mTORC1 Crosstalk With Stress Granules in Aging and Age-Related Diseases

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a master regulator of metabolism and aging. A complex signaling network converges on mTORC1 and integrates growth factor, nutrient and stress signals. Aging is a dynamic process characterized by declining cellular survival, renewal, and fertility. Stressors elicited by aging hallmarks such as mitochondrial malfunction, loss of proteostasis, genomic instability and telomere shortening impinge on mTORC1 thereby contributing to age-related processes. Stress granules (SGs) constitute a cytoplasmic non-membranous compartment formed by RNA-protein aggregates, which control RNA metabolism, signaling, and survival under stress. Increasing evidence reveals complex crosstalk between the mTORC1 network and SGs. In this review, we cover stressors elicited by aging hallmarks that impinge on mTORC1 and SGs. We discuss their interplay, and we highlight possible links in the context of aging and age-related diseases.


INTRODUCTION
The mechanistic target of rapamycin (MTOR) is a serine/threonine protein kinase conserved across all eukaryotes (Tatebe and Shiozaki, 2017). MTOR constitutes a central hub that integrates metabolic signals and adapts cellular processes to extrinsic and intrinsic changes and stressors in health, disease, and aging (Papadopoli et al., 2019;Liu and Sabatini, 2020).
MTOR resides in two complexes, MTOR complex 1 and 2 (mTORC1 and mTORC2), each of which regulates distinct functions in metabolic control throughout the life course [reviewed by Liu and Sabatini (2020) and Papadopoli et al. (2019)]. The two complexes exhibit different sensitivities to the macrolide rapamycin that gave MTOR its name (Heitman et al., 1991;Brown et al., 1994;Sabatini et al., 1994). Rapamycin directly binds and inhibits mTORC1 (Yang et al., 2013;Aylett et al., 2016) whereas long-term rapamycin exposure indirectly inhibits also mTORC2 (Sarbassov et al., 2006). From yeast to mammals, rapamycin extends lifespan, highlighting the fundamental role of MTOR as a regulator of longevity and aging (Weichhart, 2018;Papadopoli et al., 2019). Aging is a dynamic process whereby physiological functions needed for survival, renewal, and fertility deteriorate over time. Its pace differs among species and individuals due to differences in molecular networks and in stochastic damage of cellular components (Khan et al., 2017). Mitochondrial malfunction, loss of proteostasis, genomic instability and telomere shortening, dysregulated nutrient sensing, and altered cell communication are considered as hallmarks of aging (Lopez-Otin et al., 2013) and are directly regulated by MTOR [reviewed in detail by Papadopoli et al. (2019)]. Conversely, MTOR also responds to their dysregulation, thus contributing to age-related processes upstream and downstream of aging hallmarks. In this review, we cover stressors elicited by aging hallmarks that impinge on the signaling network converging on mTORC1. We highlight the complex crosstalk of mTORC1 with the formation of stress granules (SGs), a stress-dependent non-membranous cellular compartment, and we discuss the impact of their interplay in aging and age-related diseases.
Not only mTORC1 but also the TSC protein complex shuttles between the cytoplasm and the lysosomal surface. Recent evidence has shed light on the underlying molecular mechanisms: 1) TSC2 is tethered by the G3BP stress granule assembly factors 1 and 2 (G3BP1 and G3BP2, G3BPs) to the cytoplasmic portion of the lysosomal associated proteins 1 and 2 (LAMP1/2) (Prentzell et al., 2021) (Figure 1). Also the RHEB and RRAG GTPases are required for lysosomal TSC2 recruitment (Demetriades et al., 2014;Menon et al., 2014;Carroll et al., 2016;Prentzell et al., 2021). 2) TSC1 binds lysosomal phosphatidylinositol-3,5-bisphosphate (PI3,5P2) via its N-terminal domain (Fitzian et al., 2021). The interplay of TSC2 and TSC1 tethering mechanisms in the lysosomal recruitment of the TSC complex remains to be determined. While the TSC complex is widely recognized as a transducer of insulin signals to RHEB and mTORC1 (Inoki et al., 2002;Hoxhaj and Manning, 2020), TSC complex association with the RRAG GTPases suggests also a responsiveness to amino acids (Demetriades et al., 2014;Menon et al., 2014;Carroll et al., 2016). Furthermore, several stressors including hypoxia, osmotic, pH and glycolytic stress enhance lysosomal recruitment of the TSC complex (Plescher et al., 2015;Demetriades et al., 2016). Hence, the control of the lysosomal localization of the TSC complex emerges as a central regulatory event that balances mTORC1 activity in response to growth factors, amino acids, and stresses.

mTORC1 and Age-Related Stressors
Next to growth factors and amino acids, mammalian mTORC1 responds to a variety of stressors, including oxidative, DNA and unfolded protein stress (Heberle et al., 2015;Su and Dai, 2017;Ma et al., 2018). These stressors are connected to hallmarks of aging: 1) oxidative stress, promoted by the accumulation of reactive oxygen species (ROS), arises from dysfunctional oxidative phosphorylation in mitochondria (Desler et al., 2011), oxidative protein folding in the endoplasmic reticulum (ER) (Marciniak et al., 2004;Margittai and Sitia, 2011;Yoboue et al., 2018), and peroxisome metabolism (Titorenko and Terlecky, 2011). ROS accumulation results in oxidative damage of biomolecules including proteins and DNA (Liguori et al., 2018). 2) DNA stress is attributed to DNA damage, genome instability and telomere attrition (Maynard et al., 2015). DNA stress arises intrinsically from insufficient repair of replication errors or spontaneous hydrolytic reactions and telomere shortening during DNA replication (Maynard et al., 2015;Yousefzadeh et al., 2021), and upon damage by extrinsic agents, including electromagnetic radiation and chemical agents (Yousefzadeh et al., 2021). 3) ROS and DNA stress both promote proteasomal stress (loss of proteostasis) (Malhotra and Kaufman, 2007;Gonzalez-Quiroz et al., 2020), arising from imbalanced protein synthesis, folding, and turnover (declining autophagy and proteasome function) and resulting in an accumulation of unfolded proteins in the cytoplasm and/or in the ER (unfolded protein stress). The complex molecular mechanisms via which these stressors impinge on the mTORC1 network have been reviewed in detail by Heberle et al. (2015), Su and Dai (2017) and Ma et al. (2018).
Unfolded protein stress inhibits mTORC1 via AKT1repression (Qin et al., 2010;Kato et al., 2012;Li et al., 2018), for example through the negative AKT1 regulator TRIB3 (tribbles pseudokinase 3) (Ohoka et al., 2005). Prolonged unfolded protein stress triggered by the ER-stress inducers thapsigargin or tunicamycin inhibits AKT1 and phosphorylation of TSC2 at threonine 1462 and results in mTORC1 inactivation. In contrast, short-term unfolded protein stress mildly enhances AKT1 activity and phosphorylation of TSC2 (Di Nardo et al., 2009). Hence, activating and inhibitory cues converge on the TSC complex, depending on the stress duration and level. Interestingly, the RRAG GTPases may also contribute to unfolded protein stress sensing by mTORC1, as the ER stress inducer tunicamycin promotes RRAGC expression (Guha et al., 2017).

Control of SG Formation by mTORC1
SGs are cytoplasmic non-membranous assemblies of proteins and mRNAs whose interaction involves liquid-liquid phase separation (LLPS) (Ivanov and Anderson, 2019). A rapidly growing field investigates the molecular mechanisms underlying SG formation (Van Treeck and Parker, 2018;Alberti et al., 2019;Mathieu et al., 2020;Peran and Mittag, 2020;Hofmann et al., 2021;Wiedner and Giudice, 2021). SG formation has been linked with different physiological consequences that are context-dependent and are currently under debate. Depending on the stress and its duration, SGs are rapidly turned over or they persist over long periods of time (Aulas et al., 2017;Markmiller et al., 2018). SGs buffer cellular stress by minimizing energy consumption [reviewed by Mahboubi and Stochaj (2017)] and by anti-apoptotic mechanisms (Arimoto et al., 2008;Takahashi et al., 2013;Thedieck et al., 2013). Such protective functions have been assigned to short-lived SGs (Reineke and Neilson, 2019). However, SGs might also exert pro-apoptotic effects Aulas et al., 2018;Reineke and Neilson, 2019;Amen and Kaganovich, 2020) and they contribute to the formation of pathogenic protein aggregates (Aulas et al., 2018;Jeon and Lee, 2021). Chronic SG assembly has been linked with age-related disorders including neurotoxicity and cancer cell survival (Reineke and Neilson, 2019;Advani and Ivanov, 2020;Alberti and Hyman, 2021).
SG formation is intimately linked with translation inhibition. When translation is inhibited, polysomes run off their mRNAs and "naked" mRNAs assemble with SG nucleating proteins to undergo LLPS (Kedersha et al., 2013;Hofmann et al., 2021). Under non-stress conditions, cap-dependent translation is initiated by the assembly of the EIF4F complex (EIF4E, EIF4G, EIF4B and EIF4A) at the 5′ 7-methylguanosine cap (5′ cap) of mRNAs (Sonenberg and Hinnebusch, 2009). One key regulatory event of EIF4F complex assembly is the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1, also known as 4E-BP1) (Thoreen, 2017;Roux and Topisirovic, 2018;Tahmasebi et al., 2018). EIF4EBP1 competes with EIF4G for EIF4E binding, and prevents EIF4F complex assembly (Sonenberg and Hinnebusch, 2009). EIF4EBP1 phosphorylation by mTORC1 prevents EIF4EBP1-EIF4E binding and promotes EIF4F complex formation (Roux and Topisirovic, 2018) and recruitment of the 43S pre-initiation complex, consisting of the small ribosomal subunit (40S) bound to the eukaryotic translation initiation factor-2 complex (EIF2), GTP and Met-tRNA i Met FIGURE 2 | Crosstalk between stress granules (SGs) and mTORC1 signaling. SG formation is triggered by EIF2S1 phosphorylation at serine 51 or at the level of the EIF4F complex. HRI/EIF2AK1, PKR/EIF2AK2, PERK/EIF2AK3 and GCN2/EIF2AK4 phosphorylate EIF2S1-S51 in response to different stresses. Under oxidative stress, also the mTORC1-S6Ks axis enhances phosphorylation of EIF2S1-S51. Upon Bortezomib (unfolded protein stress), SGs are induced by mTORC1-driven phosphorylation of EIF4EBP1 to sustain EIF4F complex assembly. PI3K and MAPK14 activate mTORC1 to promote SG formation. Autophagy is inhibited by mTORC1 and mediates SG clearance and composition. SGs inhibit mTORC1 by sequestration of MTOR and RPTOR and via the HSP90-DYRK3 axis. SGs also recruit TSC2 and S6K1 and 2 (S6Ks). See main text for details and abbreviations.
Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 761333 (Sonenberg and Hinnebusch, 2009). This complex is required for ribosome assembly and translation initiation (Merrick and Pavitt, 2018). EIF2 is a heterotrimeric complex consisting of alpha (EIF2S1), beta (EIF2S2), and gamma (EIF2S3) subunits (Merrick and Pavitt, 2018). EIF2S1 phosphorylation at serine 51 inhibits EIF2 (Sonenberg and Hinnebusch, 2009). Four kinases (HRI or EIF2AK1, PKR or EIF2AK2, PERK or EIF2AK3, GCN2 or EIF2AK4) phosphorylate EIF2S1-S51 in response to different stress situations (Donnelly et al., 2013) (Figure 2). This is considered as one of the main regulatory events for translation inhibition and SG initiation (Kedersha et al., 1999;Kedersha et al., 2002;Hofmann et al., 2021). However, SG formation can also be EIF2-independent, e.g. upon translation inhibition at the level of EIF4F complex assembly or activity (Hofmann et al., 2021). mTORC1 inhibition has been proposed to initiate SG formation in mammalian cells Hofmann et al., 2012;Panas et al., 2016) as it prevents the phosphorylation of EIF4EBP1 and thus the assembly of the EIF4F complex. In line with this idea, EIF4EBP1 shifts in size or is dephosphorylated, and increases its binding to the mRNA 5′ cap upon several stressors (H 2 O 2 , cold shock, selenite, nitric oxide) (Emara et al., 2012;Fujimura et al., 2012;Hofmann et al., 2012;Aulas et al., 2018). Based on observations that EIF4EBP1 or EIF4E inhibition by knockdown impairs SG formation upon selenite  or H 2 O 2 (Emara et al., 2012) stress, respectively, EIF4EBP1-cap association has been proposed to enhance SG formation (Emara et al., 2012;Fujimura et al., 2012;Panas et al., 2016). Thus, unphosphorylated active EIF4EBP1 may promote SG assembly. However, none of the studies tested by inhibitors or knockdowns whether this process depends on mTORC1 (Emara et al., 2012;Fujimura et al., 2012;Hofmann et al., 2012). EIF4EBP1 is targeted by several kinases (Qin et al., 2016) and phosphatases (Kolupaeva, 2019) other than mTORC1. It remains thus open whether EIF4EBP1-mediated SG formation relates to inactive mTORC1, or if it is mediated by inactivation of another kinase or by a phosphatase. Sfakianos et al. (2018) showed that without stress, neither RPTOR knockdown nor rapamycin induced SGs. Hence, mTORC1 inhibition by itself is not sufficient to initiate SG formation. In contrast, mTORC1 inhibition has been shown by several studies to reduce SG formation upon heat shock, arsenite, and the proteasome inhibitor Bortezomib (Fournier et al., 2013;Mazan-Mamczarz et al., 2015;Sfakianos et al., 2018;Heberle et al., 2019). Conversely, TSC2 deficiency, known to hyperactivate mTORC1, increases the number of SGs formed upon arsenite or heat stress (Kosmas et al., 2021). As discussed above, mTORC1 activity under stress is enhanced-at least in part-by PI3Ks and MAPK14 (Heberle et al., 2019). Those kinases promote SG formation (Brown et al., 2011;Heberle et al., 2019), further supporting that a stress-activated signaling network converging on mTORC1 promotes SG formation.
All molecular mechanisms known so far to mediate mTORC1-driven SG formation impinge on the translation machinery. Upon arsenite, the kinases S6K1 and 2 (S6Ks) downstream of mTORC1 promote EIF2S1-S51 phosphorylation in mammalian cells (Sfakianos et al., 2018) (Figure 2), and this mechanism is conserved upon heat stress in the nematode Caenorhabditis elegans. Mammalian S6Ks enhanced SGs only under moderate arsenite stress. Higher concentrations abolished the S6Ks' impact on SG formation, although it still depended on mTORC1 (Sfakianos et al., 2018). This might be explained by findings of Fournier et al. (2013) who showed that mTORC1-driven phosphorylation of EIF4EBP1 preserves EIF4E-EIF4G interaction, consequently enhancing SG formation upon high concentrations of arsenite as well as Bortezomib (Figure 2). Thus, mTORC1 enhances SG assembly via phosphorylation of S6Ks and EIF4EBPs, both events that are known to enhance translation (Roux and Topisirovic, 2018;Liu and Sabatini, 2020). This indicates that next to translation arrest (Van Treeck and Parker, 2018; Reineke and Neilson, 2019) activating signals to the translation machinery also contribute to SG formation.
Whether and which SGs form independently of mTORC1 remains to be investigated. To the best of our knowledge this has been so far claimed twice, for UV (Ying and Khaperskyy, 2020) and heat stress (Brown et al., 2011), based on the finding that MTOR inhibitors partially inhibit SG formation, but a certain fraction of cells with SGs remains. This observation is in agreement with several studies under different stresses (Fournier et al., 2013;Mazan-Mamczarz et al., 2015;Sfakianos et al., 2018;Heberle et al., 2019). The properties of SGs that are refractory to mTORC1 inhibition are therefore an intriguing topic for future studies. Where does mTORC1 control SG formation? mTORC1's best described site of activity are the lysosomes (Rabanal-Ruiz and Korolchuk, 2018;Carroll, 2020). Recent evidence shows that SGs physically associate with lysosomes (Liao et al., 2019). Thus, lysosomal mTORC1 may enhance SG assembly. It is interesting to note that the core SG proteins G3BP1 and 2 have non-granule functions as mTORC1 suppressors at lysosomes (Prentzell et al., 2021). It remains open whether the G3BP pools at lysosomes and SGs are separate, or whether G3BPs shuttle between these two compartments. Lysosomes have also been linked to SGs in the context of autophagy: as autophagy degrades aggregated proteins (Yang and Klionsky, 2020) it is straightforward to assume that autophagy contributes to SG clearance. Indeed this has been reported in mammals (Ryu et al., 2014;Marrone et al., 2018;Silva et al., 2019), Saccharomyces cerevisiae (Buchan et al., 2013) and Caenorhabditis elegans . mTORC1 is a key suppressor of autophagy . Thus, mTORC1 may enhance SG assembly, at least in part, by inhibiting autophagy. Furthermore, autophagy not only controls SG turnover (Buchan et al., 2013;Lee, 2015) but also their composition (Seguin et al., 2014;Advani and Ivanov, 2020) (Figure 2). This raises the possibility that mTORC1 affects SG composition by inhibiting autophagy.
On a broader level, SG clearance and composition is affected not only by autophagy but also by chaperones, RNA helicases, the proteasomal machinery and unmixing of LLPS condensates (Alberti et al., 2017;Alberti and Hyman, 2021). The cooperation of these processes in SG dynamics is currently investigated by a highly active and growing field of research. The interplay of mTOR with the proteasome is also a matter of active scientific debate (Adegoke et al., 2019). It will be intriguing to link these fields and unravel mTOR's role in SG turnover.

SGs Inhibit mTORC1
Not only does mTORC1 regulate SG formation and clearance. Conversely, SGs also inhibit mTORC1 by several mechanisms in yeast as well as in mammalian cells (Takahara and Maeda, 2012;Thedieck et al., 2013;Wippich et al., 2013;Mediani et al., 2021). In mammalian cells, SG recruitment of RPTOR is mediated by SPAG5 (sperm associated antigen 5, also known as astrin) and leads to the disassembly and inhibition of mTORC1 (Thedieck et al., 2013) (Figure 2). MTOR localizes to SGs too (Wippich et al., 2013), but the molecule mediating this recruitment is unknown (Figure 2). Likewise, the Saccharomyces cerevisiae RPTOR orthologue KOG1 and TOR1 localize to SGs (Takahara and Maeda, 2012). Also the TSC subunit TSC2 (Kosmas et al., 2021) and the mTORC1 substrates S6K1 and 2 (Sfakianos et al., 2018) have been recently reported at SGs (Figure 2), which might impinge on mTORC1 activity as well. It will be interesting to delineate the coordination of the recruitment and disassembly of the TSC and mTORC1 complexes and their substrates at SGs. SGs also regulate mTORC1 via the kinase DYRK3 (dual specificity tyrosine phosphorylation regulated kinase 3) (Wippich et al., 2013;Mediani et al., 2021). DYRK3 binds HSP90 (heat shock protein 90 family) an essential chaperone which regulates the folding and stability of many clients including stress response factors important to resolve a variety of proteotoxic stresses [reviewed in detail by Schopf et al. (2017), Calderwood (2018), Moran Luengo et al. (2019), Lang et al. (2021)]. Under non-stress conditions, HSP90 keeps DYRK3 in an active confirmation. DYRK3 phosphorylates the mTORC1 inhibitor AKT1S1 at threonine 246, thus de-repressing mTORC1 (Wippich et al., 2013). Upon stress or HSP90 inhibition (Wippich et al., 2013;Mediani et al., 2021), inactive DYRK3 is recruited to SGs via its N-terminal IDR, resulting in AKT1S1 activation and mTORC1 inhibition. SG-localized DYRK3 also stabilizes SGs, thereby enhancing their inhibitory effect on mTORC1. As mTORC1 is inhibited by SGs, mTORC1-driven SG formation may constitute a negative feedback mechanism that restricts mTORC1 activation by stress, contributing to the fine-tuning of cellular anabolism and catabolism that maintains cellular homeostasis under stress.

Linkage of MTOR and SGs in Aging
Stresses linked to hallmarks of aging (see mTORC1 and Age-Related Stressors) impinge on mTORC1 activity and SG formation (see SGs and mTORC1 Signaling). Conversely, enhanced mTORC1 signaling (Papadopoli et al., 2019) and SG formation (Cao et al., 2020) have been linked to age-related processes, and MTOR and SG levels often correlate with the severity of age-related diseases (Papadopoli et al., 2019;Liu and Sabatini, 2020;Chrienova et al., 2021). However, the crosstalk between mTORC1 and SG formation in the context of aging progression is poorly explored.
Aging is characterized by increased numbers of senescent cells that have been assigned to stress stimuli which result in irreversible cell cycle arrest (Lopez-Otin et al., 2013;Papadopoli et al., 2019). Senescent cells are impaired in their proliferative capacity, but they maintain an active metabolism (Papadopoli et al., 2019) and exhibit reduced apoptosis (Wanner et al., 2020) despite stress-induced damage (Song et al., 2020). Although SG formation is mainly recognized to counteract senescence (Omer et al., 2018;Cao et al., 2020), senescent cells can form SGs (Lian and Gallouzi, 2009). Interestingly, senescent cells exhibit mTORC1 uncoupling from its suppressors, resulting in mTORC1 hyperactivity . Hence, chronic mTORC1 activity might sensitize senescent cells to SG formation. In line with this, senescent cells present a higher number of SGs upon acute stress (Lian and Gallouzi, 2009) and show slowed SG disassembly after stress recovery (Gallouzi, 2009;Lian and Gallouzi, 2009). SGs enhance survival by sequestering pro-apoptotic proteins (Arimoto et al., 2008). mTORC1-driven SG formation may also exert a negative feedback on mTORC1 that restricts its excessive activity, known to result in apoptosis (Appenzeller-Herzog and Hall, 2012;Thedieck et al., 2013). Hence, SG formation might contribute to the survival and increased presence of senescent cells in aging tissues by sequestering pro-apoptotic factors and by dampening mTORC1 activity.
Neurodegeneration and cancer are age-related diseases associated with senescence (Baker and Petersen, 2018;Sikora et al., 2021). Both aberrant mTORC1 activity (Chrienova et al., 2021;Querfurth and Lee, 2021) and chronic SG formation (Wolozin and Ivanov, 2019;Asadi et al., 2021) link with neurodegenerative diseases including ALS (amyotrophic lateral sclerosis), FTD (frontotemporal dementia), AD (Alzheimer's disease) or PD (Parkinson's disease). However, their crosstalk in neurodegenerative diseases is largely unknown. It is conceivable that hyperactive mTORC1 drives chronic SG formation, and thereby promotes the progression of neurodegeneration. In cancer, MTOR is widely recognized as a key driver and drug target (Mossmann et al., 2018;Liu and Sabatini, 2020), whereas the importance of SG proteins for tumorigenesis and treatment response is only beginning to emerge (Anderson et al., 2015;Gao et al., 2019). Many SG proteins are dysregulated in cancer (Adjibade et al., 2015;Somasekharan et al., 2015;Vilas-Boas Fde et al., 2016;Sim et al., 2019) and their altered expression has been linked with drug response and disease outcome (Gao et al., 2019). The role of mTORC1-SG crosstalk in neurodegeneration and cancer therefore deserves in depth investigation regarding its role in therapy response and to develop new therapy concepts.

CONCLUSION
The mTORC1 cascade and SGs are key mediators of cell growth and survival that are closely intertwined in a network whose Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 761333 complexity we are only beginning to understand. Current research on MTOR signaling and on SGs is largely confined to separate fields. Comparatively few studies tackle their interplay and are often correlative in nature. Toward a comprehensive understanding, the challenge of investigating mechanistic inhibitory and activating links between the SG and MTOR networks will be key to identify causal relationships between them. These mechanisms may provide leads for treatments that account for specific metabolic alterations and stresses in agerelated conditions such as cellular senescence, cancer, and neurodegeneration.