Consolidating multiple evolutionary theories of ageing suggests a need for new approaches to study genetic contributions to ageing decline

Understanding mechanisms of ageing remains a complex challenge for biogerontologists, but recent adaptations of evolutionary ageing theories offer a compelling lens in which to view both age-related molecular and physiological deterioration. Ageing is commonly associated with progressive declines in biochemical and molecular processes resulting from damage accumulation, yet the role of continued developmental gene activation is less appreciated. Natural selection pressures are at their highest in youthful periods to modify gene expression towards maximising reproductive capacity. After sexual maturation, selective pressure diminishes, subjecting individuals to maladaptive pleiotropic gene functions that were once beneficial for developmental growth but become pathogenic later in life. Due to this selective ‘shadowing ’ in ageing, mechanisms to counter such hyper/ hypofunctional genes are unlikely to evolve. Interventions aimed at targeting gene hyper/hypofunction during ageing might, therefore, represent an attractive therapeutic strategy. The nematode Caenorhabditis elegans offers a strong model for post-reproductive mechanistic and therapeutic investigations, yet studies examining the mechanisms of, and countermeasures against, ageing decline largely intervene from larval stages onwards. Importantly, however, lifespan extending conditions frequently impair early-life fitness and fail to correspond-ingly increase healthspan. Here, we consolidate multiple evolutionary theories of ageing and discuss data supporting hyper/hypofunctional changes at a global molecular and functional level in C. elegans , and how classical lifespan-extension mutations alter these dynamics. The relevance of such mutant models for exploring mechanisms of ageing are discussed, highlighting that post-reproductive gene optimisation represents a more trans-latable approach for C. elegans research that is not constrained by evolutionary trade-offs. Where some genetic mutations in C. elegans that promote late-life health map accordingly with healthy ageing in humans, other widely used genetic mutations that extend worm lifespan are associated with life-limiting pathologies in people. Lifespan has also become the gold standard for quantifying ‘ageing ’ , but we argue that gerospan compression (i. e., ‘healthier ’ ageing) is an appropriate goal for anti-ageing research, the mechanisms of which appear distinct from those regulating lifespan alone. There is, therefore, an evident need to re-evaluate experimental approaches to study the role of hyper/hypofunctional genes in ageing in C. elegans .


Introduction
Ageing remains the largest socio-economic burden in humans and the greatest risk factor for developing life-threating diseases (Garmany et al., 2021).Gerontological fields have proposed numerous potential explanations for ageing including oxidative stress and damage accumulation (Harman, 1956) and multiple developmental/ evolutionary theories (Williams, 1957;de Magalhaes and Church, 2005;Medawar, 1952;Kirkwood, 1977;Cutler, 1979;Blagosklonny, 2006;Kern, 2022).However, many of these lack the ability to explain key aspects of ageing deterioration as standalone models (Gems, 2022), and some have recently been largely discredited to explain causal pathophysiological events seen in ageing (Blagosklonny, 2010;Gladyshev, 2014).The current available explanations seem to fall exclusively into two categories; ageing caused by accumulative molecular damage, or programmatic events that define the ageing projection.Probabilistically, ageing is a combination of both processes, but defining the precise onset and mechanisms of the proximate causes of ageing remains a significant challenge.
An appreciated view is that key Hallmarks of ageing have a hierarchical sequence of events that drives ageing pathologies (Lopez-Otin et al., 2023).For example, genomic instability, epigenetic alterations, loss of proteostasis and telomere attrition are proposed 'primary' drivers of ageing, with loss of mitochondrial function, nutrient sensing and chronic inflammation consequences of perturbations in primary features.Although these events undoubtedly play roles in ageing progression, this view is still somewhat comprised of individualistic components at its core and tells us little about the causal events that dictate primary hallmark collapse.Broadly speaking, experimental approaches continue to leverage conventional frameworks, measuring molecular, morphological and metabolite changes in aged populations under the premise these aberrant signature changes must promote ageing.Significant progress has been made in our understanding of ageing 'hallmarks' using such methods, yet the precise onset of, and underlying initial causes leading to hallmark manifestation, remain poorly defined.
Propositions that ageing is caused by the accumulation of damaged genetic material undermines the possibility of wild-type gene (hyper) function driving ageing pathology manifestation later in life.Two recent ageing reviews have explored key evolutionary frameworks in detail, namely, hyperfunction (HF) and developmental theories of ageing (DTA) (Gems, 2022;Lemaitre et al., 2024).These works challenge current understandings on proximate causes of ageing and provide frameworks for how evolutionary processes might explain age-related physiological deterioration.Despite a re-emerging interest for evolutionary theories in ageing research, current use of model organisms in the laboratory present crucial limitations for testing them.There is thus a need to design pre-clinical/ invertebrate research that is compatible for testing evolutionary theories of ageing.C. elegans have contributed considerably to our understanding of ageing decline and tissue-specific genomic and proteomic deterioration across its short 3-week life-course (Herndon et al., 2002;Narayan et al., 2016;Wang et al., 2022).With a fully mapped genome, ease of genetic manipulation techniques and a translucent outer cuticle allowing in vivo protein surveillance, C. elegans are an established invertebrate model for studying conserved mechanisms of ageing.Seminal findings have shown that insulin-like growth factor receptor mutants (IGF-1/daf-2 in worms) live up to twice as long as wild-type C. elegans (Kenyon et al., 1993), evidence that reductions in growth-related/anabolic pathways attenuate ageing.Indeed, C. elegans researchers continue to exploit loss-of-function mutation models or life-long gene silencing of such pathways to study ageing.Given the significant loss of early-life fitness frequently observed with life-long developmental pathway mutations, the translatability of these findings to human ageing are limited.Late-age genetic or pharmacological manipulation (i.e., initiated during vs preceding the onset of healthspan decline) of pleiotropic pathways should be of priority when assessing the roles they play in ageing.Similarly, the ongoing focus of lifespan extension is likely less relevant to reducing the societal ageing burden than understanding and improving health in older age.Nonetheless, the optimal strategy remains to be established (Bansal et al., 2015), where precise scaling of lifespan and healthspan needs to be more closely evaluated to support such claims.
In this review, we examine recent advances in evolutionary theories, highlighting the growing experimental evidence from model organisms that supports biological programmatic 'run-on' as a framework for understanding the causes of ageing.We focus on C. elegans as a candidate model to evidence the age-related hyperfunction of the transcriptional/ translational machinery and associated cellular (tissue/ organ) processes.The majority of 'healthy' ageing research to date has been performed using long-lived mutants given their robust and consistent lifespan-extension phenotypes.Thus, the predominance of data here discussing the myriad attenuations in developmental/growth-related pathway hyperfunctions centers around these models.Whilst we claim that the physiological relevance of these data might be bound within evolutionary trade-off's and pleiotropic constraints, these works have laid fundamental understandings for improving the study of optimising gene function.Thus, we further propose that new experimental approaches can more effectively test optimised gene function for healthy ageing when applied post-reproductively into older age.

Molecular damage as a driver or symptom of ageing?
Defining the cause of organismal ageing has consumed biogerontologists for centuries.A pivotal point in the gerontological field was D. Harman's observation that ageing and disease are likely a result of free radical attack (namely, hydroperoxyl and hydroxyl radicals) on cells constituents and connective tissue (Harman, 1956).Such a hypothesis is reasonable given their endogenous production across the tree of life, the increasing intracellular accumulation with progressive age, declines in the expression of radical buffering systems, and elevated mRNA / protein oxidation levels evident in older age (Finkel and Holbrook, 2000;Tan et al., 2018;Tanaka and Chock, 2021).This provided a testable framework for studying ageing experimentally: reduce the levels of intracellular radical species and ageing attenuation should follow.Harman reported promising data in rodents, observing that free radical inhibitors such as 2-mercaptoethylamine hydrochloride extended the mean lifespan of AKR (a strain highly susceptible to Leukemia) and C3H mice (highly prone to developing mammary tumors) (Harman, 1957), and later showed 2-mercaptoethylamine hydrochloride and butylated hydroxytoluene extended mean lifespan of male LAF 1 (an outbred strain) mice when supplemented into food sources (Harman, 1968).In response, the ageing field invested substantial time manipulating radical species by both genetic and pharmacological means, with a substantial number of these experiments performed in C. elegans.
Whilst administration of extremely high concentrations of prooxidants (i.e., arsenite, tert-butylhydroperoxide, hydrogen peroxide, paraquat, juglone) has been shown to shorten the worm lifespan, affirming Harman's free radical theory of ageing, such aggressive oxidant exposures poorly reflect natural ageing conditions.Treating worms with anti-oxidant molecules (to reduce endogenous oxidation levels) was thus a preferred strategy to test Harman's theory.Results from α-tocopherol and Trolox (a primary isoform and analog of vitamin E, respectively) treatments have shown lifespan extending effects (Ishii et al., 2004), but have struggled to extend life in other hands (Adachi and Ishii, 2000).Importantly, where others have confirmed vitamin E treatments extend C. elegans lifespan, these animals display significantly impaired reproductive rates and delayed development (Harrington and Harley, 1988), raising the possibility that lifespan extension is coupled to fitness trade-offs, not reduced molecular damage.Further, others have shown that reduced glucose availability promotes the formation of reactive oxygen species that extends lifespan, and vitamin C or N-acetylcysteine (NAC) ablate lifespan-extension effects (Schulz et al., 2007).More recently, it has been shown that lifelong NAC and glutathione exposures shortened C. elegans lifespan, and late-life treatment of NAC (under the premise oxidation becomes deleterious in later ages) slightly reduced lifespan (Gusarov et al., 2021).Employing genetic approaches to test damage theories, Raamsdonk and Hekimi showed that isolated mutations in the five C. elegans SOD genes (encoding mitochondrial superoxide dismutase proteins) did not affect lifespan, and mutation in sod-2 actually extended lifespan despite significantly higher protein oxidation levels (Van Raamsdonk and Hekimi, 2009).Further, they showed that mutations in all five SOD genes within the same genotype did not affect lifespan (Van Raamsdonk and Hekimi, 2012).Mouse models overexpressing key antioxidant enzymes such as catalase, mitochondrial superoxidase dismutase, or combinations of the two also failed to show any extension of lifespan (Jang et al., 2009;Perez et al., 2009).Thus, these data, together with growing knowledge that oxidant molecules play pivotal roles in coordinating signalling events sought to maintain physiological homeostasis such as adaptations to exercise, upregulation of immune responses and metabolic signalling (Sies and Jones, 2020), have raised concerns that molecular oxidative damage might be limited to a symptom, rather than driver of ageing.

Ageing as a trade-off for early-life fitness
Ageing, defined as the increasing risk of mortality with chronological time, results in the progressive and irreversible decline in physiological function of all organ systems (Lopez-Otin et al., 2013).That lifespans differ greatly amongst species (Cohen, 2018) suggests a genetic, programmable aspect to ageing trajectories.Furthermore, wild environments pose significant threat to survival (predation, infection, starvation, competition), thus genes which benefit early reproduction are evolutionarily preferred strategies to assure gene survival into subsequent populations before death from unpredictable and extrinsic factors.Whilst the rates of ageing and late-life manifestations of organismal deterioration might differ across species, evolutionary selection of genetic mutations that contribute to reproductive success is a common operative programme across all phylum (Charlesworth, 2004).
In 1930, R.A Fisher first suggested that ageing is shaped by evolutionary forces (Fisher, 1930).Medawar in 1952, developed a first fully-fledged evolutionary theory of ageing by suggesting that the forces of natural selection decline from the onset of sexual maturity eventually becoming non-existent at death (Medawar, 1952).With this onset of selection 'shadowing' as stated by Medawar (the reduction in the ability of selection to modulate the genome with advancing age), his formulation of the mutation accumulation (MA) theory was born: mutations that do not negatively impact early reproductive fitness could be expressed in the germline escaping purifying selection, contributing later to physiological perturbation when selection has diminished (Medawar, 1952).Following a similar approach, Williams later suggested that selection can favour alleles that increase fitness in early-life but exert detrimental, pleiotropic effects later in life (antagonistic pleiotropy (AP) theory of ageing (Williams, 1957))).These genes will be continually selected for despite any negative effects in older age, given that selection processes are 'blinded' to the effects of gene function in later life after reproduction.Both MA and AP were confirmed mathematically by Hamilton in his seminal 1966 paper (Hamilton, 1966), suggesting that evolutionary processes shape organismal senescence.These theories laid previously unknown insights into how ageing and senescence evolves across species, contributing to subsequent developments in evolutionary ageing theory (see Table 1 for an overview of core evolutionary theories of ageing).
In line with Williams' AP theory, T.B.L Kirkwood suggested a tradeoff between fecundity and ageing itself, based on resource limitations: organisms devoting more finite resources towards reproduction would exhibit shorter lifespans via a reduction in late-life resource allocation towards somatic repair processes (disposable soma (DST) theory of ageing (Kirkwood, 1977))).While Kirkwood considered the DST as separate from AP theory (Kirkwood, 1977(Kirkwood, , 2008)), it is generally considered that DST is a special case of AP, where antagonistically pleiotropic genes regulate resource allocation (Lemaitre et al., 2024;Hughes and Reynolds, 2005).DST suggests that reducing the investment into reproduction in early life, or delaying reproductive onset, will slow organismal ageing rates.Studies in D. pseudoobscura (Wattiaux, 1968), and later in D. melanogaster (Rose andCharlesworth, 1980, 1981), provide support for AP theory through laboratory selection on early vs late reproduction, showing that multi-generational artificial selection for late reproducing flies reduced early-life fecundity, lengthened total reproductive periods and extended lifespan.These findings were striking, given the widely held view that older parental ages likely contribute negatively to offspring health -known as the Lansing effect (Lansing, 1947) -resulting from genomic instability, increased mutation levels, reduced gamete quality and shortened telomeres, amongst others

Table 1
Consolidated theories of evolutionarily-driven ageing programmers.

Evolutionary theory
Core tenets Mutation accumulation (Medawar, 1952) Neutrally acting and slowly developing genetic mutations not affecting reproductive success in early life can accumulate within organisms into old age, given natural selection is blinded after reproductive success.These mutations then ultimately become deleterious to physiology in older age but can no longer be selected against, and mitigative genetic programmes cannot evolve.Antagonistic pleiotropy (Williams, 1957) Genetic variants that contribute to reproductive success are selected for despite any harmful effects they elicit in later ages, given that natural selection forces decline after reproductive years.Thus, genes can exhibit beneficial effects in younger organisms, but the same gene may become detrimental when expressed in older organisms.Disposable Soma (Kirkwood, 1977) Organisms that allocate more finite resources (e.g., biochemical energy) to reproduction will experience higher mortality rates in later years due to a reduced 'pool' of energy stores to undertake damage repair and somatic maintenance.i.e., the ageing process is a consequence of energy trade-off's, where more resource is allocated into early fitness vs late-life repair.Developmental theory (Cutler, 1979) Ageing is a consequence of the inappropriate continuation of developmental programmes once beneficial to early-life fitness and reproductive success, but detrimental to latelife physiological health.Selection shadowing in later years means the expression of developmental programmes cannot be optimised for late life.Hyperfunction (Blagosklonny, 2006) With diminished selection pressure in latelife, no biological mechanism has evolved to optimise the late-life function of early-acting genes.Ageing is thus caused by the 'hyper' activation of wild-type genes that inappropriately function in old age, where reducing their activity would attenuate physiological decline Hypofunction (Maklakov and Chapman, 2019) Physiological decline results from an inability to increase the expression of certain genes that would enable organismal adaptation to aged environments due to selection shadowing in late-life Biological constraint (Cohen et al., 2019;Gems & Kern, 2022) That genes often exhibit pleiotropic functions depending on organismal ages suggests a biological constraint resulting from the complex interconnection of biological systems.Traits thus cannot be evolutionarily optimised for the unique benefit of a single trait alone.

Authors integrated view of the core evolutionary theories of ageing
Evolution optimises the function of genes and allocation of resources to benefit earlylife reproductive success.The decline in natural selection pressures after sexual maturation means evolutionary optimisation of gene expression to promote the physiological health of older organisms is weakened.Thus, the evolution of genetic mutations and/or the optimal expression levels of early acting genes cannot be foreseen by natural selection, leaving the optimisation of genes in the hands of late-life targeted therapeutics.
(reviewed extensively elsewhere (Monaghan et al., 2020)).Following studies artificially postponed the time to reproduction over 15 generations starting from 28-day-old laid eggs, reaching 70-day-old upon the 15th generation (Rose, 1984).When compared to base populations (eggs selected from the first generation), selection for postponed reproduction extended mean lifespan by 10 days, decreasing early reproductive output by a similar 10 days to base stocks, with subsequent increases in late-life fecundity (Rose, 1984).Such fly experiments have been validated in the hands of others, confirming lengthened lifespans in the descendants of late reproducing parents (Partridge and Fowler, 1992;Partridge et al., 1999).However, it must be noted that while these studies provide support for AP theory, the support for the DST is indirect as no mechanism of resource reallocation has been identified in any of these studies.Others have used a unique genetic approach to test DST of ageing in C. elegans.Specifically, by using glp-1(e2141) mutant worms that lack a functional germline, the authors show forced re-investment of resources from the germ line to the soma increases proteasome activity, the clearance of damaged proteins and extends animal lifespan and healthspan (Vilchez et al., 2012).However, the relationship between germline removal and lifespan is complex.For example, germline removal extends lifespan in male but not female C. remanei (Lind et al., 2024), a congener of C. elegans, with the same sex-specific effects evident in Killifish (Moses et al., 2024).This suggests that resource allocation may explain only a part of the germline removal effect on lifespan and ageing.

Fine tuning of developmental programmes is evolutionarily constrained
As an extension to the AP theory, operating under the premise that selection pressure diminishes with ageing, Cutler proposed that developmental genes / programmes required for reproductive success later define the pace of ageing (de Magalhaes and Church, 2005;Cutler, 1979).Optimising the expression of developmental genes for post-reproductive life is, therefore, beyond reach for selection processes, where programmatic 'run-on' (i.e., the continued expression of genetic programmes that benefit reproduction, but become pathogenic when operational in older animals) of developmental pathways might explain the proximate beginnings of physiological deterioration.Examples for this developmental theory of ageing (DTA) are presbyopia, the continual growth of the eye lens eventually causing farsightedness in humans (Strenk et al., 2005), or the continuation of needs to decrease brain plasticity in pre-adulthood that eventually lead to neurodegeneration in old age (de Magalhaes and Sandberg, 2005).More recently, Blagosklonny (2006) provided an extension to the DTA, suggesting that selection shadowing allows for the inappropriate continuation of wild-type gene function, particularly those associated with growth (i.e., mTORC1) and nutrient-sensing (i.e., IGF-1) pathways (Blagosklonny, 2006).A core tenet to the hyperfunction (HF) theory, therefore, is that ageing is caused not by molecular damage (reserved by Blagosklonny for exacerbated disease states such as cancer (Blagosklonny, 2021a)), but a persistent, overactivation of wild-type genes that begin to drive tissue and organ failure leading to diseases of ageing.Interestingly, in humans, the most effective anti-ageing lifestyle interventionphysical exercise trainingrepresses mTOR activation signatures (rapamycin-associated pathways) in those with the largest gains in performance (Phillips et al., 2013).As such, halting the 'run-on' of cell growth pathways appears to benefit human health, implying some applicability of the DTA theory to people.
Conversely, the notion that ageing is accompanied by reduced molecular functions is evident in both worms and people: loss of mitochondrial homeostasis represents a conserved feature of ageing (Migliavacca et al., 2019;Gaffney et al., 2018), and disordered cytoskeletal integrity, muscle architecture and neuromuscular function are prominent in older organisms (Etheridge et al., 2015;Martinez-Hernandez et al., 2022).Therefore, the same evolutionary neglect that allows developmental programme 'run-on' and HF of wild-type genes also gives rise to genetic hypofunction, an insufficiency of molecular machinery to maintain organismal healthspan at later ages (Maklakov and Chapman, 2019).Examples of evolutionary hypofunction have been postulated previously, namely, the inability of elderly elephants to grow new molar teeth, where the wear-and-tear of the preceding set reach such deterioration that feeding becomes limited and death from starvation is common (Kern, 2022).Simply put, biological systems achieve a final form that is optimised for early life, but do not permit malleable adaptations to changing environments in older age.For example, a particular gene expression level in early life might be sufficient to promote infection resistance or produce sufficient biochemical energy for successful reproduction, however, this same expression level can become inadequate (i.e., hypofunctional) to meet the requirements for organismal health in later-life, whilst also being untouched by the selective pressures of evolutionary optimisation (Williams, 1957;Medawar, 1952) (see Fig. 1 for an overview of theoretical gene expression changes in ageing).
Another intriguing observation is that developmental exposure to toxicants that induce ROS formation (paraquat) has been shown to increase the reproductive rate of young adult C. elegans (Smith et al., 2014).Further, higher levels of endogenous developmental ROS can predict longer lifespans of individual worms (Bazopoulou et al., 2019).Therefore, where higher developmental ROS can provide an early-life advantage to reproductive fitness, it could be that maintaining a low expression in genes that buffer these endogenous oxidant molecules might also provide early reproductive benefit.However, in post-reproductive life stages that are associated with excessive and aberrant levels of ROS (forming a central proponent of the hallmarks of ageing (Lopez-Otin et al., 2023)), organisms might suffer the consequences of natural selection being unable to optimise/ upregulate the expression in ROS buffering genes in later life, representing an example of absolute hypofunction (Fig. 1D).
In addition to these new concepts, several theoretical issues remain.Specifically, is it the overactivation or diminishing function of genes that contributes most to ageing pathology?How do these two programmes, undergoing distinct expression-direction changes, interact with each other?What are the proximate molecular alterations (at organismal / tissue-specific levels) resulting from evolutionary neglect that instigate the onset of physiological deterioration?In this review, we postulate on these questions, with a focus on frameworks pertaining to AP/ DTA (i.e., hyper/ hypofunction) and the common ways these are addressed experimentally.

Pleiotropic gene function modulates C. elegans ageing rates
Following the developments of evolutionary theory and selectionbased life extension in flies, Klass (1983) bought promising new insights in the late 20th century: single gene mutations were sufficient to significantly extend C. elegans lifespan (Klass, 1983).Shortly after, and following from the work of Klass, Johnson and Friedman (1988) showed, for the first time, that mutations in the hx546 allele of the age-1 gene (PI3K; component of the IGF-1 pathway) increased C. elegans mean and maximal lifespan by up to 40 and 60 %, respectively (Friedman andJohnson, 1988a, 1988b).Shortly after, mutations in the e1370 allele of the daf-2 gene (IGF-1 receptor) were found to double the lifespan of C. elegans (Kenyon et al., 1993;Gems et al., 1998;Larsen et al., 1995), providing the most profound manipulation of lifespan in a metazoan species.These data stimulated the quest to curate numerous single mutation, long-lived C. elegans strains under the premise that understanding the mechanisms governing long life might contribute new insights into physiological decline across species.Indeed, there exist numerous other well-characterised mutations that elicit significant longevity in the worm: the orthologue of mTORC1 (let-363) (Vellai et al., 2003), the family of Clock genes (clk-1, clk-2, clk-3 and gro-1) (Lakowski and Hekimi, 1996), mutations impairing neuromuscular feeding activity (eat-2) (Lakowski and Hekimi, 1998) and upstream regulators of the mTORC1 complex (RagA GTPase/raga-1) (Schreiber et al., 2010).Eloquent studies further revealed roles for the life-long inhibition of other mRNA translation components (ifg-1 / eIF4G, rsks-1 / S6 kinase) in C. elegans, and the additive lifespan-extending effects by coupling RNAi inhibition of longevity genes into separate long-lived loss-of-function mutant backgrounds (Pan et al., 2007).For example; RNAi inhibition of ifg-1 in let-363 mutants resulted in additive lifespan extension, ifg-1 inhibition in rsks-1 mutants extends lifespan by a further 46 % to rsks-1 mutants alone, but let-363 RNAi in rsks-1 mutants significantly shortened lifespan compared to wild-type animals (Pan et al., 2007).Interestingly, life extension from mutations in the daf-2(e1370) gene are dependent on the activation of the downstream forkhead transcription factor daf-16 (FoxO) (Lin et al., 1997).However, additive lifespan extension from RNAi inhibition of ifg-1 and rsks-1 in the daf-2(e1370) background are not dependent on daf-16 activity (Pan et al., 2007), suggesting the physiological mechanisms controlling daf-2-mediated life extension are not necessary for the effects of translation-inhibition on lifespan.Importantly, a critical commonality is present amongst these mutant strains: significant developmental delays to maturity and severe impairments in reproduction (Pan et al., 2007;Smith et al., 2023;Chen et al., 2007).It is now well known these mutant strains follow the predictions of AP and DST: mutations that promote longevity do so at the cost of early-life fitness (i.e., reproductive output), tying mutant longevity to physiological trade-off.Whilst others have claimed that particular genetic mutations (e.g., age-1 [hx546]) do not exhibit reproductive trade-off's (Walker et al., 2000), these do appear under environmental conditions that mimic the wild (Walker et al., 2000).Additionally, delayed reproduction in early life, but increased late-life reproduction (resembling that of long-lived flies bred for late reproduction (Rose and Charlesworth, 1980;Rose, 1984) in age-1 mutants does not compensate for reduced early-life performance, leading to overall fitness declines in this mutant (Maklakov et al., 2017).The early-life fitness defects evident in daf-2, raga-1, let-363, and clk mutants raises the question of whether physiological decline is a direct effect of chronological ageing or an indirect response to evolutionary trade-off's (i.e., DST).
These early works were successful in two critical areas: 1) Cementing the roles for evolutionary mechanisms regulating ageing processes, and 2), providing well-characterised genetic models to study mechanisms controlling long-life in vivo.Thus, the vast breadth of literature to date investigating ageing mechanisms in C. elegans originate from these lossof-function mutation models, providing reliable, experimentally tractable genetic models for gerontological research.As such, any review of Fig. 1.Multiple scenarios of suboptimal gene expression patterns in ageing resulting from diminishing post-reproductive selection pressure.Red lines portray true gene expression, as observed in wild-type animals, that would promote early fitness at the expense of late-life physiological health.Blue dashed lines represent a hypothetical optimal gene expression pattern to enhance late-life health.Examples where gene expression does not increase, but programme continuation leads to relative hyperfunction (A) or relative hypofunction (B) compared to expression levels in early-life.C) Gene activation that promotes early-life fitness, whose suppressed expression in older age could benefit health (e.g., mTOR, IIS).D) Gene inactivation that promotes early-life fitness, whose activation in older age could benefit health (e.g., ROS-buffering genes).E) Hyperfunctional genes can cause hypofunction of interconnected genes (e.g., the ELT circuit in C. elegans).Conversely, gene set hypofunction can cause hyperfunction of interconnected genes via a decline in the expression of suppressive genes (e.g., loss of miRNA's in ageing), or through an inability of suppressive genes to evolve in the first place (e.g., impossibility constraint, [Gems and Kern, 2022]).
the C. elegans literature inherently focuses on long-lived genetic mutants.However, several caveats exist, for example, life-long mutations in genes that form the basis of developmental programmes (daf-2, let-363, ifg-1 etc.) significantly alters developing physiology, directly hinders reproduction and, importantly, ablates the expression of these genes throughout reproductive periods where selection is known to have its greatest effect.Thus, studying the role of any loss-of-function gene mutation in ageing as a consequence of programmatic 'run-on' / HF is impossible.In the ageing context, it is likely more relevant to study the effects of manipulating their genetic function in later ages (i.e., after reproductive periods), potential options for which are discussed later in this review.

What constitutes a hyperfunctional gene?
HF theory posits that ageing results from the overactivation of cellular functions that drive diseases of ageing, with functional and molecular decline a secondary consequence to programmatic hyperfunctions (Blagosklonny, 2021a).HF is rooted within AP / DTA frameworks that imply multiple counter-opposing effects of a gene on fitness separated by life stages.For AP genes to evolve, they must provide a fitness benefit in early life that can be progressively re-populated by selection through means of reproduction.Thus, whilst physiological aberrations ultimately arise from the pleiotropic functions of genes in late-life, these genesand their associated expression level at any given life stage -are, in principle, wild-type.For the assumptions of HF-induced ageing to be met experimentally, attenuations in the decline of physiological function should become apparent by the inactivation of a (wild-type) HF gene after developmental / reproductive periods.Numerous genome-wide RNAi screens have been performed in C. elegans to identify potential longevity genes (Hamilton et al., 2005;Hansen et al., 2005;Kamath et al., 2003;Lee et al., 2003).Although novel lifespan-extending genes were identified, encompassing gene expression, protein turnover, mitochondria and metabolism-related biological functions, the inactivation of many of these genes led to developmental arrest and impaired progeny production, i.e: they play critical roles in normal animal development and contribute to successful reproduction.With such genes fitting AP criteria (e.g., positive effects on development / reproduction place them under strong evolutionary selection pressures), Kapahi's group sought to inhibit the expression in 54 of these pleiotropic genes that arrest development, but exclusively in adult worms (Chen et al., 2007a).Whilst RNAi in 42 % of these genes resulted in lifespan extension, gene silencing from the first day of adulthood led to significant impairments in reproductive output in all genes except 1 (let-611/ C48E7.2).Thus, inactivation of wild-type genes critical for development (that have ultimately become pleiotropic / HF) can extend lifespan, but these data remain constrained within DST / AP trade-off mechanisms.For HF theory to be best tested experimentally (and to isolate from trade-off effects), manipulation of gene function should occur to not alter any aspect of normal developmental processes or reproductive activity.Only few studies meeting these criteria have been reported, and these are discussed below.

Genetic hyperfunction drives ageing pathology accumulation in C. elegans
Much debate exists surrounding the importance of measuring changes in lifespan as a measure of organismal ageing (Keshavarz et al., 2023).Indeed, healthspan (for example, physical movement capacity as a validated readout of animal health in worms (Bansal et al., 2015)) extension can occur independently of increased lifespan extension (Roux et al., 2023;Vintila et al., 2023), and long-lived animals can spend longer periods of their life in states of frailty (Bansal et al., 2015).Ageing, under normal conditions (i.e., non-cancerogenic), is likely to occur from the accumulation of multiple negative ageing pathologies that drive organ failure, ultimately causing death.Thus, assessing tissue/ organ morphology represents an appealing approach to study the contribution of genes to known ageing phenotypes that are particularly well-mapped in worms (Gems and de la Guardia, 2013).
Detailed structural analyses of individual animals found that C. elegans exhibit an excessive cuticular overgrowth in late-life and accumulation of yolk proteins throughout the animal (Herndon et al., 2002).Specifically, expression of the vitellogenin gene vit-2 (measured using VIT-2::GFP) remains highly ordered within the intestine and oocytes of young worms rich in sperm.Upon sperm depletion however, yolk begins to accumulate extensively throughout the animal representing HF of reproductive biosynthetic systems that, when inhibited (i.e., vit-2 RNAi), can extend C. elegans lifespan (Murphy et al., 2003).Furthermore, despite the termination of reproduction (representing a time-point of strong natural selection decline), oocytes continue to develop within the C. elegans gonad becoming highly crowded and contribute significantly to the age-related hypertrophy in the adult germline (Jud et al., 2008).Others have shown similar results, where the formation of teratoma-like tumors in C. elegans results from HF of endoreduplication programmes, driving unfertilized oocyte entry into the uterus leading to detrimental cellular hypertrophy via endoreduplicated chromatin masses (Wang et al., 2018).
There also exists pleiotropic function of autophagic processes between early and late-life ages (Wilhelm et al., 2017), and others have shown that the continued activation of autophagic programmes that promote fitness at younger ages cause severe intestinal atrophy in aged animals (Ezcurra et al., 2018).Specifically, autophagic conversion of intestinal biomass to yolk in C. elegans contributes to successful oogenesis and egg development, exampling a candidate biological process vulnerable to AP / HF with age.Indeed, programmatic run-on of autophagy-driven gut-to-biomass conversion promotes intestinal atrophy in older animals, and intestine-specific inhibition of autophagic processes slows morphological pathology and extends lifespan (Ezcurra et al., 2018).Similarly, apoptosis of germ cells throughout embryogenesis contributes to the provision of developing oocytes and promotes fitness in early adulthood, although continuation of this pathway causes gonad degeneration and ageing decline in older animals (de la Guardia et al., 2016).
The loss of neuromuscular and functional health with age has typically been considered a consequence of gradual and progressive declines in gene and tissue functions.Teleologically, the impaired physical activity of organisms in older age could result, in part, from declines in e. g., muscle fiber integrity, postsynaptic junction activity and axonal signalling, associations evident in both worms and people (Gaffney et al., 2018;Khosa et al., 2019).The notion that HF of neuronal networks potentially contributes to physical decline, however, is less widely appreciated.In fact, neuronal integrity is maintained in older C. elegans (Herndon et al., 2002), suggesting that neuromuscular decline in worms (occurring much earlier, where movement rates significantly decline after ~ 5 days post-adulthood) is not caused by neuronal perturbation.More recently however, others have shown that neuronal 'outgrowth' occurs in C. elegans with age, and is amplified or attenuated in short-lived (jnk-1/MAPK8) and long-lived (daf-2/IGF-1) mutants, respectively (Tank et al., 2011).Much like the programmatic run-on of autophagic, embryonic, and yolk-producing pathways, the HF of neurite branching processes could be involved in impaired neuromuscular health in C. elegans, and post-reproductive inactivation of key member genes should be further explored.

Post-reproductive inactivation of hyperfunctional genes extends C. elegans lifespan.
RNAi against the daf-2 gene when applied only in adulthood (days 1 through 6 of adulthood) extended lifespan in C. elegans without impairing reproductive rates (Dillin et al., 2002a), confirming daf-2's importance throughout development but suboptimal wild-type HF expression in later life.Importantly, this study evidenced that both early and late-life Darwinian fitness can be optimised via late age inhibition of a key HF gene.The first comprehensive post-reproductive RNAi screen knocked-down over 800 chromatin and transcription-regulating genes in C. elegans (Wilhelm et al., 2017).With RNAi onset from day 9 of adulthood (the onset of substantial physiological decline in worms), 36 lifespan-extending candidate genes were identified (30 of which represented previously unknown modulators of lifespan).Interestingly, the authors show that genes integral to early-life autophagic flux become detrimental to animal lifespan and healthspan in later life, and that post-reproduction inhibition of neuronal autophagosome nucleation-regulating genes extends lifespan, implying vulnerability of the autophagic system to AP in old animals (Wilhelm et al., 2017).
More recently, Maklakov's group tested age-specific optimisations of known lifespan-extending genes in C. elegans.Specifically, genes regulating nutrient-sensing (age-1/PI3K), mTOR (raga-1/Ras-related GTPase RagA), global protein synthesis (ifg-1/eIF4G), somatic-cell protein synthesis (ife-2/eIF4E) and mitochondrial respiration (nuo-6/ NDUFB4) were inhibited with RNAi across three life-stages: life-long (from L1 larval stage), adulthood, or post-reproduction (Lind et al., 2021).Pertaining to classical DST, nuo-6 inhibition showed a strong negative correlation between reproductive rates and lifespan extension.Interestingly, both age-1 and ife-2 inhibition from all three life-stages extended lifespan without any reproductive fitness costs, evidencing that lifespan can be uncoupled from reproductive trade-off's.Whilst suggesting that some genes might confer the ideal scenario of concurrently optimising early-and late-life Darwinian fitness, potential complications from the use of RNAi-expressing bacteria could play a role.For example, detectable gene silencing can be seen within 24-48-hours, however, disparity in expression levels between genes, efficiencies in RNAi uptake, and age-related variances in bacterial consumption can dramatically alter predicted timings (Kamath et al., 2003;Fraser et al., 2000;Timmons and Fire, 1998).Nonetheless, observations for raga-1 and ifg-1 supported HF predictions.For example, life-long raga-1 inhibition decreased lifespan (despite no change in reproduction), whereas adulthood and post-reproductive inactivation extended lifespan.Both life-long and adulthood-onset RNAi caused dramatic reproductive decline in ifg-1 knock-downs, providing no beneficial trade-off in lifespan, but post-reproductive inactivation caused significant lifespan extension (Lind et al., 2021).Thus, lifespan extension by inhibiting HF-related genes associated with developmental / growth-related pathways supports HF frameworks.

Expression profiles of interconnected gene networks can be intricately linked
HF theory has been viewed with resistance over the years as an opposing view to long-standing damage accumulation and molecular decline theories (Blagosklonny, 2021b).Whilst Blagosklonny remains firm in his view that ageing ensues from the HF of developmental programmes, it is true that aged organisms display widespread accumulations in damage and impaired functions in the 'hallmarks' of cytoprotective pathways (Lopez-Otin et al., 2023).Debate is ongoing regarding the roles that molecular insufficiency (i.e., hypofunction) vs hyperactivity plays in ageing (Gladyshev, 2016), a phenomenon not discussed by Blagosklonny.Whilst both hyper/hypofunctional changes can arise independently as a consequence of the selection shadow (see Fig. 1A -1D), the interconnected effects between genes with counter-opposing expression profiles also likely affect each other directly.In fact, in budding yeast, proteins with known regulatory roles in the ageing process share greater levels of connectivity than those thought not to be involved in ageing deterioration (Promislow, 2004).Thus, whilst the selection shadow might be responsible for the initial failure to upregulate hypofunctional genes in later life, this can have knock-on consequences for genes that suppress downstream hyperfunctional targets during ageing, (e.g., the hypofunctional expression patterns of micro-RNA's in ageing tasked with degrading accumulating mRNA transcripts; see Fig. 1E).
An example of hyperfunction-induced hypofunction is the GATA transcriptional circuit in C. elegans (Vintila et al., 2023;Budovskaya et al., 2008;Hu et al., 2017;McGhee et al., 2009).Specifically, elt-5 and elt-6 are crucial transcriptional regulators of intestinal seam-cell development throughout larval growth stages.Their inactivation promotes embryonic and larval lethality, causing the fusion of seam cells with the syncytia that impairs molting phases (Koh and Rothman, 2001).Both elt-5 and elt-6 guide the expression levels of elt-3, a constituent member gene responsible for regulating thousands of downstream cytoprotective targets (Hu et al., 2017).With age, the expression profiles of elt-5/6 continue progressively (another potential example of HF), causing a direct suppression of elt-3 that inhibits the upregulation of its downstream components (Budovskaya et al., 2008).RNAi or pharmacological inhibition of elt-5/6 alleviates the suppression on elt-3, promotes the upregulation of its regulatory gene sets, increases stress resistance and extends lifespan and healthspan (Vintila et al., 2023;Budovskaya et al., 2008).Importantly, these effects are ablated when performed in an elt-3 null mutant strain, exemplifying the physiological importance of elt-3 expression-alleviation.This highlights that HF of developmental programmes can restrict the expression of interconnected genes, leading to molecular and physiological insufficiency at older ages.

Overactivation of growth pathways can inhibit evolved systems that counter gene hyperfunction
A vast portion of the C. elegans transcriptome displays age-related declines in gene expression vs an upregulation of transcript abundance (Vintila et al., 2023).However, these hypofunctional changes might not be proximate causes of ageing deterioration themselves, but rather, reflect both transcriptional drift (the tendency for gene expression-direction changes to diverge from younger profiles) and suppressive inhibition from upstream pathways.Specifically, the PI3K/AKT/mTOR axis is known to suppress mitochondrial/metabolic function (Johnson et al., 2013;Ramanathan and Schreiber, 2009), and a sizeable portion of genes decreasing with age in humans and in worms encode for mitochondrial and metabolic pathways (Migliavacca et al., 2019;Vintila et al., 2023).This suggests a possibility that HF of growth-related pathways are involved in the onset of mitochondrial dysfunction, and perhaps biochemical resource allocation that remains a priority within growth-related pathways can impair energy provision required for the regulation of metabolic pathways.HF of biosynthetic pathways (for example, the mTOR complex, a primary nutrient-sensing complex) would signal energy-rich states for the continuation of protein biosynthesis, one of the cells most energetically expensive tasks.This deleterious loop of mitochondrial function-inhibition caused by mTOR HF, but an increasing demand for biochemical energy provision for somatic maintenance highlights a detrimental positive feedback loop that is bound within AP mechanisms.Indeed, genetic inhibition of components upstream of mTOR (raga-1/ RagA) attenuates the loss in mitochondrial structural decline with age in C. elegans (Zhang et al., 2019), and has been shown to restore muscle mitochondrial ultrastructure in rodent models of myopathy (Civiletto et al., 2018), suggesting optimisation of late-life HF pathways can alleviate suppressive restraint on declining metabolic pathways.
Similarly, organisms have evolved gene regulatory mechanisms that are in place to control aberrant mRNA synthesis.Micro-RNAs (miRNA) are small, non-coding RNA molecules that play pivotal roles in posttranscriptional gene regulation (O'Brien et al., 2018).They function by binding to the 3′ untranslated region (UTR) of target mRNAs, leading to mRNA degradation (O'Brien et al., 2018).Of the small RNA family, miRNA's are the only subtype that show an age-dependent decrease, where ribosomal RNA's, small nucleolar RNA's, transfer RNA's and small ribosomal RNA's show a consistent accumulation with ageing (Kato et al., 2011).Importantly, it has been shown that targeted mutations in TSC1 (a negative regulator of mTOR function) decreases miRNA biogenesis (Ye et al., 2016).Accordingly, genetic inhibition of Raptor (a key component required for mTOR function) increased the levels of miRNA synthesis, both results suggesting that mTOR HF is involved in the suppression of miRNA synthesis required for reducing mRNA accumulation in ageing.

Hypofunction can result from an inability of late-acting genes to evolve
It has been recently proposed that 'biological constraint' can be a proximate cause of ageing (Kern, 2022), proposing a refinement to the ideas of Williams AP theory (Williams, 1957).Specifically, as a demonstration for AP, a gene regulating Ca 2+ production that would provide early benefits for bone development, but contribute to the deposition of Ca 2+ into vascular tissue in late-life, therein driving atherosclerotic plaque formation (Williams, 1957).Williams suggested that a gene regulating Ca 2+ could theoretically evolve to optimise later-life Ca 2+ levels, but this would not occur due to the declining selection strength after reproduction (Medawar, 1952;Hamilton, 1966).In his biological constraint essay, Gems has proposed that the widespread prevalence of AP genes in biological systems highlights an inability to evolutionarily optimise gene function of interconnected traits (Kern, 2022;Acerenza, 2016).Additionally, hypofunction resulting from impossibility constraint is also proposed: that genes are not always underperforming in late-life due to the inability to optimise for both early and late-life functions (i.e., interconnected constraint leading to AP), but sometimes from the inability of genes to evolve in the first case.This was supported by enzymological examples from Acerenza's essay (Acerenza, 2016), where an enzyme regulating the chemical combination of two ethanol molecules would provide an effective means to synthesise n-butanol and release energy, yet no enzyme exists.Thus, with limited enzyme catalysis reactions, it might not be chemically achievable for such as enzyme to evolve, rather than an inability of evolution to select for such enzymatic traits (Kern, 2022;Acerenza, 2016).
Therefore, both overactivation of wild-type genes and molecular and phenotypic insufficiency can result from many causes: continued gene function that once promoted fitness throughput development (DTA / HF), reproductive vs somatic trade-off's (DST), inabilities to optimise genes for both early-and late-life (AP), hypofunction resulting from the suppression of upstream hyperfunctional genes (HF), and the impossibility of corrective genes to evolve in the first instance (impossibility constraint).Growing bodies of work provided evidence for testing DTA, DST, AP and HF ageing mechanisms, and robust data exist for highthroughout approaches to screen for ageing-promoting HF gene candidates (Lee et al., 2003;Chen et al., 2007a;Wilhelm et al., 2017), as well as tissue/ organ-centric approaches to study HF in ageing (Herndon et al., 2002;Jud et al., 2008;Wang et al., 2018;Ezcurra et al., 2018;de la Guardia et al., 2016).However, to truly untangle the roles of hyperfunction and hypofunction in ageing (and their interconnections), we propose a more systematic and data driven approach combining multi-omics with temporal and spatial manipulations of putative HF genes for healthspan screening, and will discuss this below.

A remodelling of transcriptional apparatus towards a hyperfunctional state
The C. elegans transcriptome displays well-established alterations with ageing, with consistent declines in genes/pathways regulating mitochondrial function and metabolic processes, and increases in stress response, DNA damage repair and innate immunity-regulating pathways (Wang et al., 2022;Vintila et al., 2023).Up to 6000 genes can be seen to significantly change in expression profiles from young-adult to aged timepoints in the whole worm (Rangaraju et al., 2015).The assumption made using common pathway/ gene-set analysis pipelines is that these genetic alterations directly contribute to functional physiological changes.Whilst a proportion of gene expression changes inevitably contributes to healthspan deterioration (e.g., decreased gene expression and synthesis of mitochondrial proteins, a hallmark of ageing (Lopez-Otin et al., 2023)), upstream molecular systems that regulate the transcriptional apparatus are subject to age-related dysfunction (Ham et al., 2022), suggesting a class of genetic changes that neither promote nor deter ageing progression but rather reflect a coordinative loss of internal gene regulatory systems.
Recent data support this hypothesis, highlighting that transcriptional drift (where genes within a functional group change their expression in opposing directions vs youthful profiles) increases with ageing, and that mRNA stoichiometry between genes within the same functional group begin to conflict and disrupt homeostasis in C. elegans (Rangaraju et al., 2015).Specifically, individual genes that comprise a unit (i.e., a functional pathway) responsible for regulating a given biological process change their expression profile late in life compared to youthful ages.This opposing expressional 'switch' has been shown to dysregulate the communication within, and regulative capacity of functionally related signalling pathways (Rangaraju et al., 2015).Mechanistic explanations for transcriptional drift remain indefinable and, whilst being a difficult phenomenon to address experimentally, the role of diminishing selective pressure likely plays a role in allowing such widespread transcriptional dysregulation.Separately, others revealed that RNA polymerase II (RNAPII) transcription-elongation speeds increased progressively with chronological age in worms and in tissue from higher mammals (Debes et al., 2023).As a result, the time for splicing events are compromised, and increased amounts of circular RNA's, rare protein isoforms and intron-retaining transcripts become prevalent.Genetic inhibition of subunits required for RNAPII function (ama-1/POLR2A loss-of-function mutation strain) in C. elegans slowed elongation speeds, reduced circular RNA formation and extended lifespan and healthspan (Debes et al., 2023).This represents a similar phenomenon to the HF of developmental/ growth-related genes in ageing previously demonstrated within single genes and pathways, however, at the level of global transcriptional apparatus (see Fig. 2).Further work understanding how changes in the elongation speeds of RNAPII influences transcriptomic drift in ageing could reveal new insights into ways of attenuating transcriptomic integrity in ageing.
Additionally, alterations in RNA/mRNA processing genes could be an underlying cause of aberrant transcriptional regulation with ageing.Ham and colleagues recently showed that age-associated increased usage of distal (vs proximal) alternative 3' (A3) splice sites led to an increase in non-coding RNA (ncRNA) formation that accumulate with ageing in C. elegans (Ham et al., 2022).This increase in unprocessed RNA's containing both exons and introns (i.e., mis-spliced transcripts) supports the notion that normal RNA processing is lost with ageing and contributes to significant increases in mis-spliced genes and RNA isoform accumulation.Despite the potential impact of these findings for understanding ageing progression, proximate causes that lead to these impairments in RNA processing are poorly understood.Increased RNAPII speeds are known to impair splicing events and increase intron-retaining transcripts by reducing the time for splicing decisions (Debes et al., 2023), suggesting that HF of RNAPII could be a cause of aberrant splicing in ageing (see Fig. 2).Indeed, life-long genetic manipulations in core subunits of RNAPII in worms and flies attenuates the formation of circular RNA's from mis-spliced events (Debes et al., 2023).

Hyperfunction of transcriptional apparatus suppresses metabolic health
Recent work in C. elegans explored age-associated increases in transcription rates and its associated metabolic costs (Sharifi et al., 2024).RNA polymerase I (RNAPI) accounts for a significant portion of (caption on next page) L. Slade et al. Ageing Research Reviews 100 (2024) transcription and pre-rRNA processing, and the transcription initiation factor 1 A (TIF-1A) interacts with RNAPI and converges across IIS, mTOR and AMPK pathways (Sharifi et al., 2024).Genetic depletion of TIF-1A in worms via RNAi reduced pre-rRNA levels and extended animal lifespan.Further, inhibition of TIF-1A was shown to attenuate the ageing decline in mitochondrial ribosome components, and mitochondrial ATP production was restored back to young-adult levels.Interestingly, overexpression of TIF-1A increased pre-rRNA levels, promoted growth and neuromuscular fitness early in life, but at the cost of a shorter lifespan (Sharifi et al., 2024).As a similar model to TIF-1A overexpression, RNAi inhibition of NCL-1 (an RNA-binding protein that inhibits pre-rRNA processing) elevated pre-rRNA levels, increased young-adult body size and improved neuromuscular performance in early-life (day 2 of adulthood).However, older NCL-1 animals (day 12) showed impaired neuromuscular performance and lived significantly shorter than wild-types, both examples demonstrating the control of lifespan and healthspan via trade-off's in developmental growth pathways.
Similarly, the authors note early-life reductions in the neuromuscular performance following TIF-1A inhibition, but improvements later in life.
Transcriptional processes thus exhibit pleiotropic functions throughout the ageing spectrum, i.e., activity of the global transcriptional apparatus is prerequisite for early-life fitness, but, if left unabated, can become deleterious later in life.A caveat with the approach utilised in this study is the knock-down of TIF-1A by RNAi across two generations before animals were collected for experimentation.Inhibition of particular genes are well known to have transgenerational effects on the physiology of subsequent generations in C. elegans (Greer et al., 2011).Thus, transgenerational repression of transcription regulators could impact reproductive capacity in following generations (but was not reported in this work), where reports of lifespan extension and neuromuscular healthspan-promotion are confounded by potential underlying trade-offs in reproductive effort and soma regulation (as discussed earlier, conforming to the DST).However, adult-onset RNAi of rpoa-2 (RNAPI subunit POLR1B) from days 0, 6 and 8 of adulthood significantly extends lifespan and improves neuromuscular health, suggesting late-life optimisation of transcription rates can improve healthy ageing in C. elegans.Thus, with transcriptomic drift, improper splicing events and the HF of multiple RNA polymerase subunits that comprise healthspan, deeper understandings of the precise molecular changes that underpin the turning point for transcriptomic dysregulation is an important next step in ageing research.

The C. elegans ageing proteome
Whilst numerous studies have explored transcriptomic remodeling with ageing in C. elegans, limited reports have explored changes in the C. elegans ageing proteome.Despite this, significant improvements in mass-spectrometry resolution and fractionation techniques for reduced sample complexity and improved protein coverage have developed our understandings into the C. elegans ageing proteome.Additionally, targeted downstream interrogation of identified proteins using fluorescent reporter strains is beginning to couple global molecular changes to physiological function in vivo.Over >5000 proteins have been profiled across days 1, 6, 12, 17 and 22 post-adulthood using the stable-isotope labelling with amino acids in cell culture (SILAC) approach (Walther et al., 2015).One-third of the worm proteome altered at least 2-fold with ageing, where 50 % of these changes were progressively increased abundance up to day 22 (Walther et al., 2015).Proteasomal components encompass a majority of proteins with increased ageing expression, and small heat-shock proteins were upregulated up to 90-fold with age.This stark accumulation of protein degradative systems could reflect a facilitatory response to protein accumulation to promote effective clearance and avoid proteotoxic cell stress.Moreover, comparing proteins with increased abundance between days 6 and 22 post-reproduction with transcript levels in previously published dicer mutants, defective in miRNA biosynthesis (Welker et al., 2007) revealed up to 40 % of accumulating proteins identified display transcriptional upregulation in dicer mutants.This suggests that loss of miRNA function with relation to transcriptional/translational processes can contribute to ageing-induced protein accumulation.Whilst others have suggested mRNA levels are weakly correlated with protein abundances (Gygi et al., 1999;Washburn et al., 2003;Ideker et al., 2001) this highlights the complex coupling of (post)transcriptional and proteomic dysregulation in driving protein accumulation in ageing (see Fig. 2).
Subsequently, Kenyon's group performed deep analysis of the ageing proteome using SILAC covering >9300 proteins across days 1, 5 and 10 post-adulthood (Narayan et al., 2016).Of these, 627 significantly changed in abundance across the three time points, with proteins linearly increasing with ageing and enriched for terms that include nucleosome assembly, cellular macromolecular assembly tissue morphogenesis and response to stress.Conversely, protein groups declining in abundance with ageing were enriched for fatty acid metabolic processes, organic acid biosynthetic processes and oxidation reduction.These data began to highlight distinct biological differences in deteriorating vs accumulating pathways in C. elegans, with a loss in metabolic processes and continual accumulation in growth related processes.To note, this decline in metabolism, but increase in proteins of growth-related pathways fits accordingly with both DST and HF theories of ageing, respectively.
More recently, age-related changes in protein ubiquitination at the C. elegans proteome level have been examined (Koyuncu et al., 2021).Ubiquitination marks proteins for degradation by proteasomal units by tagging lysine residues with ubiquitin for identification.The decline in proteasomal degradation is well established in ageing (Hipp et al., 2019), ensuring its place within the hallmarks of ageing as a potential causal player in physiological decline (Lopez-Otin et al., 2023).Deep profiling of proteome-wide ubiquitination, however, illustrated that declines in ubiquitination and proteasomal activity with age are not simply a passive byproduct of ageing as commonly understood.With the significant decline (1813) vs increase (350) in ubiquitinated peptides in day 15 vs day 5 worms, but no clear changes in the expression levels of ubiquitin-encoding genes, the authors showed a theme for increased protein expression of de-ubiquitinating proteins (Koyuncu et al., 2021).Consequently, the increased de-ubiquitination of proteins with ageing led to impaired site recognition: many proteasomal targets contained the Lys48-linked poly-ubiquitin tail but not the Lys63-linked chain, Fig. 2. Hyperfunction of global transcriptional and translational machinery as a proximate cause of age-related changes in molecular function.In developmental and reproductive life stages, rates of mRNA transcription and protein synthesis are evolutionarily programmed to favour early-life fitness and achieve successful propagation.Declines in selection pressure, commencing after sexual maturity, promote persistent and unoptimized functions of transcriptional and translational machinery that underlie molecular features of ageing associating with poor health and/or early death.Hyperfunction in RNA polymerase subunits drive increased elongation rates of pre-mRNA's, accumulating mRNA, and impair the efficiency of splicing events, which could contribute to the widespread drifti.e., ageing gene expression profiles that oppose young adult counterparts -of transcriptomic co-expression signatures.Hyperfunction of translational machinery can increase mRNA mis-translation, the effects of which can be exacerbated by parallel declines in protein degradation systems (e.g., proteasomes) and loss of regulatory miRNA gene expression.Inhibiting the function of key growth/developmental genes (e.g., daf-2 mutants) reverses the myriad of transcriptional and translational aberrations that extend lifespan and healthspan.The extent to which these molecular and physiological improvements result from evolutionary trade-off's (in the context of daf-2 allelic mutants), however, requires a deeper understanding.
reducing their recognition by proteasomal sites and subsequent degradation.The authors further show that proteasomal subunit proteins requisite for wild-type C. elegans development and early-life fitness increase in expression with ageing, and inhibition of their encoding genes restores ubiquitination levels in aged worms, extends lifespan, and augments neuromuscular health and sarcomeric muscle organisation.Strikingly, inhibition of individual de-ubiquitination genes exhibiting HF can restore proteome-wide ubiquitination levels, associating with augmentations in healthspan (Koyuncu et al., 2021).
Contrary to classical views that protein degradation systems passively decline with ageing, these data suggest that programmatic runon of de-ubiquitination processes underlies an impairment in ubiquitination and degradation of accumulating proteins in older age.

Downregulation of protein biosynthesis attenuates ageing physiological decline
With the continuation of macromolecular/ tissue biosynthetic processes and an accumulation of proteins at the proteome level, it appears intuitive that slowing translational rates might improve organismal ageing.As a key regulator, inhibition of mTOR has become a routine approach for studying the effects of reduced protein translation.Initial studies found that life-long genetic downregulation of mTOR regulating protein targets (ifg-1, rict-1, rsks-1, let-363) extended lifespan in C. elegans, exhibiting slower mRNA translation rates by reductions in 35 S-methionine incorporation (Pan et al., 2007).Others have shown that either pharmacological (Rapamycin) or genetic (via rsks-1 RNAi) inhibition of mTOR from adulthood reduced the accumulation of insoluble protein aggregates in older C. elegans extending lifespan (Yee et al., 2021).More recently, others used cycloheximide exposures (used to block translation-elongation) to examine the roles of systemic translation-inhibition on C. elegans ageing.Cycloheximide beginning from the first day of adulthood significantly extended lifespan across a range (0.1, 1 and 10 µM) of concentrations (Takauji et al., 2016).Using β-galactosidase staining as a readout of senescence, cycloheximide also reduced the intensity of age-associated β-galactosidase staining in worms subjected to inhibition of protein synthesis.Mild protein synthesis restriction in normal (TIG-7) and tumor-derived human cells of the cervix also reduced senescent features such as cell swelling and replicative senescence (Takauji et al., 2016).Given that cycloheximide exposure began on day-1 of adulthood in C. elegans, it is plausible that reproductive rates were lower in these animals, however this was not assessed.Further work exploring the effects of post-reproductive cycloheximide exposures would help to uncouple lifespan extension and ageing protein accumulation from trade-offs in reproductive fitness.Indeed, whilst life-long inhibition and/or mutation in translational (and transcriptional) components provides the greatest physiological effects to explore causal mechanisms, there is an experimental need to employ similar interventions in post-reproductive and aged animals.

The interconnection of core transcriptional and translational machinery
That both transcriptional apparatus and protein biosynthetic pathways display HF in ageing suggests a potential interaction between the two.With both playing fundamental roles in the successful development and reproductive performance of organisms, genes central to the operation of these pathways are vulnerable to AP / HF effects.It is known that mTORC1 plays roles in controlling the activity of RNA polymerase subunits via the phosphorylation of regulatory transcription factors that determine gene expression (Laplante and Sabatini, 2013;Sukumaran et al., 2020).Thus, whilst the selection shadow might explain how mTOR can be continually activated in late-life, the communicative feedback loop with transcription apparatus thereafter might also contribute to biosynthetic protein accumulation in ageing.Whether RNA polymerase and mTORC1 HF occur in isolation or act synergistically to drive aberrant mRNA / protein accumulation in ageing remains poorly understood, and mechanistic approaches to untangle this relationship are needed.Others have, however, demonstrated that core regulatory kinases of RNAPII operates alongside mTORC1 to regulate specialised translational networks essential for chromosome stability (Choi et al., 2019), further implicating the importance of understanding how this relationship changes after reproductive years.

Long-lived C. elegans mutants slow age-associated hyperfunction across multiple biological systems
The apparent HF of multiple transcriptional programmes in ageing wild-type C. elegans prompts the question of how these are affected by genetic mutations that extend lifespan.The daf-2(e1370) mutant model has become an invaluable tool to explore transcriptomic and proteomic signatures associated with longer life.Importantly, daf-2(e1370) mutants exhibit an attenuation of HF-related changes in transcriptional, translational and morphological states observed wild-type ageing C. elegans (discussed above).

Attenuation of mRNA accumulation, elongation speed and aberrant splicing in daf-2 mutants
Polysome profiling of daf-2 mutants shows a significant decline in the number of actively translated mRNA's compared to wild-types, and poly-(A) + tail trapping assays highlighted a reduction in the total amount of mRNA compared to wild-type (Stout et al., 2013).Contrary to previously mentioned work, total protein levels were unaltered between the two when assessed by a simple spectrophotometric approach, which could suggest that an accumulation of pre-mRNA transcripts vs translated proteins are burdensome to wild-type ageing, supporting earlier findings showing inhibition of mRNA translation to extend lifespan (Pan et al., 2007;Hansen et al., 2007) and reducing pre-mRNA levels via RNAPI inhibition promotes healthy ageing (Sharifi et al., 2024).This slowing of mortality rates by inhibiting transcriptional apparatus central to gene expression and protein synthesis appears somewhat counterintuitive for organismal adaptation.However, as central molecular regulators of early-life fitness and reproductive success, core components of the transcriptional machinery meet the criteria for being vulnerable to AP / HF changes in older ages.That RNAPII activity becomes hyperfunctional with age across species, driving increases in mRNA elongation speeds (Debes et al., 2023), suggests RNAPII HF could be a proximate cause of excessive mRNA transcription in aged organisms.Mutations in core RNAPII subunits (ama-1/ POLR2A) extends lifespan/ healthspan, and daf-2(e1370) mutants have significantly slower elongation speeds in aged day-14 animals compared to wild-type (Debes et al., 2023).Thus, inhibiting the function of a central developmental growth-related pathway that is not directly involved in RNAPII activity can slow the HF of transcriptional processes in C. elegans (see Fig. 2 & 3).
The spliceosome of ageing wild-type animals also shows extensive remodelling, retaining higher amounts of intron-containing transcripts and showing increased usage of distal vs proximal A3' splice sites and higher prevalence of skipped exons (Ham et al., 2022).Whilst splice site usage was not mechanistically explored, these data suggest a further scenario where RNAPII HF could be a proximate cause of inefficient splicing.Specifically, ribosomes bind to pre-mRNA transcripts and read nucleotide sequences from 5' ends to 3' ends of introns (Bentley, 2014).Thus, HF of RNAPII that accelerates ribosomal elongation speeds along the transcript could reduce the time for splice site recognition, forcing increased usage of distal (i.e,.further away from 5' ends) vs proximal A3' splice sites.Indeed, the number of rare splicing events and accumulation of mis-spliced circular RNA's increases in day-14 vs day-1 wild-type animals: both of which are attenuated by RNAPII inhibition (Debes et al., 2023).Importantly, daf-2 mutants exhibit attenuations in the usage of distal A3' splice sites and number of exon skipping events, potentially linking the lowered RNAPII activity in these mutants to improvements in mRNA splicing with age (Ham et al., 2022).Impaired splicing fidelity of aged animals is also associated with a decline in the expression of RNA-processing transcripts, providing an additional and alternative mechanism of splicing impairments in wild-type ageing (Ham et al., 2022).The same authors show an attenuation in the gene expression of RNA-processing genes in daf-2 mutants (plrg-1 [catalytic spliceosome component], pfs-2 [mRNA polyadenylation] and F30A10.9[small subunit rRNA processome]), where RNAi-inhibition of these genes ablates the lifespan-extension phenotype of these animals.

Transcriptomic drift is diminished in daf-2 mutants
In wild-type ageing, genes begin to display divergent expression profiles (i.e., significantly increasing or decreasing) compared to younger ages, termed transcriptomic drift (Rangaraju et al., 2015).Similarly, the co-expression profiles of genes within a function group (e. g., a biological pathway such as oxidant response genes) also begin to differ greatly amongst each other with ageing, impairing mRNA stoichiometry, co-expression signalling and physiological function (Rangaraju et al., 2015).Interestingly, RNAi against the daf-2 gene (maintained from larval stages and throughout adulthood) suppressed transcriptome-wide drift, where day-6 daf-2 adults showed profiles similar to those of day-1 wild-types (Rangaraju et al., 2015).When combinations of daf-2 and daf-16 (the downstream target of daf-2) RNAi were tested, attenuations in transcriptional drift were not only abolished but elevated compared to wild-type animals (Rangaraju et al., 2015).These data implicate the FoxO transcription-factor as a central regulator of the ageing transcriptome, in line with others reports that daf-16 expression stabilises the C. elegans transcriptome (Li et al., 2019).Mechanistic evaluation into the mechanisms governing daf-2-mediated improvements in transcriptomic drift are yet to be established, and should prove a fruitful area of future investigation.

Organism-wide protein accumulation is lowered in daf-2 mutants
The wild-type proteome sees an extensive increase in the abundance of proteins throughout ageing (Walther et al., 2015), conforming to the HF of translational machinery.Full proteomic analysis in long-lived (daf-2) and two short-lived (daf-16 and hsf-1/HSF1) mutants spanning 27 days of the worm life-course found that the wild-type ageing increase in protein abundance was significantly decreased in daf-2 mutants, and elevated in short-lived mutants (Walther et al., 2015).Others have previously shown that translational flux and mRNA levels are lower in daf-2 mutants compared to wild-type, controlled by daf-16 expression (Depuydt et al., 2016).Combined with observations of lowered translational flux and mRNA levels in daf-2 mutants, protein aggregation might be anticipated to be lower in these long-lived mutants.However, daf-2 animals accumulate more insoluble proteins vs wild-type, particularly enriched for small heat-shock species and proteasomal complexes (Walther et al., 2015).Interestingly, aggregate-forming proteins were less hydrophobic, more charged and displayed higher disorder compared to wild-type aggregating proteins, suggesting that the accumulation of potentially toxic protein species could augment global proteostasis by preventing their presence within the soluble proteome, demonstrated by daf-2's improved ability to maintain the expression of a metastable FlucDM-GFP protein.Importantly, decreasing protein abundancy is suggested to aid in the solubility of proteins, where excessive protein formation can drive insoluble aggregate formation by overcrowding (Ellis and Minton, 2006), highlighting the importance of suppressing excessive protein accumulation in ageing (see Fig. 2).

Protein turnover changes in daf-2 mutants
More recently, protein turnover rates and protein half-lives have been investigated in wild-type and daf-2(e1370) mutants at day 2 postadulthood.The assumption of these authors was that protein accumulation and potentially their longer 'dwell' times could be detrimental to protein homeostasis.In one study, 54 % of analysed peptides showed slower turnover rates in daf-2 mutants, particularly in proteins involved in regulating the translation machinery, with a near doubling of average protein half-life time from 103 to 173-hours in daf-2 vs controls (Dhondt et al., 2016).Gene ontology enrichment showed those with reduced turnover and increased half-lives were enriched for mitochondrial, stress-response and cytoskeletal/muscle-related proteins.Protein translation is one of the cells most expensive energetic tasks (Buttgereit and Brand, 1995), thus, reductions in protein translation and accumulation together with suppressed turnover rates of cytoprotective proteins could represent a potential energy-saving phenomenon whilst allowing the levels of protective proteins to remain for longer within cellular environments, as suggested by the authors (Dhondt et al., 2016).How a single gene mutation could permit such a desirable effect, however, is not clear, and cytoprotective proteins with slower turnover rates identified by the authors were not mechanistically tested/ implicated to be a requirement of the extended lifespan of daf-2 mutants.Moreover, protein would only accumulate if the rate of synthesis exceeded that of degradation.If reductions in protein translational are met with a similar reduction in their turnover rates, then the level of protein remaining within the cell is unaffected.It is known that protein translational rates are lower in daf-2(e1370) mutants (Depuydt et al., 2016), therefore viewed together, alterations in protein accumulation might not reflect a key molecular feature to the extended lifespan/ healthspan of these mutants.
This work was published alongside others that explored the role of protein turnover in ageing and mutant models of longevity and protein aggregation.The findings, however, were mostly contrasting to the aforementioned study (Visscher et al., 2016).Using daf-2(e1370) and human (muscle-driven) α-synuclein overexpression strains (as an established Parkinsonian model), the authors used a heavy nitrogen labelling approach to show that protein turnover in daf-2(e1370) mutants was slower in developing animals (L4 larvae) but faster in day-5 adults, and slower at both ages in α-synuclein overexpressing animals compared to controls (Visscher et al., 2016).This represents a crucial difference to findings in the aforementioned work (Dhondt et al., 2016) that could lead to largely different interpretations.If protein turnover is faster in daf-2(e1370) mutants, coupled to the slowing of protein translation rates in these mutants, a mechanism involving the rapid turnover and replacement of damaged proteins could be in operation.
Adding further complication, the turnover rates in α-synuclein overexpressing animals were reduced proportionally across the proteome at both ages.When considered with the previous work, the role slower protein turnover plays in controlling organismal ageing, thus, becomes unclear.For example, both daf-2 mutants and α-synuclein animals exhibit a slowing of protein turnover in younger age, making the connection between reduced turnover and longevity (as suggested in the previous work) limited at best.
Importantly, there are few considerations that should be made when comparing these data.Although turnover rates in daf-2 mutants were, on average, shown to be significantly slower in day-2 adults in one study (Dhondt et al., 2016) and significantly faster in day-5 adults on the other (Visscher et al., 2016), there are 3-days of adulthood that separate these data that span a period of significant growth in C. elegans.One critical difference is the use of combined glp-4 (a temperature-sensitive sterile mutant) and daf-2 mutations in the work showing a slowing of turnover rates (Dhondt et al., 2016), meaning this study analysed turnover rates of proteins from somatic tissues, as previously performed (Vilchez et al., 2012).Further, the temperature for glp-4;daf-2 mutants was 4 degrees higher than that of single daf-2 mutations: the lifespan and healthspan of C. elegans is known to be drastically altered between such temperatures (Gaffney et al., 2018).Lastly, of the group showing faster turnover rates in day-5 daf-2 mutants, there was no 5-fluro-2′-deoxyuridine (FUdR) used in these experiments, a chemical used to inhibit the enzyme thymidylate synthetase interfering with DNA synthesis that blocks reproductive capacity (Mitchell et al., 1979).Therefore, important differences form using reproductively active (Visscher et al., 2016) and sterile (Dhondt et al., 2016) daf-2(e1370) mutants might explain the opposing effects these two groups see in the turnover rate of proteins in adult daf-2 mutants.This highlights a key message of our work: modulations of normal reproductive periods can substantially alter the molecular environments of organisms.These small but important differences between these studies can have profound effects on the interpretation of such data.There is certainly a need for future studies exploring the roles of protein turnover in contributing to wild-type ageing pathologies.
Therefore, within this section, we show that continued expression of growth pathways such as daf-2/IGF-1 in later life appear to contribute to the negative physiological effects of chronological ageing, and limiting the expression/activity of such pathways can positively effect lifespan/ healthspan via an attenuation of age-related HF across transcriptional/ translational systems (see Fig. 2).

Long-lived mutants for healthspan researcha complex story
The vast majority of C. elegans ageing research to date has exploited mutants harbouring stable genetic mutations integrated into the genome, providing life-long inhibition of desired genetic candidates for mechanistic research.Similarly, the use of life-long RNAi against target genes provides a robust and simple to use platform for genetic inhibition studies.Such models have provided paradigm shifts in our understanding of genes that control the rate of organismal ageing.However, fundamental limitations of using such models exist when exploring physiological processes underlying age-related deterioration, particularly, when studying the contribution of gene function to physiological decline at different life stages.Specifically, DTA, AP and HF theories maintain that ageing is a consequence of the inappropriate hyper/hypofunction of genes once beneficial for early-life fitness, but pathogenic in older age.Many of these genes are related to organismal growth, development and nutrient sensing, and loss-of-function in these genes from birth lead to physiological defects, embryonic lethality and developmental arrest (see Fig. 3).Although allelic substitution (e.g., e1370 allele in daf-2 mutants) can produce viable adults for experimentation, the high prevalence of severe reproductive and growth defects amongst many mutations makes separating trade-off's from physiological augmentation very complicated.Supporting this, animal healthspan in well-characterised long-lived mutants (e.g., daf-2, clk-1, ife-2 and eat-2) does not scale with the extent of lifespan extension (Bansal et al., 2015;Chen et al., 2007) i.e., animals spend longer in a state of poor health.Conversely, others have recently refuted lifespan/healthspan dissociation in daf-2(e1370) mutants, due to the presence of elevated odorant receptor expression, which drives food consumption vs exploration (Hahm et al., 2015).When life-course measures of neuromuscular health are carried out in the absence of food, daf-2 mutants display a comparable scaling of healthspan to wild-types whilst retaining the benefit of significantly longer lifespan.Nevertheless, this feature appears exclusive to daf-2 mutants, where many other lifespan extending mutations inflict significant fitness costs when scaled to lifespan extension and early-life fitness impairments (Bansal et al., 2015;Chen et al., 2007;Maklakov et al., 2017).Additionally, despite healthspan improvements under favourable (i.e., food odorant receptor inhibition) conditions in daf-2 mutants, this does not circumvent the early fitness defects in these mutants such as reduced fecundity, egg retention, growth defects and germline shrinkage (Venz Fig. 3. Multiple mechanisms of improved health in long-lived C. elegans mutants highlights a need for adult-onset anti-ageing therapeutic strategies.In accordance with the disposable soma theory of ageing, a finite pool of resources (i.e., biochemical energy) are available for reproductive or soma-maintenance processes.In earlylife stages of C. elegans (i.e., development and young-adulthood) the physiological health of wild-type animals is high, and energy demands for somatic maintenance are low.Thus, preferential allocation of resources into wild-type reproduction promotes early-life success, yet depletes resources required for somatic maintenance processes in older age when physiological health is low.Long-lived mutants display significant impairments in reproductive rates compared to wild-type animals, reducing early-life success, but increasing available resources for late-life somatic maintenance to mitigate health decline and delay death.Additionally, under wildtype conditions, high levels of reproductive activity engage the genes required for embryogenesis, representing gene sets vulnerable to programme hyperfunction.Late-life physiological burden can, therefore, arise from both energy deficit and gene hyperfunction paradigms, both of which are attenuated by mutations in developmental/growth-related genes that extend lifespan.The heatmap bar on the right reflects the hypothetical levels of developmental gene-expression from low (pink) to high (red).Physiological burden pertains to the formation of tissue/ organ pathologies that cause physiological impairments and ultimately death.et al., 2021).Indeed, these early life defects likely, at least in part, underpin lifespan extension in these animals (and others such as clk-1 (Chen et al., 2007)), in line with DST and DTA (see Fig. 3).
A further complication for the proper study of evolutionary theory in ageing C. elegans, is the widespread use of FUdR.DTA posits that reproductive programmes drive ageing pathology.Ablating reproduction therefore interferes with the normal function of developmental genes, confounding interpretations of improved physiological function from genetic and/or pharmacological interventions.Whilst early studies showed FUdR exposure in late-developing animals does not alter C. elegans lifespan (Gandhi et al., 1980), others have shown that FUdR can alter C. elegans lifespan and ageing pathology accumulation, which are dependent on exposure timings (Wang et al., 2019).Specifically, introducing FUdR at pre-adulthood stages (L4 larvae) shortens lifespan and facilitates intestinal atrophy.Exposure commencing in adulthood (days 2 and 3 post L4 stage), however, extends lifespan, improves pharyngeal degeneration and reduces the formation of uterine tumors (Wang et al., 2019) both key ageing pathologies driving C. elegans mortality (Wang et al., 2018(Wang et al., , 2019)).Thus, whilst the effects of FUdR on organismal health can be multifaceted, the presence of this compound can dramatically alter physiology, adding further complication to ageing studies in nematodes (see Fig. 4).Of note, the vast majority of studies evaluated in this review demonstrating attenuations in ageing employ the use of FUdR.Therefore, in addition to existing complications of life-long mutations in developmental genes (e.g., daf-2, let-363, age-1), reproduction-inhibition might contribute to late-life phenotypic improvements.For example, cycloheximide treatments from L4 larvae (under FUdR exposure) extends lifespan and attenuates senescence (Takauji et al., 2016), yet, if these same effects would be seen when energetic resource allocation is required within normal reproductive programmes is not known.Similar arguments can be made for that of ama-1/ RNAPII life-long mutations (Debes et al., 2023): restriction of global transcription might only benefit organisms when the normal function of costly developmental programmes are abolished.The reproductive output of animals in these studies was not measured, making claims of AP-related effects difficult.If the roles of AP and HF in ageing are to be addressed experimentally it is, in our opinion, crucial that conditions permit wild-type reproductive capacity.The advent of newer genetic technologies (i.e., auxin-inducible degron mutants) should prove useful for studying the healthspan effects of late-life gene optimisation.

Protein-degron systems represent a new candidate approach for antagonistic pleiotropy and hyperfunction research
The Auxin-inducible degron (AID) system was first identified in plants (Nishimura et al., 2009), but has been adapted for use in mammalian cell lines and animal models such as C. elegans, Drosophila, zebrafish and mice (reviewed previously (Phanindhar and Mishra, 2023)).Briefly, proteins are engineered to include small AID peptide tags for recognition by auxin upon its introduction into the system.These proteins are then subject to ubiquitin-induced degradation and removal of the protein from the system, commonly with ~90 % efficiency, a stark improvement on the inconsistent knock-down of genes using RNAi approaches (Phanindhar and Mishra, 2023).This allows for tight temporal control of protein levels either within the entire organism to examine system biological effects, or within tissue-specific regions (by expressing the ubiquitin ligase substrate recognition subunit, TIR1, in the tissue of interest (Roy et al., 2022)) using promoters for tissue-targeted degradation of AID-tagged proteins.AID can also be coupled to GFP motifsthis technique applied to (e.g.) C. elegans allows in vivo conformation of protein degradation using fluorescence microscopy.A further important technological aspect of this method is that the effects of AID are quickly and easily reversible upon the removal of auxin, permitting acute temporal switching of target protein expression at any life stage Post-developmental AID-based degradation of DAF-2 at the global, non-tissue specific level caused 70-135 % lifespan extension, surpassing that of both daf-2 (e1368) and daf-2(e1370) mutants, suggesting degradation of wild-type daf-2 protein elicits greater effects than allelic manipulation of daf-2 (Venz et al., 2021).When performed specifically in neurons or the intestine (but not muscle) AID degradation of DAF-2 Fig. 4. Reproduction-inhibiting chemicals routinely employed in ageing experiments mask physiological processes that form the basis of late-life hyperfunctional pathways.C. elegans ageing experiments almost exclusively exploit the reproduction inhibitor 5-fluro-2′-deoxyuridine (FUdR) to limit potential progeny contamination within populations.However, if AP and hyperfunction theories are correct, genes required during sexual maturation are bound for late-life overactivation due to selection shadowing, with run-on expression detrimental to organismal health via senescent pathology accumulation.In accordance with the disposable soma theory, inhibition of costly (i.e., biochemically speaking) developmental processes could mitigate the accumulation of senescent pathologies via a 'sparing' of resources for soma regulation.Mitigating the widespread use of FUdR in ageing C. elegans experiments is thus vital for understanding true, physiological functions of late-acting genes.
still induced lifespan extension but to lesser extents than daf-2(e1370).This greater magnitude of life extension in daf-2(e1370) vs neuronal or intestinal DAF-2 protein loss, could highlight AP-related soma tradeoffs, where this mutant (but not tissue-specific AID against DAF-2) has a marked reduction in reproductive investment.An important observation from this work was the extent of lifespan extension when systemic AID of DAF-2 was induced from days 10 (48-72 % increase) and 12 (49-57 % increase) post-adulthood.Because this contradicts earlier reports of reduced lifespan caused by daf-2 RNAi when applied onwards of day 6 post-adulthood (Dillin et al., 2002a), this possibly indicates experimental limitations of double-stranded RNA interference when investigating AP/DST ageing theories (Dillin et al., 2002a).Moreover, AID starting from days 21 and 25 post-adulthood (very old age for the worm and a timepoint where up to 75 % of the population had died) extended animal life for a further 22 and 36 days, respectivelya major increase in longevity that, on its own, is as long as a full wild-type lifespan.
Subsequent work also employed DAF-2 AID in C. elegans, recapitulating findings (Venz et al., 2021) that neuronal and intestinal, but not muscle AID, was sufficient to extend lifespan (Roy et al., 2022).Extending these observations, epidermal and germline depletion was insufficient to extend lifespan.Despite the ability of intestinal and neuronal DAF-2 AID to improve lifespan, neuromuscular performance was not correspondingly improved in later life, reflecting a negative gerospan extension model.Conversely, DAF-2 depletion in muscle did not affect lifespan, but significantly improved late-life neuromuscular performance, representing compression of gerospan and a positive outcome for 'healthy ageing' (gerospan compression models are discussed later, see Fig. 6).
The AID system was also recently exploited for global somatic inhibition of RAGA-1 and LET-363, as well as neuron-specific depletion of both protein targets in C. elegans (Smith et al., 2023).Severe impairments in early-life fitness are well known for both of these genes in loss-of-function mutants (Vellai et al., 2003;Smith et al., 2023).Accordingly, global somatic AID of RAGA-1 from day 1 of adulthood impairs reproduction and developmental growth but leads to extended lifespan, whereas neuronal-specific RAGA-1 AID extends life whilst avoiding perturbations in developmental fitness.Conversely, day 1-onset of global somatic AID-induced degradation of LET-363 impairs survival and induces a severe reproductive defect leading to internal hatching, but neuron-specific LET-363 AID significantly extends life and avoids impairments in developmental and reproductive fitness (Smith et al., 2023).Whilst neuromuscular health was not assessed, these findings support that HF of growth-related complexes (e.g., IIS and mTOR) negatively affects lifespan (and where data permits, ageing functional health (Venz et al., 2021)).Combined, this data further supports the potential for lifespanhealthspan dissociation upon manipulation of core AP/DST pathways, and extends this by highlighting the need for careful consideration of tissue specificity and temporal onset of hyper/hypofunctional interventions.Importantly, AID systems represent a powerful method for the post-reproductive temporal and spatial optimisation of gene function in vivo, and can be effective in geriatric animals where AP is known to onset and ageing pathologies to accumulate from the HF of wild-type genes.

Using systematic multi-omics approaches to identify hyperfunctional programmes
Hyperfunction research, particularly in C. elegans, largely focuses on well-established pathways of cell growth/development to elicit pleiotropic effects in older age (i.e., IIS, mTORC1).Although late-life manipulations of these pathways remains underexplored, mapping the myriad of hyper/hypofunctional genetic and proteomic changes using systematic approaches stands to broaden the HF horizon.Mapping an age-related increase in gene/protein expression to physiological perturbation can be challenging in many ex vivo and cell culture models.However, C. elegans offer a unique opportunity to precisely manipulate gene/protein function at any stage of the lifespan to study the consequences on physiological function in real-time (e.g., movement capacity, survival).Thousands of transcripts are known to have increased expression profiles in aged C. elegans.Whilst many of these might reflect adaptive responses (i.e., increased expression of autophagic genes to combat biomass accumulation), many could be a consequence of continued developmental programmes (de Magalhaes and Church, 2005;Cutler, 1979).Indeed, that inhibition of central ROS-buffering genes can extend worm lifespan (Van Raamsdonk andHekimi, 2009, 2012), or that late-life inactivation of autophagic genes attenuates healthspan in old worms (Wilhelm et al., 2017) initially contradicted long-held believes in the field.Thus, a non-biased and systematic approach for uncovering hyperfunctional molecular profiles is fundamental to inform future functional -omic screens.Illustration for such experimental workflows are presented in Fig. 5.
That hyperfunctional genes can elicit direct hypofunction of interconnected genes via suppression restraint highlights the advantages of systematic omic approaches.Specifically, transcription-factor binding site and/or protein-protein interaction analyses can inform putative upstream regulators of differentially downregulated gene clusters (e.g., as performed in (Vintila et al., 2023)), providing a condensed pipeline to enrich HF candidates for late-life inhibition screens.Much like that of the ELT-5/6 HF-induced constraint on ELT-3 expression, it is plausible that numerous other interconnected networks exist within a similar operative framework.Thus, such an approach will also help us to understand how much of the ageing transcriptome is hypofunctional because of HF gene-induced suppression in later-life (e.g., if hyperfunctional transcription factors are causally reducing expression of a downstream hypofunctional gene network, inhibiting such regulatory transcription factors would be anticipated to increase the expression of the hypofunctional gene set).
The advent of protein degron mutants in C. elegans provides an exciting new approach for late-life targeting of pleiotropic genetic candidates.Unlike RNAi feeding approaches, genomic integration of degron tags selectively degrades target proteins within hours of auxin administration, allowing temporal and reversible control of protein function throughout the organism, or within distinct tissue-specific regions (Zhang et al., 2015).Understanding how degron-inhibition of well-established HF candidates (i.e., mTORC1, IGF-1, RagA) within individual tissues affects healthspan decline is gaining traction (Smith et al., 2023;Zhang et al., 2019;Venz et al., 2021;Roy et al., 2022), despite continuing to employ protein inhibition from early in the life-course (first day of adulthood), with a single exception (Venz et al., 2021).
Efforts to study late-life onset (such as > day 10 post-reproduction) will provide new insights into the effectiveness of optimising gene HF both ubiquitously and within specific tissues that better mimic human intervention timings.Performing high-throughput functional genomic screens for late-life inactivation of novel HF targets will also delineate between true adaptive vs HF gene expression in ageing organisms (see Fig. 5).Advancements in machine learning and artificial intelligencebased drug prediction models are also beginning to show promise for uncovering potentially gero-protective molecules (Chen et al., 2023a;Vidovic et al., 2023).A combinatorial workflow of multi-omic pleiotropic analyses, functional in vivo validations and computational drug-prediction pipelines could provide a strong scheme to advance the study and translation of mitigative therapies to combat HF in ageing.

Temporal considerations for therapeutic interventions
Much like late-life gene optimisation research, there is an appreciable lack of experimental data exploring the effectiveness of late-life drug treatments in C. elegans.It is likely that this research focus has been driven by the goal of maximizing any physiological effects of drug treatments.Indeed, known gero-protective molecules can become ineffective when administered later in the C. elegans life-course (Rangaraju et al., 2015;Cabreiro et al., 2013;Espada et al., 2020).In humans, anti-ageing treatments typically begin in later-life, i.e., once sub-cellular and physiological decline have already manifested.Thus, while life-long treatments in C. elegans have been insightful, understanding how later-life drug treatments improve healthspan should perhaps now be the focus of the ageing research field.Many essential biochemical processes are established during C. elegans development: developmental starvation imprints adult foraging behavior (Pradhan et al., 2019), mitochondrial dynamics can determine adult respiration levels and lifespan (Dillin et al., 2002b) and individual variances in levels of ROS through development can predict adult longevity (Bazopoulou et al., 2019).Developmental genetic / pharmacological exposures, therefore, likely interact with these process, all of which play central roles in adult health, therein confounding the applicability of such ageing studies for forward-translation.As such, post-adulthood interventions will likely be pivotal for translational research, since the mechanisms governing healthspan attenuation from life-long drug exposures likely differ substantially from those administered later in life.Limited studies have implemented post-reproductive drug treatments in C. elegans, and most of these only investigate drug treatment onset at the first day of adulthood, rather than critical geriatric periods of the worm life-course.
For example, the anti-diabetic drug metformin has been proposed as an anti-ageing candidate in C. elegans (Onken and Driscoll, 2010).However, these data, and the many that followed, employed metformin exposure from young-adulthood to yield positive effects on lifespan/ healthspan (Chen et al., 2017;Banerjee et al., 2016;Saewanee et al., 2021;De Haes et al., 2014;Xiao et al., 2022;Mor et al., 2020).In both C. elegans and mice, metformin treatments starting at geriatric periods (56-60 weeks in mice, and day-8 in worms) fail to show significant lifespan extension (Anisimov et al., 2011;Alfaras et al., 2017), and can become toxic when administered at later ages in both organisms (Cabreiro et al., 2013;Thangthaeng et al., 2017).Given the known mitochondria-targeting properties of metformin, others have demonstrated that toxicity in day-8 treated worms is underpinned by impaired metabolic plasticity and mitochondrial function (Espada et al., 2020).That phenotypic effects from late-life metformin treatments in C. elegans matches those seen in higher mammals highlights the usefulness of this model for geriatric drug screens and the importance in considering intervention timings to improve the translatability of drug screening.Similar patterns have been noted with the gero-protective anti-depressant molecule mianserin, where 50 µM treatment from day 1 of adulthood extends lifespan by 46 %, acting through powerful suppression of age-related transcriptomic drift (altered expression of ~80 % of the transcriptome) (Rangaraju et al., 2015).When administered in post-reproductive (day 5) adults however, the lifespan extension effects are completely abolished (8 % lifespan reduction), and the degree of transcriptomic drift mirrors wild-type untreated animals.Concerns regarding the uptake of drug molecules when administered in aged worms have been previously raised.However, Petrascheck's group showed constituent genes within xenobiotic-response pathways were equally upregulated (~1,000-fold expression increase) from day-1 and day-5 treatments, refuting claims for reduced compound uptake with age (Rangaraju et al., 2015).Others have shown the nicotinic acid-based psychostimulant, Arecoline, can significantly increase neuromuscular performance when acutely treated (assessed 10-minutes after exposure) in aged (day-9 and day-15 post-adulthood) C. elegans (Liu et al., 2013), highlighting that effective compounds can continue to preserve healthspan decline towards the end of the worm lifespan.
To our knowledge, there is a dearth of research implementing postreproductive and late-life drug treatments in C. elegans.Rodents studies have examined compounds that mitigate the age-related HF of mTORC1 and IGF-1 pathways.Rapamycin treatment starting from the 600th day of life in male and female mice significantly extended late-life survivability compared to untreated controls (Harrison et al., 2009).Although healthspan was not assessed, these lifespan effects were more prominent when treatment began from 600 days (~60 y in humans) vs 270 days of life.More recently, transient rapamycin treatments (8 mg/kg injection for 3-months) in middle-aged male mice significantly Fig. 5. Uncovering putative hyperfunctional molecular regulators of healthy ageing.Transcriptomic and proteomic analyses at the systems biological (in vivo) and tissue-specific levels, sampled at key stages of the C. elegans lifespan, can characterise molecular clusters with increasing/ decreasing expression during and after reproductive periods.Overlapping gene/protein targets can then be identified to establish the strongest candidates for functional genomic screening in vivo.Utilising auxin-inducible degron mutants allows ubiquitous and/or tissue-specific inhibition of target genes at defined timepoints post-reproduction to study the effects of reversing late-life gene hyperfunction on animal survival and healthspan.To study the activation of hypofunctional targets, transcription-factor binding site analyses can identify predicted upstream regulators of hypofunctional gene sets.Degron-inhibition screens would, in-turn, identify whether hyperfunction of putative regulatory transcription factors causes suppression-induced hypofunction of downstream targets.extended lifespan and healthspan (Bitto et al., 2016).Delivery of monoclonal antibodies against the IGF-1 receptor in 78-week-old mice was also sufficient to extend lifespan and healthspan (Mao et al., 2018).Given that mutations in genes of these pathways causes lethality in rodents (Powell-Braxton et al., 1993), these data lend further support to the spectrum of evolutionary theories, that gene expression of fundamental developmental pathways continue unoptimised in aged organisms.
In C. elegans, post-adulthood treatments with mitochondria-targeted hydrogen sulfide treatments to study healthspan across the life-course have been performed (Vintila et al., 2023).Hydrogen sulfide (H 2 S) is a gaseous signalling molecule produced endogenously in invertebrates and higher mammals (Szabo et al., 2014), and is a conserved post-translational regulator of mitochondrial function across species (Zivanovic et al., 2019).The age-related decline in H 2 S levels is associated with multiple diseases (Slade et al., 2024), and exogenous supplementation from L1 larval stage throughout life can extend lifespan, healthspan and ameliorate numerous ageing and disease-related phenotypes (Miller and Roth, 2007;Qabazard et al., 2014;Ng et al., 2020).However, the application of life-long exposure raised the possibility that lifespan extension is caused by developmentally-induced hormetic mechanisms (Sokolov et al., 2021).However, when mitochondrial H 2 S is administered to young adult or day 4 post-adulthood animals, healthspan was increased but lifespan was unaltered.Importantly, adult-onset, but not developmental drug treatments significantly restored ageing transcriptomic profiles in late-life via the downregulation of ELT-6 gene/protein expression, allowing the upregulation of genes controlled by ELT-3 GATA transcription factor circuit (Vintila et al., 2023).Additionally, mitochondrial health and muscle-structural integrity were maintained for longer periods in adult vs developmentally treated animals.These greater transcriptomic and sub-cellular improvements of putative ageing-related targets in adult vs developmental H 2 S treatments raise the question: is lifespan extension a fundamental requirement for interventional success in C. elegans studies?Could healthspan extension up to the limit of existing animal lifespans (i.e., gero-compression) be a more biologically meaningful finding for translational research?Similarly, two transcription factors, lin-1 and ztf-28, with increased age-related expression (Roux et al., 2023) extended animal healthspan following RNAi knockdown, without affecting lifespan.It may, therefore, be that lengthened lifespan is a dispensable phenotype for effective healthspan improvements in C. elegans.

Lifespan extension or gero-compression?
Living longer without simultaneous improvements in physiological health is a major societal burden, increasing time periods spent in frail conditions (Chen et al., 2023b).Recent mathematical projections suggest that a compression of morbidity, rather than an extension of lifespan, would provide greater socio-economic gain (Scott et al., 2021).Despite this, an overwhelming majority of lower organism research focuses on genetic and/or therapeutic strategies to extend animal lifespan, infrequently corroborating these findings with life-course measures of healthspan or an offset in senescent phenotypes, with the assumption these readouts are inextricably linked.Importantly, we know there are distinct mechanisms controlling lifespan vs healthspan in C. elegans (Bansal et al., 2015).Thus, the absence of lifespan extension in response to genetic/pharmacological intervention in isolation does not necessarily indicate ineffective healthspan effects.The coordinative scaling of healthspan with lifespan extension is a non-trivial requirement to make the claim of ageing attenuation (Bansal et al., 2015).Consequently, could interventions that extend healthspan up to the limits of natural lifespan be preferential over those that extend both lifespan and healthspan?
For example, if i) the onset of physical deterioration was suppressed until later in life vs normal ageing, albeit with identical lifespans, healthspan is sufficiently extended via a compression of gerospan (Fig. 6B).Similarly, if ii) such intervention significantly extended lifespan and the onset of deterioration was further delayed proportionally to longer life, the time spent in gerospan is further compressed relative to lifespan (Fig. 6 C).Importantly, iii) lifespan and healthspan extension where the onset of deterioration does not scale with longer life would reduce time in gerospan compared to normal ageing projections, however, would be less effective compared to both previous examples (Fig. 6D).Lastly, iv) were lifespan extension to occur, but with no delayed onset of physiological deterioration, the relative time sent in gerospan is exacerbated beyond normal ageing periods (Fig. 6E).Thus, small but meaningful differences in relative lifespan/healthspan ratios induced by genetic or pharmacological interventions are an important, but somewhat overlooked aspect for translational research.
Recent individual ageing experiments in C. elegans lend support for this concept (Zhang et al., 2016).Specifically, isogenic populations of C. elegans show uniform levels of physiological fitness in early adulthood, but lifespan and healthspan begin to diverge across individuals of the same age throughout ageing, an event previously believed to be stochastic in nature (Herndon et al., 2002), but might in fact involve genetic (Zhu et al., 2023) and programmatic (Rando and Wyss-Coray, 2021) control.Importantly, some individuals that live longer exhibit lower rates of physiological decline, however, undergo a disproportionately extended period of frailty (Zhang et al., 2016).In humans, medical advancements have contributed significantly to increased life expectancies, however, improvements in sanitation, vaccinations and nutrition encompassed the vast majority of longer life (Garmany et al., 2021).Therefore, extending the human lifespan to similar degrees is an unlikely near-term possibility, where it has recently been suggested that humans are approaching their maximal lifespan, and further extension would require interventions beyond improvements in healthspan (Dong et al., 2016).Thus, we argue interventions that can extend healthspan up to species-specific lifespan limits in C. elegans (e.g., a gerospan compression model) will identify the most promising forward-translational candidates.It is therefore crucial that animal healthspan (e.g., thrashing rate, crawling speed) is analysed in combination with lifespan to provide a more accurate assessment of changes in organismal health across the life-course.The continual development of high-throughput microfluidic platforms will be central to the widespread adoption of dual lifespan/ healthspan measures by significantly reducing the time burden of traditional plate-based assays (Rahman et al., 2020).17.From worms to humansthe need for better models

Mutations in nutrient-sensing genes attenuate worm ageing, but promote human pathology
As discussed in detail here, the daf-2(e1370) mutant represents the most commonly used genetic model for slowing ageing rates in worms, however, coupled to trade-offs in early-life fitness.Critically, such findings do not map to human phenotypes, where impaired IGF-1 function is associated with diabetes and skeletal muscle loss in people (Lu et al., 2022;Hata et al., 2021;Lewitt et al., 2014).In fact, elevated circulating levels of IGF-1 have been suggested to plays protective roles against the development of glucose intolerance in both male and female individuals aged between 46 and 65 years of age (Sandhu et al., 2002).That life-long mutations in an allele of the worm IGF-1 receptor extend lifespan and delay physiological decline, therefore, contradict the requirements for healthy ageing in people.This does lend further support to our claim within this work: ageing attenuations from life-long loss-of-function mutations in nutrient-sensing developmental genes likely result from evolutionary trade-offs.This exemplifies the need for more appropriate models (e.g., the use of degron mutants) to study how tissue-specific manipulations in these receptors affect C. elegans ageing rates.For example, Venz and colleagues showed that AID of daf-2 within the muscle of C. elegans did not extend lifespan or healthspan, in accordance with the known importance for the IGF-1 system in maintaining muscle mass in humans, whereas intestinal degradation of daf-2 extended both lifespan and healthspan whilst evading any early-life fitness defects (Venz et al., 2021).

Healthy ageing strategies in humans elicit a repression of growthrelated signatures
Currently, exercise remains the most effective healthy ageing regime in humans, and muscular strength is one of the greatest predictors of allcause mortality (Ruiz et al., 2008).mTOR upregulation is a requisite response for muscle protein synthesis in humans, ablated by rapamycin treatments (Dickinson et al., 2011).However, the importance of mTOR upregulation for acute vs chronic adaptations to resistance exercise remain debated (Terzis et al., 2008).Interestingly, Phillips and colleagues showed that (human) individuals displaying the greatest hypertrophic responses to a 20-week resistance training programme demonstrate an inhibited mTOR activation signature, with a concomitant downregulation of 70 ribosomal RNA's (Phillips et al., 2013).This represents a paradoxical finding for muscle anabolism/adaptation to resistance exercise, yet aligns with the positive effects of suppressing growth-related pathways on ageing as demonstrated in C. elegans, Drosophila and rodents (detailed above).Critically, the positive effects of mTOR inhibition on worm healthspan are maintained when inhibited within specific tissues that circumvent early-fitness defects seen with life-long mutations (Smith et al., 2023).Of course, many of the benefits of resistance exercise are explained by improvements in insulin sensitivity, mitochondrial biogenesis, and epigenetic modifications (Plaza-Diaz et al., 2022), however, it is interesting to consider (exercise-induced) repression of growth pathways as a contributor to healthy ageing in humans and how this associates with healthspan improvements in lower organisms.Thus, optimisation of gene function (genetically or pharmacologically) in these pleiotropic pathways might converge across similar molecular networks to those of resistance exercise regimes.With 50-75 % of individuals aged 75+ years deemed physically inactive (AgeUK, 2019), gene optimisation could represent an alternative mimetic strategy for healthy ageing regimes in elderly individuals unwilling or otherwise unable to participate in regular physical activity.
Together, the slowing of organismal ageing rates from life-long mutations in AP genes does not always match that of healthy ageing signatures in humans.We believe transitioning to more recently developed genetic models that allow tissue-specific and temporal manipulation of hyper/hypofunctional targets later in the worm life-course will provide rich information on putative programmes driving ageing pathologies.

Conclusions and future directions
Long-standing frameworks that ageing is caused by a passive accumulation of molecular damage have been challenged in recent years.The emergence of proposed ageing hallmarks motivated substantial scientific efforts to combat these myriad of molecular perturbations, however, yielding limited success.Understanding if the proposed hallmarks of ageing are causally involved in the physiological decline of organisms, or if they represent mere secondary symptomologies to ageing deterioration, remains an ongoing effort.Indeed, considerable model organism research suggests these traits to be poor predictors of healthy ageing.Thus, whilst features of molecular damage, oxidative stress and mitochondrial dysfunction are sure to plays important roles in exacerbating healthspan decline, proximate molecular events that underpin the onset of healthspan decline remain largely elusive and difficult to study.Evolutionary theory has long maintained that declines in the force of natural selection after sexual maturity allow the sub-optimal expression and function of fitness-promoting genes in late-life.Thus, genomes have evolved for developmental and reproductive success, not healthy ageing.This suggests that evolutionary neglect encompasses the proximate cause of ageing onset, yet is unable to elucidate precisely which late-acting (pleiotropic) genes contribute to physiological decline.Identifying the entirety of late-acting hyperfunctional genes will, therefore, allow thorough investigations into optimising their expression levels at the organismal and tissue-specific level at geriatric life stages.We propose that combinations of untargeted multi-omics and late-life degron inhibition healthspan screening is the preferred strategy for studying the roles of hyperfunction in normal physiological ageing.
Furthermore, loss-of-function and/or substitution mutations (i.e., in let-363 and daf-2) have played central roles in advancing our Example of lifespan and healthspan extension where health decline onset is not scaled to longer life.Thus, the proportion of time in gerospan is higher than scenarios (B) and (C), yet relative gerospan is still compressed vs. wild-type ageing (scenario A).E) Interventions where there is no delay in the onset of physical deterioration, coupled with an extended lifespan, increases the period of time spent in gerospan.This scenario is the most commonly observed amongst lifespan-extending mutations in C. elegans.Bars graphs on right: a theoretical summary of the total time spent in gerospan for each scenario (i.e., the total length of gerospan arrows relative to the duration of lifespan).
understanding on single gene mutations that can alter the rate of organismal ageing across the life-course.Yet, there are many complexities in the interpretation of these data with regards to developmental timings, early-life fitness defects, progeny production and reproductionsoma trade off's when understanding the roles of pleiotropy/ hyperfunction in the wild-type genome.With the advent of novel geneticmanipulation technologies (e.g., degron mutants), such confounding variables can be circumvented, allowing temporal and controllable optimisations in protein function.To summarise, the majority of critical proof-of-concept experiments have been accomplished with mutant models, and transition to new tools is likely to greatly advance mechanistic understandings.

Fig. 6 .
Fig. 6.Modulation of lifespan with varying changes in healthspan can have distinct and meaningful effects on relative time spent in gerospan.A) Schematic of wildtype ageing progression.B) Example of an intervention where lifespan remains unaltered, but healthspan improvements delay the onset of physiological decline, compressing the relative time spent in gerospan.C) Dual lifespan and healthspan extension, where functional decline is proportionally delayed with longer life.D)Example of lifespan and healthspan extension where health decline onset is not scaled to longer life.Thus, the proportion of time in gerospan is higher than scenarios (B) and (C), yet relative gerospan is still compressed vs. wild-type ageing (scenario A).E) Interventions where there is no delay in the onset of physical deterioration, coupled with an extended lifespan, increases the period of time spent in gerospan.This scenario is the most commonly observed amongst lifespan-extending mutations in C. elegans.Bars graphs on right: a theoretical summary of the total time spent in gerospan for each scenario (i.e., the total length of gerospan arrows relative to the duration of lifespan).