The Logic of Transgenerational Inheritance: Timescales of Adaptation.

Myriad mechanisms have evolved to adapt to changing environments. Environmental stimuli alter organisms' physiology to create memories of previous environments. Whether these environmental memories can cross the generational barrier has interested scientists for centuries. The logic of passing on information from generation to generation is not well understood. When is it useful to remember ancestral conditions, and when might it be deleterious to continue to respond to a context that may no longer exist? The key might be found in understanding the environmental conditions that trigger long-lasting adaptive responses. We discuss the logic that biological systems may use to remember environmental conditions. Responses spanning different generational timescales employ different molecular machineries and may result from differences in the duration or intensity of the exposure. Understanding the molecular components of multigenerational inheritance and the logic underlying beneficial and maladaptive adaptations is fundamental to understanding how organisms acquire and transmit environmental memories across generations.

The role of the Cer1 transposon in horizontal transfer of transgenerational memory.

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. C. elegans protects itself from pathogens by "reading" bacterial small RNAs, using this information to both induce avoidance and transmit memories for four generations. Here, we found that memories can be transferred from either lysed animals or from conditioned media to naive animals via Cer1 retrotransposon-encoded virus-like particles. Moreover, Cer1 functions internally at the step of transmission of information from the germline to neurons and is required for learned avoidance. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small-RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

Piwi/PRG-1 Argonaute and TGF-ß Mediate Transgenerational Learned Pathogenic Avoidance.

The ability to inherit learned information from parents could be evolutionarily beneficial, enabling progeny to better survive dangerous conditions. We discovered that, after C. elegans have learned to avoid the pathogenic bacteria Pseudomonas aeruginosa (PA14), they pass this learned behavior on to their progeny, through either the male or female germline, persisting through the fourth generation. Expression of the TGF-ß ligand DAF-7 in the ASI sensory neurons correlates with and is required for this transgenerational avoidance behavior. Additionally, the Piwi Argonaute homolog PRG-1 and its downstream molecular components are required for transgenerational inheritance of both avoidance behavior and ASI daf-7 expression. Animals whose parents have learned to avoid PA14 display a PA14 avoidance-based survival advantage that is also prg-1 dependent, suggesting an adaptive response. Transgenerational epigenetic inheritance of pathogenic learning may optimize progeny decisions to increase survival in fluctuating environmental conditions.

Genome Sequencing Fishes out Longevity Genes.

Understanding the molecular basis underlying aging is critical if we are to fully understand how and why we age-and possibly how to delay the aging process. Up until now, most longevity pathways were discovered in invertebrates because of their short lifespans and availability of genetic tools. Now, Reichwald et al. and Valenzano et al. independently provide a reference genome for the short-lived African turquoise killifish, establishing its role as a vertebrate system for aging research.

For longevity, perception is everything.

Aging is a risk factor for chronic diseases, and identifying targets for intervention is a goal of the aging field. Burkewitz et al. now describe a mechanism that mediates the specific role for AMPK in longevity, whereby its activity in neurons modulates metabolism and mitochondrial integrity in peripheral tissues.

PQM-1 complements DAF-16 as a key transcriptional regulator of DAF-2-mediated development and longevity.

Reduced insulin/IGF-1-like signaling (IIS) extends C. elegans lifespan by upregulating stress response (class I) and downregulating other (class II) genes through a mechanism that depends on the conserved transcription factor DAF-16/FOXO. By integrating genome-wide mRNA expression responsiveness to DAF-16 with genome-wide in vivo binding data for a compendium of transcription factors, we discovered that PQM-1 is the elusive transcriptional activator that directly controls development (class II) genes by binding to the DAF-16-associated element (DAE). DAF-16 directly regulates class I genes only, through the DAF-16-binding element (DBE). Loss of PQM-1 suppresses daf-2 longevity and further slows development. Surprisingly, the nuclear localization of PQM-1 and DAF-16 is controlled by IIS in opposite ways and was also found to be mutually antagonistic. We observe progressive loss of nuclear PQM-1 with age, explaining declining expression of PQM-1 targets. Together, our data suggest an elegant mechanism for balancing stress response and development.

A natural bacterial pathogen of C. elegans uses a small RNA to induce transgenerational inheritance of learned avoidance.

C. elegans can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans' natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans' natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in P. vranovensis, that has a 16-nucleotide match to an exon of the C. elegans gene maco-1. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in C. elegans' natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

Aging research: A field grows up.

Breakthroughs in the longevity field over the past few decades have led to major shifts in how we attack the problem of aging. What have been the most important of these shifts in our perspectives, aims, and approaches that will likely guide future research?

C. elegans interprets bacterial non-coding RNAs to learn pathogenic avoidance.

Caenorhabditis elegans must distinguish pathogens from nutritious food sources among the many bacteria to which it is exposed in its environment1. Here we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance in the treated worms and in four subsequent generations of progeny. The RNA interference (RNAi) and PIWI-interacting RNA (piRNA) pathways, the germline and the ASI neuron are all required for avoidance behaviour induced by bacterial small RNAs, and for the transgenerational inheritance of this behaviour. A single P. aeruginosa non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. Our results suggest that this non-coding-RNA-dependent mechanism evolved to survey the microbial environment of the worm, use this information to make appropriate behavioural decisions and pass this information on to its progeny.

TGF-ß and insulin signaling regulate reproductive aging via oocyte and germline quality maintenance.

Reproductive cessation is perhaps the earliest aging phenotype that humans experience. Similarly, reproduction of Caenorhabditis elegans ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here, we show that TGF-ß Sma/Mab and Insulin/IGF-1 signaling regulate C. elegans reproductive aging by modulating multiple aspects of the reproductive process, including embryo integrity, oocyte fertilizability, chromosome segregation fidelity, DNA damage resistance, and oocyte and germline morphology. TGF-ß activity regulates reproductive span and germline/oocyte quality noncell-autonomously and is temporally and transcriptionally separable from its regulation of growth. Chromosome segregation, cell cycle, and DNA damage response genes are upregulated in TGF-ß mutant oocytes, decline in aged mammalian oocytes, and are critical for oocyte quality maintenance. Our data suggest that C. elegans and humans share many aspects of reproductive aging, including the correlation between reproductive aging and declining oocyte quality and mechanisms determining oocyte quality.

Oocyte mitophagy is critical for extended reproductive longevity.

Women's reproductive cessation is the earliest sign of human aging and is caused by decreasing oocyte quality. Similarly, C. elegans' reproduction declines in mid-adulthood and is caused by oocyte quality decline. Aberrant mitochondrial morphology is a hallmark of age-related dysfunction, but the role of mitochondrial morphology and dynamics in reproductive aging is unclear. We examined the requirements for mitochondrial fusion and fission in oocytes of both wild-type worms and the long-lived, long-reproducing insulin-like receptor mutant daf-2. We find that normal reproduction requires both fusion and fission, but that daf-2 mutants utilize a shift towards fission, but not fusion, to extend their reproductive span and oocyte health. daf-2 mutant oocytes' mitochondria are punctate (fissioned) and this morphology is primed for mitophagy, as loss of the mitophagy regulator PINK-1 shortens daf-2's reproductive span. daf-2 mutants maintain oocyte mitochondria quality with age at least in part through a shift toward punctate mitochondrial morphology and subsequent mitophagy. Supporting this model, Urolithin A, a metabolite that promotes mitophagy, extends reproductive span in wild-type mothers-even in mid-reproduction-by maintaining youthful oocytes with age. Our data suggest that promotion of mitophagy may be an effective strategy to maintain oocyte health with age.

Feeding the germline.

Nucleotides are required in order to replicate DNA in the developing germline. Here, Chi and colleagues (pp. 307-320) have used Caernohabditis elegans to identify a GLP-1-dependent checkpoint that senses food (bacterially)-supplied nucleotide levels, arresting reproductive development in the absence of sufficient nucleotide supplies.

Metformin rescues Parkinson's disease phenotypes caused by hyperactive mitochondria.

Metabolic dysfunction occurs in many age-related neurodegenerative diseases, yet its role in disease etiology remains poorly understood. We recently discovered a potential causal link between the branched-chain amino acid transferase BCAT-1 and the neurodegenerative movement disorder Parkinson's disease (PD). RNAi-mediated knockdown of Caenorhabditis elegans bcat-1 is known to recapitulate PD-like features, including progressive motor deficits and neurodegeneration with age, yet the underlying mechanisms have remained unknown. Using transcriptomic, metabolomic, and imaging approaches, we show here that bcat-1 knockdown increases mitochondrial respiration and induces oxidative damage in neurons through mammalian target of rapamycin-independent mechanisms. Increased mitochondrial respiration, or "mitochondrial hyperactivity," is required for bcat-1(RNAi) neurotoxicity. Moreover, we show that post-disease-onset administration of the type 2 diabetes medication metformin reduces mitochondrial respiration to control levels and significantly improves both motor function and neuronal viability. Taken together, our findings suggest that mitochondrial hyperactivity may be an early event in the pathogenesis of PD, and that strategies aimed at reducing mitochondrial respiration may constitute a surprising new avenue for PD treatment.

Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant.

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants' long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock, and we directly perform the tensile test on isolated cuticles using microfluidic technology. Using these tools, we find that the cuticle, not the muscle, is responsible for changes in the "stretchiness" of C. elegans, and that cuticle stiffness is highly nonlinear and anisotropic. We also found that collagen mutations alter the integrity of the cuticle by significantly changing the elasticity. In addition, aging stiffens the cuticle under mechanical loads beyond the cuticle's healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.

The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators.

Insulin/insulin-like growth factor signalling (IIS) is a critical regulator of an organism's most important biological decisions from growth, development, and metabolism to reproduction and longevity. It primarily does so through the activity of the DAF-16 transcription factor (forkhead box O (FOXO) homologue), whose global targets were identified in Caenorhabditis elegans using whole-worm transcriptional analyses more than a decade ago. IIS and FOXO also regulate important neuronal and adult behavioural phenotypes, such as the maintenance of memory and axon regeneration with age, in both mammals and C. elegans, but the neuron-specific IIS/FOXO targets that regulate these functions are still unknown. By isolating adult C. elegans neurons for transcriptional profiling, we identified both the wild-type and IIS/FOXO mutant adult neuronal transcriptomes for the first time. IIS/FOXO neuron-specific targets are distinct from canonical IIS/FOXO-regulated longevity and metabolism targets, and are required for extended memory in IIS daf-2 mutants. The activity of the forkhead transcription factor FKH-9 in neurons is required for the ability of daf-2 mutants to regenerate axons with age, and its activity in non-neuronal tissues is required for the long lifespan of daf-2 mutants. Together, neuron-specific and canonical IIS/FOXO-regulated targets enable the coordinated extension of neuronal activities, metabolism, and longevity under low-insulin signalling conditions.

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

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

Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression.

The biology and behavior of adults differ substantially from those of developing animals, and cell-specific information is critical for deciphering the biology of multicellular animals. Thus, adult tissue-specific transcriptomic data are critical for understanding molecular mechanisms that control their phenotypes. We used adult cell-specific isolation to identify the transcriptomes of C. elegans' four major tissues (or "tissue-ome"), identifying ubiquitously expressed and tissue-specific "enriched" genes. These data newly reveal the hypodermis' metabolic character, suggest potential worm-human tissue orthologies, and identify tissue-specific changes in the Insulin/IGF-1 signaling pathway. Tissue-specific alternative splicing analysis identified a large set of collagen isoforms. Finally, we developed a machine learning-based prediction tool for 76 sub-tissue cell types, which we used to predict cellular expression differences in IIS/FOXO signaling, stage-specific TGF-ß activity, and basal vs. memory-induced CREB transcription. Together, these data provide a rich resource for understanding the biology governing multicellular adult animals.

An insulin-to-insulin regulatory network orchestrates phenotypic specificity in development and physiology.

Insulin-like peptides (ILPs) play highly conserved roles in development and physiology. Most animal genomes encode multiple ILPs. Here we identify mechanisms for how the forty Caenorhabditis elegans ILPs coordinate diverse processes, including development, reproduction, longevity and several specific stress responses. Our systematic studies identify an ILP-based combinatorial code for these phenotypes characterized by substantial functional specificity and diversity rather than global redundancy. Notably, we show that ILPs regulate each other transcriptionally, uncovering an ILP-to-ILP regulatory network that underlies the combinatorial phenotypic coding by the ILP family. Extensive analyses of genetic interactions among ILPs reveal how their signals are integrated. A combined analysis of these functional and regulatory ILP interactions identifies local genetic circuits that act in parallel and interact by crosstalk, feedback and compensation. This organization provides emergent mechanisms for phenotypic specificity and graded regulation for the combinatorial phenotypic coding we observe. Our findings also provide insights into how large hormonal networks regulate diverse traits.

EGF signalling activates the ubiquitin proteasome system to modulate C. elegans lifespan.

Epidermal growth factor (EGF) signalling regulates growth and differentiation. Here, we examine the function of EGF signalling in Caenorhabditis elegans lifespan. We find that EGF signalling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. As animals enter adulthood, EGF signalling upregulates the expression of genes involved in the ubiquitin proteasome system (UPS), including the Skp1-like protein SKR-5, while downregulating the expression of HSP16-type chaperones. Using reporters for global UPS activity, protein aggregation, and oxidative stress, we find that EGF signalling alters protein homoeostasis in adults by increasing UPS activity and polyubiquitination, while decreasing protein aggregation. We show that SKR-5 and the E3/E4 ligases that comprise the ubiquitin fusion degradation (UFD) complex are required for the increase in UPS activity observed in adults, and that animals that lack SKR-5 or the UFD have reduced lifespans and indications of oxidative stress. We propose that as animals enter fertile adulthood, EGF signalling switches the mechanism for maintaining protein homoeostasis from a chaperone-based approach to an approach involving protein elimination via augmented UPS activity.

C. elegans positive olfactory associative memory is a molecularly conserved behavioral paradigm.

While it is thought that short-term memory arises from changes in protein dynamics that increase the strength of synaptic signaling, many of the underlying fundamental molecular mechanisms remain unknown.Our lab developed a Caenorhabditis elegans assay of positive olfactory short-term associative memory (STAM), in which worms learn to associate food with an odor and can remember this association for over 1h. Here we use this massed olfactory associative assay to identify regulators of C. elegans short-term and intermediate-term associative memory (ITAM) processes. We show that there are unique molecular characteristics for different temporal phases of STAM, which include: learning, which is tested immediately after training, short-term memory, tested 30min after training, intermediate-term memory, tested 1h after training, and forgetting, tested 2h after training. We find that, as in higher organisms, C. elegans STAM requires calcium and cAMP signaling, and ITAM requires protein translation. Additionally, we found that STAM and ITAM are distinct from olfactory adaptation, an associative paradigm in which worms learn to disregard an inherently attractive odor after starvation in the presence of that odor. Adaptation mutants show variable responses to short-term associative memory training. Our data distinguish between shorter forms of a positive associative memory in C. elegans that require canonical memory pathways. Study of STAM and ITAM in C. elegans could lead to a more general understanding of the distinctions between these important processes and also to the discovery of novel conserved memory regulators.