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The recent unprecedented progress in ageing research and drug discovery brings together fundamental research and clinical applications to advance the goal of promoting healthy longevity in the human population. We, from the gathering at the Aging Research and Drug Discovery Meeting in 2023, summarised the latest developments in healthspan biotechnology, with a particular emphasis on artificial intelligence (AI), biomarkers and clocks, geroscience, and clinical trials and interventions for healthy longevity. Moreover, we provide an overview of academic research and the biotech industry focused on targeting ageing as the root of age-related diseases to combat multimorbidity and extend healthspan. We propose that the integration of generative AI, cutting-edge biological technology, and longevity medicine is essential for extending the productive and healthy human lifespan.

Little is known about the possibility of reversing age-related biological changes when they have already occurred. To explore this, we have characterized the effects of reducing insulin/IGF-1 signaling (IIS) during old age. Reduction of IIS throughout life slows age-related decline in diverse species, most strikingly in the nematode Caenorhabditis elegans. Here we show that even at advanced ages, auxin-induced degradation of DAF-2 in single tissues, including neurons and the intestine, is still able to markedly increase C. elegans lifespan. We describe how reversibility varies among senescent changes. While senescent pathologies that develop in mid-life were not reversed, there was a rejuvenation of the proteostasis network, manifesting as a restoration of the capacity to eliminate otherwise intractable protein aggregates that accumulate with age. Moreover, resistance to several stressors was restored. These results support several new conclusions. (1) Loss of resilience is not solely a consequence of pathologies that develop in earlier life. (2) Restoration of proteostasis and resilience by inhibiting IIS is a plausible cause of the increase in lifespan. And (3), most interestingly, some aspects of the age-related transition from resilience to frailty can be reversed to a certain extent. This raises the possibility that the effect of IIS and related pathways on resilience and frailty during aging in higher animals might possess some degree of reversibility., (© 2024. The Author(s).)

The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin I DC , leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/I DC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin I DC . C. elegans condensin I/I DC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Investigations into human longevity are increasingly focusing on healthspan enhancement, not just lifespan extension. Lifestyle modifications and nutritional choices, including food supplements, can significantly affect aging and general health. Phytochemicals in centenarians' diets, such as those found in Timut pepper, a Nepalese spice with various medicinal properties, may contribute to their longevity. Similarly, Sichuan pepper, a related species, has demonstrated anti-inflammatory and neuroprotective activities. With the broader purpose of uncovering a novel treatment to address aging and its comorbidities, this study aims to investigate the potential lifespan- and healthspan-promoting effects of Timut pepper using the model organism Caenorhabditis elegans . We show that Timut pepper extract extends C. elegans' lifespan at different maintenance temperatures and increases the proportion of active nematodes in their early adulthood. In addition, we show that Timut pepper extract enhances speed and distance moved as the nematodes age. Finally, Timut pepper extract assures extracellular matrix homeostasis by slowing the age-dependent decline of collagen expression.

The intersection of metabolic processes and epigenetic regulation during embryogenesis is crucial yet not fully understood. Through a candidate RNAi screen in Caenorhabditis elegans , we identified metabolic enzymes ALDO-2 and PDHB-1 as potential epigenetic regulators. Mild alteration of the chromatin remodeler LET-418 /Mi2 activity rescues embryonic lethality induced by suppressing aldo-2 or pdhb-1 , suggesting a critical role for glucose and pyruvate metabolism in chromatin remodeling during embryogenesis. Given the conservation of central metabolic pathways and chromatin modifiers across species, our findings lay the foundation for future mechanistic investigations into the interplay between epigenetics and metabolism during development and upon disease., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Traf2- and Nck-interacting kinase (TNIK) has emerged as a key regulator of pathological metabolic signaling in several diseases and is a promising drug target. Originally studied for its role in cell migration and proliferation, TNIK possesses several newly identified functions that drive the pathogenesis of multiple diseases. Specifically, we evaluate TNIK's newfound roles in cancer, metabolic disorders, and neuronal function. We emphasize the implications of TNIK signaling in metabolic signaling and evaluate the translational potential of these discoveries. We also highlight how TNIK's role in many biological processes converges upon several hallmarks of aging. We conclude by discussing the therapeutic landscape of TNIK-targeting drugs and the recent success of clinical trials targeting TNIK., Competing Interests: Declaration of interests C.Y.E. declares no competing financial or non-financial interests. F.E.P., S.W.Y.L., F.W.P., A.A., F.R., and A.Z. are affiliated with Insilico Medicine, a commercial company developing and using generative artificial intelligence (AI) and other next-generation AI technologies and robotics for drug discovery, drug development, and aging research. Utilizing its generative AI platform and a range of deep aging clocks, Insilico Medicine has developed a portfolio of multiple therapeutic programs targeting fibrotic diseases, cancer, immunological diseases, and a range of age-related diseases., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with β-galactosidase (β-gal) ex vivo. Here, we describe a progressive accumulation of β-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of β-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating β-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated β-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes., (© 2023. The Author(s).)

Numerous anti-amyloid therapies have seen recent clinical development and approval, such as the monoclonal antibodies aducanumab and lecanemab. However, in Alzheimer's disease patients, amyloid-β (Aβ) plaques are found embedded in the extracellular matrix and surrounded by collagens, which might hinder these antibodies from targeting the plaques. We reasoned that various different nutraceutical and pharmaceutical agents might induce collagen and extracellular matrix turnover and removal of these collagen-embedded amyloid-β (Aβ) plaques. To address this idea, here, we used a transgenic C. elegans strain, LSD2104 , expressing fluorescent human Aβ 1-42 as an in-vivo model for secreted amyloid aggregation in the extracellular matrix. We performed a screen of various nutraceuticals and pharmaceuticals along with different combinations, and we found that quercetin 350 µM and rifampicin 75 µM successfully cleared the extracellular amyloid plaque burden compared to the 0.2% DMSO control group, with a combination of the two agents producing the maximum effect compared to either drug alone. These results may implicate the exploration of combination therapeutics of nutraceuticals and pharmaceuticals in the clearance of amyloid-β (Aβ) plaques in Alzheimer's disease., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Dysfunctional extracellular matrices (ECM) contribute to aging and disease. Repairing dysfunctional ECM could potentially prevent age-related pathologies. Interventions promoting longevity also impact ECM gene expression. However, the role of ECM composition changes in healthy aging remains unclear. Here we perform proteomics and in-vivo monitoring to systematically investigate ECM composition (matreotype) during aging in C. elegans revealing three distinct collagen dynamics. Longevity interventions slow age-related collagen stiffening and prolong the expression of collagens that are turned over. These prolonged collagen dynamics are mediated by a mechanical feedback loop of hemidesmosome-containing structures that span from the exoskeletal ECM through the hypodermis, basement membrane ECM, to the muscles, coupling mechanical forces to adjust ECM gene expression and longevity via the transcriptional co-activator YAP-1 across tissues. Our results provide in-vivo evidence that coordinated ECM remodeling through mechanotransduction is required and sufficient to promote longevity, offering potential avenues for interventions targeting ECM dynamics., (© 2024. The Author(s).)

The mammalian innate immune system is sex-dimorphic. Neutrophils are the most abundant leukocyte in humans and represent innate immunity's first line of defense. We previously found that primary mouse bone marrow neutrophils show widespread sex-dimorphism throughout life, including at the transcriptional level. Extracellular matrix [ECM]-related terms were observed among the top sex-dimorphic genes. Since the ECM is emerging as an important regulator of innate immune responses, we sought to further investigate the transcriptomic profile of primary mouse bone marrow neutrophils at both the bulk and single-cell level to understand how biological sex may influence ECM component expression in neutrophils throughout life. Here, using curated gene lists of ECM components and unbiased weighted gene co-expression network analysis [WGCNA], we find that multiple ECM-related gene sets show widespread female-bias in expression in primary mouse neutrophils. Since many immune-related diseases (e.g., rheumatoid arthritis) are more prevalent in females, our work may provide insights into the pathogenesis of sex-dimorphic inflammatory diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 McGill et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Food protein amyloid fibrils have superior technological, nutritional, sensorial, and physical properties compared to native monomers, but there is as yet insufficient understanding of their digestive fate and safety for wide consumption. By combining SDS-PAGE, ELISA, fluorescence, AFM, MALDI-MS, CD, microfluidics, and SAXS techniques for the characterization of β-lactoglobulin and lysozyme amyloid fibrils subjected to in-vitro gastrointestinal digestion, here we show that either no noticeable conformational differences exist between amyloid aggregates and their monomer counterparts after the gastrointestinal digestion process (as in β-lactoglobulin), or that amyloid fibrils are digested significantly better than monomers (as in lysozyme). Moreover, in-vitro exposure of human cell lines and in-vivo studies with C. elegans and mouse models, indicate that the digested fibrils present no observable cytotoxicity, physiological abnormalities in health-span, nor accumulation of fibril-induced plaques in brain nor other organs. These extensive in-vitro and in-vivo studies together suggest that the digested food amyloids are at least equally as safe as those obtained from the digestion of corresponding native monomers, pointing to food amyloid fibrils as potential ingredients for human nutrition., (© 2023. The Author(s).)

The amyloid beta (Aβ) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aβ aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo, we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aβ 1-42 . This secreted Aβ forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aβ aggregation regulators, we identified different collagens to ameliorate or potentiate Aβ aggregation. We show that a disintegrin and metalloprotease a disintegrin and metalloprotease 2 (ADM-2), an ortholog of ADAM9, reduces the load of extracellular Aβ aggregates. ADM-2 is required and sufficient to remove the extracellular Aβ aggregates. Thus, we provide in vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aβ aggregate removal., Competing Interests: EJ, AG, JM, CE No competing interests declared, (© 2023, Jongsma et al.)

A real-time, noninvasive, and clinically applicable aging test in humans has yet to be established. Herein we propose a sweat- and wearable-based test to determine biological age. This test would empower users to monitor their aging process and take an active role in managing their lifestyle and health., Competing Interests: Declaration of interests No interests are declared by the authors., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging., Competing Interests: Competing interests C.Y.E. is a co-founder and shareholder of Avea Life AG, and is on the Scientific Advisory Board of Maximon AG, Biotein, Longaevus Technologies Ltd. and Galyan Bio, Inc., (© 2023. Published by The Company of Biologists Ltd.)

The extracellular matrix (ECM), composed of interlinked proteins outside of cells, is an important component of the human body that helps maintain tissue architecture and cellular homeostasis. As people age, the ECM undergoes changes that can lead to age-related morbidity and mortality. Despite its importance, ECM aging remains understudied in the field of geroscience. In this review, we discuss the core concepts of ECM integrity, outline the age-related challenges and subsequent pathologies and diseases, summarize diagnostic methods detecting a faulty ECM, and provide strategies targeting ECM homeostasis. To conceptualize this, we built a technology research tree to hierarchically visualize possible research sequences for studying ECM aging. This strategic framework will hopefully facilitate the development of future research on interventions to restore ECM integrity, which could potentially lead to the development of new drugs or therapeutic interventions promoting health during aging.

Breakthroughs in medical research in the last century have led to a significant extension of the human lifespan, resulting in a shift towards an elderly population worldwide. Due to the ongoing progress of global development towards elevated standards of living, this study specifically examines Switzerland as a representative nation to explore the socioeconomic and healthcare ramifications associated with an ageing population, thereby highlighting the tangible impact experienced in this context. Beyond the exhaustion of pension funds and medical budgets, by reviewing the literature and analysing publicly available data, we observe a "Swiss Japanification". Old age is associated with late-life comorbidities and an increasing proportion of time spent in poor health. To address these problems, a paradigm shift in medical practice is needed to improve health rather than respond to existing diseases. Basic ageing research is gaining momentum to be translated into therapeutic interventions and provides machine learning tools driving longevity medicine. We propose that research focus on closing the translational gap between the molecular mechanisms of ageing and a more prevention-based medicine, which would help people age better and prevent late-life chronic diseases.

The biomechanical properties of extracellular matrices (ECM) and their consequences for cellular homeostasis have recently emerged as a driver of aging. Here we review the age-dependent deterioration of ECM in the context of our current understanding of the aging processes. We discuss the reciprocal interactions of longevity interventions with ECM remodeling. And the relevance of ECM dynamics captured by the matrisome and the matreotypes associated with health, disease, and longevity. Furthermore, we highlight that many established longevity compounds promote ECM homeostasis. A large body of evidence for the ECM to qualify as a hallmark of aging is emerging, and the data in invertebrates is promising. However, direct experimental proof that activating ECM homeostasis is sufficient to slow aging in mammals is lacking. We conclude that further research is required and anticipate that a conceptual framework for ECM biomechanics and homeostasis will provide new strategies to promote health during aging.

The mechanistic target of rapamycin (mTOR) kinase is one of the top drug targets for promoting health and lifespan extension. Besides rapamycin, only a few other mTOR inhibitors have been developed and shown to be capable of slowing aging. We used machine learning to predict novel small molecules targeting mTOR. We selected one small molecule, TKA001, based on in silico predictions of a high on-target probability, low toxicity, favorable physicochemical properties, and preferable ADMET profile. We modeled TKA001 binding in silico by molecular docking and molecular dynamics. TKA001 potently inhibits both TOR complex 1 and 2 signaling in vitro. Furthermore, TKA001 inhibits human cancer cell proliferation in vitro and extends the lifespan of Caenorhabditis elegans , suggesting that TKA001 is able to slow aging in vivo.

The extracellular matrix (ECM) is earning an increasingly relevant role in many disease states and aging. The analysis of these disease states is possible with the GWAS and PheWAS methodologies, and through our analysis, we aimed to explore the relationships between polymorphisms in the compendium of ECM genes (i.e., matrisome genes) in various disease states. A significant contribution on the part of ECM polymorphisms is evident in various types of disease, particularly those in the core-matrisome genes. Our results confirm previous links to connective-tissue disorders but also unearth new and underexplored relationships with neurological, psychiatric, and age-related disease states. Through our analysis of the drug indications for gene-disease relationships, we identify numerous targets that may be repurposed for age-related pathologies. The identification of ECM polymorphisms and their contributions to disease will play an integral role in future therapeutic developments, drug repurposing, precision medicine, and personalized care.

Transgenic overexpression of collagen col-120 increases the lifespan of C. elegans . However, whether post-developmental enhancement of collagen expression could also increase the lifespan is unknown. Recently, we described a method to induce the expression of a target gene using catalytically dead Cas9 (dCas9)-engineered C. elegans via ingestion of bacteria expressing a pair of promoter-specific single guide RNAs (sgRNA). Here, we cloned col-120 promoter-specific sgRNA oligo pair into L4440-Biobrick-sgRNA and fed these bacteria to dCas9::VP64 transgenic C. elegans . We observed a similar percentage of lifespan extension by post-developmentally dCas9-induced expression of col-120 , as previously reported through transgenic overexpression of col-120 . Consistent with this result is that induction of another previously shown longevity-promoting collagen, col-10 , also increased lifespan. Furthermore, we found an enhanced resilience to heat stress and increased expression of hsp-16.2 upon dCas9-activated col-120 expression. Together, these results provide an orthogonal method to validate longevity by enhancing col-120 expression and point towards a potential role of collagen enhancement in thermotolerance., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

Collagen has been postulated to be the most abundant protein in our body, making up one-third of the total protein content in mammals. However, a direct assessment of the total collagen levels of an entire mammal to confirm this estimate is missing. Here we measured hydroxyproline levels as a proxy for collagen content together with total protein levels of entire mice or of individual tissues. Collagen content normalized to the total protein is approximately 0.1% in the brain and liver, 1% in the heart and kidney, 4% in the muscle and lung, 6% in the colon, 20-40% in the skin, 25-35% in bones, and 40-50% in tendons of wild-type (CD1 and CB57BL/6) mice, consistent with previous reports. To our surprise, we find that collagen is approximately 12% in females and 17% in males of the total protein content of entire wild-type (CD1 and CB57BL/6) mice. Although collagen type I is the most abundant collagen, the most abundant proteins are albumin, hemoglobulin, histones, actin, serpina, and then collagen type I. Analyzing amino acid compositions of mice revealed glycine as the most abundant amino acid. Thus, we provide reference points for collagen, matrisome, protein, and amino acid composition of healthy wild-type mice., (© 2023. The Author(s).)

Repurposing drugs capable of extending lifespan and health span has a huge untapped potential in translational geroscience. Here, we searched for known compounds that elicit a similar gene expression signature to caloric restriction and identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating Caenorhabditis elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, health span, and lifespan benefits of rilmenidine treatment in C. elegans are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to calorically restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Moreover, transcriptional changes similar to caloric restriction were observed in liver and kidney tissues in mice treated with rilmenidine. Together, these results reveal a geroprotective and potential caloric restriction mimetic effect by rilmenidine that warrant fresh lines of inquiry into this compound., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Disrupted biological function, manifesting through the hallmarks of aging, poses one of the largest threats to healthspan and risk of disease development, such as metabolic disorders, cardiovascular ailments, and neurodegeneration. In recent years, numerous geroprotectors, senolytics, and other nutraceuticals have emerged as potential disruptors of aging and may be viable interventions in the immediate state of human longevity science. In this review, we focus on the decrease in nicotinamide adenine dinucleotide (NAD+) with age and the supplementation of NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), in combination with other geroprotective compounds, to restore NAD+ levels present in youth. Furthermore, these geroprotectors may enhance the efficacy of NMN supplementation while concurrently providing their own numerous health benefits. By analyzing the prevention of NAD+ degradation through the inhibition of CD38 or supporting protective downstream agents of SIRT1, we provide a potential framework of the CD38/NAD+/SIRT1 axis through which geroprotectors may enhance the efficacy of NAD+ precursor supplementation and reduce the risk of age-related diseases, thereby potentiating healthspan in humans.

Currently, there are no therapies available to modify the disease progression of Huntington's disease (HD). Recent clinical trial failures of antisense oligonucleotide candidates in HD have demonstrated the need for new therapeutic approaches. Here, we developed a novel in-silico fragment scanning approach across the surface of mutant huntingtin (mHTT) polyQ and predicted four hit compounds. Two rounds of compound analoging using a strategy of testing structurally similar compounds in an affinity assay rapidly identified GLYN122. In vitro, GLYN122 directly binds and reduces mHTT and induces autophagy in neurons. In vivo, our results confirm that GLYN122 can reduce mHTT in the cortex and striatum of the R/2 mouse model of Huntington's disease and subsequently improve motor symptoms. Thus, the in-vivo pharmacology profile of GLYN122 is a potential new preclinical candidate for the treatment of HD., (© 2022. The Author(s).)

Muscle mass and force are key for movement, life quality, and health. It is well established that resistance exercise is a potent anabolic stimulus increasing muscle mass and force. The response of a physiological system to resistance exercise is composed of non-modifiable ( i.e., age, gender, genetics) and modifiable factors ( i.e., exercise, nutrition, training status, etc.). Both factors are integrated by systemic responses ( i.e., molecular signaling, genetic responses, protein metabolism, etc.), consequently resulting in functional and physiological adaptations. Herein, we discuss the influence of non-modifiable factors on resistance exercise: age, gender, and genetics. A solid understanding of the role of non-modifiable factors might help to adjust training regimes towards optimal muscle mass maintenance and health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Viecelli and Ewald.)

Aging is one of the largest risk factors for cancer, type 2 diabetes, osteoarthritis, cardiovascular diseases, and other age-related pathologies. Here, we give a detailed description of the interplay of chronic age-related pathologies with the remodeling of the extracellular matrix during disease development and progression. Longevity-promoting signaling pathways slow or prevent age-related diseases. In particular, we focus on the mTOR signaling pathway, sirtuins, and canonical longevity-promoting transcription factors, such as FOXO, NF-κB, and Nrf2. We extend our analysis using chromatin immunoprecipitation (ChIP) sequencing and transcriptomic data and report that many established and emerging longevity-promoting transcription factors, such as CREB1, FOXO1,3, GATA1,2,3,4, HIF1A, JUN, KLF4, MYC, NFE2L2/Nrf2, RELA/NF-κB, REST, STAT3,5A, and TP53/p53, directly regulate many extracellular matrix genes and remodelers. We propose that modulation of these pathways increases lifespan and protects from age-related diseases in part due to their effects on extracellular matrix remodeling. Therefore, to successfully treat age-related diseases, it is necessary to better understand the connection between extracellular matrix components and longevity pathways., Competing Interests: TV is employed by Tinka Therapeutics. CYE is a co-founder and shareholder of Avea Life AG and is on the Scientific Advisory Board of Maximon AG and Galyan Bio, INC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vidović and Ewald.)

Inhibition of gene expression in Caenorhabditis elegans, a versatile model organism for studying the genetics of development and aging, is achievable by feeding nematodes with bacteria expressing specific dsRNAs. Overexpression of hypoxia-inducible factor 1 (hif-1) or heat-shock factor 1 (hsf-1) by conventional transgenesis has previously been shown to promote nematodal longevity. However, it is unclear whether other methods of gene overexpression are feasible, particularly with the advent of CRISPR-based techniques. Here, we show that feeding C. elegans engineered to stably express a Cas9-derived synthetic transcription factor with bacteria expressing promoter-specific single guide RNAs (sgRNAs) also allows activation of gene expression. We demonstrate that CRISPR activation via ingested sgRNAs specific for the respective promoter regions of hif-1 or hsf-1 increases gene expression and extends lifespan of C. elegans. Furthermore, and as an in silico resource for future studies aiming to use CRISPR activation in C. elegans, we provide predicted promoter-specific sgRNA target sequences for >13,000 C. elegans genes with experimentally defined transcription start sites. We anticipate that the approach and components described herein will help to facilitate genome-wide gene overexpression studies, for example, to identify modulators of aging or other phenotypes of interest, by enabling induction of transcription by feeding of sgRNA-expressing bacteria to nematodes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human centenarians and longevity mutants of model organisms show lower incidence rates of late-life morbidities than the average population. However, whether longevity is caused by a compression of the portion of life spent in a state of morbidity, i.e ., "sickspan," is highly debated even in isogenic Caenorhabditis elegans . Here, we developed a microfluidic device that employs acoustophoretic force fields to quantify the maximum muscle strength and dynamic power in aging C. elegans . Together with different biomarkers for healthspan, we found a stochastic onset of morbidity, starting with a decline in dynamic muscle power and structural integrity, culminating in frailty. Surprisingly, we did not observe a compression of sickspan in longevity mutants but instead observed a temporal scaling of healthspan. Given the conservation of these longevity interventions, this raises the question of whether the healthspan of mammalian longevity interventions is also temporally scaled., Competing Interests: The authors have no competing interests to declare., (© 2022 The Author(s).)

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H 2 S) production. This H 2 S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H 2 S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H 2 S levels, or enhancing mechanisms that H 2 S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition., (© 2022. The Author(s).)

We profiled the liver transcriptome, proteome, and metabolome in 347 individuals from 58 isogenic strains of the BXD mouse population across age (7 to 24 months) and diet (low or high fat) to link molecular variations to metabolic traits. Several hundred genes are affected by diet and/or age at the transcript and protein levels. Orthologs of two aging-associated genes, St7 and Ctsd, were knocked down in C. elegans, reducing longevity in wild-type and mutant long-lived strains. The multiomics data were analyzed as segregating gene networks according to each independent variable, providing causal insight into dietary and aging effects. Candidates were cross-examined in an independent diversity outbred mouse liver dataset segregating for similar diets, with ∼80%-90% of diet-related candidate genes found in common across datasets. Together, we have developed a large multiomics resource for multivariate analysis of complex traits and demonstrate a methodology for moving from observational associations to causal connections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59's localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation., Competing Interests: The authors have declared that no competing interests exist.

Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. Reducing insulin/insulin-like growth factor (IGF)-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode Caenorhabditis elegans , we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system. This allowed for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi become ineffective. Using this system, we found that AID-mediated depletion of DAF-2 protein surpasses the longevity of daf-2 mutants. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg retention, and reduced brood size. By contrast, AID-mediated depletion of DAF-2 post-reproduction, or specifically in the intestine in early adulthood, resulted in an extension of lifespan without these deleterious effects. Strikingly, at geriatric ages, when 75% of the population had died, AID-mediated depletion of DAF-2 protein resulted in a doubling in lifespan. Thus, we provide a proof-of-concept that even close to the end of an individual's lifespan, it is possible to slow aging and promote longevity., Competing Interests: RV, TP, IK, RC, CE No competing interests declared, (© 2021, Venz et al.)

Discovering compounds that promote health during aging ("geroprotectors") is key to the retardation of age-related pathologies and the prevention of chronic age-related diseases. In in-silico and model organisms' lifespan screens, chondroitin sulfate has emerged as a geroprotective compound. Chondroitin sulfate is a glycosaminoglycan attached to extracellular matrix proteins and is naturally produced by our body. Oral supplementation of chondroitin sulfate shows a high tolerance in humans, preferable pharmacokinetics, a positive correlation with healthy human longevity, and efficacy in deceleration of age-related diseases in randomized clinical trials. We have recently shown that chondroitin sulfate supplementation increases the lifespan of C. elegans . Thus, chondroitin sulfate holds the potential to become a geroprotective strategy to promote health during human aging. This review discusses the two major potential mechanisms of action, extracellular matrix homeostasis and inhibition of inflammation, that counteract age-related pathologies upon chondroitin sulfate supplementation., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ewald.)

The identification and validation of drugs that promote health during aging ("geroprotectors") are key to the retardation or prevention of chronic age-related diseases. Here, we found that most of the established pro-longevity compounds shown to extend lifespan in model organisms also alter extracellular matrix gene expression (i.e., matrisome) in human cell lines. To harness this observation, we used age-stratified human transcriptomes to define the age-related matreotype, which represents the matrisome gene expression pattern associated with age. Using a "youthful" matreotype, we screened in silico for geroprotective drug candidates. To validate drug candidates, we developed a novel tool using prolonged collagen expression as a non-invasive and in-vivo surrogate marker for Caenorhabditis elegans longevity. With this reporter, we were able to eliminate false-positive drug candidates and determine the appropriate dose for extending the lifespan of C. elegans. We improved drug uptake for one of our predicted compounds, genistein, and reconciled previous contradictory reports of its effects on longevity. We identified and validated new compounds, tretinoin, chondroitin sulfate, and hyaluronic acid, for their ability to restore age-related decline of collagen homeostasis and increase lifespan. Thus, our innovative drug screening approach-employing extracellular matrix homeostasis-facilitates the discovery of pharmacological interventions promoting healthy aging., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The gpa-4 promoter-driven expression is described as specific for ASIL and ASIR chemosensory neurons in the nematode Caenorhabditis elegans , yet this was mostly examined in adult animals. Here we used a recombination-mediated reporter to test the previously used gpa-4 promoter expression. This reporter highlights all cells in which the gpa-4 promoter has been active at one point or another during development. We show that the gpa-4 promoter is indeed active in ASI, yet to our surprise, thispromoter drives also expression in many other cell types, including the somatic gonad, the seam cells, a subset of tail and head neurons, and muscle cells, demonstrating a widespread activity of this transgenic gpa-4 promoter during embryonic and post-embryonic development., (Copyright: © 2021 by the authors.)

Although genetic approaches have identified key genes and pathways that promote longevity, systems-level approaches are less utilized. Here, we took advantage of the wealth of omics data characterizing the BXD family of mice. We associated transcript and peptide levels across five tissues from both female and male BXD isogenic lines with their median lifespan. We identified over 5000 genes that showed a longevity correlation in a given tissue. Surprisingly, we found less than 1% overlap among longevity-correlating genes across tissues and sex. These 1% shared genes consist of 51 genes, of which 13 have been shown to alter lifespan. Only two genes - Coro7 and Set - showed a longevity correlation in all tissues and in both sexes. While differential regulation of aging across tissues and sex has been reported, our systems-level analysis reveals two unique genes that may promote healthy aging in unique sex- and tissue-agnostic manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vitiello, Dakhovnik, Statzer and Ewald.)

Significant non-genetic stochastic factors affect aging, causing lifespan differences among individuals, even those sharing the same genetic and environmental background. In Caenorhabditis elegans, differences in heat-shock response (HSR) are predictive of lifespan. However, factors contributing to the heterogeneity of HSR are still not fully elucidated. Here, the authors characterized HSR dynamics in isogenic C. elegans expressing GFP reporter for hsp-16.2 for identifying the key contributors of HSR heterogeneity. Specifically, microfluidic devices that enable cross-sectional and longitudinal measurements of HSR dynamics in C. elegans at different scales are developed: in populations, within individuals, and in embryos. The authors adapted a mathematical model of HSR to single C. elegans and identified model parameters associated with proteostasis-maintenance of protein homeostasis-more specifically, protein turnover, as the major drivers of heterogeneity in HSR dynamics. It is verified that individuals with enhanced proteostasis fidelity in early adulthood live longer. The model-based comparative analysis of protein turnover in day-1 and day-2 adult C. elegans revealed a stochastic-onset of age-related proteostasis decline that increases the heterogeneity of HSR capacity. Finally, the analysis of C. elegans embryos showed higher HSR and proteostasis capacity than young adults and established transgenerational contribution to HSR heterogeneity that depends on maternal age., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

Transcriptomic signatures based on cellular mRNA expression profiles can be used to categorize cell types and states. Yet whether different functional groups of genes perform better or worse in this process remains largely unexplored. Here we test the core matrisome - that is, all genes coding for structural proteins of the extracellular matrix - for its ability to delineate distinct cell types in embryonic single-cell RNA-sequencing (scRNA-seq) data. We show that even though expressed core matrisome genes correspond to less than 2% of an entire cellular transcriptome, their RNA expression levels suffice to recapitulate essential aspects of cell type-specific clustering. Notably, using scRNA-seq data from the embryonic limb, we demonstrate that core matrisome gene expression outperforms random gene subsets of similar sizes and can match and exceed the predictive power of transcription factors. While transcription factor signatures generally perform better in predicting cell types at early stages of chicken and mouse limb development, i.e., when cells are less differentiated, the information content of the core matrisome signature increases in more differentiated cells. Moreover, using cross-species analyses, we show that these cell type-specific signatures are evolutionarily conserved. Our findings suggest that each cell type produces its own unique extracellular matrix, or matreotype, which becomes progressively more refined and cell type-specific as embryonic tissues mature., (© 2021 The Author(s).)

Although C. elegans is one of the best-studied model organisms, an estimate of its cell sizes and tissues is missing. Here we used the Virtual Worm that is based on electron microscopy images to calculate a zeroth-order approximation of cell and tissue sizes of C. elegans . We conclude that the intestine is the largest tissue, followed by the hypodermis, gonads, body wall muscles, pharynx, and neurons. Thus, we provide an approximation of tissue volumes of young adult C. elegans ., (Copyright: © 2021 by the authors.)

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7 th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1 st to 4 th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8 th ARDD meeting that is scheduled for the 31 st of August to 3 rd of September, 2021, at Columbia University, USA.

Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. We performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi in C. elegans to identify members of the lipid bilayer stress response. AID-mediated degradation of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, induced the activation of lipid bilayer stress-sensitive reporters. We screened through most C. elegans kinases and transcription factors by feeding RNAi. We discovered nine genes that suppressed the lipid bilayer stress response in C. elegans These suppressor genes included drl-1 /MAP3K3, gsk-3 /GSK3, let-607 /CREB3, ire-1 /IRE1, and skn-1 /NRF1,2,3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Thus, we demonstrated proof-of-concept for combining AID and RNAi as a new screening strategy and identified eight conserved genes that had not previously been implicated in the lipid bilayer stress response., (Copyright © 2020 Venz et al.)

Extracellular matrices are essential for cellular and organismal function. Recent genome-wide and phenome-wide association studies started to reveal a broad spectrum of phenotypes associated with genetic variants. However, the phenome or spectrum of all phenotypes associated with genetic variants in extracellular matrix genes is unknown. Here, we analyzed over two million recorded genotype-to-phenotype relationships across multiple species to define their extracellular matrix phenomes. By using the previously defined matrisomes of humans, mice, zebrafish, Drosophila, and C. elegans , we found that the extracellular matrix phenome comprises of 3-10% of the entire phenome. Collagens ( COL1A1 , COL2A1 ) and fibrillin ( FBN1 ) are each associated with >150 distinct phenotypes in humans, whereas collagen COL4A1 , Wnt- and sonic hedgehog (shh) signaling are predominantly associated in other species. We determined the phenotypic fingerprints of matrisome genes and found that MSTN, CTSD, LAMB2, HSPG2 , and COL11A2 and their corresponding orthologues have the most phenotypes across species. Out of the 42,551 unique matrisome genotype-to-phenotype relationships across the five species with defined matrisomes, we have constructed interaction networks to identify the underlying molecular components connecting with orthologues phenotypes and with novel phenotypes. Thus, our networks provide a framework to predict unassessed phenotypes and their potential underlying molecular interactions. These frameworks inform on matrisome genotype-to-phenotype relationships and potentially provide a sophisticated choice of biological model system to study human phenotypes and diseases., Competing Interests: The authors have no competing interests to declare. Correspondence should be addressed to C. Y. E., (© 2020 The Author(s).)

Accumulation of damage is generally considered the cause of aging. Interventions that delay aging mobilize mechanisms that protect and repair cellular components. Consequently, research has been focused on studying the protective and homeostatic mechanisms within cells. However, in humans and other multicellular organisms, cells are surrounded by extracellular matrices (ECMs), which are important for tissue structure, function, and intercellular communication. During aging, components of the ECM become damaged through fragmentation, glycation, crosslinking, and accumulation of protein aggregation, all of which contribute to age-related pathologies. Interestingly, placing senescent cells into a young ECM rejuvenates them. Furthermore, we found that many longevity-assurances pathways reactivate de novo synthesis of ECM proteins during aging. This raises the question of what constitutes a young ECM to reverse aging or maintain health? In order to make inroads to answering this question, I suggest a systems-level approach of quantifying the matrisome or ECM compositions reflecting health, pathology, or phenotype and propose a novel term, the "matreotype," to describe this. The matreotype is defined as the composition and modification of ECM or matrisome proteins associated with or caused by a phenotype, such as longevity, or a distinct and acute physiological state, as observed during aging or disease. Every cell type produces its unique ECM. Intriguingly, cancer-cell types can even be identified based on their unique ECM composition. Thus, the matreotype reflects cellular identity and physiological status. Defined matreotypes could be used as biomarkers or prognostic factors for disease or health status during aging with potential relevance for personalized medicine. Treatment with biologics that alter ECM-to-cell mechanotransduction might be a strategy to reverse age-associated pathologies. An understanding of how to reverse from an old to a young matreotype might point toward novel strategies to rejuvenate cells and help maintain tissue homeostasis to promote health during aging., (© 2019 S. Karger AG, Basel.)

The aging of the human brain in the absence of diseases is accompanied by subtle changes of neuronal morphology, such as dendrite restructuring, neuronal sprouting, and synaptic deteriorations, rather than neurodegeneration or gross deterioration. Similarly, the nervous system of Caenorhabditis elegans does not show neurodegeneration or gross deterioration during normal aging, but displays subtle alterations in neuronal morphology. The occurrence of these age-dependent abnormalities is stochastic and dynamic, which poses a major challenge to fully capture them for quantitative comparison. Here, we developed a semi-automated pipeline for quantitative image analysis of these features during aging. We employed and evaluated this pipeline herein to reproduce findings from previous studies using visual inspection of neuronal morphology. Importantly, our approach can also quantify additional features, such as soma volume, the length of neurite outgrowths, and their location along the aged neuron. We found that, during aging, the soma of neurons decreases in volume, whereas the number and length of neurite outgrowths from the soma both increase. Long-lived animals showed less decrease in soma volume, fewer and shorter neurite outgrowths, and protection against abnormal sharp bends preferentially localized at the distal part of the dendrites during aging. We found a correlation of sharp bends with neurite outgrowth, suggesting the hypothesis that sharp bends might proceed neurite outgrowths. Thus, our semi-automated pipeline can help researchers to obtain and analyze quantitative datasets of this stochastic process for comparison across genotypes and to identify correlations to facilitate the generation of novel hypothesis., (Copyright © 2019 Hess et al.)

Proteins are the building blocks of life. While proteins and their localization within cells and sub-cellular compartments are well defined, the proteins predicted to be secreted to form the extracellular matrix - or matrisome - remain elusive in the model organism C. elegans . Here, we used a bioinformatic approach combining gene orthology and protein structure analysis and an extensive curation of the literature to define the C. elegans matrisome. Similar to the human genome, we found that 719 out of ~20,000 genes (~4%) of the C. elegans genome encodes matrisome proteins, including 181 collagens, 35 glycoproteins, 10 proteoglycans, and 493 matrisome-associated proteins. We report that 173 out of the 181 collagen genes are unique to nematodes and are predicted to encode cuticular collagens, which we are proposing to group into five clusters. To facilitate the use of our lists and classification by the scientific community, we developed an automated annotation tool to identify ECM components in large datasets. We also established a novel database of all C. elegans collagens (CeColDB). Last, we provide examples of how the newly defined C. elegans matrisome can be used for annotations and gene ontology analyses of transcriptomic, proteomic, and RNAi screening data. Because C. elegans is a widely used model organism for high throughput genetic and drug screens, and to study biological and pathological processes, the conserved matrisome genes may aid in identifying potential drug targets. In addition, the nematode-specific matrisome may be exploited for targeting parasitic infection of man and crops., (© 2018 The Author(s).)