Extending Healthy Lifespan by Systematically Targeting Ageing Pathways Synergies (PHD Thesis)

This covers some of the research on the most common longevity supplements and drugs. I’ve excerpted a section that I think likely is of most interest to people here - I’ve moved the “rapamycin” and “resveratrol” sections to the top for easier access:

Author: Diogo Goncalves Barardo

Advisors: Gruber, Jan

Associate Professor Jan Gruber, Main Supervisor
Associate Professor Thilo Hagen, Co-Supervisor

Examiners: Professor Brian Kennedy

Publisher: National University of Singapore (Singapore)

2.2 - Drugs Literature Review

In the following subsections, I will analyze the available literature for each drug that is part of my library plus all 5 drugs that were used in our previous work 70a The methodology for identifying the relevant literature for each compound was the following: I started by extracting the literature referred in DrugAge* 71*; I then checked the abstracts of literature that cite the previously selected DrugAge references; additionally, I included new literature that is yet to be included in DrugAge but that I was aware of.

2.2.15 - Rapamycin

Rapamycin is arguably the most famous anti-aging drug; therefore, this FDAapproved

immunosuppressant drug is vastly studied, with one of the major

aging pathways being named after it - mammalian target of rapamycin (mTOR).

A systematic review of rapamycin potential in biogerontology is outside the

scope of this dissertation, and this section provides only the upmost relevant

information. The curious reader is advised to read the cited body of work.

In fruit flies, rapamycin slightly extends mean lifespan in a gender-independent

manner, although at a significant fecundity cost148. Both these effects have been

reproduced and shown to be modulated specifically through the complex 1

branch of the TOR pathway (TORC1), including the downstream upregulation

of autophagy149. Furthermore, it is accompanied by increase stress resistance

and it is efficacious even on long/lived IIS mutant and DR flies.

The literature in C. elegans shows that the lifespan extension from rapamycin

treatment150–154 is dependent on SKN-1, but not on DAF-16155,156 or DAF-2157.

Additionally it induces mitochondrial unfolded protein response and increases

respiration158, and ameliorates the age-related decline of pharyngeal pumping

rate156. The fact that rapamycin treatment does not extend lifespan of eat-2

mutants and that it elicits a gene expression profile resembling that of a DR

state, might suggest that it could be considered a calorie restriction

mimetic156[6], although evidence in mice disputes this line of reasoning159[13].

Surprisingly, the majority of studies with rapamycin as an anti-aging

intervention have been done on Mus musculus (e.g.159,160).


On the ITP, whether fed at 270 or 600 days of age, rapamycin extends median

and maximal lifespan in both genders of genetically heterogenous mice161. In

old mice, rapamycin restores the self-renewal and hematopoiesis of

hematopoietic stem cells and boosts immune function162.

Considering that mTOR is an evolutionary conserved aging pathway, in mice

too, rapamycin administration reduces its activity, albeit not in all tissues163.

Nonetheless, it is important to mention that muscle mass was maintained163.

Interestingly, since rapamycin improves age-related phenotypes even in young

mice, its lifespan-extension might be dissociated from the aging process


Due to the side effects of inducing insulin resistance and immune suppression,

several intermittent schemes of rapamycin have been attempted. A 2 weeks per

month intermittent administration, is sufficient to increase lifespan, inhibit agerelated

weight gain, and delay spontaneous cancer incidence in inbred female

mice165. Moreover, a once every 5 days rapamycin treatment regimen was

shown to have no impact on blood glucose, while it still resulted on lifespan

extension in 20-months old female mice166. Although, such regiment was

elsewhere shown to not completely negate neither the immunosuppressive

effects of rapamycin, nor the associated decrease in testis weight167. Hopefully

complementary to the intermittent regimens, a 3-months transient rapamycin

treatment extends the median lifespan of middle-age mice by up to 60%168.


2.2.16 - Resveratrol

Resveratrol is a natural polyphenol found in grapes and red wine, and it is the

compound for which there are the most lifespan assays169.

Resveratrol activates sirtuin genes and extends lifespan in yeast170–175,

worm38,154,182,183,158,170,176–181, flies170,184, fish185–188 and bees189. When nutrients

are restricted, this effect is abrogated which suggests that it is related with

caloric restriction170,171.

In worms the lifespan extension is fully dependent on SIR-2.1176,179,190 (at least

sometimes177), AAK-2190 and autophagy178, but it is independent from DAF-

16176,190. In this model organism, resveratrol treatment also induces mitonuclear

protein imbalance and activates the mitochondrial unfolded protein response158.

In Nothobranchius furzeri, a short-lived fish, it causes a 56% and 59% increase

in median and maximum lifespan, respectively. Additionally, the treated group

exhibits ameliorated decay of locomotor and cognitive function. The initial

lower survival of the treated group for the first few weeks, led the authors to

propose that resveratrol might be an hormetic compound185. In another

vertebrate model, the fish Nothobranchius guentheri, the lifespan-extending

effects and increased cognitive and locomotor function caused by resveratrol

administration were conserved. Additionally, it was shown that there was less

accumulation of lipofuscin and senescence (as measured by beta-galactosidase

activity)186. In this species too, resveratrol decreases oxidative stress188.

An additional mode of action to the hypothesis that resveratrol is a CR mimetic

acting through activating sirtuins, namely because it was contested that


resveratrol can increase sirtuins in vivo in either worms or flies191a, is as an

antioxidant188. For example, in worms resveratrol increased mean and

maximum lifespan and also oxidative stress resistance183 (these effects have

been reproduced elsewhere179), but also there is work showing that resveratrol

does not have free-radical scavenging activity in vivo180 (albeit in this particular

work it also did not extended lifespan).

This antioxidant role it is not how resveratrol acts in bees, as the mean and

maximum lifespan extension are abolished in hyperoxic stress and the honey

bees ingest fewer quantities of food189.

After the previous brief description of the lifespan-extending effects of

resveratrol, it is also now crucial to underline the plethora of negative results.

In the ITP and in the only lifespan assay in a mammal, resveratrol did not

extended lifespan100. Also, in the model crustacean Daphnia, resveratrol has no

effect or even significantly decreases lifespan192. Furthermore, even though

resveratrol was tested in both genders of a tephritid fruit fly species and under

24 different diets it still did not elicit any lifespan extension193. In D.

melanogaster, using different strains, gender and diets, the pro-longevity effects

of resveratrol were very variable, and eventually consider dubious177. Wang et

al., reported that resveratrol has effect only on female D. melanogaster and

depending on the diet in question184. Staas et al. that dietary resveratrol had

absolutely no effect on male and female w1118 D. melanogaster in terms of

a In yeast that is clearly not the case as resveratrol does increase SIRT1 12-fold172, and

certainly there is evidence that allows a counter-argument to be made170,190.


lifespan, locomotor activity, body composition, stress response and longevityassociated

gene expression194.

In sum, I consider that resveratrol achieves its pro-longevity effects through

caloric restriction mimicry or antioxidant capacity, in a species-specific manner.

Moreover, I consider it perhaps the most unclear anti-aging compound: the

mode of action and efficacy are highly debated, but either way it is extremely

sensitive to experiment conditions. The incredibly unreliability of resveratrol

treatment can be predicted if its main mode of action is as an antioxidant or

mitohormetic compound, as both of these are known to has an inverted Ushaped

dose-response on longevity38.

2.2.4 - Captopril

Captopril is an FDA-approved treatment for high blood pressure. It is an

angiotensin-converting enzyme (ACE) inhibitor, which is of importance as the


ACE gene has been conserved from bacteria to mammals97 and it is one of the

few genes with genetic polymorphisms robustly associated with longevity in


It has only been explored once under the biogerontology paradigm and was

shown to extend the mean and maximum lifespan of C. elegans97. Moreover, its

pro-longevity effect was robust in 3 distinct culture temperatures and to live and

heat-killed bacteria feeding conditions; but with no effect on brood size,

reproductive span, and pharyngeal pumping rate. The same authors replicated

these effects using acn-1a RNAi-treated worms, with the exception that the

RNAi treatment was able to increase the pharyngeal pumping rate from days

12th to 20th of adulthood.

There is substantial evidence that the pro-longevity MoA of captopril is the

inhibition of the ACN-1 gene: combined treatment of acn-1 RNAi with

captopril, did not have and addictive effect on lifespan and both interventions

have the same results in distinct genetic backgrounds. Going into more detail

on the later point, reducing the activity of ACN-1: has additive pro-longevity

benefits with caloric restriction (eat-2), mitochondrial insufficiency (isp-1) and

with long-lived IIS pathway mutants (daf-2 and age-1); it is a lifespan-extension

pathway independent of TOR (rict-1), proteotoxic stress (hsf-1)b and of sir-2.1

activity; and it is toxic to daf-16 worms, reducing their lifespan.

a the C. elegans homologue of the ACE gene.

b although it is also revealed that acn-1 RNAi increases resistance to heat-stress.


Even though captopril has not been assayed for longevity in mammals, its prolongevity

MoA is tractable and evolutionary conserved in humans98 and fliesa,

robust to different conditions and additive to the main pro-longevity genetic

interventions (which indicates its potential to be combined with other drugs).

2.2.5 - Curcumin

Curcumin is a component of turmeric in Indian curry. The metabolite of

curcumin, tetrahydrocurcumin, extends the mean and maximum lifespan of

male C57BL/6 mice when treatment is initiated at 13-months of age, but is of

no effect when started at the 19th month of age99. However, in under the ITP

these benefits were not reproduced100. Despite failing in the gold standard

testing program, curcumin is still consideration from a biogerontology

perspective due to its very robust results in all the other model organisms tested,

e.g. in worms, curcumin does promote lifespan101b. The rest of supporting

literature was conducted in flies.

Curcumin robustly extends lifespan of male and female D. melanogaster from

two distinct wild-type strain, confers additional protection against oxidative

stress and improves climbing ability102. In yet another fly strain, a female and a

male group of flies exhibited increased mean lifespan and activity of the

superoxide dismutase103. Moreover, several works reproduced the gender and

strain-independent pro-longevity effect of curcumin in flies104–106. Other

beneficial effects include enhanced progeny viability and parental reproductive

a another FDA-approved ACE inhibitor, lisinopril, extends the lifespan of 3 D.

melanogaster strains through the same MoA307.

b I am disregarding results in which the control group had a mean lifespan of less than

9 days. Albeit there is still lifespan-extension in this work308.

fitness107. Larval feeding of curcumin in flies did not yield any additional benefit

on CR flies, which indicates potential overlapping modes of action between

these two pro-longevity interventions. Furthermore, age-specific lifespan assays

reveal that curcumin treatment effects are incredibly age-dependent, going from

benefic to harmful; accordingly to if the treatment is stated at the healthspan to

the senescent span, respectively104,106. An additional factor influencing the

magnitude of lifespan-extension caused by curcumin treatment is temperature.

Through regulation of heat shock proteins, the higher the environmental

temperature, in other words, heat stress, the larger the effect size relative to

untreated flies, independently of gender108.

2.2.6 - Epigallocathecin gallate

The first trial of the popular green tea polyphenol epigallocatechin gallate

(EGCG), resulted in no effects on the survival of worms, but improvement in

healthspan-associated traits including augmented resistance to oxidative stress,

attenuated decline of pharyngeal pumping rate and enhance chemotaxis index

in old age109. Three years after, the potent oxidative stress resistance induced by

EGCG was replicated, but this time it was accompanied the a reported mean

lifespan-extending effect of 10%110.

The unclear role of EGCG as a lifespan-extending compound in worms was

resolved by Liu Gui Xiong et al.111 which showed that this effect is robust to

different temperatures and genetic backgrounda, but highly sensitive to

a two distinct wild-type strains were used.

concentrationa. EGCG induces longevity by causing a transient increase in

reactive oxygen species, that stimulates the endogenous detoxification defense

system. More importantly for my approach, co-administration of n-acetyl-lcysteine

abolished EGCG-mediated lifespan and oxidative stress resistance

benefits and further supports that EGCG is a case of mitohormesis112. Due to

the transient induction of reactive oxygen species, and consequent defense

response, declining with age, EGCG treatment is progressively blunted with

age. This work also reiterated the requirement of the DAF-16 pathway for the

EGCG-induced longevity that was reported in a previous manuscript113.

The proposed mode of action of EGCG seems to be conserved in mammals, as

male weaning Wistar rats fed EGCG display increased median lifespan,

improved age-associated oxidative stress and superior activation of the FoxO3a

longevity factor114.

For the sake of completeness I must mention that there is an alternative mode

of action for EGCG based on results obtained in flies that proposes that

reduction in glucose metabolism is the key contributor for the superior fitness

and lifespan observed in EGCG-treated D. melanogaster115.

2.2.7 - Icariin

At the optimal dosage of 45μM when under 25ºC, icariin extends the mean

lifespan of worms by 25%116. Icariin is not the active biological form, and this

flavonoid is eventually hydrolyzed to icariside II. The direct administration of

a the reader might notice that this is common for compounds that act mainly by

antioxidant mechanisms. For the relevant literature please read the subsection about nacetyl-



icariside II resulted in increased tolerance to thermo and oxidative stress and

slowed the locomotion (as measured by swimming bends) decline in late

adulthood. In terms of the genetic pathways involved, both icariin and icariside

II fail to extend the mean lifespan of daf-16(mu86) and daf-2(e1370) mutants,

but they did prolong the lifespan of eat-2(ad1116) and rsks-1(ok1255) strains;

suggesting that icariin depends on the IIS pathway and it is not a caloric

restriction mimetic116. Furthermore, the expression of SOD-3 increased as

confirmed by PCR and fluorescent marking.

The results from mice are in agreement with the ones obtained in worms: there

is a mean lifespan extended by 8% but no maximal lifespan; and the SOD gene

expression is increased, moreover it is indistinguishable from youth levels117.

Treated mice were lighter than the control individuals, even though they

consumed more calories. Healthspan improved as measured by the Morris water

maze, rotarod and bone mineral density (worth notice that bone mineral density

is conserved at youth levels). The icariin-treated mice also display less DNA

damage as indicated by the decreased expression of gamma-H2AX.

2.2.8 - Lipoic Acid

Lipoic acid was first shown to extend the lifespan of female and male D.

melanogaster, by 12% and 4%, respectively118. Very recently, this effect on

female flies was replicated even when started only at day 26 (mid-aged)119. The

pro-longevity effect of lipoic acid are associated with the prevention of ageassociated

functional decline and hyperproliferation of intestinal stem cells,

through the activation of the endocytosis-autophagy network119.


The literature in C. elegans reveals that lipoic acid administration results in an

increase in the mean and maximum lifespan, with no effect on pharyngeal

pumping rate, but with the enhancement of the chemotaxis indexa in aged

worms. The lifespan-extending effect of lipoic acid on this model organism has

been confirmed only in regard to mean lifespan, but not maximum120.

Male rats fed an ad libitum diet supplemented with lipoic acid display similar

survival profiles as the control animals121.

2.2.9 - Lithium

The first initial report of lithium as a lifespan-extending drug showed that this

effect was independent of DR, FOXO/IIS or germline signaling pathways122.

Additionally, it was observed a trade-off between longevity and fertility, as

lithium treated animals produced less eggs, and most eggs that were laid

subsequently failed to hatch. Due to the lithium status as an FDA-approved drug

for the treatment of psychiatric disorders123 as a classic glycogen synthase

kinase-3 (GSK-3) inhibitor, the authors conducted lifespan assays of lithium

under different genetic interventions in the worm orthologue of this gene.

Inhibition of GSK-3β is not sufficient for lifespan extension (mutants live

shorter), and lifespan extension by lithium actually requires GSK-3β (because

it further decreases the short lifespan of gsk3 mutants).

The beneficial effects of lithium on the mean lifespan of worms were

reproduced, and there were no changes seen in maximum lifespan. Although,

a it measures the fraction of worms able to display goal-oriented motor responses from

the transformation of specific sensory stimuli.


lithium treatment did not significantly slow the rate of aging as measured by

mortality rate doubling time. The slower decline in locomotor phenotype at all

ages, together with the previous mention studies, shows that lithium treatment

in worms as a mixed effect on healthspan assays. Moreover, the authors create

a dynamical model and supporting it by several lines of evidence conclude that

lithium elicits a greater increase in autophagy than in mitochondrial biogenesis,

and that this dictates the cellular respiratory capacity by influencing the ratio of

functional and dysfunctional mitochondria. In sum, the increase in the ATP

levels by lithium is due to a higher ratio of functional to dysfunctional


The lifespan-extending of lithium in C. elegans were reproduced in yet two

other studies and with a similar dose-response curve125,126, giving rise to the

view that lithium is a robust pro-longevity treatment. Lithium therapy as a

biphasic dose-dependent effect on lifespan, seen at similar concentrations in in

vitro127, worms and flies128. After a minimum threshold to elicit a therapeutic

effect, lithium treatment eventually becomes toxic at higher concentrations122.

On top of the biphasic lifespan extension effects being reproduced in flies, in

this model organism these are independent from gender and genetic

background128. Lithium is arguably even more benefic than in worms because

it elicits a significant improvement and protection against age-related locomotor

decline, but this time without compromising feeding behavior or fecundity.

With relevancy for the translational potential of lithium as an anti-aging therapy,

this work validated that lithium extends lifespan even when administration starts

only in mid-life or just with a short-term treatment in the case of young flies.

Like in worms, lithium extends the lifespan of DR flies, but it seems that it does


so by inhibiting the fly orthologue of GSK-3. Also, in opposition to what was

suggested based on worm experiments, lithium does not induce or require

autophagy to promote longevity in flies. These authors propose a model in

which lithium inhibits GSK-3, which indirectly leads to the activation of the

transcription factor nuclear factor erythroid 2-related factor (NRF-2)a.

Very exciting form a translational point-of-view, are two studies showing

correlation evidence that lithium concentration in drinking water is associated

with reduced all-cause mortality, in distinct human populations, and with a

similar dosage therapeutic window to the aforementioned pre-clinical


As discussed in the known drug synergies section (section X), lithium as a prolongevity

treatment can be paired with rapamycin or trametinib for additional

benefits, which can be even more powerful by combining all these drugs as a

triple pharmaceutical treatment37.

2.2.10 - N-acetyl-L-cysteine

N-acetyl-L-cysteine (NAC) is a natural source of cysteine which in turn is used

in the synthesis of glutathione, and as such, it is a potent antioxidant in vivo. It

was initially discovered to increase the mean and maximum lifespan of flies by

26.6%130. Albeit this effect seems to be only conserved in the male gender of

genetically heterogenous mice, and it is marked by an accentuated weight loss,

which might hint at the induction of a partially DR-like state131.

a since GSK-3 is an NRF-2 inhibitor.


In C. elegans, it was first shown that liposomal delivery of NAC improves

survival, in opposition with the lack of effect seen from its direct

administration132. In contrast, it was later shown that direct NAC administration

extended the mean and maximum lifespan, by up to 30.5% and 8 days,

respectively. Furthermore, it was found that NAC increases the number of

progeny, and resistance to radiation and heat shock stress133. These discrepant

evidence can be conciliated by the recent results showing that NAC has a highly

sensitive dose-response curve. NAC is a canonical antioxidant and as such its

efficacy is slave to the inverted U-shaped dose-response relation between

reactive oxygen species levels and lifespan38.

2.2.11 - Metformin

Metformin is a biguanide drug commonly used to treat type-2 diabetes.

In worms it extends median lifespan134–137 and delays the age-related decay of

mobility134,138[2,11]. The lifespan extension seems to be independent of the IIS

pathway (DAF-16, DAF-2, and AGE-1). The lack of effect of metformin in eat-

2(ad1116) mutants coupled with the display of several phenotypes associated

with DR (lower lipofuscin accumulation138, slimmer bodies, extended period of

egg-laying) and the genetic requirement of SKN-1 and AAK-2 strongly

suggests that metformin is a CR mimetic. Important from a translational

perspective, metformin did not impaired feeding134. An alternative model for

the mode of action of metformin is mitohormesis. Metformin increases reactive

oxygen species production, metabolic heat production and respiration, and,

crucial to our paradigm of combinatorial drug interventions, the antioxidant

NAC abolishes its benefits135a. These are the two main ones, but not the only

models seeking to elucidate the mode of action of metformin137,138.

In an attempt to confirm that metformin promotes longevity in an evolutionary

conserved manner in flies, indeed it was observed a robust activation of AMPK

and reduced body fat, but no lifespan extension in either gender. Moreover,

metformin was toxic in some of the concentrations tested139.

Metformin has also been tested in some less common animal models. In crickets

metformin extends survivorship and maximal longevity86. Metformin

significantly reduced growth rates and delayed maturation in crickets of both

genders. The same authors also tried the aspirin plus metformin drug pair, but

the lifespan-extension was less than in any of the single drugs. Furthermore,

metformin prolongs the lifespan of in short-lived fish and delays several

markers of aging, including lipofuscin, inflammagingb, cell senescence and

cognitive decline140.

Results in middle-aged male mice of two different strains are more

encouraging141. Metformin improves general fitness and lifespan at a low

dosagec, but it is significantly nephrotoxic (renal failure) at a higher one.

Animals in the low-dose long-term metformin treatment are initially slimmer

(even though they consumed more calories than the control animals, like in

worms), but tended to preserve their bodyweight with advancing age.

Intermittent treatment regimens of every-other week or two consecutive weeks

a the requirement of AAk-2 would equally be justified under this alternative model.

b the chronic low-grade inflammation that its characteristic of advanced age.

c this dosage still corresponds to serum levels an order of magnitude higher than those

used in the treatment diabetic human patients.

43 per month were tested, with the aim of bypassing the toxicity associated with

high levels (relative to the human therapeutic dosages) of long-term metformin

treatment. Unfortunately, neither extend mean or maximum lifespan in mice142.

In rats, metformin supplementation replicates the reduced food intake and body

weight seen in the caloric restricted group but did not extended lifespan at any


2.2.12 - Myricetin

The two works with lifespan assays using the naturally occurring flavonol

myricetin were conducted in worms. Their findings agree: on its pro-longevity

effects (18%144 and 33%145 mean lifespan increase, 22% maximum lifespan

increase144), on its anti-oxidant capacity and that it elicits the nuclear

translocation of the daf-16 protein. They do diverge, however, in the effects of

myricetin on the daf-16(mu86) genetic background. On one there is a prolongevity

effect on this background144, while on the other this is completely

abolished145. It is suggested that the differences might be consequence of the

markedly different culturing conditions used, solid medium at 20ºC versus

liquid medium at 25ºC, respectively.

The great anti-oxidant potential of myricetin might explain its reduction on the

accumulation of lipofuscin145, a biomarker of aging, if one takes into

consideration that lipofuscin are highly oxidized crossed-linked proteins.

Additionally, myricetin does not increase tolerance to heat-stress, pharyngeal

pumping rate or body size, which are all evidence against a possible role as a

caloric restriction mimetic145.


2.2.17 - Rifampicin

The FDA-approved antibiotic rifampicin (RIF) or rifampin extends the lifespan

of C.elegans by almost 45%, even in heat-killed bacterial lawns, which clearly

indicates that its pro-longevity effects are not due to its bactericidal

properties195. The very same work showed that rifampicin acts as a potent

glycation inhibitor in vivo, reducing the age-relate accumulation of advanced

glycation end products. It is worth mentioning that rifampicin treatment

extended lifespan even when initiated at only day 9 of adulthood.

Besides its anti-glycation effects, rifampicin pro-longevity effects depend on

the DAF-16 gene, as it was shown to activate daf-16 protein translocation into

the nucleus, had no influence on the longevity of the daf-16(mgdf50) null

mutant strain, and led to an increase in the expression of SOD-3 (which is a

direct target of DAF-16). Although the targets downstream of DAF-16


modulated by rifampicin seem to be a separate subset from those regulated by

the IIS pathway195.

2.2.18 - Spermidine

Spermidine is a polyamine naturally present in humans, and its intracellular

levels are known to decline during the human aging process. Therapeutic

quantities are hard to be obtain through diet alone, but its supplementation is

regarded as safe196.

Research of spermidine in murine models is compelling. Four-months old

C57BL/6J wild-type female mice subjected to a lifelong supplementation of

spermidine in their drinking water, have their median lifespan extended. Even

more relevant from a translational medicine perspective, there is still a 10%

increase of the median lifespan of a group of male and female mice when

spermidine supplementation is started later in life, on the 18th month197.

Spermidine caused no changes in food and water consumption, and on body

weight and composition, so the possibility of CR-like state being in play can be

rejected. Instead, spermidine delays the aging process (namely cardiac aging)

by eliciting autophagy. The same authors also report an inverse correlation

between dietary spermidine intake and human cardiovascular disease197. I note

that the corresponding spermidine human dosage to the one used in this mice

study would be too high to be achieved easily by diet198, which suggests that

extra spermidine supplementation might have unexplored cardioprotective

potential in humans. The pro-longevity effects of life-long intake of spermidine,

at the same dosage, were replicated on a different wild-type mouse strain with


a median lifespan extension of up to 25%, and the autophagy-based mode of

action was further substantiated199. In middle-aged Sprague-Dawley male rats

spermidine supplementation fails to extend mean or maximum lifespan200, but

the dosage given was only 72% of the converted mice dosage198. Nonetheless,

it still extended healthspan (if only very slightly), as measured by improved

kidney tubules, liver, and heart morphology; increased exploratory behavior;

and diminished expression of neuroinflammatory markers (once again the

autophagy process was enhanced)200.

Regarding other model organisms, spermidine retards chronological aging and

rejuvenates replicative old yeast cells, and enhances the lifespan in D.

melanogaster, C. elegans and human peripheral blood mononuclear cells. In

yeast, mice and human cells, this spermidine-induced longevity is correlated

with hypoacetylation of histone H3. Furthermore, and in completely agreement

with the rest of the literature, it is shown that spermidine induces autophagy,

and that this process is required for its lifespan-extension effect in yeast, flies

and worms201.

2.2.19 - Thioflavin-T

The intuition that compounds traditionally used in histopathology to stain

amyloid in tissues might be candidate drugs for delaying the aging process

comes from evidence that such compounds do bind and slow the aggregation of

such protein aggregates in vitro202, and that loss of proteostasis is one of the

main hallmarks of aging203. Thioflavin T is an amyloid-binding dye that was

first explored under this rationale in C. elegans, with an impressive 70% median


lifespan extension and 43%-78% maximum lifespan extension204a. These are

perhaps the largest drug-induced pro-longevity effects ever reported for an

adult-onset monotherapy at the standard culturing conditions of 20ºC in solid

medium15. The efficacy of thioflavin T requires HSF-1 and SKN-1, but it is

partly independent of caloric restriction, as it prolongs lifespan (albeit not to the

same degree) in nutrient-based models of CR and eat-2(ad1116) mutants.

With this effect size on lifespan, it is not very surprising that thioflavin T was

the most robust compound assayed in the CITP, showing significant media

lifespan-extension in 5 of the 6 strains of Caenorhabditis tested83. Not only that,

but it was also the most potent convincingly reproducing the initial result. The

robustness and potency of thioflavin T suggest that its mode of action is wellconserved

and of major importance among aging processes, respectively.

2.2.20 - Ursolic Acid

Ursolic acid is a lipophilic pentacyclic triterpenoid of botanical origins that was

first shown to increase the mean and maximum lifespan of treated worms by up

to 30%, although these effects were relative to a control group that had a mean

lifespan of less than 15 days205. Later, the same group, with equally relatively

short-lived controls, replicated their initial results and complement them with

healthspan assays206. These latter also showcase the anti-aging potential of

ursolic acid including in delaying the accumulation of lipofuscin, protecting

against heat-shock, achieving an healthier chemotaxis index, and improving

motility. Regarding the mode of action, since there is similar lifespan extension

a it also decreased age-specific mortality across all ages.


on the daf-16(mgDf50) and daf-2(e1370) genetic backgrounds, the mode of

action is thought to be independent of the IIS pathway. Instead, the failure to be

of benefit to a JNK-1 mutant strain and its predicted binding affinity with the

jnk-1 protein supports that it is through the modulation of JNK-1 that ursolic

acid exerts its pro-longevity effect205. Another additional mode of action is the

initiation of a DR-like, supported by the observation that treated worms are

slimmer and that there are no effects on the lifespan of eat-2(ad1116) animals206.

In w1118 Drosophila melanogaster flies, ursolic acid is of benefit to males only.

In this gender it increases the mean and maximum lifespan and healthspan (as

measured by climbing ability and immune function), without any fertility tradeoff.

However, it does not increase oxidative stress resistance or affect gut health.

Interestingly, total body weight remains the same even though ursolic acid-fed

male flies ate significantly more food than controls. This makes sense in light

that removal of the microbiome negates the anti-aging effects of ursolic acid.

Which to me suggests that the treated flies could be benefiting from a

microbiome-derived metabolite that is produced from the extra calories and

activated by ursolic acid.

Full PHD Thesis:


BarardoDGExtendingHealthyLifespanThesis.pdf (2.5 MB)


Seems to be the same info found in the publication below, although the focus is on Sirt6.