PolyQ length-based molecular encoding of vocalization frequency in FOXP2.

The transcription factor FOXP2, a regulator of vocalization- and speech/language-related phenotypes, contains two long polyQ repeats (Q1 and Q2) displaying marked, still enigmatic length variation across mammals. We found that the Q1/Q2 length ratio quantitatively encodes vocalization frequency ranges, from the infrasonic to the ultrasonic, displaying striking convergent evolution patterns. Thus, species emitting ultrasonic vocalizations converge with bats in having a low ratio, whereas species vocalizing in the low-frequency/infrasonic range converge with elephants and whales, which have higher ratios. Similar, taxon-specific patterns were observed for the FOXP2-related protein FOXP1. At the molecular level, we observed that the FOXP2 polyQ tracts form coiled coils, assembling into condensates and fibrils, and drive liquid-liquid phase separation (LLPS). By integrating evolutionary and molecular analyses, we found that polyQ length variation related to vocalization frequency impacts FOXP2 structure, LLPS, and transcriptional activity, thus defining a novel form of polyQ length-based molecular encoding of vocalization frequency.

Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans.

In several long-lived Caenorhabditis elegans strains, such as insulin/IGF-1 receptor daf-2 mutants, enhanced proteostatic mechanisms are accompanied by elevated intestinal lipid stores, but their role in longevity is unclear. Here, while determining the regulatory network of the selective autophagy receptor SQST-1/SQSTM1, we uncovered an important role for lipid droplets in proteostasis and longevity. Using genome-wide RNAi screening, we identified several SQST-1 modulators, including lipid droplets-associated and aggregation-prone proteins. Expansion of intestinal lipid droplets by silencing the conserved cytosolic triacylglycerol lipase gene atgl-1/ATGL enhanced autophagy, and extended lifespan. Notably, a substantial amount of ubiquitinated proteins were found on lipid droplets. Reducing lipid droplet levels exacerbated the proteostatic collapse when autophagy or proteasome function was compromised, and significantly reduced the lifespan of long-lived daf-2 animals. Altogether, our study uncovered a key role for lipid droplets in C. elegans as a proteostatic mediator that modulates ubiquitinated protein accumulation, facilitates autophagy, and promotes longevity.

Neuronal HLH-30/TFEB modulates peripheral mitochondrial fragmentation to improve thermoresistance in Caenorhabditis elegans.

Transcription factor EB (TFEB) is a conserved master transcriptional activator of autophagy and lysosomal genes that modulates organismal lifespan regulation and stress resistance. As neurons can coordinate organism-wide processes, we investigated the role of neuronal TFEB in stress resistance and longevity. To this end, the Caenorhabditis elegans TFEB ortholog, hlh-30, was rescued panneuronally in hlh-30 loss of function mutants. While important in the long lifespan of daf-2 animals, neuronal HLH-30/TFEB was not sufficient to restore normal lifespan in short-lived hlh-30 mutants. However, neuronal HLH-30/TFEB rescue mediated robust improvements in the heat stress resistance of wildtype but not daf-2 animals. Notably, these mechanisms can be uncoupled, as neuronal HLH-30/TFEB requires DAF-16/FOXO to regulate longevity but not thermoresistance. Through further transcriptomics profiling and functional analysis, we discovered that neuronal HLH-30/TFEB modulates neurotransmission through the hitherto uncharacterized protein W06A11.1 by inducing peripheral mitochondrial fragmentation and organismal heat stress resistance in a non-cell autonomous manner. Taken together, this study uncovers a novel mechanism of heat stress protection mediated by neuronal HLH-30/TFEB.

Exportin 1 modulates life span by regulating nucleolar dynamics via the autophagy protein LGG-1/GABARAP.

Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.

Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging.

Efficient proteostasis is crucial for somatic maintenance, and its decline during aging leads to cellular dysfunction and disease. Selective autophagy is a form of autophagy mediated by receptors that target specific cargoes for degradation and is an essential process to maintain proteostasis. The protein Sequestosome 1 (p62/SQSTM1) is a classical selective autophagy receptor, but it also has roles in the ubiquitin-proteasome system, cellular metabolism, signaling, and apoptosis. p62 is best known for its role in clearing protein aggregates via aggrephagy, but it has recently emerged as a receptor for other forms of selective autophagy such as mitophagy and lipophagy. Notably, p62 has context-dependent impacts on organismal aging and turnover of p62 usually reflects active proteostasis. In this review, we highlight recent advances in understanding the role of p62 in coordinating the ubiquitin-proteasome system and autophagy. We also discuss positive and negative effects of p62 on proteostatic status and their implications on aging and neurodegeneration. Finally, we relate the link between defective p62 and diseases of aging and examine the utility of targeting this multifaceted protein to achieve proteostatic benefits.

Reduced ech-6 expression attenuates fat-induced lifespan shortening in C. elegans.

Deregulated energy homeostasis represents a hallmark of aging and results from complex gene-by-environment interactions. Here, we discovered that reducing the expression of the gene ech-6 encoding enoyl-CoA hydratase remitted fat diet-induced deleterious effects on lifespan in Caenorhabditis elegans, while a basal expression of ech-6 was important for survival under normal dietary conditions. Lipidomics revealed that supplementation of fat in ech-6-silenced worms had marginal effects on lipid profiles, suggesting an alternative fat utilization for energy production. Transcriptomics further suggest a causal relation between the lysosomal pathway, energy production, and the longevity effect conferred by the interaction between ech-6 and fat diets. Indeed, enhancing energy production from endogenous fat by overexpressing lysosomal lipase lipl-4 recapitulated the lifespan effects of fat diets on ech-6-silenced worms. Collectively, these results suggest that the gene ech-6 is potential modulator of metabolic flexibility and may be a target for promoting metabolic health and longevity.

Location, location, location: subcellular protein partitioning in proteostasis and aging.

Somatic maintenance and cell survival rely on proper protein homeostasis to ensure reliable functions across the cell and to prevent proteome collapse. Maintaining protein folding and solubility is central to proteostasis and is coordinated by protein synthesis, chaperoning, and degradation capacities. An emerging aspect that influences proteostasis is the dynamic protein partitioning across different subcellular structures and compartments. Here, we review recent literature related to nucleocytoplasmic partitioning of proteins, nuclear and cytoplasmic quality control mechanisms, and their impact on the development of age-related diseases. We also highlight new points of entry to modulate spatially-regulated proteostatic mechanisms to delay aging.

C. elegans to model autophagy-related human disorders.

Autophagy is a highly conserved degradation process that clears damaged intracellular macromolecules and organelles in order to maintain cellular health. Dysfunctional autophagy is fundamentally linked to the development of various human disorders and pathologies. The use of the nematode Caenorhabditis elegans as a model system to study autophagy has improved our understanding of its regulation and function in organismal physiology. Here, we review the genetic, functional, and regulatory conservation of the autophagy pathway in C. elegans and we describe tools to quantify and study the autophagy process in this incredibly useful model organism. We further discuss how these nematodes have been modified to model autophagy-related human diseases and underscore the important insights obtained from such models. Altogether, we highlight the strengths of C. elegans as an exceptional tool to understand the genetic and molecular foundations underlying autophagy-related human diseases.

Autophagy in aging and longevity.

Our understanding of the process of autophagy and its role in health and diseases has grown remarkably in the last two decades. Early work established autophagy as a general bulk recycling process which involves the sequestration and transport of intracellular material to the lysosome for degradation. Currently, autophagy is viewed as a nexus of metabolic and proteostatic signalling that can determine key physiological decisions from cell fate to organismal lifespan. Here, we review the latest literature on the role of autophagy and lysosomes in stress response and longevity. We highlight the connections between autophagy and metabolic processes, the network associated with its regulation, and the links between autophagic dysfunction, neurodegenerative diseases, and aging.

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein. The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.).

Emerging topics in C. elegans aging research: Transcriptional regulation, stress response and epigenetics.

Key discoveries in aging research have been made possible with the use of model organisms. Caenorhabditis elegans is a short-lived nematode that has become a well-established system to study aging. The practicality and powerful genetic manipulations associated with this metazoan have revolutionized our ability to understand how organisms age. 25 years after the publication of the discovery of the daf-2 gene as a genetic modifier of lifespan, C. elegans remains as relevant as ever in the quest to understand the process of aging. Nematode aging research has proven useful in identifying transcriptional regulators, small molecule signals, cellular mechanisms, epigenetic modifications associated with stress resistance and longevity, and lifespan-extending compounds. Here, we review recent discoveries and selected topics that have emerged in aging research using this incredible little worm.

Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan.

Transcriptional modulation of the process of autophagy involves the transcription factor HLH-30/TFEB. In order to systematically determine the regulatory network of HLH-30/TFEB, we performed a genome-wide RNAi screen in C. elegans and found that silencing the nuclear export protein XPO-1/XPO1 enhances autophagy by significantly enriching HLH-30 in the nucleus, which is accompanied by proteostatic benefits and improved longevity. Lifespan extension via xpo-1 silencing requires HLH-30 and autophagy, overlapping mechanistically with several established longevity models. Selective XPO1 inhibitors recapitulated the effect on autophagy and lifespan observed by silencing xpo-1 and protected ALS-afflicted flies from neurodegeneration. XPO1 inhibition in HeLa cells enhanced TFEB nuclear localization, autophagy, and lysosome biogenesis without affecting mTOR activity, revealing a conserved regulatory mechanism for HLH-30/TFEB. Altogether, our study demonstrates that altering the nuclear export of HLH-30/TFEB can regulate autophagy and establishes the rationale of targeting XPO1 to stimulate autophagy in order to prevent neurodegeneration.

Visible light reduces C. elegans longevity.

The transparent nematode Caenorhabditis elegans can sense UV and blue-violet light to alter behavior. Because high-dose UV and blue-violet light are not a common feature outside of the laboratory setting, we asked what role, if any, could low-intensity visible light play in C. elegans physiology and longevity. Here, we show that C. elegans lifespan is inversely correlated to the time worms were exposed to visible light. While circadian control, lite-1 and tax-2 do not contribute to the lifespan reduction, we demonstrate that visible light creates photooxidative stress along with a general unfolded-protein response that decreases the lifespan. Finally, we find that long-lived mutants are more resistant to light stress, as well as wild-type worms supplemented pharmacologically with antioxidants. This study reveals that transparent nematodes are sensitive to visible light radiation and highlights the need to standardize methods for controlling the unrecognized biased effect of light during lifespan studies in laboratory conditions.

Autophagy-mediated longevity is modulated by lipoprotein biogenesis.

Autophagy-dependent longevity models in C. elegans display altered lipid storage profiles, but the contribution of lipid distribution to life-span extension is not fully understood. Here we report that lipoprotein production, autophagy and lysosomal lipolysis are linked to modulate life span in a conserved fashion. We find that overexpression of the yolk lipoprotein VIT/vitellogenin reduces the life span of long-lived animals by impairing the induction of autophagy-related and lysosomal genes necessary for longevity. Accordingly, reducing vitellogenesis increases life span via induction of autophagy and lysosomal lipolysis. Life-span extension due to reduced vitellogenesis or enhanced lysosomal lipolysis requires nuclear hormone receptors (NHRs) NHR-49 and NHR-80, highlighting novel roles for these NHRs in lysosomal lipid signaling. In dietary-restricted worms and mice, expression of VIT and hepatic APOB (apolipoprotein B), respectively, are significantly reduced, suggesting a conserved longevity mechanism. Altogether, our study demonstrates that lipoprotein biogenesis is an important mechanism that modulates aging by impairing autophagy and lysosomal lipolysis.

Transcriptional and epigenetic regulation of autophagy in aging.

Macroautophagy is a major intracellular degradation process recognized as playing a central role in cell survival and longevity. This multistep process is extensively regulated at several levels, including post-translationally through the action of conserved longevity factors such as the nutrient sensor TOR. More recently, transcriptional regulation of autophagy genes has emerged as an important mechanism for ensuring the somatic maintenance and homeostasis necessary for a long life span. Autophagy is increased in many long-lived model organisms and contributes significantly to their longevity. In turn, conserved transcription factors, particularly the helix-loop-helix transcription factor TFEB and the forkhead transcription factor FOXO, control the expression of many autophagy-related genes and are important for life-span extension. In this review, we discuss recent progress in understanding the contribution of these transcription factors to macroautophagy regulation in the context of aging. We also review current research on epigenetic changes, such as histone modification by the deacetylase SIRT1, that influence autophagy-related gene expression and additionally affect aging. Understanding the molecular regulation of macroautophagy in relation to aging may offer new avenues for the treatment of age-related diseases.

Guidelines for monitoring autophagy in Caenorhabditis elegans.

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans.

Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity-extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans.

Autophagy genes are required for normal lipid levels in C. elegans.

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec- 1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.

Lessons from C. elegans: signaling pathways for longevity.

Recent research using model organisms such as the nematode Caenorhabditis elegans has highlighted a crucial role for several conserved signaling pathways in longevity determination. Here, we review three major endocrine- and nutrient-sensing signaling pathways with influence on lifespan, the insulin/insulin-like growth factor (IGF), target of rapamycin (TOR), and germline signaling pathways. Although these pathways engage distinct sets of transcription factors, the three pathways appear to modulate aging in C. elegans through partially overlapping effector mechanisms, including lipid metabolism and autophagy. This review highlights the latest advances in our understanding of how the insulin/IGF-1, TOR, and germline signaling pathways utilize different transcription factors to modulate aging in C. elegans with special emphasis on the role of lipid metabolism and autophagy.