Guidelines for minimal information on cellular senescence experimentation in vivo.

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.

Cellular Senescence: Defining a Path Forward.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.

Nuclear Genomic Instability and Aging.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

An aged immune system drives senescence and ageing of solid organs.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

miR-96-5p expression is sufficient to induce and maintain the senescent cell fate in the absence of stress.

Senescence is a cell fate driven by different types of stress that results in exit from the cell cycle and expression of an inflammatory senescence-associated secretory phenotype (SASP). Here, we demonstrate that stable overexpression of miR-96-5p was sufficient to induce cellular senescence in the absence of genotoxic stress, inducing expression of certain markers of early senescence including SASP factors while repressing markers of deep senescence including LINE-1 and type 1 interferons. Stable miR-96-5p overexpression led to genome-wide changes in heterochromatin followed by epigenetic activation of p16Ink4a, p21Cip1, and SASP expression, induction of a marker of DNA damage, and induction of a transcriptional signature similar to other senescent lung and endothelial cell types. Expression of miR-96-5p significantly increased following senescence induction in culture cells and with aging in tissues from naturally aged and Ercc1-/Δ progeroid mice. Mechanistically, miR-96-5p directly suppressed expression of SIN3B and SIN3 corepressor complex constituents KDM5A and MORF4L2, and siRNA-mediated knockdown of these transcriptional regulators recapitulated the senescent phenotype. In addition, pharmacologic inhibition of the SIN3 complex suppressed senescence and SASP markers. These results clearly demonstrate that a single microRNA is sufficient to drive early senescence in the absence of genotoxic stress through targeting epigenetic and transcriptional regulators, identifying novel targets for the development of senotherapeutics.

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

microRNA-449a reduces growth hormone-stimulated senescent cell burden through PI3K-mTOR signaling.

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including neurodegeneration, atherosclerosis, and metabolic disease. Therefore, investigating novel methods to reduce or delay the accumulation of senescent cells during aging may attenuate age-related pathologies. microRNA-449a-5p (miR-449a) is a small, noncoding RNA down-regulated with age in normal mice but maintained in long-living growth hormone (GH)-deficient Ames Dwarf (df/df) mice. We found increased fibroadipogenic precursor cells, adipose-derived stem cells, and miR-449a levels in visceral adipose tissue of long-living df/df mice. Gene target analysis and our functional study with miR-449a-5p have revealed its potential as a serotherapeutic. Here, we test the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli. We demonstrated that GH downregulates miR-449a expression and accelerates senescence while miR-449a upregulation using mimetics reduces senescence, primarily through targeted reduction of p16Ink4a, p21Cip1, and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and the progression of age-related pathologies.

Senolytic Drugs: Reducing Senescent Cell Viability to Extend Health Span.

Senescence is the consequence of a signaling mechanism activated in stressed cells to prevent proliferation of cells with damage. Senescent cells (Sncs) often develop a senescence-associated secretory phenotype to prompt immune clearance, which drives chronic sterile inflammation and plays a causal role in aging and age-related diseases. Sncs accumulate with age and at anatomical sites of disease. Thus, they are regarded as a logical therapeutic target. Senotherapeutics are a new class of drugs that selectively kill Sncs (senolytics) or suppress their disease-causing phenotypes (senomorphics/senostatics). Since 2015, several senolytics went from identification to clinical trial. Preclinical data indicate that senolytics alleviate disease in numerous organs, improve physical function and resilience, and suppress all causes of mortality, even if administered to the aged. Here, we review the evidence that Sncs drive aging and disease, the approaches to identify and optimize senotherapeutics, and the current status of preclinical and clinical testing of senolytics.

Osteoarthritis gene therapy in 2022.

PURPOSE OF REVIEW: To assess the present status of gene therapy for osteoarthritis (OA). RECENT FINDINGS: An expanding list of cDNAs show therapeutic activity when introduced into the joints of animals with experimental models of OA. In vivo delivery with adenovirus or adeno-associated virus is most commonly used for this purpose. The list of encoded products includes cytokines, cytokine antagonists, enzymes, enzyme inhibitors, growth factors and noncoding RNA. Elements of CRISPR-Cas have also been delivered to mouse knees to ablate key genes. Several human trials have been initiated, using transgenes encoding transforming growth factor-ß1, interleukin-1 receptor antagonist, interferon-ß, the NKX3.2 transcription factor or variant interleukin-10. The first of these, using ex vivo delivery with allogeneic chondrocytes, gained approval in Korea which was subsequently retracted. However, it is undergoing Phase III clinical trials in the United States. The other trials are in Phase I or II. No gene therapy for OA has current marketing approval in any jurisdiction. SUMMARY: Extensive preclinical data support the use of intra-articular gene therapy for treating OA. Translation is beginning to accelerate and six gene therapeutics are in clinical trials. Importantly, venture capital has begun to flow and at least seven companies are developing products. Significant progress in the future can be expected.

A report on the International Society for Cell & Gene Therapy 2022 Scientific Signature Series, "Therapeutic advances with native and engineered human extracellular vesicles".

The International Society for Cell & Gene Therapy Scientific Signature Series event "Therapeutic Advances With Native and Engineered Human EVs" took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.

Aging and chronic inflammation: highlights from a multidisciplinary workshop.

Aging is a gradual, continuous series of natural changes in biological, physiological, immunological, environmental, psychological, behavioral, and social processes. Aging entails changes in the immune system characterized by a decrease in thymic output of naïve lymphocytes, an accumulated chronic antigenic stress notably caused by chronic infections such as cytomegalovirus (CMV), and immune cell senescence with acquisition of an inflammatory senescence-associated secretory phenotype (SASP). For this reason, and due to the SASP originating from other tissues, aging is commonly accompanied by low-grade chronic inflammation, termed "inflammaging". After decades of accumulating evidence regarding age-related processes and chronic inflammation, the domain now appears mature enough to allow an integrative reinterpretation of old data. Here, we provide an overview of the topics discussed in a recent workshop "Aging and Chronic Inflammation" to which many of the major players in the field contributed. We highlight advances in systematic measurement and interpretation of biological markers of aging, as well as their implications for human health and longevity and the interventions that can be envisaged to maintain or improve immune function in older people.

Cellular Senescence in Obesity and Associated Complications: a New Therapeutic Target.

PURPOSE OF REVIEW: Obesity has increased worldwide recently and represents a major global health challenge. This review focuses on the obesity-associated cellular senescence in various organs and the role of these senescent cells (SnCs) in driving complications associated with obesity. Also, the ability to target SnCs pharmacologically with drugs termed senotherapeutics as a therapy for these complications is discussed. RECENT FINDINGS: Several studies have shown a positive correlation between obesity and SnC burden in organs such as adipose tissue, liver, and pancreatic-ß-cells. These SnCs produce several secretory factors which affect other cells and tissues in a paracrine manner resulting in organ dysfunction. The accumulation of SnCs in adipocytes affects their lipid storage and impairs adipogenesis. The inflammatory senescence-associated secretory phenotype (SASP) of SnCs downregulates the antioxidant capacity and mitochondrial function in tissues. Senescent hepatocytes cannot oxidize fatty acids, which leads to lipid deposition and senescence in ß-cells decrease function. These and other adverse effects of SnCs contribute to insulin resistance and type-2 diabetes. The reduction in the SnC burden genetically or pharmacologically improves the complications associated with obesity. The accumulation of SnCs with age and disease accelerates aging. Obesity is a key driver of SnC accumulation, and the complications associated with obesity can be controlled by reducing the SnC burden. Thus, senotherapeutic drugs have the potential to be an effective therapeutic option.

Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation.

Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model.

Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho.

Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Previously, we demonstrated that intratumoral delivery of adenoviral vector encoding single-chain (sc)IL-23 (Ad.scIL-23) was able to induce systemic antitumor immunity. Here, we examined the role of IL-23 in diabetes in nonobese diabetic mice. Intravenous delivery of Ad.scIL-23 did not accelerate the onset of hyperglycemia but instead resulted in the development of psoriatic arthritis. Ad.scIL-23-treated mice developed erythema, scales, and thickening of the skin, as well as intervertebral disc degeneration and extensive synovial hypertrophy and loss of articular cartilage in the knees. Immunological analysis revealed activation of conventional T helper type 17 cells and IL-17-producing γδ T cells along with a significant depletion and suppression of T cells in the pancreatic lymph nodes. Furthermore, treatment with anti-IL-17 antibody reduced joint and skin psoriatic arthritis pathologies. Thus, these Ad.scIL-23-treated mice represent a physiologically relevant model of psoriatic arthritis for understanding disease progression and for testing therapeutic approaches.-Flores, R. R., Carbo, L., Kim, E., Van Meter, M., De Padilla, C. M. L., Zhao, J., Colangelo, D., Yousefzadeh, M. J., Angelini, L. A., Zhang, L., Pola, E., Vo, N., Evans, C. H., Gambotto, A., Niedernhofer, L. J., Robbins, P. D. Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Signal Transduction, Ageing and Disease.

Ageing is defined by the loss of functional reserve over time, leading to a decreased tissue homeostasis and increased age-related pathology. The accumulation of damage including DNA damage contributes to driving cell signaling pathways that, in turn, can drive different cell fates, including senescence and apoptosis, as well as mitochondrial dysfunction and inflammation. In addition, the accumulation of cell autonomous damage with time also drives ageing through non-cell autonomous pathways by modulation of signaling pathways. Interestingly, genetic and pharmacologic analysis of factors able to modulate lifespan and healthspan in model organisms and even humans have identified several key signaling pathways including IGF-1, NF-κB, FOXO3, mTOR, Nrf-2 and sirtuins. This review will discuss the roles of several of these key signaling pathways, in particular NF-κB and Nrf2, in modulating ageing and age-related diseases.

Treating Age-Related Diseases with Somatic Stem Cells.

Life expectancy in the developed world has advanced beyond the number of years in which healthy tissue homeostasis can be maintained, and as a result, the number of persons with severe and debilitating chronic illnesses, including cancer, diabetes, osteoarthritis, osteoporosis, neurodegenerative and cardiovascular disease has continued to rise. One of the key underlying causes for the loss in the ability to replenish damaged tissues is the qualitative and quantitative decline in somatic stem cell populations. A concerted effort to understand why aging adult stem cells fail to maintain "stem" potential while simultaneously developing new strategies and therapeutic interventions to prevent or reverse age-dependent stem cell decline is required to improve the overall healthspan of our rapidly aging population. This review focuses on what drives stem cell dysfunction with age, the contribution of stem cell dysfunction in driving aging and therapeutic approaches using stem cells to treat aging.

Arsenic Promotes NF-Κb-Mediated Fibroblast Dysfunction and Matrix Remodeling to Impair Muscle Stem Cell Function.

Arsenic is a global health hazard that impacts over 140 million individuals worldwide. Epidemiological studies reveal prominent muscle dysfunction and mobility declines following arsenic exposure; yet, mechanisms underlying such declines are unknown. The objective of this study was to test the novel hypothesis that arsenic drives a maladaptive fibroblast phenotype to promote pathogenic myomatrix remodeling and compromise the muscle stem (satellite) cell (MuSC) niche. Mice were exposed to environmentally relevant levels of arsenic in drinking water before receiving a local muscle injury. Arsenic-exposed muscles displayed pathogenic matrix remodeling, defective myofiber regeneration and impaired functional recovery, relative to controls. When naïve human MuSCs were seeded onto three-dimensional decellularized muscle constructs derived from arsenic-exposed muscles, cells displayed an increased fibrogenic conversion and decreased myogenicity, compared with cells seeded onto control constructs. Consistent with myomatrix alterations, fibroblasts isolated from arsenic-exposed muscle displayed sustained expression of matrix remodeling genes, the majority of which were mediated by NF-κB. Inhibition of NF-κB during arsenic exposure preserved normal myofiber structure and functional recovery after injury, suggesting that NF-κB signaling serves as an important mechanism of action for the deleterious effects of arsenic on tissue healing. Taken together, the results from this study implicate myomatrix biophysical and/or biochemical characteristics as culprits in arsenic-induced MuSC dysfunction and impaired muscle regeneration. It is anticipated that these findings may aid in the development of strategies to prevent or revert the effects of arsenic on tissue healing and, more broadly, provide insight into the influence of the native myomatrix on stem cell behavior.