Aging causes a functional decline in various components of the protein translation system, as well as age-related regulatory changes (Walther et al., 2015; Dhondt et al., 2017; Anisimova et al., 2018). The net effect is that the rate and the frequency of protein translation, and by extension protein synthesis, decreases with age, with many of these age-associated changes often having detrimental impacts on health and lifespan (Syntichaki et al., 2007b; Dhondt et al., 2017). This observed correlation has previously led to the speculation of a possible relationship between a decreased translation rate and the progression of age (Tavernarakis and Driscoll, 2002). However, contrary to this expectation, a number of studies have since shown that decreased protein synthesis increases lifespan in both normal aging and long-lived paradigms (Syntichaki et al., 2007b; Depuydt et al., 2016; Dhondt et al., 2017). Thus, studies regarding protein translational rates focus not only on the characterization and understanding of translation pathways and regulators in respect to health and lifespan, but also on how these pathways can be exploited for potential therapeutic benefit.

There are primarily two non-conflicting theories often put forward to rationalize the lifespan improvement observed with the inhibition of protein synthesis. The first is that a decrease in protein synthesis allows a greater allocation of metabolic energy towards cellular maintenance and repair. This is broadly in line with the principles of the disposable Soma theory of aging (Jin, 2010), which postulates that there is a fitness cost in the growth and development of an organism through the diversion of resources away from cellular maintenance (Wieser and Krumschnabel, 2001; Anisimova et al., 2018). The second theory is that decreased protein synthesis also broadly reduces the expression of aggregation-prone proteins, thereby partially alleviating the buildup of toxic aggregates and reducing the burden placed on aggregate-clearance pathways (Silva et al., 2011; Kim and Strange, 2013; Solis et al., 2018). From a molecular perspective, there are a wide array of causes behind this decrease in protein synthesis. This includes a broad decrease in transfer RNAs (tRNAs) availability, downregulation and functional impairment of ribosomes, as well as several hormonal and transcriptional changes (Gonskikh and Polacek, 2017).

Although there is ongoing debate as to how significantly translation fidelity changes with age, cumulative evidence firmly indicates that the accurate synthesis of proteins defines organismal lifespan and is essential for organismal health (Tavernarakis and Driscoll, 2002; Anisimova et al., 2018; Ke et al., 2018; Francisco et al., 2020). Excluding genomic causes, these errors primarily arise due to inaccurate start and stop codon recognition by the ribosome, amino acid mismatch during the aminoacylation of tRNAs by aminoacyl-tRNA synthetases, and inaccurate aminoacyl-tRNA selection by the ribosomes. One study showed that the lifespan of C. elegans could be improved by increasing translational fidelity, through the use of pharmacological anti-aging treatments such as rapamycin, trametinib and torin 1 (Martinez-Miguel et al., 2021). This study further demonstrated that a fidelity-improving mutation to the ribosomal 40S subunit RPS23 could likewise improve lifespan primarily by decreasing erroneous stop-codon read-through, with this lifespan extension reproducible across multiple species, including C. elegans. Crucially, this mutation did not impact the rate of translation, allowing translational accuracy to be studied in a manner isolated from the effects on translation rate (Martinez-Miguel et al., 2021). Another study showed that knocking out efk-1, the C. elegans, orthologue of the elongation factor eEf2K, decreases translation fidelity and lifespan (Xie et al., 2019). Furthermore, depletion of multiple aminoacyl-tRNA synthetases (ARSs) including leucyl, arginyl, asparaginyl and methionyl ARSs similarly caused a decrease in lifespan through higher levels of amino acid misincorporation (Xie et al., 2019). It has also become increasingly apparent that errors in protein translation may arise from faulty RNA splicing, where it has been shown that RNA splicing fidelity decreases during aging, and that this decline is ameliorated by caloric restriction (Heintz et al., 2017). In addition, the exposure to reagents such as cadmium lead to disruption in RNA splicing and contribute to aging (Wu et al., 2019).

Other studies in C. elegans have found that inhibition of some ARS can instead have pro-longevity effects. In these lines, RNAi-mediated knockdown of the tyrosine ARS yars-2 is necessary for the longevity phenotype of daf-2 mutant worms, a genetic model of reduced IIS signaling (Son et al., 2017). Similarly, another study showed that the leucine ARS lars-1 activates the mTOR pathway, which as discussed previously, negatively impacts lifespan (Nakamura et al., 2016). These effects on lifespan appear to be independent of changes to translational fidelity, and are a relatively recent discovery. As such, much of the regulatory roles played by ARSs and tRNAs remains unclear. Indeed, despite evidence that the misaminoacylation of tRNAs can lead to diseases that involve protein aggregation, misaminoacylation has recently been acknowledged to play an important functional role in various cellular processes and stress responses (Schimmel, 2018). Although studies investigating the role of misaminoacylation in aging and disease with C. elegans remains sparse, there are an increasing number of studies using C. elegans to study how ARSs and the loss of tRNA into small tRNA-derived fragments contributes to aging and disease, with detailed studies and extensive reviews into this subject available elsewhere (Kato et al., 2011; Shin et al., 2021; Zhou et al., 2021).

From studies in C. elegans, there is now clear evidence that translational fidelity is important for the preservation and maintenance of proteome integrity, and by extension both longevity and healthspan. Moreover, these studies identified new sources of translational error, and have begun to clarify previously unknown regulatory roles of the different components of the translational machinery. These new findings may have significant consequences for therapeutic development, and as such, there is ample room for further research.

The chaperome network is formed by chaperones and co-chaperones that have an integral role in enabling the assembly of proteins into a functional state (Brehme et al., 2014). As such, the chaperome network stabilizes folding intermediates of newly synthesized or unfolded proteins, and further prevents the denaturation or irreversible aggregation of many proteins (Heintz et al., 2017). Chaperones can be broadly classified as ATP dependent, or ATP independent (Mogk et al., 2018). ATP-dependent chaperones such as HSP70 are responsible for both protecting proteins against aggregation while also using the chemical energy provided by ATP to overcome thermodynamically unfavorable intermediates during folding. ATP-independent chaperones such as small heat shock chaperones, also called holdases, likewise bind to unfolded proteins to prevent their aggregation, but most are not thought to contribute to protein folding (Mogk et al., 2018). Due to their essential role, chaperones must be ever-present in cells for proteome maintenance and protein folding. However, with age, misfolded and aggregated proteins accumulate and exceed the stabilizing capacity of available chaperones, diverting many chaperones away from other crucial functions such as regulating the proper folding of nascent proteins (Labbadia and Morimoto, 2015) (Figure 3). In addition, there is evidence from studies in C. elegans showing that chaperone expression becomes increasingly impaired with age, further compounding the burden of insults to the proteome (Labbadia and Morimoto, 2015).

The overexpression of several chaperones, such as small heat shock proteins and members of Hsp70 family, decreases aggregate formation and extends lifespan in C. elegans (Yokoyama et al., 2002; Walker and Lithgow, 2003; Morley and Morimoto, 2004). Accordingly, the disruption of chaperone complexes can be deleterious to lifespan and health. Indeed, the TRiC/CCT complex, a chaperonin that promotes the folding of 10% of the proteome, is essential for regulation of longevity (Noormohammadi et al., 2016). Disruption in the assembly of TRiC/CCT complex causes cellular defects, while increasing TRiC/CCT assembly through the overexpression of the subunit CCT8 leads to an extended lifespan in C. elegans and reduces the neurotoxicity of aggregation-prone polyglutamine (polyQ) (Noormohammadi et al., 2016). Accordingly, one paradigm is that protein aggregates are invariably deleterious to organism health and lifespan. However, this has been challenged by the proteomic analysis of long lived daf-2 mutant worms, which found that long-lived adult daf-2 worms possess a higher chaperone-associated aggregate load compared to wild-type worms (Walther et al., 2015). These aggregates were comparatively chaperone-rich, and were proposed to be a feature of a “protective aggregation response” that sequestered surplus and dysfunctional proteins in order to alleviate the burden to the proteostasis system. Interestingly, while there were no changes in Hsp70 and Hsp90 expression, small heat shock protein levels increased significantly, meaning this response prioritized sequestration and isolation of unfolded proteins, rather than repair and folding (Walther et al., 2015). Although there are several studies in other organisms that investigate the protective potential of aggregation (Saad et al., 2017), further work is required to fully characterize this potential stress response and how it impacts aging.

It has also become increasingly apparent that individual chaperones possess important regulatory functions outside protein stabilization and folding, and that these unique functions are not compensated for by other types of chaperones. For instance, RNAi-mediated knockdown of daf-21/Hsp90 chaperone in non-neuronal tissues decreases lifespan in both wild-type and long-lived daf-2 worms. Notably, this lifespan attenuation revealed that Hsp90 ensures DAF-16 isoform A nuclear translocation and function, but this process does not rely on any of the protein stabilizing properties of Hsp90 (Somogyvári et al., 2018). Although there is much more to uncover in regards to other functions of individual chaperones, it is clear that chaperones are primary mediators of proteostasis and therefore lifespan. Studies in C. elegans, have begun to uncover, but also explore their role as the primary effector of numerous stress responses.

The addition of a phosphoryl group from adenosine triphosphate (ATP) to serine, threonine, or tyrosine residues serves as the most common PTM for proteins (Santos and Lindner, 2017). Phosphorylation is often necessary to facilitate functional conformational changes or provide a chemical moiety required for catalytic activity and protein-protein interactions (Yaffe, 2002). Numerous studies have demonstrated that protein phosphorylation regulates longevity in C. elegans (Derisbourg et al., 2021b; Huang et al., 2018; Lin et al., 2001; Li et al., 2021). One well studied mechanism of longevity regulation is in how phosphorylation controls various aspects of the IIS pathway (Kenyon et al., 1993; Lin et al., 2001). The foremost example arises when insulin-like ligands bind to the insulin/IGF-1 receptor DAF-2, which initiates a phosphorylation cascade culminating in the phosphorylation of the transcription factor DAF-16. Like its analogue FOXO in humans, the phosphorylation of DAF-16 results in its inactivation and retention in the cytoplasm, negatively regulating longevity by preventing the transcription of lifespan-extending effectors (Tatar et al., 2003). Targeted RNAi screens for different serine/threonine protein phosphatases to further study the role of the kinases in the IIS pathway have subsequently identified other novel longevity regulators, such as pptr-1 (Padmanabhan et al., 2009). PPTR-1 negatively regulates the phosphorylation of AKT-1, preventing AKT-1 from inhibiting DAF-16. The modulation of pptr-1 regulates a wide range of reduced IIS-related phenotypes including longevity, the activation of stress responses, and entry into the dauer state (Padmanabhan et al., 2009). A more recent study, similarly focused on IIS-dependent phosphorylation, demonstrated that there are 476 differentially regulated phosphosites in daf-2 mutant worms (W.-J. Li et al., 2021). Their analysis also indicated that casein kinase 2 (CK2) negatively modulates longevity (Li et al., 2021).

These numerous studies thus cement phosphorylation as a fundamental component of the IIS pathway, however phosphorylation has also been found to play a crucial role beyond IIS. Phosphoproteomics analysis of C. elegans at two different temperatures (20 and 25°C) has revealed that phosphoprotein GTBP-1 modulates longevity at both temperatures, and promotes resistance to heat and oxidative stresses (Huang et al., 2018). This study also indicated that the kinases CK2, MAPK, and CAMK2 may similarly modulate aging through their kinase activity (Huang et al., 2018). Other studies have found that the phosphorylation of actin binding protein drebrin through the kinase ataxia-telangiectasia mutated (ATM) regulates lifespan and stress tolerance by improving the stability of drebrin and dynamics of actin remodeling (Kreis et al., 2019). Furthermore, phosphorylation of AMP-activated protein kinases has been found to be crucial for the regulation of cellular energy metabolism and cellular homeostasis, and by extension lifespan, across numerous studies (Hwang et al., 2014; Chang et al., 2017; Park et al., 2020). For example, one recent study demonstrates that the nuclear protein kinase vaccinia-related kinase (VRK-1) promotes lifespan extension through phosphorylation and activation of AMP-kinases (Park et al., 2020).

Thus, both preventing phosphorylation, as with DAF-16, or promoting phosphorylation, through phosphoproteins such as GTBP-1 or the phosphorylation of various AMP-kinases, can drastically increase lifespan, and firmly demonstrates that PTMs are key determinants of longevity.

Acetylation is the addition of an acetyl group to a nitrogen molecule of a target protein (Santos and Lindner, 2017). Numerous studies into the impact of protein acetylation on aging focus on age-associated changes in histone acetylation. During aging, histone acetylation is modulated, and is coupled to changes in metabolic activity and gene expression (Peleg et al., 2016). Sirtuin proteins are histone deacetylases, and act as important regulators of histone acetylation (Grabowska et al., 2017). Increased histone deacetylase activity of silent information regulator 2 (Sir2) extends lifespan (Tissenbaum and Guarente, 2001). Moreover, DAF-16 nuclear localization, which is essential for longevity and stress signal regulation, can be modulated by acetylation through the sirtuin SIR 2.4 protein (Chiang et al., 2012). Sirtuin activators such as oligonol also prolong lifespan in C. elegans infected with lethal Vibrio cholera (Park et al., 2016). Another study has indicated that early stage exposure to heat stress results in increased histone acetylation and helps the establishment of epigenetic “memory”, leading to an extended stress response and longevity in C. elegans (Zhou et al., 2019). Crucially, proteomics analysis comparing young worms to aged worms revealed the accumulation of acylated proteins, particularly in mitochondria (Hong et al., 2016). The accumulation of acylated proteins in mitochondria has been further shown to cause mitochondrial dysfunction and contribute to aging (Hong et al., 2016).

Although the detrimental effects of acetylated protein accumulation remains poorly understood, these studies nonetheless show that epigenetic and metabolic regulation by acetylation modifications have an important role in longevity.

Protein SUMOylation is the covalent attachment of a small ubiquitin like modifier (SUMO) to lysine residues of target proteins (K. A. Wilkinson and Henley, 2010). This SUMOylation then facilitates, or prevents, the interaction of the modified protein with its interaction partner. SUMOylation is involved in several biological processes including development, DNA damage stress-responses, and mitochondrial dynamics (Flotho and Melchior, 2013). These diverse roles of SUMOylation are well studied, in particular, its role in development (Broday et al., 2004; Zhang et al., 2004; Kaminsky et al., 2009; Pelisch et al., 2017). However, many recent studies also highlight the role of SUMOylation in the regulation of longevity (Moll et al., 2018; Princz et al., 2020). Of note is how reduced IIS can modulate protein SUMOylation, where IIS driven SUMOylation of the germline RNA binding protein CAR-1 was found to shorten lifespan in C. elegans. Conversely, the expression of a mutant CAR-1, one which cannot be SUMOylated, promotes enhanced proteostasis and lifespan extension (Moll et al., 2018). The role of SUMOylation in IIS was further expanded in a recent study showing that SUMOylation of DAF-16 regulates mitophagy and mitochondrial dynamics, affecting lifespan (Princz et al., 2020). Furthermore, the same study showed a tissue-specific dependency on SUMOylation, where the RNAi-mediated knockdown of the small ubiquitin-like modifier gene, smo-1, shortened lifespan, while the overexpression of smo-1, specifically in intestine tissue, was sufficient to extend lifespan (Princz et al., 2020). Moreover, SUMOylation also regulates essential biological processes for healthspan and lifespan (Lim et al., 2014; Baytek et al., 2021). SUMOylation modulates the UPRER in C. elegans by regulating calreticulin gene expression in an XBP-1-dependent manner (Lim et al., 2014). SUMOylation also regulates chromatin dynamics by regulating protein activity of the chromodomain factor MRG-1 (Baytek et al., 2021). SUMOylation targets have previously been identified through gene ontology analysis, with most of these SUMOlyated proteins playing a role in metabolism (Drabikowski et al., 2018), and indeed evidence has been found that SUMOylation plays a role under calorie restricted conditions, by modulating the NHR-49 transcription factor (Drabikowski, 2020). These studies demonstrate that SUMOylation plays a decisive role in the proteostasis network for determining aging and longevity in C. elegans, in particular through modulation nodes of the IIS. However, as the majority of SUMOylation targets remain poorly characterized or understood, further research is necessary before the true impact of SUMOylation can be appreciated.

Ubiquitination is the covalent conjugation of ubiquitin, a small highly-conserved protein, to a lysine residue or N-terminal methionine of a target protein (Yau and Rape, 2016). Ubiquitination is a multistep reaction reliant on the coordination of E1, E2, and E3 enzymes (Hochstrasser, 2006). The process begins with ubiquitin-activating enzyme (E1), which activates ubiquitin through an ATP-dependent mechanism. The activated ubiquitin then transfers to the ubiquitin-conjugating enzyme (E2). Finally, a specific ubiquitin-protein ligase (E3) mediates the attachment of ubiquitin from the E2 enzyme to the target protein (Hochstrasser, 1996). Conversely, deubiquitinating enzymes (DUBs) can remove ubiquitin molecules and thereby unmark proteins (K. D. Wilkinson, 2000).

Ubiquitination can determine the fate of a given protein in several ways; it can mark it for degradation by the proteasome or autophagy pathways, regulate their activity, modulate protein-protein interactions and intracellular localization (Hershko and Ciechanover, 1998). Ubiquitination thus has a central regulatory role across a wide range of biological process, including signal transduction, transcriptional regulation, the DNA damage response, and the immune response (Hershko and Ciechanover, 1998). Furthermore, ubiquitination also regulates various stress responses and can also influence protein aggregation (Brehme et al., 2014) (Vilchez et al., 2014; Koyuncu et al., 2018). There is ample evidence that ubiquitination plays a crucial role in aging and longevity (Li et al., 2007; Powers et al., 2009; Kevei and Hoppe, 2014; Tawo et al., 2017; Koyuncu et al., 2021), however the underlying mechanisms are only now becoming clearer. The ubiquitination of a target protein is primarily achieved by the activity of E3 ubiquitin ligases, and can be reversed with deubiquitinase enzymes (Hochstrasser, 2006). We now understand that several of these E3 ligases and deubiquitinases modulate longevity (W. Li et al., 2007; Mehta et al., 2009). Several E3 ligases have been reported as regulators of the IIS pathway. For instance, the E3 ubiquitin ligase CHIP regulates the levels of DAF-2 through its ubiquitination and degradation, which modulates longevity (Tawo et al., 2017). Downregulation of RLE-1 E3 ligases results in less polyubiquitination of DAF-16, and increased DAF-16 transcriptional activation which results in extended lifespan (W. Li et al., 2007). The WWP-1 E3 ligases are required for the regulation of lifespan under caloric restricted conditions (Carrano et al., 2009). The lifespan regulation by WWP-1 depends on E3 Ubiquitin ligase activity and also interactions with the E2 ubiquitin conjugating enzyme UBC-18 (Carrano et al., 2009). Moreover, it has been shown the interactions of ubiquitin-selective chaperone CDC-48 and ATX-3 deubiquitinase modulate longevity through IIS (Kuhlbrodt et al., 2011). A decrease in both cdc-48.1 and atx-3 enhances their substrate stability and longevity by up to 50% (Kuhlbrodt et al., 2011). Recently, we have explored the role of ubiquitination in maintaining proteostasis and regulating longevity by analyzing system-wide ubiquitination changes that occur during aging (Koyuncu et al., 2021). Our findings revealed a global loss of ubiquitination during aging, which is ameliorated by longevity pathways, such as caloric restriction and reduced IIS (Koyuncu et al., 2021). This remodeling of ubiquitination patterns throughout the proteome is a result of elevated deubiquitinase activity. Remarkably, dysregulation of ubiquitination leads to the selective accumulation of various proteasome targets such as the intermediate filament, IFB-2 and EPS-8, a modulator of RAC signaling (Di Fiore and Scita, 2002; Geisler et al., 2019; Koyuncu et al., 2021). Accumulation of IFB-2 leads to loss of intestinal integrity while increased EPS-8 hyperactivates RAC signaling in muscle and neurons causing changes in actin cytoskeleton and hyperactivation of protein kinase JNK. Therefore, the dysregulation in the ubiquitination of structural and regulatory proteins across tissues contributes to aging features and regulate longevity (Koyuncu et al., 2021).

Ubiquitination thus has a much more significant role in aging than previously thought, and further research may allow us to therapeutically exploit parts of the ubiquitination network for anti-aging purposes.

The UPS is the main system for selective degradation of proteins, determining the half-life of multiple regulatory proteins and controlling the clearance of damaged and unnecessary proteins (Pickart, 2001). Repeated addition of ubiquitin creates a polyubiquitin chain, marking the target protein for recognition and processing by the proteolytic machinery of the UPS, the 26S proteasome (Glickman and Ciechanover, 2002). A Lys48-linked polyubiquitin chain is the primary signal for recognition and degradation by the 26S proteasome. The 26S proteasome itself is composed of a 20S core catalytic particle and 19S regulatory particles (Saez and Vilchez, 2014).

The UPS declines after development in C. elegans, as observed by in vivo imaging strategy following the levels of chimeric green fluorescent protein fused to a non-cleavable ubiquitin moiety (Segref et al., 2011). Studies into the relationship between proteasome activity and longevity have shown that elevated UPS activity, mediated by elevated assembly or activity through induction of proteasome subunits, leads to an extension in lifespan (Vilchez et al., 2012; Chondrogianni et al., 2015). Reverting this age-related decrease in proteasome activity through the overexpression of 19S proteasome subunit rpn-6.1 increased survival rate and heat stress resistance (Vilchez et al., 2012). Moreover, long-lived glp-1 mutants, which lack a germline, have enhanced proteasome activity upon DAF-16 activation (Vilchez et al., 2012). Another study has shown that overexpression of the psb-5 catalytic subunit of the 20S proteasome results in an extension of lifespan and resistance to oxidative stress in a DAF-16 dependent manner (Chondrogianni et al., 2015).

Similarly, epidermal growth factor (EGF) signaling has been reported to positively modulate proteasome activity by upregulating the expression of genes involved in the UPS such as Skp1-like protein SKR-5 (Liu et al., 2011). EGF signaling also regulates longevity by upregulating UPS activity, where animals lacking SKR-5 also have shorter lifespans (Liu et al., 2011). Other recent studies also support the role of proteasome activity on the regulation of lifespan, where defects in the import of mitochondrial proteins results in proteasome activation and lifespan extension (Sladowska et al., 2021).

These studies into the impact of proteasomal regulation on longevity highlights the importance of the UPS on aging and longevity. Thus, the proteasome can be an important target to find novel interventions to promote healthy aging.

Autophagy is an evolutionary conserved degradation pathway, in which cellular components including defective organelles and protein aggregates are sequestered in double-membrane vesicles and delivered to the lysosome for degradation (He and Klionsky, 2009). More than 30 proteins (encoded by ATG genes) are recruited at different steps of the autophagy process (Klionsky et al., 2016). There are three well-characterized types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In macroautophagy, cytoplasmic components are first encapsulated into a double membrane-bound vesicle, called an autophagosome. This autophagosome then fuses with the lysosome to form an autolysosome, in which the sequestered cytoplasmic cargo is degraded with hydrolases, glycosidases, nucleotidases, lipases and proteases (Klionsky et al., 2016). In microautophagy, cytosolic cargo is delivered directly to the lysosome for degradation. In CMA, chaperones such as HSP70 promote the lysosomal degradation of targeted proteins (Glick et al., 2010).

Like the UPS, there is now substantial evidence that the autophagy-lysosome pathway is linked to the regulation of aging and age-related diseases. Dysfunctional autophagy during aging has been observed across a diverse range of species, including C. elegans (Aman et al., 2021). Genetic manipulation experiments of selective and non-selective autophagy pathway components have demonstrated an important role of autophagy in lifespan and healthspan (Tóth et al., 2008; Hansen et al., 2018; Kumsta et al., 2019). Either the knockdown or inactivating mutations of several autophagy components such as bec-1 (orthologue of the mammalian APG6/VPS30/beclin1), lgg-1, Igg-3, unc-51, or atg-7 leads to an accelerated aging phenotype and shortened lifespan (Tóth et al., 2008; Hansen et al., 2018). Moreover, several studies indicate that the upregulation of autophagy is mechanistically crucial for the extension of lifespan by different pro-longevity pathways (Meléndez et al., 2003; Hansen et al., 2008; Alberti et al., 2010; Gelino and Hansen, 2012). For instance, the RNAi-mediated downregulation of bec-1 suppresses the extension of lifespan in daf-2 mutants (Meléndez et al., 2003). In addition, the knockdown of autophagy-regulating transcriptional factors such as hlh-30 and daf-16 shortens the lifespan of both wild type and daf-2 mutant worms (Lin et al., 2018). It has been shown that worms exposed to dietary restriction have increased levels of the autophagy marker LGG-1 (the orthologue of ATG8) in their hypodermis, and require functional autophagy promoting genes for longevity (Mörck and Pilon, 2006; Hansen et al., 2008; Tóth et al., 2008).

Overexpression of the key regulator of autophagy sqst-1/p62 induces autophagy in distinct tissues of C. elegans, leading to an extension of lifespan, and an overall improvement in organismal fitness (Kumsta et al., 2019). Overexpression of autophagy regulators such as AMPK enhances autophagy activity and extends lifespan (Hansen et al., 2018). Additionally, the induction of autophagy with different pharmacological agents such as spermidine, resveratrol, and metformin prolongs lifespan in C. elegans (Mariño et al., 2011; Gillespie et al., 2019). Pharmacological inhibition of XPO-1 similarly leads to increased autophagy and lifespan by nuclear enrichment of HLH-30 in C. elegans (Silvestrini et al., 2018).

These studies consistently demonstrate that maintaining and upregulating autophagy can be beneficial for lifespan. Mechanistically, this longevity effects may arise from the persistent clearance of misfolded and aggregated proteins.

Proteotoxic stress, such as heat stress, can upregulate the expression of many specialist chaperones as a part of the HSR. Moreover, binding of damaged and misfolded proteins to chaperones leads to the liberation of heat shock transcriptional factors (HSFs) from chaperone complexes, which further upregulates the transcription of additional chaperones. The chaperones upregulated by the HSR principally act to stabilize and refold thermally denatured proteins, however the HSR also regulates a broad range of genes involved in normal aging, including the small heat shock protein sip-1, and cyp-35B1, a member of cytochrome P450 family (Hsu et al., 2003).

The HSR is mainly regulated by HSFs, specifically HSF-1 in C. elegans (Akerfelt et al., 2010). In worms, the activation of HSR by HSF-1 is also controlled by thermosensory neurons that sense temperature changes, in addition to the basal levels of HSF-1 present in chaperone complexes (Prahlad et al., 2008). Activation of HSF-1 induces the transcription of several chaperones, including HSP70, HSP90 family members, and small heat shock proteins (Hsu et al., 2003). There is substantial evidence that HSF-1 is also a regulator of aging, where HSR activation enhances longevity and stress tolerance by utilizing aspects of longevity-enhancing mechanisms similar to reduced IIS, caloric restriction and suppression of mTOR activity (Morley and Morimoto, 2004; Steinkraus et al., 2008; Seo et al., 2013; Kovács et al., 2019). Overexpression of hsf-1 prolongs lifespan and decreases age-associated protein aggregation of disease-related proteins. Moreover, upregulation of HSF-1-target genes under unstressed conditions similarly extends longevity (Walker and Lithgow, 2003). Accordingly, RNAi-mediated knockdown of hsf-1 shortens lifespan (Hsu et al., 2003; Morley and Morimoto, 2004). Interestingly, overexpression of a modified version of HSF-1, incapable of inducing the expression of HSPs, was found to also prolong lifespan (Baird et al., 2014). Thus HSF-1 can likely upregulate lifespan-extending genes outside what is considered the subset of HSR target genes. This is supported by other studies, where transcriptomics analysis revealed that several distinct longevity associated genes including pha-4, lys-7 and dod-3 are upregulated in HSF-1-dependent long-lived strains (Sural et al., 2019). Likewise, a recent study has also shown that mitochondrial stress can result in HSF-1 dephosphorylation, which induces the upregulation of lifespan extending holdases (Williams et al., 2020), further exemplifying the role of HSF-1 in mediating lifespan extending pathways. In addition, the upregulation of HSF-1 in neurons leads to the activation of DAF-16 in other tissues, making neuronal HSF-1 essential for longevity in a cell non-autonomous manner (Douglas et al., 2015).

Overall, studies in C. elegans have not only shown that the HSR is necessary for thermal protection of the proteome, but also for normal function under unstressed conditions. Moreover, HSF-1 is an essential modulator of aging and longevity through the activation of the HSR and other pathways.

The ISR is an important central stress response in eukaryotic cells, which is induced by a broad range of physiological and environmental changes (Pakos-Zebrucka et al., 2016). The ISR is primarily activated by the phosphorylation of a serine of eIF2α. This reaction is catalyzed by eIF2α kinases after stress stimuli such as viral infection, hypoxia and amino acid deprivation (Harding et al., 2003; Wek et al., 2006; García et al., 2007). The phosphorylation of eIF2α results in a broad decrease in protein translation, while increasing the translation of selected survival genes, such as activating transcription factor 4 (ATF4). If this adaptive response proves insufficient to counteract the stress, additional components of the ISR are activated to induce cell death, preventing potential cellular dysfunctions from impacting organismal health. After the stress stimulus disappears, or is mitigated, eIF2α is dephosphorylated, stopping the ISR and thereby allowing translation and other cellular process to return to normal levels (Novoa et al., 2003; Donnelly et al., 2013). In worms, there are two main eIF2α kinases; the general control nonderepressible 2 (GCN2) kinase, and the PKR-like endoplasmic reticulum kinase (PERK) (Derisbourg et al., 2021b). Previous studies have suggested that ISR is induced with age in different organisms (Derisbourg et al., 2021a), whereas enhanced ISR activation is already observed from early adulthood in C. elegans (Derisbourg et al., 2021b).

As discussed previously (section 2.1.2), although the decrease in protein synthesis induced by the ISR may be expected to be lifespan extending, the ISR is detrimental to lifespan in C. elegans. The cause behind this impairment to lifespan has only recently become to be understood. Through a large-scale mutagenesis screen, it was found that lifespan extending mutations to eIF2 inhibited the ISR, and these mutations relied on the putative kinase kin-35. Crucially, the lifespan extension mediated by kin-35 was found to be independent of any changes to protein synthesis (Derisbourg et al., 2021b). This finding indicates that the ISR may decrease lifespan due to the selective translation of key detrimental genes. Furthermore, contrary to previous studies that observed that knockouts of ISR kinases gcn-2 and pek-1 do not have impact on longevity, this study also showed that single inhibitory amino acid substitutions to GCN-2 and PEK-1 lead to lifespan extensions (Henis-Korenblit et al., 2010; Baker et al., 2012). There is still further investigation needed to fully understand the transcriptional changes caused by the ISR, and why this decreases lifespan in C. elegans.

The unfolded protein response of the endoplasmic reticulum (UPR^ER).

The endoplasmic reticulum (ER) houses and regulates many of the chaperones that aid protein folding, as well as many enzymes that are responsible for the maintenance of proteostasis (Schönthal, 2012). The protein folding capacity of the ER is monitored by the unfolded protein response (UPR^ER) signaling pathway. This pathway is conserved from yeasts to mammals, and is activated by the accumulation of unfolded and misfolded proteins in the ER lumen (Hipp et al., 2019). To maintain protein folding fidelity, the UPR^ER regulates mRNA translation to decrease the further accumulation of misfolded proteins, while also upregulating folding chaperones in the ER (Taylor and Hetz, 2020). In the metazoan ER, there are three identified activators for the different signaling pathway sub-branches of the UPR^ER, i.e. IRE1 (inositol-requiring enzyme), PERK (protein kinase RNA-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6).

Activation of the UPR^ER by external stress declines with age, and this decline is associated with several age-related diseases (Taylor and Dillin, 2011). By studying the metazoan UPR^ER-component analogues in C. elegans, we have begun to understand what contributes to this decline in UPR^ER responsiveness, and how this impacts organismal aging. Genetic manipulation of the ER stress response has shown that the UPR^ER has an important role in the modulation of lifespan and healthspan (Klionsky et al., 2016). Further studies in C. elegans, demonstrated that overexpression of the stress-activated transcriptional factor XBP-1 in neurons prolongs lifespan in a cell non-autonomous manner (Taylor and Dillin, 2013). In addition to neuronal UPR^ER activation, intestinal UPR^ER activation enhanced longevity through an increase in lipophagy (Imanikia et al., 2019; Daniele et al., 2020). A recent study further demonstrated that constitutive activation of the UPR^ER with XBP-1 in astrocyte-like glia prolongs lifespan and stress resistance (Frakes et al., 2020). Moreover, a wide range of longevity paradigms such as reduced insulin signaling and caloric restriction require the UPR^ER (Chen et al., 2009; Henis-Korenblit et al., 2010; Matai et al., 2019). Thus, the consensus between these studies is that activation of the UPR improves lifespan, and is most likely mediated through the increased presence of chaperones preventing aggregate formation, as well as some contribution from upregulation of various metabolic processes such as lipophagy.

Interestingly, although the UPR^ER is not activated in the long-lived daf-2 mutant worms, an enhancement to lifespan through reduced insulin signaling requires the presence of IRE1α/XBP1 (Henis-Korenblit et al., 2010). Conversely, caloric restriction of worms induces a higher basal level of UPR^ER activity, and the increased lifespan phenotype requires the ER stress branch IRE-1 (Matai et al., 2019). This provides clear evidence that the IIS and UPR^ER are in some way linked by a common regulatory element, and that the UPR^ER contributes to the lifespan enhancement observed under calorie restricted conditions. However, the precise mechanism behind how caloric restriction, or the IIS pathway, influence or utilize the UPR^ER remains unclear.

It has been also shown that the activation of the hexosamine pathway, which leads to enhanced UPR^ER activity, increases lifespan through improvement of ER-associated protein degradation (Denzel et al., 2014). Another study has also reported that enhancing lifespan with vitamin D treatments requires IRE1α and XBP1 (Mark et al., 2016). Additionally, treatment with activators of the UPR^ER, such as tunicamycin, prolongs lifespan based on IRE1α branch of UPR^ER (Matai et al., 2019). Accordingly, mutations in IRE1α or XBP1 has been found to shorten lifespan (Taylor and Hetz, 2020). Collectively, these studies show that UPR^ER activation is necessary for both normal and enhanced organismal lifespan, that multiple regulatory and nutrient sensing pathways converge on the UPR^ER, and also that various components of the UPR show promise as therapeutic targets for anti-aging outcomes.

The unfolded protein response of the mitochondria (UPR^mt).

The mitochondria provides cellular energy and regulates a broad range of metabolic events (Dilberger et al., 2019). As a consequence of the process of oxidative phosphorylation (OXPHOS), mitochondria produce reactive oxygen species (ROS) (Sies and Cadenas, 1985). Elevated levels of ROS can lead to cellular damage, with such damage also called oxidative stress (Sies and Cadenas, 1985). Impairment of the mitochondria is associated with cellular dysfunction and is considered a hallmark of aging. Damage to mitochondrial integrity induces transcriptional responses, including the mitochondrial unfolded protein response (UPR^mt) which is regulated by mitochondrial-to-nuclear communication. The UPR^mt induces the recovery of mitochondrial networks through mitochondrial biogenesis and metabolic adaptations, promoting cell survival under stress conditions (Münch and Harper, 2016; Shpilka and Haynes, 2018). Whereas an acute stress in the mitochondria can lead to cell dysfunction and death, a reduced amount of mitochondrial stress can be beneficial for organismal longevity in a process known as mitohormesis. Mitohormesis also encompasses the activation of the UPR^mt.

In C. elegans, the UPR^mt is regulated by the transcription factor ATFS-1 and the co-factors DVE-1 and UBL-5, where ATFS-1 is activated by disruptions to mitochondrial proteostasis as well as ROS produced by OXPHOS. This activation leads to transcriptional regulation to regulate survival and mitochondrial stress through ATFS-1 (Shpilka and Haynes, 2018). It is important to note that although ATSF-1-regulated genes are upregulated in long-lived worms, chronic expression of ATFS-1 itself is not sufficient to extend lifespan (Soo et al., 2021). However, activation of the UPR^mt is known to positively regulate longevity in C. elegans (Dillin et al., 2002; Durieux et al., 2011; Ito et al., 2021). In these lines, RNAi-mediated knockdown of mitochondrial OXPHOS components such as complexes I, III and IV promotes longevity through activation of the UPR^mt (Feng et al., 2001; Dillin et al., 2002). Decreasing mitochondrial protein translation by knockdown of mitochondrial ribosomal protein S5 (mrps-5) likewise extends lifespan through enhanced UPR^mt (Houtkooper et al., 2013). Furthermore, changes in mitochondrial dynamics due to impaired fission or fusion has been shown to decrease mitochondrial translation, upregulating the UPR^mt and extending lifespan (Y. J. Liu et al., 2020).

Pharmacological agents can similarly extend lifespan through targeting activators and inhibitors of the UPR^mt. Antimycin, an inhibitor of mitochondrial ETC complex III, extends lifespan (Dillin et al., 2002). Likewise, Metolazone, a blocker of Na + -Cl− cotransporters, including NKCC-1, also prolongs lifespan by activating the UPR^mt. However how NKCC-1 activates the UPR^mt in C. elegans is still unclear (Ito et al., 2021). Another study has shown that NAD⁺ level decreases with age in C. elegans, and restoration of NAD⁺ levels with NAD + boosters increases sir-2.1 (sirtuin homolog) activity, which in turn improves lifespan through activation of the UPR^mt (Mouchiroud et al., 2013). In addition, the mitochondrial chaperone prohibitin is an important part of the UPR^mt in longevity regulation (Gatsi et al., 2014). Interestingly, depletion of prohibitin, which induces UPR^mt, shortens lifespan in wild type worms whereas this depletion extends lifespan in metabolically compromised worms (Artal-Sanz and Tavernarakis, 2009; Gatsi et al., 2014).

The lifespan extension afforded by the UPR^mt relies, at least in part, on cell non-autonomous interactions between different tissues. It has been shown that neuronal induction of UPR^mt by the accumulation of expanded-polyQ aggregates leads to UPR^mt induction in the intestine, and depends on the neuronal release of serotonin and long-range Wnt signaling pathway (Berendzen et al., 2016; Q.; Zhang et al., 2018). In addition to the nervous system, other tissues can also communicate their proteostasis status and induce the UPR^mt in distal tissues (Calculli et al., 2021). For instance, aggregation of the germ granule component PGL-1 triggers intracellular changes in the mitochondrial network of C. elegans germline cells. In turn, the germline releases long-range WNT ligands that induce an overactivation of the UPR^mt in somatic tissues, promoting somatic mitochondrial fragmentation and aggregation of proteins linked with age-related neurodegenerative diseases such amyotrophic lateral sclerosis and Huntington’s (Calculli et al., 2021).

Beyond the UPR^mt, other factors could be involved in mitohormesis. The most prominent example is the contribution of ROS towards aging in respect to oxidative damage and regulatory roles. The Harman Free Radical Theory of Aging postulates that cellular aging is driven by the formation of mitochondrial ROS. These ROS induce a damage to distinct components of the cell, including DNA, the proteome, and the mitochondria itself. This has been challenged by studies in C. elegans, which have found that lifespan can be decoupled from oxidative damage or oxidant sensitivity. In fact, it has been proposed that low levels of ROS may be beneficial for longevity, where ROS could potentially act as signaling molecules that promote mitohormesis (Ristow and Schmeisser, 2014). For example, it has been reported that deletion of the mitochondrial superoxide dismutase sod-2 increases ROS production, yet prolongs lifespan despite the increased levels of oxidative damaged proteins (Van Raamsdonk and Hekimi, 2009). Moreover, worms treated with low amounts of the oxidant reagent paraquat also live longer (Yang and Hekimi, 2010). One study showed that a mild reduction of respiration extends longevity through ROS mediated activation of the hypoxia-inducible factor HIF-1 (Lee et al., 2010). Glucose restriction also leads to the formation of ROS due to enhanced mitochondrial respiration, prolonging lifespan in C. elegans (Schulz et al., 2007). The knockdown of the hydroxylase clk-1 was also found to enhance longevity despite elevated levels of ROS production (Lakowski and Hekimi, 1996; Lee et al., 2010) Together, these studies indicate that the role of ROS in mitohormesis and aging is less clear as previously thought, and further demonstrate possible roles of ROS as both signal molecules and cellular stress factors. Further studies are required to fully assess the contribution of ROS and oxidative damage towards organism health and longevity. Nevertheless, studies in C. elegans support that the UPR^mt can be a powerful promoter of longevity, and a promising therapeutic target for pharmacological intervention.