G C A T

T A C G
G C A T
genes
Review
The Target of Rapamycin Signalling Pathway
in Ageing and Lifespan Regulation
Ivana Bjedov 1, * and Charalampos Rallis 2, *
1 UCL Cancer Institute, Paul O’Gorman Building, 72 Huntley Street, London WC1E 6DD, UK
2 School of Life Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK
* Correspondence: [email protected] (I.B.); [email protected] (C.R.)




Received: 18 August 2020; Accepted: 30 August 2020; Published: 3 September 2020


Abstract: Ageing is a complex trait controlled by genes and the environment. The highly conserved
mechanistic target of rapamycin signalling pathway (mTOR) is a major regulator of lifespan in
all eukaryotes and is thought to be mediating some of the effects of dietary restriction. mTOR is
a rheostat of energy sensing diverse inputs such as amino acids, oxygen, hormones, and stress
and regulates lifespan by tuning cellular functions such as gene expression, ribosome biogenesis,
proteostasis, and mitochondrial metabolism. Deregulation of the mTOR signalling pathway is
implicated in multiple age-related diseases such as cancer, neurodegeneration, and auto-immunity.
In this review, we briefly summarise some of the workings of mTOR in lifespan and ageing through
the processes of transcription, translation, autophagy, and metabolism. A good understanding of the
pathway’s outputs and connectivity is paramount towards our ability for genetic and pharmacological
interventions for healthy ageing and amelioration of age-related disease.

Keywords: ageing; Drosophila; yeast; nutrient-response; autophagy; translation; transcription;

metabolism; TORC1; TORC2

1. Introduction
The nutrient-sensing and evolutionarily conserved mechanistic target of rapamycin (mTOR)
signalling pathway is a pro-ageing signalling hub regulating stress, growth and metabolism from
yeast to human [1–3]. The centrepiece of the pathway is the phosphatidylinositol 3-kinase-related TOR
serine/threonine kinase, firstly discovered in budding yeast, that associate with two (in most eukaryotes),
structurally and functionally distinct, multiprotein complexes termed TORC1 and TORC2 [1–4].
TORC complexes coordinate a plethora of basic cellular organisation and metabolism processes
(Figure 1A) such as transcription, translation, autophagy, metabolism, as well as cytoarchitecture and
have multiple interactions with other pathways, for instance the Insulin Growth Factor (IGF) and
AMP-activated protein kinase (AMPK) signalling pathways [3,5,6].

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(A)TORC1
Figure 1. (A) TORC1and
andTORC2
TORC2subunits
subunitsand
andprocesses
processescontrolled
controlled
byby
thethe complexes.
complexes. (B) (B) Target
Target Of
Of Rapamycin
Rapamycin Complexes
Complexes (TORC1
(TORC1 and TORC2)
and TORC2) subunits
subunits in various
in various modelmodel organisms
organisms used inused in
ageing
ageing research.
research.

TORC1
TORC1 isisshown showntotocontrol
control global
global translation
translation through
throughtwo two
distinct mechanisms.
distinct mechanisms. Firstly, via direct
Firstly, via
phosphorylation
direct phosphorylation of the translation regulators eukaryotic initiation factor 4E-binding protein 11
of the translation regulators eukaryotic initiation factor 4E-binding protein
(4E-BP1)
(4E-BP1) [7,8][7,8] and
and ribosomal
ribosomal protein protein S6 S6 kinase
kinase 11 (S6K1)
(S6K1) [9–13]. Secondly, through
[9–13]. Secondly, through regulation
regulation of of
transcription
transcription (dependent
(dependentonon allall
three RNA
three RNA polymerases)
polymerases) of genes codingcoding
of genes for ribosomal proteinsproteins
for ribosomal [1,9,14],
ribosome biogenesis
[1,9,14], ribosome factors [15]
biogenesis and tRNAs
factors [15] and [16–20].
tRNAs Apart fromApart
[16–20]. translation-related genes TORC1
from translation-related has
genes
major positive impact on gene expression related to growth and metabolism
TORC1 has major positive impact on gene expression related to growth and metabolism [2,18] and a [2,18] and a negative action
on genes coding
negative action foronproteins related to
genes coding forcellular
proteinsstress [21–23].toGenetic
related cellularor stress
pharmacological
[21–23]. Geneticinhibitionor
of mTOR activity stimulates autophagy, a conserved lysosomal
pharmacological inhibition of mTOR activity stimulates autophagy, a conserved lysosomal catabolic catabolic pathway controlling the
degradation and turnover
pathway controlling the ofdegradation
macromolecules andand organelles
turnover of and maintaining metabolic
macromolecules and organelleshomeostasisand
at
maintaining metabolic homeostasis at both the cellular and whole-organism level [24,25]. On but
both the cellular and whole-organism level [24,25]. On the other hand, TORC2 has reverse the
coordinated
other hand, TORC2 roles to hasTORC1 [4,26]
reverse butand is implicated,
coordinated rolesamong
to TORC1others, in transcription,
[4,26] and is implicated, cell survival,
among
DNA
others,damage, telomere length,
in transcription, gene silencing,
cell survival, DNA damage, stress response,
telomerecytoskeletal
length, gene organisation lipogenesis
silencing, stress and
response,
glucose transport [6,27–31]. TORC2 is activated in both a growth factor-dependent
cytoskeletal organisation lipogenesis and glucose transport [6,27–31]. TORC2 is activated in both a and -independent
fashion
growth [32]. These effects are
factor-dependent andmediated through fashion
-independent phosphorylation
[32]. These of AGC kinase
effects arefamily members
mediated such
through
as AKT, SGK1 andofPKC
phosphorylation AGC[32]. Traditionally,
kinase family members TORC1such has been
as AKT,associated
SGK1 and withPKC temporal
[32]. while TORC2
Traditionally,
with spatial aspects of cellular growth. Nevertheless, recent evidence
TORC1 has been associated with temporal while TORC2 with spatial aspects of cellular growth. shows that such distinctions have
become somewhat
Nevertheless, recentobscure
evidence [3].shows
Both thatTORC1 suchand TORC2 complexes
distinctions have become andsomewhat
their upstreamobscure regulators
[3]. Both
such as the TSC1–TSC2 (hamartin and tuberin, respectively) protein
TORC1 and TORC2 complexes and their upstream regulators such as the TSC1–TSC2 (hamartin and complex that represses TORC1 by
affecting Rheb, a G-protein that acts as positive regulator of this complex
tuberin, respectively) protein complex that represses TORC1 by affecting Rheb, a G-protein that acts [33], show great conservation
within eukaryotes
as positive regulator (Figure
of this1B).
complex [33], show great conservation within eukaryotes (Figure 1B).
In most organisms,
In most organisms, mTOR mTOR signalling
signallingcan becan inhibited by rapamycin,
be inhibited a macrolidea from
by rapamycin, Streptomyces
macrolide from
hygroscopicus
Streptomyces hygroscopicus bacteria living within the soil on the Rapa Nui or Easter Island exhibits
bacteria living within the soil on the Rapa Nui or Easter Island [34,35]. Rapamycin [34,35].
broad anti-proliferative
Rapamycin exhibits broad properties and is a potentproperties
anti-proliferative anti-tumour and andisimmunosuppressant
a potent anti-tumour drug [36].
and
Rapamycin directly binds FKBP12 and the complex then
immunosuppressant drug [36]. Rapamycin directly binds FKBP12 and the complex then binds andbinds and inhibits the TOR kinase [37]
with beneficial
inhibits the TOReffects
kinase on [37]both
withhealth
beneficial span and on
effects lifespan of allspan
both health cellular and organismal
and lifespan systems
of all cellular and
tested [5]. Intense research, so far, has shown that genetic or pharmacological
organismal systems tested [5]. Intense research, so far, has shown that genetic or pharmacological interventions that
lower mTOR activity
interventions resultmTOR
that lower in upregulation
activity result of stressin responses,
upregulation alteration
of stressof mitochondrial
responses, alterationrespirationof
and metabolism,
mitochondrial slower ageing
respiration and rates and prolongment
metabolism, slower ageing of lifespan
rates[5,18,22,25,38,39].
and prolongment In this review,
of lifespan
we briefly presentIna this
[5,18,22,25,38,39]. snapshot
review, of we
roles of the
briefly mTORa pathway
present snapshot in of ageing
roles ofand the lifespan
mTOR pathway regulation,in
focusing on four basic cellular processes: gene transcription, protein
ageing and lifespan regulation, focusing on four basic cellular processes: gene transcription, protein translation, autophagy and
metabolism presenting specific
translation, autophagy examples.presenting
and metabolism Accumulating specificevidence
examples.and Accumulating
increasing understanding evidence and of
increasing understanding of the mechanisms of mTOR-related processes in ageing will help towards
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the mechanisms
the design of interventions that will
of mTOR-related increase
processes health will
in ageing spanhelp
andtowards
ameliorate mTOR-dependent
the design age-
of interventions
related conditions.
that will increase health span and ameliorate mTOR-dependent age-related conditions.

2. Lifespan
2. Lifespan Effects of mTOR
Effects of mTOR through
through Regulation
Regulation of
of Gene
Gene Transcription
Transcription and
and Metabolism
Metabolism
mTOR exerts
mTOR exerts many
many of of its effects through
its effects through the
the regulation
regulation Pol
Pol I,
I, Pol
Pol IIII and
and Pol
Pol III
III transcription
transcription
realms [5,40,41].
realms [5,40,41]. For
For example,
example, TORC1
TORC1 regulates
regulates autophagy
autophagy partly through phosphorylation
partly through phosphorylation and and
inactivation of the nuclear translocation of the transcription factor EB (TFEB) which
inactivation of the nuclear translocation of the transcription factor EB (TFEB) which controls genes forcontrols genes
for lysosomal
lysosomal biogenesis
biogenesis andautophagy
and the the autophagy machinery
machinery [42–44].[42–44]. Recent
Recent work work in Drosophila
in Drosophila has
has identified
identifiedand
REPTOR REPTOR and REPTOR-Binding
REPTOR-Binding Protein (REPTOR-BP)
Protein (REPTOR-BP) as transcription
as transcription factors downstream
factors downstream of TORC1
of TORC1 required for ∼90%
required for ∼90% of the transcriptional induction that occurs upon TORC1 inhibition inhibition
of the transcriptional induction that occurs upon TORC1 [45].
[45]. However,
However,
a REPTORahomologue
REPTOR homologue or functionally
or functionally related transcription
related transcription factor in mammals
factor in mammals and yeastand yeast
is yet to
is yet to be identified. The localisation of mTOR kinases and complexes is the focus
be identified. The localisation of mTOR kinases and complexes is the focus of a recent review [46]. of a recent review
[46]. Here,
Here, we concentrate
we concentrate on examples
on examples of mTOR-dependent
of mTOR-dependent ageing-related
ageing-related transcriptional
transcriptional effects ineffects
diversein
diversesystems
model model systems from
from yeast to yeast
human to cells
human cells 2).
(Figure (Figure 2).

Figure
Figure 2. Transcription factors
2. Transcription factors downstream
downstream of
of TORC1,
TORC1, input
input linked
linked to
to their
their action
action and
and cellular
cellular
processes affected.
processes affected.
2.1. GATA Factors, Maf1 and tRNA Gene Regulation Downstream of mTOR
2.1. GATA Factors, Maf1 and tRNA Gene Regulation Downstream of mTOR
TORC1 is thought to mediate part of the life-extending effects of dietary restriction [47].
TORC1 analysis
Systematical is thought to mediate part
of transcription of the life-extending
via RNA-seq across organseffects of dietary
of Drosophila, restriction
subjected [47].
to dietary
Systematical analysis of transcription via RNA-seq across organs of Drosophila,
restriction or lowered mTOR and subsequent prediction of transcription regulators, identified the subjected to dietary
restriction or lowered
evolutionarily conserved mTOR
GATAand subsequent
factors prediction
[48]. Lifespan of transcription
and fitness regulators,
analyses showed identified the
that knockdown of
the GATA transcription factor srp in specific fly tissues recapitulated the benefits but notknockdown
evolutionarily conserved GATA factors [48]. Lifespan and fitness analyses showed that the costs of
of the GATA
dietary transcription
restriction [48]. These factor srpindicated
results in specificthatflyGATA
tissuesfactors
recapitulated
mediate the benefits
effects but not
of dietary the acids
amino costs
of dietary
on lifespanrestriction
and placing [48].
themThese results
as target forindicated
longevitythat GATA factors
interventions mediate
[49]. GATA effects
factors of dietary
have amino
been linked to
acids on lifespan and placing them as target for longevity interventions [49].
ageing downstream of mTOR in Caenorhabditis elegans. ChIP-seq data screening towards identification GATA factors have been
linked
of to ageing
transcription downstream
factors of mTOR
that regulate in Caenorhabditis
to age-related elegans.elt-2,
genes identified ChIP-seq
a gene data screening
responsible towards
for inducing
identification of transcription factors that regulate to age-related genes identified
expression of intestinal genes during embryogenesis. Interestingly expression of ELT-2 protein decreases elt-2, a gene
responsible for inducing expression of intestinal genes during embryogenesis.
during ageing, persistent ELT-2 expression is a common feature of long-lived animals [50] and is found Interestingly
expression
to be required of for
ELT-2 protein decreases
TORC1-dependent during
lifespan ageing,inpersistent
extension ELT-2 expression
hypoxic conditions [51]. is a common
feature of long-lived animals [50] and is found to be required for TORC1-dependent
Nitrogen is a limiting element required for fuelling cellular growth and anabolic processes. lifespan
extension in hypoxic conditions [51].
Nitrogen utilization is employed by the alleviation of Nitrogen Catabolite Repression (NCR) which
ensuresNitrogen
the useisofanon-preferential
limiting elementorrequired
alternative fornitrogen
fuelling sources
cellularwhen
growth and anabolic
preferential processes.
sources are not
Nitrogen utilization is employed by the alleviation of Nitrogen Catabolite Repression (NCR) which
ensures the use of non-preferential or alternative nitrogen sources when preferential sources are not
Genes 2020, 11, 1043 4 of 21

available. In yeasts, TORC1 has been linked with nitrogen catabolite repression and GATA-dependent
transcription. When cells are cultured under nitrogen rich conditions TORC1 activation results
in Gln3 and Gat1 cytoplasmic sequestration and transcription of genes required to scavenge poor
nitrogen sources are highly repressed. Pharmacological or nutritional repression of TORC1 leads to
PP2A-dependent translocation of Gln3 and Gat1 to the nucleus [52]. Fission yeast Gat1 orthologue,
Gaf1, has similar roles. TORC1 phosphorylates and inhibits Gaf1 and upon TORC1 down-regulation
Gaf1 is dephosphorylated by the PP2A-like phosphatase Ppe1 [53]. This allows it to enter the nucleus
where it upregulates amino acid permeases including isp7 (coding for an oxygenase controlling amino
acid permeases) and negatively regulates ste11 (coding for a transcription factor implicated in mating
and sporulation) [54,55]. Upregulated genes in gaf1 mutant cells grown in nitrogen-rich medium
are enriched in nitrogen starvation-responsive genes Ste11 targets [54]. While budding yeast GATA
transcription factors are not directly implicated in lifespan regulation, a recent study [18] indicated
that, similarly to the C. elegans elt-2, the fission yeast gaf1 is an anti-ageing gene. Cells lacking gaf1 are
short-lived and Gaf1 is partially required for Torin1-induced lifespan extension [18]. Gene expression,
DNA binding and downstream analyses showed that Gaf1 defines a transcriptional control of protein
translation mechanism, tuning the production of components required for protein translation according
to mTORC1 activity. Importantly, the study showed that Gaf1 binds and directly suppresses all tRNA
genes [18] and mediates both Pol II- and Pol III-dependent transcription downstream of mTORC1.
Inhibition of Pol III, which transcribes the tRNAs, prolongs lifespan in yeast, worms, and flies,
and is required for the lifespan extension mediated by TORC1 inhibition [40]. Pol III transcription
is regulated by a plethora of basic transcription factors (such as TFIIIB, TFIIIC, or TBP) and several
other factors without direct DNA binding [56], including the coactivator PNRC as well as MYC which
interacts with the Pol III basal apparatus [57–59] and the conserved Pol III inhibitor Maf1. The latter
is negatively regulated via phosphorylation by the mTOR pathway and tailors 5S rRNA and tRNA
production in response to various environmental cues [60]. Recent data have shown that the Maf1
binding site on Pol III overlaps with that of TFIIIB in the preinitiation complex [61]. Deletion of
maf1 in the budding yeast Saccharomyces cerevisiae but not its homologue mafr-1 in C. elegans prevents
dietary restriction or caloric restriction to extend lifespan. Interestingly, mafr-1 deletion increases stress
tolerance and extends lifespan due to an enhancement of stress response, including oxidative stress
response, mitochondrial unfolded protein response (UPRmt) and autophagy [62]. Recent data in
fission yeast suggest that Maf1-dependent inhibition of tRNA synthesis controls lifespan by preventing
genomic instability that arises at tRNA genes [63]. Overall, numerous studies provide evidence that
GATA factors regulate a multitude of metabolic processes downstream of mTOR through the action of
all three polymerases.

2.2. Metabolic Homeostasis and Lipid Synthesis through ATF4 and SREBPs
The general amino acid signalling pathway harmonises availability of amino acids with protein
translation (discussed below). Amino acid starvation leads to TORC1 activity repression and lifespan
extension. In the transcriptional level, following TORC1 inactivation, the expression of the leucine
zipper transcription factor Gcn4 (in yeast) [64] or ATF4 (activating transcription factor 4) in mammalian
cells [65,66] is induced. ATF4/Gcn4 induces amino acid transporters and metabolic enzymes [67–72],
as well as autophagy factors [73]. Interestingly TORC1 can be inhibited through ATF4-dependent
mechanisms upon leucine, arginine, or glutamine deprivation with the mechanism in long-term
deprivation including SESTRIN2 [74,75]. On the other hand, TORC1 activation leads to de novo
purine synthesis through the ATF4-dependent expression of MTHFD2, a bifunctional enzyme and
key component of the mitochondrial tetrahydrofolate cycle that provides one carbon units for purine
synthesis [76]. Purine metabolism has recently been highlighted as a longevity target with dietary
sugar intake strongly predicting circulating purine levels in humans. In addition, high-sugar diets
promote accumulation of uric acid, an end-product of purine catabolism [77] and regulating uric acid
production impacts on lifespan in a water-dependent manner [77]. Synthesis of pyrimidines is also
Genes 2020, 11, 1043 5 of 21

promoted by TORC1, via its down-stream effector S6K, which phosphorylates and activates CAD
(carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a rate limiting
enzymes in the first steps of pyrimidine de novo synthesis [78,79].
Sterol regulatory element-binding proteins (SREBPs) are key basic helix–loop–helix leucine zipper
(bHLH-Zip) transcription factors that govern fat metabolism [80]. They are synthesized as inactive
precursors bound to the endoplasmic reticulum (ER) [81]. Mammalian genomes have two SREBP
genes, SREBP-1, and SREBP-2. SREBP-1 has two distinct isoforms, SREBP-1a and -1c, differing in
their first exons. SREBP-1a is a potent activator of all SREBP-responsive genes in proliferating cells,
including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides [82]. SREBP-1c
regulates de novo lipogenesis and plays a major role in the nutritional regulation of fatty acid and
triglyceride (TG) synthesis in lipogenic organs, such as the liver, while SREBP-2 ubiquitously regulates
sterol synthesis in tissues [83]. Upon its activation, mTORC1 promotes lipid synthesis through
SREBPs contributing to cellular and organ growth, although the connections of the TORC1 and TORC2
complexes with the activation of SREBPs might be more complicated as crosstalk with other pathways
that regulate mTOR, such as the AMP-activated protein kinase (AMPK), are involved [84]. The roles
of SREBPs in ageing are not clear yet: results using tissue culture models suggest that enhanced
lipogenesis via SREBP-1 induction is involved in senescence [85]. Blocking lipogenesis with fatty
acid synthase (FAS) inhibitors and via siRNA-mediated silencing of SREBP-1 and ATP citrate lyase
attenuated H2 O2 -induced senescence. In addition, aged human fibroblasts were effectively reversed to
young-like cells by challenging with FAS inhibitors [85]. Calorie restriction in rats enhances fatty acid
(FA) biosynthesis, and SREBP-1c, a master regulator of FA synthesis, has been identified as a mediator
of some of the beneficial effects of calorie restriction [86]. SREBP-1c knockout mice do not exhibit
extended lifespan following calorie restriction and fail to upregulate factors involved in FA biosynthesis.
SREBP-1c is shown to be implicated in calorie restriction-associated mitochondrial activation through
the upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master
regulator of mitochondrial biogenesis [86]. In C. elegans, the homolog of mammalian SREBP-1
(sbp-1) in complex with MDT-15 (mediator-15) prevents life-shortening effects of a glucose-rich diet.
The upregulation of the SREBP-1/MDT-15 transcription factor complex was necessary and sufficient
to mitigate the detrimental effects of a glucose-rich diet to lifespan. SREBP-1/MDT-15 were able to
reduce the levels of saturated fatty acids and moderated glucose toxicity on lifespan [87]. Finally,
triple-drug combinations in nematode (that included mTOR inhibition) and effectively prolonged
lifespan and improved health span, resulted in increased levels of monounsaturated fatty acids
(MUFAs) in phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) membrane lipids [88].
The increase in MUFAs, as well as the synergistic lifespan benefits, required sbp-1 (SREBP-1) [88].
Overall, the data so far suggest interesting connections of SREBP transcription factors with lifespan and
dietary restriction downstream of mTOR. Further studies might highlight SREBP-related interventions
in age-related metabolic syndromes.

2.3. Hypoxia and HIF-1 in Lifespan Regulation Downstream of mTOR

Studies in diverse systems have shown that HIF1, a heterodimeric transcriptional complex
containing HIF-1α and HIF-1β, is a target of the TOR pathway [89–92]. Increased mTOR and
S6K activities enhance HIF-1 levels under both normoxic and hypoxic conditions [89,91,93,94].
Under hypoxia, HIF-1α is stabilized promoting cells adaptation to low-oxygen stress [95].
HIF-1 overexpression is expressed in solid tumours and inhibition of HIF-1 can prevent tumour
growth [96,97]. However, TORC1 is reported to control growth by shifting cells from oxidative
phosphorylation to glycolysis, a phenomenon occurring in cancer cells, through increase of HIF1α
translation which drives the expression of several glycolytic enzymes [98].
In C. elegans, studies have shown that HIF-1 can modulate lifespan both positively and negatively.
For instance, stabilization of HIF-1 increases lifespan and health span through a mechanism distinct
from the insulin-like signalling pathway and from dietary restriction [99,100]. HIF-1 was found to
Genes 2020, 11, 1043 6 of 21

act in parallel to the SKN-1/NRF and DAF-16/FOXO transcription factors to promote longevity [99].
On the other hand, HIF-1 is reported to modulate longevity in a nutrient-dependent manner and the
hif-1 loss-of-function mutant extends lifespan under rich nutrient conditions but fails to show lifespan
extension under dietary restriction [101]. In addition, inactivation of the maa-1 (membrane-associated
acyl-CoA binding protein 1), which is a C. elegans paralogue of ACBP (Acyl-CoA-binding protein),
promotes lifespan extension and resistance to different types of stress. HIF-1 is required for the
anti-ageing response induced by MAA-1 deficiency. This response relies on the activation of molecular
chaperones known to contribute to maintenance of the proteome [102]. The discrepancy between
studies could be explained through temperature. Indeed, stabilization of HIF-1 increased life span
under all conditions but deletion of hif-1 increased lifespan at 25 ◦ C but not at 15 ◦ C [103]. At these
low temperatures, RNAi knockdown of hif-1 impaired health span due to age-dependent loss of
vulval integrity. Interestingly, hif-1 knockdown resulted in nuclear localisation of the DAF-16 FOXO
transcription factor that was found necessary for the hif-1-dependent life span extension at all
temperatures [103].
In Drosophila, HIF-1α expression is increased in diapause-destined pupal brains and mitochondrial
biogenesis is negatively regulated through HIF-1α. This effect is mediated via c-Myc activity inhibition
through proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A
(TFAM), encoding a key mitochondrial factor for transcription and DNA replication, is activated
through direct binding of c-Myc on TFAM promoter. The HIF-1α-c-Myc-TFAM signalling pathway
is, therefore, shown to participate in the regulation of mitochondrial activity for insect diapause or
lifespan extension [104]. Changes in HIF-1α with age have been reported in rats and could be related
to age-related pathologies [105], such as neurodegeneration [106].
HIF-1α regulates mitochondrial biogenesis, and modulation of the nuclear-mitochondrial
communication during aging depends on PGC-1α [107]. Notably, TORC1 balances energy metabolism
through transcriptional control of mitochondrial oxidative function via the YY1/PGC-1α transcriptional
complex required for rapamycin-dependent repression of respiratory genes [108]. HIF-1α is found
to accumulate in the nucleus of SIRT1-silenced primary myoblasts and adult SIRT1 knockout
mice [109]. The increased HIF-1α levels observed during ageing or in response to SIRT1 knockout
activated Mxi1, encoding a c-Myc transcriptional repressor. Mxi1 restricts the interaction between
c-Myc and mitochondrial transcription factor A (TFAM) that is critical for replication, transcription,
and maintenance of mitochondrial biogenesis [110]. SIRT1 transcription is also coupled with hypoxia
during ageing and during acute hypoxia, HIF-1α and HIF-2α interact with Hsp90, and directly
bind to hypoxia response elements in the SIRT1 promoter [111]. Overall, numerous studies support
the implication of HIF factors in lifespan and their connections with mitochondrial biogenesis and
stress response.

3. Lifespan Regulation through mTOR Effects on Protein Translation

One of the principal effects of the mTOR pathway is adjusting protein synthesis and proteome
content, according to available nutrients and growth factors. mTOR exerts one of its most prominent,
mechanistic links to ageing through regulation of translation (Figure 3), which is described below.
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Figure 3.
Figure mTORC1promotes
3. mTORC1 promotesprotein
proteinsynthesis.
synthesis. Decreased
Decreasedprotein
proteinsynthesis
synthesiscorrelates
correlates with
with extension
extension
of lifespan and many mutants affected in translation initiation, elongation, and ribosomal
of lifespan and many mutants affected in translation initiation, elongation, and ribosomal proteins proteins
are long-lived.
are long-lived. Some
Someof
ofthe
theproposed
proposed mechanisms
mechanismswhereby
whereby less
less translation
translation could
could promote
promote longevity
longevity
are illustrated.
are illustrated.

During protein synthesis, mRNAs are scanned for start codon in an interaction with different
During protein synthesis, mRNAs are scanned for start codon in an interaction with different
complexes. The 43S preinitiation complex (PIC) is formed from 40S ribosomal subunit, initiation factors
complexes. The 43S preinitiation complex (PIC) is formed from 40S ribosomal subunit, initiation
eIF3, eIF1, and eIF5, while a ternary complex consists of methionyl-initiator tRNA (Met-tRNAi)
factors eIF3, eIF1, and eIF5, while a ternary complex consists of methionyl-initiator tRNA (Met-
and GTP-bound eIF2. The m7 G cap structure, situated at the 50 end of mRNAs, is recognised by
tRNAi) and GTP-bound eIF2. The m7G cap structure, situated at the 5′ end of mRNAs, is recognised
eIF4F cap-binding complex. An important feature of eIF4F complex, formed of eIF4E (cap binding),
by eIF4F cap-binding complex. An important feature of eIF4F complex, formed of eIF4E (cap
eIF4A (RNA helicase) and eIF4G (scaffolding protein), is its very low cellular concentration, which places
binding), eIF4A (RNA helicase) and eIF4G (scaffolding protein), is its very low cellular concentration,
it in a critical position to regulate translation [112]. The cap structure of mRNA is also a binding site
which places it in a critical position to regulate translation [112]. The cap structure of mRNA is also a
for different RNA-binding proteins, which can shield them from translation [113]. Some mRNAs,
binding site for different RNA-binding proteins, which can shield them from translation [113]. Some
typically from viruses and likely circular mRNAs as well, bypass mRNA scanning by using highly
mRNAs, typically from viruses and likely circular mRNAs as well, bypass mRNA scanning by using
structured internal ribosomal entry site (IRES) elements to recruit PIC to 50 UTR [114–118]. Once the
highly structured internal ribosomal entry site (IRES) elements to recruit PIC to 5′ UTR [114–118].
AUG start codon is encountered in the 50 UTR within a favourable sequence context, GTP is hydrolysed
Once the AUG start codon is encountered in the 5′ UTR within a favourable sequence context, GTP
and
is release ofand
hydrolysed eIF2-GDP
release ofaccompanied by joining by
eIF2-GDP accompanied of the 60Sofribosome
joining subunit, subunit,
the 60S ribosome allowingallowing
protein
synthesis to commence [117].
protein synthesis to commence [117].
The mTOR
The mTORpathway
pathwaypromotes
promotes translation
translation byby stimulating
stimulating assembly
assembly of eIF4F
of the the eIF4F complex
complex [5]. One[5].
One of the major mTORC1 downstream targets is inhibitory phosphorylation
of the major mTORC1 downstream targets is inhibitory phosphorylation of eIF4E-binding proteins of eIF4E-binding
proteins This
(4EBPs). (4EBPs). This4EBP
releases releases
from4EBP
bindingfrom binding
eIF4E eIF4E and
and enables enables between
interaction interaction between
eIF4E eIF4E
and eIF4G,
and eIF4G, therebyeIF4F
enhancing eIF4F complexand formation 0
and 5 cap-dependent
thereby enhancing complex formation 5′ cap-dependent translation.translation.
In the context In theof
context of ageing, and well-investigated in C. elegans, the down-regulation
ageing, and well-investigated in C. elegans, the down-regulation of eIF2β/iftb-1, eIF4G/ifg-1, and of eIF2β/iftb-1, eIF4G/ifg-1,
and eIF4E/ife-2,
eIF4E/ife-2, all extend
all extend lifespan
lifespan in worms
in worms [118].[118]. In a large
In a large RNAiRNAi
screenscreen for long-lived
for long-lived mutants,
mutants, 2700
2700 genes
genes were targeted
were targeted in worms,in worms,
and oneand oneprominent
of the of the prominent
categories categories
consistedconsisted of translation
of translation initiation
initiation
factors: factors: eIF4A/inf-1,
eIF3/eif-3, eIF3/eif-3, eIF4A/inf-1, and [119].
and eIF1B/eif-1 eIF1B/eif-1
This and[119]. This
other and other experiments
experiments strongly suggeststrongly
that
suggest that reducing translation by down-regulation of a variety of
reducing translation by down-regulation of a variety of initiation factors is an anti-ageing initiation factors is an anti-ageing
intervention [118–123].
intervention [118–123]. In Drosophila,4EBP
InDrosophila, 4EBPisisrequired
requiredforforlifespan
lifespanextension
extensionunderunderdietary
dietaryrestriction,
restriction,
and it was proposed that when 4EBP is activated, this replicates condition
and it was proposed that when 4EBP is activated, this replicates condition of mTOR downregulation of mTOR downregulation
and low
and low nutrients,
nutrients, andand favours
favours translation
translation of of mitochondrial
mitochondrial genesgenes with
with shorter
shorter and
and less
less structured
structured 5′ 50
UTRs [124].
UTRs [124]. When
When 4EBP
4EBP is is over-expressed
over-expressed in in the
the muscles,
muscles, itit preserves
preserves muscle
muscle function
function andand extends
extends
lifespan [125]. This illustrates that numerous translation initiation genes are
lifespan [125]. This illustrates that numerous translation initiation genes are good anti-ageing targets.good anti-ageing targets.
Another critical downstream target of mTORC1 that is implicated in ageing is S6K. S6K is
phosphorylated and activated by both PDK and mTORC1 [126]. In all organisms tested, from SCH9
mutants in S. cerevisiae [127], S6K/rsks-1 RNAi in C. elegans [118], S6KKQ dominant negative
Genes 2020, 11, 1043 8 of 21

Another critical downstream target of mTORC1 that is implicated in ageing is S6K. S6K is
phosphorylated and activated by both PDK and mTORC1 [126]. In all organisms tested, from SCH9
mutants in S. cerevisiae [127], S6K/rsks-1 RNAi in C. elegans [118], S6KKQ dominant negative
overexpressor flies [128], and to S6K1 null mice [129], deleting or down-regulating of S6K results
in longer lifespan. S6K is one of the kinases phosphorylating ribosomal S6 protein, and its other
translation targets include eIF4B and eEF2K, suggesting S6K can regulate both initiation and elongation
step of protein synthesis [126]. Intriguingly, in mouse cells from S6K1-/- S6K2-/- double knockout,
global translation as measured by polysomal profiles was not affected [130]. However, majority of
ribosomal biogenesis (RiBi) genes were transcriptionally down-regulated in S6K1 and S6K2 double
knockout cells [131], convincingly showing a role of S6K in transcriptional regulation of nucleolar
factors involved in ribosome maturation and assembly [131]. S6K has many other roles and targets that
are involved in mRNA processing, protein folding, metabolism and cell survival some of which could
be important in health and ageing [126,132]. For instance, under high mTOR signalling, S6K mediates
negative feedback to insulin receptor substrate 1 (IRS1). When IRS1 is phosphorylated by S6K and
mTOR, it is degraded and cells become unresponsive to insulin [133]. Interestingly, S6K1 null mice are
resistant to diet induced obesity [134], and their transcriptomic changes resemble transcriptome under
caloric restriction and AMPK activation by AICAR [129]. This suggests S6K as a promising drug target
for health benefits.
Another layer of mTOR translation regulation and coordinated translational control stems from its
effect on positively regulating translation of mRNA having 50 terminal oligopyrimidine (50 TOP) motif.
Such motifs are found in components of translation machinery, more precisely ribosomal proteins
and eEF2 [13,135]. Ribosomal profiling experiments showed that these 50 TOP mRNAs are major
targets for translation down-regulation upon mTORC1 and mTORC2 inhibition using Torin 1 [136,137].
A polysome profiling study on the other hand, found many mRNAs without 50 TOP motif to be sensitive
to mTOR pathway inhibition as well, such as cyclins and c-Myc [138]. This discrepancy is attributed to
technical details between the two methods and better sensitivity of polysome profiling to detect small
changes in translation. When ribosome profiling is used, greater sequencing depth may be required
to detect less abundant mRNAs that are being translated [139,140]. Precise underlying mechanism
of mTOR-mediated 50 TOP translation remains uncertain, and competition of eIF4F with a repressor
protein such as Larp1 for 50 TOP motif is being proposed [140,141]. Because nearly all ribosomal
mRNAs have 50 TOP motif, their mTOR-mediated regulation is interesting from a healthy ageing
perspective as it is well established that ribosomal mutants live longer [142]. In worms, when rps6,
rps11, and rps22, are down-regulated by RNAi, lifespan is extended [118]. In yeast, replicative lifespan
of 107 ribosomal gene deletion strains showed that the large 60S subunit mutants are long-lived,
a mechanism that resembles dietary restriction involving a nutrient-responsive transcription factor
Gcn4, an ATF4 homologue [143]. Another argument for the importance of translation in ageing comes
from the results of a deletion library screen, consisting of 4698 S. cerevisiae mutants. In this screen
ribosome, mitochondrial translation, and tRNA export were the most significant functional categories
among long-lived mutants [144]. It will be interesting to examine these different long-lived ribosomal
mutants in light of the discovery that cells may possess heterogeneous ribosomes that can dictate
translation of specific mRNAs [145].
In sum, there is an abundant evidence of the anti-ageing effect of down-regulation of translation.
Long-lived mutants are found among different components of the translation machinery [146], and the
mTOR pathway is the principal regulator of protein synthesis. Rapamycin, one of the first anti-ageing
drugs discovered, inhibits completely mTORC1 pathway in budding yeast, while in mammals it
inhibits less well some mTORC1-dependent processes, but its prolonged inhibition may also inhibit
mTORC2 complex through interference with the complex assembly [2,5]. Based on numerous long-lived
translation mutants, many other drugs targeting translation machinery are predicted to improve
longevity and health [147].
Genes 2020, 11, 1043 9 of 21

A key question is: Why are different translation mutants long-lived [148]? Ageing is accompanied
by reduction of translation, likely as a compensation for overall collapse of proteostasis, which consists
of protein synthesis, protein folding and degradation [146,149]. Several hypotheses have been put
forward, such as that reduced translation may be an anti-ageing intervention that saves cellular
energy resources that could then be invested in cell maintenance. Reduced translation means protein
folding and degradation machinery become more available to deal with damaged cellular components.
Another interesting possibility is that under reduced translation, a different set of proteins are being
synthesized and the proteome is more adapted for stressful conditions [148,150]. Finally, there is also
a possibility that reduced translation could improve translation fidelity and lead to fewer protein
errors [146,151], a hypothesis that was heavily debated in the past [152] but currently neglected.
Some interesting correlative evidence suggest that animals having improved translation accuracy live
longer, such asGenes
exceptionally
2020, 11, x FOR PEERlong-lived
REVIEW naked mole rat [153]. In addition, hypoaccurate 9 of 21 ribosomal
yeast mutants have shorter chronological lifespan [154], and accuracy in yeast mitochondrial ribosomes
components. Another interesting possibility is that under reduced translation, a different set of
positively correlates
proteins with
are beinglongevity
synthesized[155]
and the(Figure
proteome3).
is more adapted for stressful conditions [148,150].
Finally, there isisalso
Reduced translation anaimportant
possibility thatevolutionarily
reduced translationconserved
could improveanti-ageing
translation fidelity and lead
intervention, and there is
to fewer protein errors [146,151], a hypothesis that was heavily debated in the past [152] but currently
a plethora of different
neglected.mutants and longevity
Some interesting mechanisms
correlative evidence yet
suggest that to behaving
animals discovered
improved in this field. To improve
translation
health in the elderly,
accuracydifferent
live longer, translation-modifying
such as exceptionally long-liveddrugs could
naked mole be tested
rat [153]. forhypoaccurate
In addition, longevity and rapamycin
ribosomal yeast mutants have shorter chronological lifespan [154], and accuracy in yeast
is one of the first examples of how this approach may be successful.
mitochondrial ribosomes positively correlates with longevity [155] (Figure 3).
Reduced translation is an important evolutionarily conserved anti-ageing intervention, and
4. mTOR and Autophagy
there is a plethora of different mutants and longevity mechanisms yet to be discovered in this field.
To improve health in the elderly, different translation-modifying drugs could be tested for longevity
mTOR integrates information
and rapamycin on examples
is one of the first growthoffactors, cellularmay
how this approach nutrient status, energy level, and stress,
be successful.

in order to either promote growth

4. mTOR and Autophagy
when the environment is favourable, or trigger catabolic processes,
such as autophagy, that degrade cellular components to obtain energy and building blocks [5,156]
mTOR integrates information on growth factors, cellular nutrient status, energy level, and stress,
(Figure 4). Autophagy is,promote
in order to either together
growth with the
when the proteasome,
environment a major
is favourable, or triggercellular degradation process,
catabolic processes,
such as autophagy, that degrade cellular components to obtain energy and building blocks [5,156]
and the only process that can degrade entire organelles. There are three different types of autophagy,
(Figure 4). Autophagy is, together with the proteasome, a major cellular degradation process, and the
microautophagy, onlychaperone-mediated
process that can degrade and entire macroautophagy, which
organelles. There are three differ
different in the
types way cargo is delivered
of autophagy,
to the lysosome;microautophagy,
here we will chaperone-mediated and macroautophagy,
refer to macro-autophagy which differ in
as autophagy the way cargo is
[157,158].
delivered to the lysosome; here we will refer to macro-autophagy as autophagy [157,158].

Figure 4. mTORC1Figure 4. mTORC1

inhibitsinhibits varioussteps
various steps ofof
autophagy. mTORC1 phosphorylates
autophagy. and inhibits ULK1
mTORC1 phosphorylates and inhibits
and Atg13 which are part of the ULK complex, thereby blocking autophagy initiation step. mTORC1-
ULK1 and Atg13 which are part of the ULK complex, thereby blocking
mediated phosphorylation of Atg14, AMBRA1, and NRBF2 from the PI3KC3 complex I, which is one
autophagy initiation step.
mTORC1-mediated phosphorylation of Atg14, AMBRA1, and NRBF2 from the PI3KC3 complex I,
of the PI3KC3 complexes, negatively regulates autophagosomal nucleation step. mTORC1 also affects

which is one ofmaturation

the PI3KC3 of autophagosome through inhibitory phosphorylation of UVRAG. TFEB and TFE3 are
complexes, negatively regulates autophagosomal nucleation step. mTORC1
transcription factors critical for lysosomal biogenesis and expression of autophagy genes. Different
also affects maturation of autophagosome
steps in the autophagy through
process are illustrated inhibitory
and denoted phosphorylation
in blue. Bold of UVRAG. TFEB and
letters in different complexes
denote proteins
TFE3 are transcription that arecritical
factors phosphorylated by mTORC1. biogenesis and expression of autophagy genes.
for lysosomal
Different stepsDuring
in theautophagy,
autophagy process
a portion of theare illustrated
cytoplasm and within
is captured denoted in blue. Boldand
the autophagosome, letters
then in different
complexes denote proteins that are phosphorylated by mTORC1.
degraded by lysosomal hydrolases upon fusion with the lysosome, thereby replenishing the
Genes 2020, 11, 1043 10 of 21

During autophagy, a portion of the cytoplasm is captured within the autophagosome, and then
degraded by lysosomal hydrolases upon fusion with the lysosome, thereby replenishing the cytoplasm
with amino acids, lipids, and nucleotides [159,160]. Two ubiquitin-like conjugation systems are critical
for autophagosome formation. These systems enable covalent conjugation of Atg8, a ubiquitin-like
protein, to phosphatidylethanolamine (PE) and its incorporation into the inside and outside membranes
of the phagophore [161,162]. Atg8 is firstly cleaved by Atg4, and then lipidation is mediated by Atg7,
the E1 enzyme, and Atg3, the E2 enzyme. Its localisation to the autophagic membranes is determined by
the E3-like Atg5-Atg12 Atg16L complex, which is a second conjugation system driving the autophagy
process. Autophagy proteins were discovered in yeast and more than 30 of them are now known,
including the mammalian homologues of Atg8, LC3, and GABARAP proteins [163]. Atg8 interacts
with different proteins that have LC3 interaction regions (LIR), enabling the autophagosome to be
linked with specific cargo in a process of selective autophagy degradation, as opposed to general
autophagy where cellular components are randomly recycled [164]. There is a growing number of LIR
containing proteins, which are mainly autophagy receptors, but also components of the core autophagy
machinery and of proteins being degraded. This is critical for precise elimination of certain cellular
components, such as damaged mitochondria, in a process called mitophagy, bacteria by xenophagy,
protein aggregates by aggrephagy, lipids by lipophagy and many other cellular macromolecules and
organelles that can selectively be degraded [165]. For instance, ribosomal degradation mediated by
starvation and mTORC1-inhibition via NUFIP1 autophagy receptor liberates both amino acids and
ribonucleotides, which is important for subsequent mTOR reactivation [166]. Ribophagy has potential
to be interesting in ageing research, given that many ribosomal mutants are long-lived across different
model organisms [118,143] and that reduced ribosomal biogenesis is a well-established pro-longevity
intervention [167].
mTORC1 inhibits autophagy by regulating different autophagy steps as well as lysosome
biogenesis [168,169]. For instance, mTORC1 phosphorylates and inhibits Atg1, a homologue of
human ULK1, as well as Atg13, both of which are part of the ULK1 complex together with Atg101
and FIP200 [170]. ULK1 has many phosphorylation sites, during starvation or mTORC1 inhibition,
mTORC1 inhibitory phosphorylation is replaced by AMPK-mediated phosphorylation of ULK1 at
different sites. AMPK is an energy sensor kinase that is active when the cellular AMP/ATP ratio
increases and its phosphorylation of ULK1 promotes interaction and activity of the ULK1 complex [18].
mTORC1 also inhibits a PIK3C3/VPS34 complex, through inhibitory phosphorylation of Atg14,
thereby providing an additional break on autophagy when growth conditions are favourable [171].
Moreover, mTORC1 is shown to block late stages of autophagy as well, by negatively affecting
endosome to lysosome maturation through phosphorylation of UVRAG (UV radiation resistance
associated), an important regulator of mammalian macroautophagy/autophagy [172].
Transcription of autophagy genes and lysosomal biogenesis factors requires the transcription
factors TFEB and TFE3, and mTORC1-mediated inhibitory phosphorylation results in their cytoplasmic
retention [43,173,174]. Interestingly, overexpression of mammalian TFEB orthologue HLH30 in worms
leads to extension of lifespan that is dependent on autophagy [175]. This is one of the few direct genetic
interventions that induces autophagy and promotes health and longevity [25,176]. Others include
overexpression of the Atg8a protein in Drosophila neurons which diminished accumulation of damaged
proteins in the brain [177] and a similar lifespan-extending effect of Atg8a is observed when it is
overexpressed in fly muscles [178]. In Drosophila, it was shown that up-regulation of Atg1 can trigger
the autophagy process [179] and this is also an anti-ageing intervention [180]. In mice on the other
hand, it is Atg5 overexpression that extends lifespan and was accompanied by increased insulin
sensitivity and improved motor function [181]. Despite numerous autophagy proteins, only some of
them when overexpressed can trigger the entire process and extend lifespan. Additional evidence for
the importance of autophagy in ageing was provided by experiments in which autophagy inhibition
blocks lifespan extension of long-lived mutants, such as the C. elegans insulin receptor daf-2 mutant [176].
Given that accumulation of damage, damaged organelles, and overall loss of proteostasis is a hallmark of
Genes 2020, 11, 1043 11 of 21

ageing, it is not surprising that autophagy activation has beneficial effects and that many pro-longevity
interventions depend on increased autophagy [176]. Activation of the lysosomal pathway was also
recently associated with clearance of protein aggregates in quiescent neuronal stem cells, resulting in
their younger, healthier state [182]. However, while there are numerous studies that show enhancement
of autophagy is beneficial for ageing, we should nevertheless be cautious because if autophagy flux is
clogged during ageing, then further autophagy enhancement could be detrimental and autophagy
inhibition could be beneficial instead [183].
Overall, there are several points of interaction whereby mTORC1 signalling inhibits autophagy
under favourable growth conditions. Autophagy is continuously active at basal levels; however,
mTORC1 inhibition provides a degradation boost, cleansing the cell of damaged organelles via
autophagy, which is a particularly beneficial anti-ageing intervention. There are several anti-ageing
drugs that exert their effect by increased autophagy, such as valproic acid, lithium, trehalose, spermidine,
urolithin A, and mTOR inhibitor rapamycin [25,176,184]. Most of these drugs have additional effects,
therefore finding compounds that can specifically enhance autophagy, and even more precisely ones that
can boost the selective types of autophagy, would be of great interest. These drugs, under continuous
or an intermittent regime, would be promising candidates for improving health during ageing.

5. mTOR, Human Ageing, and Cellular Senescence

The links of mTOR with pathologies such as metabolic syndrome and neurological problems
has been reviewed elsewhere [5]. More work is required to directly link mTOR function with human
organismal lifespan. Accumulating evidence from model organisms and human cell lines provides
great promise for mTOR pharmacological interventions in human ageing. With mTOR deregulation
and associated mutations being linked to different types of cancers [2,5] the large number of clinical
trials in various phases that include rapalogues (a class of allosteric mTOR inhibitors that are derivatives
of rapamycin) and other mTOR-related pharmacological interventions and inhibitors (clinicaltrials.gov)
is not surprising. The complexity and breadth of connections of mTOR signalling pathway with other
nutrient- and growth factor-responsive pathways imposes great toxicity risks when using high doses of
inhibitors [5,185] and optimal dosages and durations of treatment are unknown factors [186]. The most
common side effects are immunosuppression, hyperglycaemia and dyslipidaemia as well as interstitial
pneumonitis [187]. Importantly, a study on human elderly patients showed that low doses of mTOR
inhibitors could improve immune function [188].
Nevertheless, significant amount of work conducted in human cellular models has shown
that mTOR inhibition supresses the senescence associated secretory phenotype (SASP), which can
disrupt tissues and contribute to age-related pathologies, including cancer [187]. For example,
mTOR regulates SASP by promoting IL1A translation, which in turn promotes NFKB1 transcriptional
activity [187,189]. Additional experiments utilising engineered human fibroblasts that can be arrested
in G1 for determination of their chronological lifespan (CLS, define as the time that postmitotic cells
can remain viable) have shown the importance of mTOR in human cellular ageing [190]. Rapamycin in
this model was able to increase CLS while the effect was related to the acidification of the medium
similarly to yeast lifespan studies [190]. In the same model, exposure to hypoxia suppressed the mTOR
pathway, prevented senescent morphology and loss of regenerative potential, maintaining reversible
quiescence [191]. Finally, dual mTORC1/C2 inhibitors such as Torin1 and Torin2 were shown to suppress
hypertrophy, senescent morphology and extend CLS of human fibroblasts even better compared to
rapamycin, suggesting that at doses lower than anti-cancer concentrations, pan-mTOR inhibitors
could be developed as anti-ageing interventions [192]. Research in delaying pathological aspects of
human ageing and, therefore, increasing health span has proven hard and elusive. Data from diverse
experimental systems including human cellular models provide the optimism that mTOR inhibitors
could provide the desired solution [193]. The complexity of the mTOR signalling hub, its diverse
inputs and outputs [194], as well as interface with multiple other signalling pathways while complicate
the matter, present the opportunity of testing pharmacological combinations or repurpose already
Genes 2020, 11, 1043 12 of 21

approved drugs. The Dog Aging Project [195], bridging the gap between invertebrate, small mammals
and human subjects as well as increasing numbers of mTOR-related trials, mark an exciting era in
biogerontology pointing to the cross-roads towards regulating human lifespan and health span.

Author Contributions: I.B. and C.R. decided the structure and content, generated the figures and wrote the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: IB acknowledges funding from ERC StG 311331, ERC PoC 842174, CRUK-UCL Centre Award
[C416/A25145], Royal Society Research Grant [RSG\R1\180431] and the Bill Lyons foundation. CR acknowledges
funding from Royal Society Research Award [grant number RGS\R1\201348]. We thank Eleanor R. Stead for
critical reading of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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