719-728.
• 63)  Schafer, M.J., White, T.A., Iijima, K., Haak, A.J., Ligresti, G., Atkinson, E.J. et al. (2017) Cellular senescence mediates fibrotic pulmonary diseases. Nat. Commun. 8, 14532.
• 64)  Kim, S.R., Puranik, A.S., Jiang, K., Chen, X., Zhu, X.Y., Taylor, I. et al. (2021) Progressive cellular senescence mediates renal dysfunction in ischemic nephropathy. J. Am. Soc. Nephrol. 32, 1987-2004.
• 65)  Bourgeois, B. and Madl, T. (2018) Regulation of cellular senescence via the FOXO4-p53 axis. FEBS Lett. 592, 2083-2097.
• 66)  Baar, M.P., Brandt, R.M., Putavet, D.A., Klein, J.D., Derks, K.W., Bourgeois, B.R. et al. (2017) Targeted apoptosis of senescent cells restores tissue homeostasis in response to chemotoxicity and aging. Cell 169, 132-147.
• 67)  Yosef, R., Pilpel, N., Tokarsky-Amiel, R., Biran, A., Ovadya, Y., Cohen, S. et al. (2016) Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL. Nat. Commun. 7, 11190.
• 68)  Vogler, M., Hamali, H.A., Sun, X.M., Bampton, E.T., Dinsdale, D., Showden, R.T. et al. (2011) BCL2/BCL-XL inhibition induces apoptosis disrupts cellular calcium homeostasis and prevents platelet activation. Blood 117, 7145-7154.
• 69)  Lai, A.C. and Crews, C.M. (2017) Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101-114.
• 70)  Burslem, G.M. and Crews, C.M. (2020) Proteolysis-targeting chimeras as therapeutics and tools for biological discovery. Cell 181, 102-114.
• 71)  He, Y., Zhang, X., Chang, J., Kim, H.N., Zhang, P., Wang, Y. et al. (2020) Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity. Nat. Commun. 11, 1996.
• 72)  Wakita, M., Takahashi, A., Sano, O., Loo, T.M., Imai, Y., Narukawa, M. et al. (2020) BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells. Nat. Commun. 11, 1935.
• 73)  Johmura, Y., Yamanaka, T., Omori, S., Wang, T.W., Sugiura, Y., Matsumoto, M. et al. (2021) Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders. Science 371, 265-270.
• 74)  Velde, C.V., Dubik, J.C.D., Alimonti, J., Brown, T., Israel, S., Hakem, R. et al. (2000) BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol. Cell. Biol. 20, 5454-5468.
• 75)  Kubasiak, L.A., Hernandez, O.M., Bishopric, N.H., and Webster, K.A. (2002) Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc. Natl. Acad. Sci. U.S.A. 99, 12825-12830.
• 76)  Roh, K., Noh, J., Kim, Y., Jang, Y., Kim, J., Choi, H. et al. (2023) Lysosomal control of senescence and inflammation through cholesterol partitioning. Nat. Metab. 5, 398-413.
• 77)  Carrasco, E., Gomez de las Heras, M.M., Gabande-Rodriguez, E., Desdin-Mico, G., Aranda, J.F. and Mittelbrunn, M. (2021) The role of T cells in age-related diseases. Nat. Rev. Immunol. 22, 97-111.
• 78)  Yousefzadeha, M.J., Flores, R.R., Zhu, Y., Schmiechen, Z.C., Brooks, R.W., Trussoni, C.E. et al. (2021) An aged immune system drives senescence and ageing of solid organs. Nature 594, 100-105.
• 79)  Krizhanovsky, V., Yon, M., Dickins R.A., Hearn, S., Simon, H., Miething, C. et al. (2008) Senescence of activated stellate cells limits liver fibrosis. Cell 134, 657-667.
• 80)  Kang, T-W., Yevsa, T., Woller, N., Hoenicke, L., Wuestefeld, T., Dauch, D. et al. (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 470, 547-551.
• 81)  Wang, T-W. and Nakanishi, M. (2024) Immune surveillance of senescence: potential application to age-related diseases. Trends Cell Biol. 35, 248-257.
• 82)  Ovadya, Y. and Krizhanovsky, V. (2018) Strategies targeting cellular senescence. J. Clin. Invest. 128, 1247-1257.
• 83)  Prata, L.G.P.L., Ovsyannikova, I.G., Tchkonia, T. and Kirkland, J.L. (2019) Senescent cell clearance by the immune system: Emerging therapeutic opportunities. Semin. Immunol. 40, 101275.
• 84)  Burton, D.G.A. and Krizhanovsky, V. (2014) Physiological and pathological consequences of cellular senescence. Cell Mol. Life Sci. 71, 4373-4386.
• 85)  Kale, A., Sharma, A., Stolzing, A., Desprez, P-Y. and Campisi, J. (2020) Role of immune cells in the removal of deleterious senescent cells. Immun. Ageing 17, 16.
• 86)  Arora, S., Thompson, P.J., Wang, Y., Bhattacharyya, A., Apostolopoulou, H., Hatano, R. et al. (2021) Invariant natural killer T cells coordinate removal of senescent cells. Med 2, 938-950.
• 87)  Hasegawa, T., Oka, T., Son, H.G., Oliver-Garcia, V.S., Azin, M., Eisenhaure, T.M. et al. (2023) Cytotoxic CD4^＋ T cells eliminate senescent cells by targeting cytomegalovirus antigen. Cell 186, 1417-1431.
• 88)  Wang, T-W., Johmura, Y., Suzuki, N., Omori, S., Migita, T., Yamaguchi, K. et al. (2022) Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes. Nature 611, 358-364.
• 89)  Onorati, A., Havas, A.P., Lin, B., Rajagopal, J., Sen, P., Adams, P.D. et al. (2022) Upregulation of PD-L1 in senescence and aging. Mol. Cell. Biol. 42, e0017122.
• 90)  Janelle, V., Neault, M., Level, M-E., De Sousa, D.M., Boulet, S., Durrieu, L. et al. (2021) p16INK4a regulates cellular senescence in PD-1-expressing human T cells. Front. Immunol. 12, 698565.
• 91)  Majewska, J., Agrawal, A., Mayo, A., Roitman, L., Chatterjee, R., Kralova, J.S. et al. (2024) p16-dependent increase of PD-L1 stability regulates immunesurveillance of senescent cells. Nat. Cell. Biol. 26, 1336-1345.
• 92)  Chaib, S., Lopez-Dominguez, J.A., Lalinde-Gutierrez, M., Prats, N., Marin, I., Boix, O. et al. (2024) The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2. Nat. Cancer 5, 448-462.
• 93)  Dahan, R., Sega, E., Engelhardt, J., Selby, M., Korman, A.J. and Ravetch, J.V. (2015) FcγRs modulate the anti-tumor activity of antibodies targeting the PD-1/PD-L1 axis. Cancer Cell 28, 285-295.
• 94)  Hall, B.M., Balan, V., Gleiberman, A.S., Strom, E., Krasnov, P., Virtuoso, L.P. et al. (2017) p16(Ink4a) and senescence-associated b-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli. Aging 9, 1867-1884.
• 95)  Ventura, A., Kirsch, D.G., McLaughlin, M.E., Tuveson, D.A., Grimm, J., Lintault, L. et al. (2007) Restoration of p53 function leads to tumour regression in vivo. Nature 445, 661-665.
• 96)  Marin, I., Boix, O., Garcia-Garijo, A., Sirois, I., Caballe, A., Zarzuela, E. et al. (2023) Cellular senescence is immunogenic and promotes antitumor immunity. Cancer Discov. 13, 410-431
• 97)  Chen, H-A., Ho, Y-J., Mezzadra, R., Adrover, J.M., Smolkin, R., Zhu, C. et al. (2023) Senescence rewires microenvironment sensing to facilitate antitumor immunity. Cancer Discov. 13, 432-453.
• 98)  Yang, D., Liu, J., Qian, H. and Zhuang, Q. (2023) Cancer-associated fibroblasts: from basic science to anticancer therapy. Exp. Mol. Med. 55, 1322-1332.
• 99)  Omori, S., Wang, T-W., Johmura, Y., Kanai, T., Nakano, Y., Kido, T. et al. (2020) Generation of a p16 reporter mouse and its use to characterize and target p16^high cells in vivo. Cell Metab. 32, 814-828.
• 100)  Meguro, S., Johmura, Y., Wang, T-W., Kawakami, S., Tanimoto, S., Omori, S. et al. (2024) Preexisting senescent fibroblasts in the aged bladder create a tumor-permissive niche through CXCL12 secretion. Nat. Aging 4, 1582-1597.
• 101)  Belle, J.I., Sen, D., Baer, J.M., Liu, X., Lander, V.E., Ye, J. et al. (2024) Senescence defines a distinct subset of myofibroblasts that orchestrates immunosuppression in pancreatic cancer. Cancer Discov. 14, 1324-135.
• 102)  Assouline, B., Kahn, R., Hodali, L., Condiotti, R., Engel, Y., Elyada, E. et al. (2024) Senescent cancer-associated fibroblasts in pancreatic adenocarcinoma restrict CD8^＋ T cell activation and limit responsiveness to immunotherapy in mice. Nat. Commun. 15, 6162
• 103)  Ye, J., Baer, J.M., Faget, D.V., Morikis, V.A., Ren, Q., Melam, A. et al. (2024) Senescent CAFs mediate immunosuppression and drive breast cancer progression. Cancer Discov 14, 1302-1323.
• 104)  Lin, D., Zhai, X., Qi, X., Zhou, Q., Liu, Y., Lin, Y. et al. (2024) Senescent cancer-associated fibroblasts facilitate tumor associated neutrophil recruitment suppressing tumor immunity. J. Transl. Med. 22, 231.
• 105)  Lamptey, R.N.L., Chaulagain, B., Trivedi, R., Gothwal, A., Layek, B. and Singh, J. (2022) A review of the common neurodegenerative disorders: current therapeutic approaches and the potential role of nanotherapeutics. Int. J. Mol. Sci. 23, 1851.
• 106)  Wilson III, D.M., Cookson, M.R., Van Den Bosch, L., Zetterberg, H., Holtsman, D.M. and Dewachter, I. (2023) Hallmarks of neurodegenerative diseases. Cell 186, 693-714.
• 107)  Nakajima, Y. and Suzuki, S. (2013) Environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner. Int. J. Mol. Sci. 14, 7771-7783.
• 108)  Kopito, R. R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524-530.
• 109)  Cuanalo-Contreras, K., Schulz, J., Mukherjee, A., Park, K-W., Armijo, E. and Soto, C. (2023) Extensive accumulation of misfolded protein aggregates during natural aging and senescence. Front. Aging Neurosci. 14, 1090109.
• 110)  Li, D., Johmura, Y., Morimoto, S., Doi, M., Nakanishi, K., Ozawa, M. et al. (2023) LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits. Nat. Aging 3, 1001-1019.
• 111)  Gur, E. and Sauer, R.T. (2008) Recognition of misfolded proteins by Lon, a AAA+ protease. Genes Dev 22, 2267-2277.
• 112)  Lee, I. and Suzuki, C.K. (2008) Functional mechanics of the ATP-dependent Lon protease-lessons from endogenous protein and synthetic peptide substrates. BBA Proteins Proteom. 1784, 727-735.
• 113)  Ciechanover, A. and Kwon, Y.T. (2017) Protein quality control by molecular chaperones in neurodegeneration. Front. Neurosci. 11, 185.
• 114)  Franceschi, C., Bonafe, M., Valensin, S., Olivieri, F., De Luca, M., Ottaviani, E. et al. (2000) Inflamm-aging. An evolutionary perspective on immunosenescence. Ann. NY Acad. Sci. 908, 244-254.
• 115)  Lopez-Otin, C., Blasco, M., Partridge, L., Serrano, M. and Kroemer, G. (2023) Hallmarks of aging: an expanding universe. Cell 186, 243-278.
• 116)  Franceschi, C., Garagnari, P., Parini, P., Giuliani, C. and Santoro, A. (2018) Inflammaging: a new immune-metabolic viewpoint for age-related diseases. Nat. Rev. Endocrinol. 14, 576-590.
• 117)  Zhang, L., Pitcher, L.E., Prahalad, V., Niedernhofer, L.J. and Robbins, P.D. (2023) Targeting cellular senescence with senotherapeutics: senolytics and senomorphics. FEBS J. 290, 1362-1383.
• 118)  Lopez-Otin, C., Blasco, M.A., Partridge, L., Serrano, M. and Kroemer, G. (2023) Hallmarks of aging: An expanding universe. Cell 186, 243-278.

Non-standard abbreviation list

ALS

amyotrophic lateral sclerosis

CAF

cancer-associated fibroblast

DNMT1

DNA methyltransferase 1

FOXO4

forkhead box protein O4

GLS1

glutaminase 1

iPS

induced pluripotent stem

NK

natural killer

PDAC

pancreatic ductal adenocarcinoma

PROTAC

proteolysis-targeting chimeras

SASP

senescence-associated secretory phenotype

UHRF-1

ubiquitin-like PHD and RING finger domain-containing protein 1

Profile

Makoto Nakanishi was born in 1960 in Nagoya. He completed his medical education at Nagoya City University Medical School, obtaining his Doctor of Medicine degree in 1985. He subsequently pursued his academic career at the Graduate School of Medical Sciences, Nagoya City University, where he obtained his PhD in 1989. From 1993 to 1995, he held positions at Jichi Medical School as an assistant professor and at Baylor College of Medicine as a research associate. From 1996 to 1999, he served as head of the Geriatric Research Division at the National Center for Geriatrics and Gerontology. Prior to assuming the position of professor at the Institute of Medical Science, The University of Tokyo in April 2016, he held the position of professor in the Department of Cell Biology at Nagoya City University Medical School. He was elected to serve as Dean of the Institute of Medical Science, The University of Tokyo in 2023. His research is primarily focused on elucidating the mechanisms that regulate organismal aging. Notably, he identified a GLS1 inhibitor as a drug that selectively eliminates senescent cells and improves age-related organ dysfunction. Furthermore, he determined that PD-L1 expression plays a pivotal role in the age-dependent accumulation of senescent cells. For his accomplishment, he received the Award for Science and Technology, the Commendation for Science and Technology from the MEXT of Japan and the Academic Award of the Mochida Memorial Foundation.