Mechanotransduction through hemidesmosomes during aging and longevity.

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.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans.

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.

Strategic outline of interventions targeting extracellular matrix for promoting healthy longevity.

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.

Ingestion of single guide RNAs induces gene overexpression and extends lifespan in Caenorhabditis elegans via CRISPR activation.

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.

AI-Predicted mTOR Inhibitor Reduces Cancer Cell Proliferation and Extends the Lifespan of C. elegans.

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.

Stochastic and Age-Dependent Proteostasis Decline Underlies Heterogeneity in Heat-Shock Response Dynamics.

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.

Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity.

Interventions that delay ageing mobilize mechanisms that protect and repair cellular components, but it is unknown how these interventions might slow the functional decline of extracellular matrices, which are also damaged during ageing. Reduced insulin/IGF-1 signalling (rIIS) extends lifespan across the evolutionary spectrum, and in juvenile Caenorhabditis elegans also allows the transcription factor DAF-16/FOXO to induce development into dauer, a diapause that withstands harsh conditions. It has been suggested that rIIS delays C. elegans ageing through activation of dauer-related processes during adulthood, but some rIIS conditions confer robust lifespan extension unaccompanied by any dauer-like traits. Here we show that rIIS can promote C. elegans longevity through a program that is genetically distinct from the dauer pathway, and requires the Nrf (NF-E2-related factor) orthologue SKN-1 acting in parallel to DAF-16. SKN-1 is inhibited by IIS and has been broadly implicated in longevity, but is rendered dispensable for rIIS lifespan extension by even mild activity of dauer-related processes. When IIS is decreased under conditions that do not induce dauer traits, SKN-1 most prominently increases expression of collagens and other extracellular matrix genes. Diverse genetic, nutritional, and pharmacological pro-longevity interventions delay an age-related decline in collagen expression. These collagens mediate adulthood extracellular matrix remodelling, and are needed for ageing to be delayed by interventions that do not involve dauer traits. By genetically delineating a dauer-independent rIIS ageing pathway, our results show that IIS controls a broad set of protective mechanisms during C. elegans adulthood, and may facilitate elucidation of processes of general importance for longevity. The importance of collagen production in diverse anti-ageing interventions implies that extracellular matrix remodelling is a generally essential signature of longevity assurance, and that agents promoting extracellular matrix youthfulness may have systemic benefit.

Clearance of extracellular human amyloid-ß aggregates in C. elegans by nutraceutical and pharmaceutical interventions.

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-ß (Aß) 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-ß (Aß) plaques. To address this idea, here, we used a transgenic C. elegans strain, LSD2104 , expressing fluorescent human Aß 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-ß (Aß) plaques in Alzheimer's disease.

In-vivo screening implicates endoribonuclease Regnase-1 in modulating senescence-associated lysosomal changes.

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 ß-galactosidase (ß-gal) ex vivo. Here, we describe a progressive accumulation of ß-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 ß-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating ß-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated ß-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes.

Timut Pepper Extract Slows Age-Dependent Decline of Mobility and Collagen Loss and Promotes Longevity.

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.

Metabolic enzymes aldo-2 and pdhb-1 as potential epigenetic regulators during C. elegans embryogenesis.

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.

TNIK's emerging role in cancer, metabolism, and age-related diseases.

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.

Longevity interventions modulate mechanotransduction and extracellular matrix homeostasis in C. elegans.

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.

Improved resilience and proteostasis mediate longevity upon DAF-2 degradation in old age.

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.

Pan-neuronal expression of APL-1, an APP-related protein, disrupts olfactory, gustatory, and touch plasticity in Caenorhabditis elegans.

Patients with Alzheimer's disease show age-related cognitive decline. Postmortem autopsy of their brains shows the presence of large numbers of senile plaques, whose major component is the ß-amyloid peptide. The ß-amyloid peptide is a cleavage product of the amyloid precursor protein (APP). In addition to the neurodegeneration associated with ß-amyloid aggregation in Alzheimer's disease patients, mutations in APP in mammalian model organisms have also been shown to disrupt several behaviors independent of visible amyloid plaque formation. However, the pathways in which APP function are unknown and difficult to unravel in mammals. Here we show that pan-neuronal expression of APL-1, the Caenorhabditis elegans ortholog of APP, disrupts several behaviors, such as olfactory and gustatory learning behavior and touch habituation. These behaviors are mediated by distinct neural circuits, suggesting a broad impact of APL-1 on sensory plasticity in C. elegans. Furthermore, we found that disruption of these three behaviors requires activity of the TGFß pathway and reduced activity of the insulin pathway. These results suggest pathways and molecular components that may underlie behavioral plasticity in mammals and in patients with Alzheimer's disease.

CRISPR-activated expression of collagen col-120 increases lifespan and heat tolerance.

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.

Sex-dimorphic expression of extracellular matrix genes in mouse bone marrow neutrophils.

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.

Extracellular Matrix Dynamics as an Emerging yet Understudied Hallmark of Aging and Longevity.

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.

Sex-dimorphic expression of extracellular matrix genes in mouse bone marrow neutrophils.

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.