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Mitochondrial clearance and increased HSF-1 activity are coupled to promote

longevity in fasted

Caenorhabditis elegans

Nikolaos Tataridas-Pallas, Yahyah Aman, Rhianna Williams, Hannah Chapman,

Kevin J.H. Cheng, Casandra Gomez-Paredes, Gillian P. Bates, John Labbadia

PII:

S2589-0042(24)01056-3

DOI:

https://doi.org/10.1016/j.isci.2024.109834

Reference:

ISCI 109834

To appear in:

ISCIENCE

Received Date: 20 July 2023
Revised Date: 27 March 2024
Accepted Date: 24 April 2024

Please cite this article as: Tataridas-Pallas, N., Aman, Y., Williams, R., Chapman, H., Cheng, K.J.H.,

Gomez-Paredes, C., Bates, G.P, Labbadia, J., Mitochondrial clearance and increased HSF-1 activity

are coupled to promote longevity in fasted

Caenorhabditis elegans

ISCIENCE

(2024), doi:

https://

doi.org/10.1016/j.isci.2024.109834

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Mitochondrial clearance and increased HSF-1 activity are coupled to promote

longevity in fasted Caenorhabditis elegans

Nikolaos Tataridas-Pallas

, Yahyah Aman

, Rhianna Williams

, Hannah Chapman

Kevin J. H. Cheng

, Casandra Gomez-Paredes

, Gillian P Bates

, and John

Labbadia

1,3

Institute of Healthy Ageing, Department of Genetics, Evolution and Environment,

Division of Biosciences, University College London, London, WC1E 6BT, UK

Huntington

’s Disease Centre, Department of Neurodegenerative Disease and UK

Dementia Research Institute at UCL, Queen Square Institute of Neurology, University

College London, London, WC1N 3BG, UK

Lead contact

* correspondence, j.labbadia@ucl.ac.uk

Summary

Fasting has emerged as a potent means of preserving tissue function with age in

multiple model organisms. However, our understanding of the relationship between

food removal and long-term health is incomplete. Here, we demonstrate that in the

nematode worm

Caenorhabditis elegans,

a single period of early-life fasting is

sufficient to selectively enhance HSF-1 activity, maintain proteostasis capacity and

promote longevity without compromising fecundity

These effects persist even when

food is returned, and are dependent on the mitochondrial sirtuin, SIR-2.2 and the

H3K27me3 demethylase, JMJD-3.1. We find that increased HSF-1 activity upon

fasting is associated with elevated SIR-2.2 levels, decreased mitochondrial copy

number and reduced H3K27me3 levels at the promoters of HSF-1 target genes.

Furthermore, consistent with our findings in worms, HSF-1 activity is also enhanced in

muscle tissue from fasted mice, suggesting that the potentiation of HSF-1 is a

conserved response to food withdrawal.

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Introduction

Maintaining a fully functional proteome is crucial for long-term cell and tissue health.

This is routinely achieved by the Proteostasis Network (PN), a collection of molecular

machines that promote accurate protein synthesis, folding, trafficking and

degradation

. In parallel, the PN also rapidly recognises and neutralises any

misfolded, mislocalised or aggregated proteins that arise from biosynthetic errors or

environmental stress

As organisms age, the capacity of the PN declines, leaving tissues vulnerable

to proteostasis collapse (i.e. the accumulation of misfolded, mislocalised and

aggregated proteins)

. This process lies at the heart of many age-associated sporadic

and inherited diseases (e.g. Alzheimer’s Parkinson’s Huntington’s), raising the

possibility that suppressing age-related proteostasis collapse could be a potent way

to simultaneously suppress multiple age-associated diseases

Studies in the nematode worm

Caenorhabditis elegans

, have revealed that the

timing and magnitude of age-related proteostasis collapse are linked to the functional

status of the germline and mitochondria early in life

4-7

. In response to impaired electron

transport chain (ETC) activity or reduced levels of the mitochondrial carrier

homologue, MTCH-1/MTCH2, cells can activate mitochondria-to-cytosolic stress

responses that converge on the transcription factor HSF-1 to up-regulate specific PN

components and protect cells against proteostasis collapse later in life

8,9

. In addition,

increased HSF-1 activity has been shown to extend lifespan through mitochondria

associated mechanisms

10,11

, suggesting a close relationship between mitochondrial

status, HSF-1 activity and proteome integrity. However, it remains unclear whether

mitochondria-to-HSF-1 communication plays a role in promoting longevity in response

to lifestyle changes that alter mitochondrial function.

Among the lifestyle interventions linked to increased lifespan, enhanced

longevity as a result of altered nutrient availability is strongly linked to changes in

mitochondrial status. In addition, activation of mitophagy (the primary mechanism

through which defective mitochondria are removed from cells) has been shown to

promote longevity and suppress age-associated disease

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Nutrient restriction can take many forms, with the chronic removal or reduction

of food intake through dietary restriction (DR) being one of the oldest lifestyle changes

associated with increased longevity across model organisms

. More recently,

intermittent fasting (IF) has emerged as a more sustainable way to extend lifespan

and prolong healthy tissue function

. IF can take many different forms, including

alternate day fasting (ADF

– the removal and restoration of food on alternating days),

the 5:2 diet (eating normally for 5 days and then limiting food intake to 500-600 calories

for 2 days a week) and time restricted fasting (TRF

– food intake is limited to short

windows on each day)

. These lifestyle interventions are sufficient to provide long-

term benefits, including extended lifespan and increased stress resistance in worms

and flies

15,16

. Furthermore, exposing worms or flies to fasting regimens exclusively

during early life is sufficient to elicit lifespan extension

15,16

Both DR and fasting are associated with mitochondrial and metabolic

remodelling

17,18

, and HSF-1 is required for DR and TRF to increase lifespan in

worms

15,19

. This raises the possibility that the beneficial effects of fasting may be

mediated by an interplay between mitochondria and HSF-1. However, it remains

unknown whether mitochondria communicate with HSF-1 in response to fasting, how

this is achieved, how long food removal is required for to elicit these effects and

whether this can protect against age-related protein aggregation.

Here we address these questions and demonstrate that a single period of

fasting early in life potentiates HSF-1 activity, suppresses age-related protein

aggregation, enhances stress resistance and extends lifespan in

C. elegans

. These

effects are dependent on mitochondrial sirtuins and the H3K27me3 demethylase,

JMJD-3.1, and are associated with a rapid reduction in mitochondrial copy number

and a decrease in H3K27me3 levels at HSF-1 target genes.

Results

Early-life fasting increases proteostasis capacity in aged tissues

TRF and 5:2 fasting have been shown to extend lifespan, prolong healthy tissue

function and increase stress resistance in both worms and flies

15,16

. In

C. elegans

, the

removal of food at the transition to adulthood results in a rapid NHR-49-dependent

starvation response

. Given that early life fasting is sufficient to elicit long-term health

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benefits, we tested whether transiently removing worms from food for 24 hours as they

transition to adulthood (Figure S1A), would be sufficient to extend lifespan and

maintain proteostasis capacity with age. We found that worms that had been fasted

for 24 hours at the L4/D1 adult stage and then returned to food, exhibited a 25%

increase in median and maximal lifespan (Figure 1A). In addition, fasted animals also

exhibited increased resistance to heat and endoplasmic reticulum (ER) stress

(Tunicamycin treatment), but less so to oxidative stress (paraquat treatment), when

returned to food for 24 hours and allowed to commence reproduction (Figure 1B, S1B

and S1C). These effects were not associated with reduced brood size, although the

onset of egg-laying was delayed in fasted animals (Figure S1D and Figure 1C).

However, a 24-hour period of fasting was required before the onset of reproductive

maturity for maximal enhancement of stress resistance (Figure 1D). These data

demonstrate that a single, 24 hour fast, prior to the onset of reproduction, is sufficient

to increase lifespan and stress resistance in

C. elegans

, even when animals are

returned to food.

Increased stress resistance in reproductively mature worms has previously

been linked to the maintenance of proteostasis capacity in adulthood and the

suppression of age-related protein aggregation

4,5

. To ascertain whether a single

period of early life fasting maintains proteostasis capacity with age, we examined the

effect of transient food removal on polyglutamine (polyQ) aggregation and toxicity in

body wall muscle and intestinal cells. Consistent with increased lifespan and stress

resistance, we found that fasting suppressed age-related polyQ aggregation in both

tissues (Figure 1E and F) and delayed the onset of muscle paralysis caused by polyQ

proteins (Figure 1G). In addition, fasting also improved muscle function in control and

Abeta1-42 expressing worms (Figure 1H).

Moreover, fasting further increased stress resistance in germline stem cell

(GSC) deficient, chronically dietary restricted (DR) and electron transport chain (ETC)

compromised mutants (Figure S1E

– G), suggesting that fasting may enhance

proteostasis capacity through different mechanisms than these interventions. Our data

demonstrate that a single, transient period of fasting early in life is sufficient to

suppress age-related proteostasis collapse in different tissues and promote longevity

without compromising reproduction.

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Fasting enhances stress resistance and increases lifespan by potentiating

HSF-1 activity and selectively remodelling the proteostasis network

Proteostasis capacity is controlled through the activity of well-established

transcription factors that balance the expression of proteostasis network components

(e.g. molecular chaperones and degradation factors) with the protein folding

requirements of the cell

1,2

. To determine which, if any, of these PN regulators are

required for fasting to enhance proteostasis capacity, we examined resistance to heat

stress in worms defective in the HSR (

hsf-1(sy441), daf-16(mu86), pqm-1(ok485)

UPR

(

xbp-1(zc12), atf-6(tm1153), pek-1(ok275)

and

ire-1(ok799)

) and UPR

(

atfs-1(tm4525)

As expected, wild-type worms exposed to fasting exhibited a 2-fold increase in

median survival following heat stress, as compared to fed controls (Figure 2A).

Although we cannot rule out the presence of adaptive changes to parallel PN pathways

in our strains, mutations in

daf-16, pqn-1, xbp-1, atf-6,

pek-1

ire-1

did not block the

ability of fasting to enhance stress resistance (Figure 2A and Figure S2B-G), while

survival was markedly increased in

atfs-1(tm4525)

mutants (Figure 2A and Figure

S2H). In contrast, elevated stress resistance was almost completely abolished in

fasted

hsf-1(sy441)

loss-of-function mutants (Figure 2A and Figure S2A). Consistent

with this, loss of HSF-1 activity also prevented fasting from increasing lifespan (Figure

2B).

To determine whether HSF-1 activity is increased in transiently fasted animals,

we measured the basal and heat-induced expression of canonical HSF-1 target genes

(

hsp-70, F44E5.4, hsp-16.2

) in worms that had been fasted for 24 hours at the L4

stage and allowed to recover for 24 hours in the presence of food. The basal

expression of HSF-1 target genes, or canonical UPR

or UPR

genes, was not

increased under basal conditions (Figure 2C and E). However, the induction of

canonical genes from all three pathways was increased 2 to 3-fold in transiently fasted

worms immediately following heat shock (Figure 2D and F). These data suggest that

transient fasting simultaneously potentiates proteostastic stress responses associated

with the cytosol/nucleus, ER and mitochondria.

Using RNA-sequencing, we asked whether fasting broadly or selectively

enhanced HSF-1 activity. Worms were fasted for 24 hours at the L4 stage and then

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collected immediately following exposure to control (basal) conditions (20



C) or heat

shock (35



C) for 30 minutes. Of the 107 genes known to be directly regulated by

HSF-1 during development or in response to heat shock

, only a small sub-set (9

significantly up-regulated and 9 significantly down-regulated) were found to be

significantly changed in fasted animals compared to fed animals, under basal

conditions (

< 0.05) (Figure 2G). In contrast, the expression of a far larger proportion

of HSF-1 targets (31 significantly up-regulated and 2 significantly down-regulated) was

significantly altered in fasted animals compared to constantly fed controls, following

heat shock (

< 0.05) (Figure 2G). Of these genes, only 5 were concordantly up-

(

F19B2.5, cdd-1, Y94H6A.10, Y22D7AL.10

) or down-regulated (

egg-5

) in fasted

animals compared to fed controls under both basal and heat shock conditions (Figure

2G).

Of the HSF-1 target genes that exhibited increased expression following heat

shock in fasted animals compared to fed controls, many encoded for core components

of key proteostasis machines, including the HSP70/DNAJ machinery (

hsp-70, dnj-12,

bag-1

), the disaggregase machinery (

hsp-1, hsp-110, dnj-12

), non ATP-dependent

chaperones (

hsp-16.1, hsp-16.11, hsp-16.2, hsp-16.41, hsp-16.48, hsp-16.49

), and

the HSC70/HSP90 complex (

hsp-1, hsp-90, hip-1, sgt-1

) (Figure 2H). We did not see

substantial changes in the expression of proteasomal subunits or components of the

autophagy machinery. Moreover, fluorescence-based reporters of proteasome

activity

or autophagic flux

revealed that neither of these pathways were enhanced

in worms that had been transiently fasted, compared to constantly fed controls (Figure

S2I-O). In fact, degradation of a UbV::GFP proteasomal substrate was reduced in

fasted animals under both basal and heat shock conditions (Figure S2M-O).

Together, our data show that

hsf-1

is required for fasting to promote longevity

and enhance proteostasis capacity, and that these beneficial effects are associated

with the increased expression of a sub-set of HSF-1 target genes, either basally, or in

response to stress. While central regulators of the UPR

and UPR

were not required

for increased resistance to heat stress (Figure 2A), it remains possible that the

potentiation of these pathways, and perhaps other PN factors/pathways, also

contributes to resistance to other stresses and/or increased longevity.

Fasting induced stress resistance is dependent on mitochondrial sirtuins and

mitophagy

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Fasting is associated with increased fatty acid beta oxidation, altered

mitochondrial morphology and elevated NAD+ levels in worms and mice

17,23,24

Consistent with previous reports, we observed increased

acs-2

p::gfp expression (a

hallmark of increased beta-oxidation), increased NAD+ levels and a more fragmented

mitochondrial network in worms that had been fasted for 24 hours and then allowed to

recover for 24 hours in the presence of food (Figure 3A and B, Figure S3 A and B). In

addition, RNA-seq data from fasted animals exhibited a strong enrichment for

metabolic pathways among differentially expressed genes (1,667 up-regulated, 999

down-regulated,

< 0.05) (Figure S3C and D).

Metabolic and mitochondrial remodelling in response to nutrient deprivation are

dependent on the transcription factor NHR-49, and the lysine deacetylase SIR-2.1

(whose activity is increased in response to elevated NAD+ levels)

17,23

. Given that both

NHR-49 and SIR-2.1 have been linked to HSF-1 activity

11,25,26

, we hypothesised that

one or both of these factors may be necessary for fasting induced stress resistance

and longevity.

To address this, we measured stress resistance in fed and transiently fasted

(24 hours fasting followed by 24 hours recovery on food) loss-of-function

nhr-

49(nr2041)

and

sir-2.1(ok434)

mutants

Surprisingly,

nhr-49

and

sir-2.1

mutants

exhibited comparable stress resistance to wild-type worms under both fed and fasted

conditions (Figure 3C and D). Given that we observed increased NAD+ levels and

altered mitochondrial homeostasis in fasted animals, we also tested whether the

mitochondrial sirtuins, SIR-2.2 and SIR-2.3

, are required for fasting to increase

stress resistance. We observed that stress resistance was mildly increased in

sir-

2.2(tm2648)

and

sir-2.3(ok444)

mutants under fed conditions, although not to the

same extent as is observed in fasted wild-type animals (Figure 3E and F). Crucially,

fasting failed to increase stress resistance or lifespan in

sir-2.2

sir-2.3

mutants

(Figure 3E-H), although it should be noted that loss of

sir-2.3

increased lifespan under

fed conditions to a level comparable with that seen following fasting (Figure 3H).

Together, our data suggest that SIR-2.2 and SIR-2.3 are required for fasting to fully

enhance stress resistance and increase longevity.

SIR-2.2 couples reduced mitochondrial copy number with increased HSF-1

activity to promote longevity

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To understand the relationship between fasting, mitochondrial sirtuins, HSF-1

activity and maintenance of proteostasis, we first asked whether

sir-2.2

was required

for potentiation of HSF-1 activity. As expected,

hsp-70, F44E5.4

and

hsp-16.2

mRNA

were increased 3- to 4-fold in transiently fasted animals (24 hours fasting followed by

24 hours recovery on food) immediately following heat shock (Figure 4A-C). In

contrast, this response was severely blunted in

sir-2.2

mutants (Figure 4A-C),

suggesting that

sir-2.2

is required for fasting to increase HSF-1 activity in response to

stress.

To determine whether SIR-2.2 or SIR-2.3 are elevated in response to fasting,

we measured

sir

-2.2 and

sir-2.3

mRNA levels in fed and fasted animals. Both

sir-2.2

and

sir-2.3

mRNA were increased 1.5-fold and 2-fold respectively, following 24 hours

of fasting (no recovery on food) (Figure 4D and E). In addition, we observed increased

fluorescence in fasted worms expressing SIR-2.2::GFP

(Figure 4F), indicating that

SIR-2.2 levels, and presumably activity, are increased by fasting.

Given that SIR-2.2 is a mitochondrial protein

, we hypothesised that increased

SIR-2.2::GFP levels may be reflective of increased mitochondrial biogenesis. To test

this, we measured SIR-2.2::GFP levels and mitochondrial copy number every 4 hours

following removal of food. SIR-2.2::GFP levels increased approximately 1.5-fold after

8 hours of fasting and persisted at this level thereafter (Figure 4G). In contrast with our

hypothesis, this was coincident with a 30-40% decrease in mitochondrial copy number

within the same time window (Figure 4H). These effects were also observed in

germline-less

glp-1(e2144ts)

mutants (Figure S4A and B), indicating that

mitochondrial copy number is rapidly reduced within the soma of fasted worms.

Furthermore, this reduction in mitochondrial copy number was suppressed in fasted

sir-2.2

mutants compared to wild-type animals (Figure 4I), suggesting that elevated

levels of SIR-2.2 may promote mitochondrial turnover in response to fasting.

Given that imbalances in copy number between the mitochondrial and nuclear

genomes can result in mitochondrial stress responses, and that

sir-2.2

is necessary

for the potentiation of HSF-1, we reasoned that mitochondrial clearance may be

coupled with HSF-1 activity to promote proteostasis capacity. Mitophagy is the primary

mechanism by which mitochondria are removed from cells if UPS-based repair is not

sufficient to correct mitochondrial defects

12,29

; to test whether mitochondrial clearance

was required for potentiation of HSF-1 activity, we measured stress resistance in

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worms with impaired lysosomal biogenesis (

hlh-30(tm1978)

) or defective PINK1 and

parkin (

pink-1(tm1779)

and

pdr-1(gk448)

), all of which are required for efficient

mitophagy

30,31

Consistent with a coupling of mitochondrial clearance and increased HSF-1

activity, we found that stress resistance was reduced (but not abolished) in

hlh-30,

pink-1

, and

pdr-1

mutant worms following transient fasting (Figure 4J and Figure S4C

and D), suggesting that mitophagy is required for transient food withdrawal to enhance

organismal robustness. However, the fact that mitophagy defective mutants remained

more stress resistant than fed counterparts suggests that pathways beyond mitophagy

may also contribute to stress resistance following temporary food withdrawal.

Together, our data support a model in which elevated levels of SIR-2.2

potentiate HSF-1 activity through mitochondrial clearance in fasted animals, thereby

enhancing proteostasis capacity and promoting longevity. However, it remains

possible that SIR-2.2 may also control mitochondrial copy number by influencing

mechanisms outside of mitophagy, such as mitochondrial biogenesis.

JMJD-3.1 promotes HSF-1 activity in response to fasting

How then, do elevated SIR-2.2 levels and reduced mitochondrial copy number

influence HSF-1 activity in the nucleus? Given that changes in mitochondrial import

drive mitophagy

, we reasoned that SIR-2.2 may translocate to the nucleus in

response to fasting. However, we did not observe any evidence for the nuclear

localisation of SIR-2.2::GFP in fasted animals (Figure S5A).

We next considered how SIR-2.2 could remotely influence HSF-1 activity.

Mitochondrial stress responses and homeostasis are dependent on the H3K27me3

demethylase JMJD-3.1 and the lysine acetyl transferase CBP-1

32,33

. In addition,

increased expression of

jmjd-3.1

promotes HSF-1 activity following germline stem cell

removal in worms

. Therefore, we hypothesised that SIR-2.2 dependent changes in

mitochondrial copy number might potentiate HSF-1 activity by increasing JMJD-3.1

activity.

To test this, we exposed wild type and

jmjd-3.1

KO worms to transient fasting

(24 hours fasting followed by 24 hours recovery on food) and then measured the

expression of HSF-1 target genes immediately following heat shock (35



C, 30 min).

We observed that transiently fasted

jmjd-3.1

mutants were unable to increase the

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expression of HSF-1 target genes in response to heat shock to the level observed in

transiently fasted wild type animals (Figure 5A and B). In addition, transiently fasted

jmjd-3.1

KO mutants were also not as stress resistance or long-lived as transiently

fasted wild-type animals (Figure 5C and D). Similarly, we found that knockdown of

cbp-1

also suppressed stress resistance in fasted animals compared to empty vector

(L4440) controls (Figure S5B). Consistent with an increase in JMJD-3.1 activity, we

also found that total H3K27me3 levels were strongly reduced in transiently fasted

animals compared to constantly fed counterparts (Figure 5E) and that H3K27me3

levels were reduced in transiently fasted worms by 20-40% at the promoters of

hsp-

70, F44E5.4 and hsp-16.2,

but not at the promoter of

cdc-42

, whose expression is not

regulated by HSF-1 or altered by fasting (Figure 5F). Surprisingly, reduced levels of

H3K27me3 did not correlate with an increase in total levels of H3K27ac and were not

due to a reduction in histone H3, the levels of which were highly elevated (Figure 5E),

suggesting a substantial change in chromatin state occurs in response to fasting.

Together, our data suggest that in response to reduced mitochondrial copy

number, JMJD-3.1 promotes HSF-1 activity in response to fasting through the

demethylation of histone H3 at the promoters of HSF-1 target genes.

Fasting selectively potentiates HSF-1 activity in mouse skeletal muscle but not

brain tissue

Various forms of fasting are associated with lifespan extension in worms, flies

and mammals

. Therefore, we tested whether fasting also increases HSF-1 activity

in mice using a brain permeable HSF1 activator (NVP-HSP990)

. We found that mice

subjected to fasting for 16 hours exhibited no increase in the basal expression of HSF1

target genes in quadricep muscle or brain tissue (cerebrum), with

Hspa1a/b, Hspb1,

Dnajb1,

and

Hsph1

mRNA decreasing in the cerebrum (Figure S6A and B). Similarly,

fasting had no effect on the fold induction of HSF1 target genes in the cerebrum

following treatment with NVP-HSP990 (Figure 6A-C and Figure S6C-E). In contrast,

the induction of

Hsp90aa1, Hspb1

and

Dnajb1

following NVP-HSP990 treatment, was

increased in the quadriceps of fasted mice compared to fed controls (Figure 6D-F).

However, this was not seen for

Hspa1a/b, Dnaja1

Hsph1

(Figure S6F-H). Together,

our data demonstrate that fasting selectively potentiates HSF1 activity in mammals,

but that these effects occur in a tissue-specific manner.

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Discussion

Here we have shown that a single, transient period of fasting, early in life, is

sufficient to enhance organismal robustness and extend lifespan. These effects were

associated with an enhanced ability to suppress polyglutamine aggregation in different

tissues, suggesting that fasting is an effective way to maintain a healthy proteome.

This is supported by earlier indications that fasting prevents the accumulation of

carbonylated proteins in aged worms

Previous fasting regimens have either continuously removed food or exposed

animals to multiple rounds of fasting and re-feeding in adulthood to extend lifespan

Surprisingly, we find that if food removal is administered prior to reproductive maturity,

a single, transient period of fasting is sufficient to elicit beneficial effects without

compromising fecundity. This suggests that as well as the duration/pattern of fasting,

the stage of life when fasting is administered is a key determinant of longevity and

health outcomes. However, it should also be noted that chronic or repeated removal

of food can still increase lifespan, even when initiated later in adulthood

15,36

C. elegans

, the transition to reproductive maturity is marked by the

repression or remodelling of numerous proteostasis pathways, including the heat

shock response

5,37,38

. Removing germline stem cells (GSCs) or compromising egg-

shell integrity prevents repression of the HSR, maintains stress resistance and

protects animals against age-related proteostasis collapse

4,7

. Chronic fasting has

been linked to defects in reproduction

; however, the fasting conditions employed

here did not lead to a reduction in total brood size, suggesting that the effects we

observe arise independently of changes to the germline. The fact that an analogous

response is also present in mouse muscle tissues further argues that the potentiation

of HSF-1 activity is a general cellular response to fasting.

An important unanswered question is, how exactly does fasting promote

longevity and tissue health? Increased lifespan in response to fasting has been linked

to altered metabolism, mitochondrial remodelling and increased activity of PN

pathways, including the ubiquitin proteasome system, the UPR

and

autophagy

35,40,41

. We find that in addition to these pathways, the potentiation of HSF-

1 activity in response to protein folding stress is also a key factor in fasting induced

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longevity. Consistent with this, HSF-1 is also necessary for lifespan extension in

response to altered nutrient signalling

15,19,42,43

, suggesting that HSF-1 is broadly

required for the coupling of nutrient signalling and lifespan.

The relationship between early-life fasting and HSF-1 appears to be complex,

with the expression of a distinct sub-set of HSF-1 target genes (sHSPs, HSP70s,

DNAJs and NEFs) being increased in response to protein folding stress but not

increased under basal conditions. In fact, our data in mice suggests that the basal

expression of some HSF-1 target genes declines in the brain under fasted conditions,

an observation that parallels those previously reported in the livers of fasted mice and

that has been linked to increased PGC1alpha activity

. In addition, HSF-1 activity was

potentiated within the muscles but not the brain tissues of fasted mice, possibly due

to the increased resistance of the brain to fasting as compared to other organs

However, we cannot exclude the possibility that the responses identified in muscle

cells are not present in neurons.

Increased HSF-1 activity has recently emerged as a key component of the

cellular response to mitochondrial disturbances

6,46,47,48

. Our data suggest that the

potentiation of HSF-1 activity and the preservation of proteostasis capacity in fasted

animals arises from altered mitochondrial homeostasis, specifically, elevated levels of

mitochondrial sirtuins and reduced mitochondrial copy number within the first 8 hours

of fasting. Mitophagy is the primary route by which mitochondria are removed from

cells and is responsive to increased NAD+ levels and lysosomal biogenesis

30,49,50

Furthermore, increased mitophagy has been shown to be necessary for lifespan

extension in response to dietary restriction, while reduced mitophagy is associated

with shortened lifespan

30,49

We find that NAD+ levels are elevated in fasted worms, and that HLH-30 (the

master regulator of lysosomal biogenesis genes), and mitophagy adapters are

necessary for full stress resistance in fasted animals. HLH-30 is required for increased

lifespan in response to altered mitochondrial dynamics and chronic DR

31,51

, and

increased neuronal HLH-30 promotes heat stress resistance through mitochondrial

fragmentation in muscle tissues

. Furthermore, interactions between HLH-30 and

HSF-1 have been proposed to govern autophagy, metabolism and stress

resistance

53,54

. Therefore, we propose that in response to fasting, HLH-30 promotes

organismal robustness through a combination of macroautophagy and mitophagy,

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which then stimulates HSF-1 activity to further enhance protein folding and

degradation.

Our finding that elevated levels of SIR-2.2 and SIR-2.3 are necessary for

mitochondrial clearance and enhanced HSF-1 activity in

C. elegans

is intriguing.

Sir-2.2

and

sir-2.3

mutants have been shown to be less resistant to oxidative stress

and have been shown to interact with mitochondrial biotin-dependent carboxylases

involved in the TCA cycle (PYC-1), amino acid catabolism (PCAA-1) and formation of

ketone bodies (MCCC-1)

. SIRT4, the mammalian orthologue of SIR-2.2/SIR-2.3,

also interacts with these enzymes and malonyl CoA decarboxylase

27,55

, suggesting

that intermediates and/or outputs from these pathways may mediate mitochondria-to-

HSF-1 signalling in fasted tissues. However, unlike our findings in worms, the levels

of SIRT4 have been shown to decrease in muscle and liver tissue of fasted mice

. In

contrast, the levels of other mammalian mitochondrial sirtuins (SIRT3 and SIRT5)

increase or remain unchanged in response to fasting in mouse tissue following

fasting

56,57

. Therefore, it is possible that different upstream processes couple

mitochondria with HSF1 activity in mammals. Alternatively, the activity of mitochondrial

sirtuins may increase in mammals during early fasting, before protein levels decline.

Lastly, our work reveals that the potentiation of HSF-1 activity, enhanced stress

resistance and increased longevity are dependent on the H3K27me3 demethylase,

JMJD-3.1. Increased JMJD-3.1 activity has been found to maintain the HSR in GSC

deficient animals and promotes the UPR

under conditions of mitochondrial stress

5,32

JMJD-3.1 has also been shown to cooperate with the H3K27 acetyltransferase, CBP-

1, to promote the UPR

mt 33

, and we also find that CBP-1 is necessary for fasting to fully

enhance stress resistance. CBP-1 is also required for dietary restriction mediated

lifespan extension in worms

, providing further support for the role of histone modifiers

in promoting HSF-1 activity in the absence of food. However, CBP-1 has also been

shown to acetylate and negatively regulate HSF-1 in worms and mammals under non-

fasted conditions

. This suggests that in the absence of food, CBP-1 switches its

focus from regulating HSF-1 to acting on histones. Similarly, components of the

nucleosome remodelling and deacetylase complex are required for both the HSR and

UPR

mt 60,61

, and mitochondrial stress is associated with widespread chromatin

reorganization via MET-2, LIN-65 and ISW-1

47,62

. Therefore, future work to determine

how fasting alters the acetylation status of HSF-1, and general chromatin organization,

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may shed further light on precisely how HSF-1 activity is potentiated at a select subset

of PN genes during fasting.

We also observed that total histone H3 levels are elevated in fasted animals,

suggesting that histone content, as well as status, are altered by fasting. Previous

studies in flies and mice have demonstrated that inhibition of mTORC1 with

Rapamycin is also associated with elevated H3 and H4 levels

63,64,65

. Therefore,

increased histone levels and altered chromatin organisation are likely a general

phenomenon of metabolic remodelling. Given that overexpression of histones also

promotes tissue health and longevity, it will be interesting to ascertain the extent to

which these effects are mediated by potentiated HSF1 activity and enhanced

proteostasis capacity.

In summary, the absence of food prior to reproductive maturity initiates a

mitochondria-to-HSF-1 signalling axis through the activity of mitochondrial sirtuins and

chromatin remodelling enzymes. This leads to an altered chromatin state that

facilitates HSF-1 binding in response to protein folding stress, thereby protecting

tissues against age-related proteostasis collapse. We propose that this mechanism

exists to preserve tissue integrity in the face of additional external stresses during

nutrient deprivation. In both

C. elegans

and mammals, this would provide greater

organismal robustness when seeking new food sources in order to maximise fitness.

Limitations of the study

While our work suggests a link between mitochondrial mass and HSF-1 activity, we

do not formally demonstrate that changes in mitochondrial copy number in fasted

animals are exclusively through clearance and not also due to reduced mitochondrial

biogenesis. In addition, while increased SIR-2.2 levels are important for fasting to

reduce mitochondrial copy number, we do not formally demonstrate that SIR-2.2 has

a direct role in mitophagy. Lastly, while we demonstrate that both JMJD-3.1 and SIR-

2.2 are necessary for transient fasting to increase lifespan, we have not resolved

whether these factors interact with one another, or act through parallel pathways to

augment longevity in response to temporary food removal.

Acknowledgements

This work was supported by grants to John Labbadia and Gillian Bates from the

BBSRC (BB/P005535/1), CHDI Foundation and the UK Dementia Research Institute,

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which receives its funding from DRI Ltd, funded by the UK Medical Research Council,

Alzheimer’s Society and Alzheimer’s Research UK. Some strains were provided by

the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40

OD010440). Some strains were provided by NBRP, which is funded by the Japanese

government.

The Graphical Abstract was created with Biorender.com. The authors

have no conflict of interest.

Author contributions

J.L. and G.P.B. acquired funding for this study. Y.A., N.T.P., C.G.P., G.P.B. and J.L.

designed the study. Y.A., N.T.P., H.C. and C.G.P. developed the methodology. N.T.P.,

Y.A., R.W., H.C., K.J.H.C, and C.G.P. performed the experiments. Y.A., N.T.P.,

K.J.H.C, and C.G.P. analysed and visualised the data. N.T.P., C.G.P., G.P.B and J.L.

wrote the manuscript and all authors read and approved the manuscript.

Declaration of interests

The authors declare no competing interests.

Figure titles and legends

Figure 1: Fasting at the transition to adulthood maintains proteostasis capacity

in aged tissues and extends lifespan

(A & B)

Survival at 20



C or following heat shock (HS) (35



C, 4 hours) in fed or fasted

worms.

(C)

Progeny produced at specific days of adulthood in fed and fasted animals.

Data plotted are mean values +/- SEM.

(D)

Survival of fed or fasted animals (starting

at L3, L4, day 1 of adulthood or day 2 of adulthood) following heat shock (35



C, 4

hours).

(E & F)

PolyQ::YFP aggregate number in fed and fasted worms on day 3 and

day 5 of adulthood in

(E)

body wall muscles and

(F)

intestines, respectively.

(G)

Proportion of motile polyQ (35) worms at different days of adulthood following constant

feeding or transient fasting. Data plotted are mean values +/- SEM.

(H)

Body bends

per minute (thrashing rate) of fed and fasted worms expressing ABeta in body wall

muscle cells. Statistical comparisons were made using Mantel-Cox Log Rank test (A

& B), Student’s unpaired t-test (E & F) or two-way ANOVA (G & H). * = p < 0.05, ****

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= p < 0.0001. Full statistics for survival curves can be found in Supplemental Table 1.

Fasting conditions were as follows: A, C, E, F, G and H - animals were removed from

food for 24 hours starting at the L4 stage and then returned to food thereafter; B -

animals were removed from food for 24 hours starting at the L4 stage and then

returned to food for 24 hours prior to HS; C

– animals were removed from food for 24

hours starting at the indicated life stage and then returned to food for 24 hours prior to

HS. See also Figure S1 and Table S1.

Figure 2: Fasting enhances proteostasis capacity and promotes longevity

through the selective potentiation of HSF-1 activity

(A)

Survival of fed and fasted wild-type (N2) or PN mutant animals following heat shock

(HS) (35



C, 4 hours).

(B)

Survival at 20



C of fed and fasted wild-type and

hsf-1(sy441)

mutant worms.

(C & D)

Relative mRNA levels of canonical HSF-1 target genes in fed

and fasted worms under

(C)

basal (20



C) and

(D)

HS (35



C, 30 minutes) conditions.

(E & F)

Relative mRNA levels of

hsp-4

and

hsp-6

in fed and fasted worms under

(E)

basal or

(F)

HS (35



C, 30 minutes) conditions.

(G)

Relative expression of all HSF-1

target genes in fed and fasted worms under basal or HS (35



C, 30 minutes) conditions.

(H)

STRING network of up-regulated (

< 0.05) HSF-1 target PN genes in fasted

worms following HS (35



C, 30 minutes). For panels C-G, worms were harvested

immediately after the heat shock conditions specified. Statistical comparisons were

made using Mantel-

Cox Log Rank test (B) or Student’s unpaired t-test with FDR

correction (C-F). ns =

> 0.05, * =

< 0.05, **** =

< 0.0001. Full statistics for heat

map and survival curves can be found in Supplemental Table 1. Fasting conditions

were as follows: A, C, D, E and F - animals were removed from food for 24 hours

starting at the L4 stage and then returned to food for 24 hours prior to HS; B - animals

were removed from food for 24 hours starting at the L4 stage and then returned to

food thereafter; G - animals were removed from food for 24 hours starting at the L4

stage and then immediately exposed to basal or HS conditions. See also Figure S2,

Table S1 and Table S4.

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Figure 3: Mitochondrial sirtuins are necessary for increased stress resistance

and longevity following early life fasting

(A)

Representative images of fed and fasted WBM321 acs-2p::gfp worms. Scale bar

= 200



(B)

Relative NAD+ levels in fed and fasted worms.

(C - F)

Survival of fed

and fasted wild-type (N2),

(C)

nhr-49(nr2041)

(D)

sir-2.1(ok434)

(E)

sir-2.2(tm2648)

and

(F)

sir-2.3(ok444)

mutant worms following heat shock (35



C, 4 hours).

(G & H)

Lifespan at 20



C of fed and fasted wild-type (N2),

(G)

sir-2.2(tm2648)

and

(H)

sir-

2.3(ok444)

mutants. Statistical comparisons were made using Student’s unpaired t-

test (B) and Mantel-Cox Log Rank test (C-H). ns =

> 0.05, * =

< 0.05, **** =

0.0001. Full statistics for survival curves can be found in Supplemental Table 1.

Survival assays in D - F, and G & H, were run in parallel. Fasting conditions were as

follows: A-F - animals were removed from food for 24 hours starting at the L4 stage

and then returned to food for 24 hours prior to imaging, collection or HS; G & H -

animals were removed from food for 24 hours starting at the L4 stage and then

returned to food thereafter. See also Figure S3 and Table S1.

Figure 4: SIR-2.2 couples mitochondrial clearance with enhanced HSF-1 activity

in fasted animals

(A - C)

Relative expression of HSF-1 target genes in fed and fasted wild-type worms

sir-2.2(tm2648)

mutants following heat shock (35



C, 30 min). Worms were

harvested immediately following heat shock.

(D & E)

Relative

sir-2.2

and

sir-2.3

mRNA

levels in fed and fasted animals.

(F)

Representative images of fed and fasted SIR-

2.2::EGFP worms. Scale bar = 200



(G & H)

Relative

(G)

SIR-2.2::EGFP levels and

(H)

mitochondrial copy number at different times post fasting.

(I)

Mitochondrial copy

number in fed and fasted wild-type or

sir-2.2(tm2648)

mutant worms.

(J)

Survival of

fed and fasted wild-type (N2) and

hlh-30

mutant worms following heat shock (35



C, 4

hours). Statistical comparisons were made using two-way ANOVA with post-analysis

pairwise comparison of groups (A-C & G-

I), Student’s unpaired t-test (D & E) and

Mantel-Cox Log Rank test (J). ns = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001,

**** = p < 0.0001. Full statistics for survival curves can be found in Supplemental Table

1. The survival assay in J was run in parallel with S4C and D. Fasting conditions were

as follows: A-C and J - animals were removed from food for 24 hours starting at the

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L4 stage and then returned to food for 24 hours prior to HS; D-F - animals were

removed from food for 24 hours starting at the L4 stage and then immediately collected

or imaged; G & H - animals were removed from food at the L4 stage for the indicated

times before collection; I - animals were removed from food at the L4 stage for 16

hours before collection. See also Figure S4, Table S1 and Table S4.

Figure 5: JMJD-3.1 promotes HSF-1 potentiation in response to fasting (see also

Supplemental Figure 5)

(A and B)

Relative mRNA of HSF-1 target genes in heat shocked (35



C, 30 min) wild-

type and

jmjd-3.1(gk384) KO

mutant worms. Worms were harvested immediately

following heat shock.

(C)

Survival of fed and fasted wild-type and

jmjd-3.1(gk384)

mutant worms following heat shock (35



C, 4 hours).

(D)

Lifespan at 20



C of wild-type

and

jmjd-3.1(gk384)

mutant worms.

(E)

Representative western blots of total histone

H3,

H3K27me3,

H3K27ac

and

tubulin

fed

and

fasted

worms.

(F)

Relative H3K27me3 levels at HSF-1 target promoters in fed and fasted worms.

Statistical comparisons were made using two-way ANOVA with post-analysis pairwise

comparison of groups (A & B), Mantel-Cox Log Rank test (C & D) and unpaired

Student’s t-test (F). ns = p > 0.05, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. Full

statistics for survival curves can be found in Supplemental Table 1. Fasting conditions

were as follows: A-C and F - animals were removed from food for 24 hours starting at

the L4 stage and then returned to food for 24 hours prior to HS or collection; D -

animals were removed from food for 24 hours starting at the L4 stage and then

returned to food thereafter; E - animals were removed from food for 24 hours starting

at the L4 stage and then immediately collected. See also Figure S5, Table S1 and

Table S4.

Figure 6: Fasting selectively potentiates HSF-1 activity in mouse skeletal muscle

but not brain tissue (see also Supplemental Figure 6)

(A - C)

QuantiGene assessment of HSF-1 target genes in brain tissue of fed and fasted

mice following treatment with vehicle or NVP-HSP990 (12 mg/kg). Values following

NVP-HSP990 treatment were normalised to corresponding vehicle controls.

– F)

QuantiGene assessment of HSF-1 target genes in quadricep muscle tissue of fed and

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fasted mice following treatment with vehicle or NVP-HSP990 (12 mg/kg). Statistical

comparisons were made using two-way ANOVA with Bonferroni correction. ***

0.001. Data were screened for outliers using a ROUT test and one mouse was

removed from both the NVP-HSP990 fed and fasted groups. Final sample numbers

were: vehicle non-fasting = 4, vehicle fasted = 6, NVP-HSP990 non-fasting = 9, NVP-

HSP990 fasted = 9. See also Figure S6, Table S2, Table S3 and Table S4.

Supplemental Table 1 (Excel File): Statistics for all lifespan and stress

resistance assays (related to Figures 1, S1, 2, S2, 3, 4, S4, 5 and S5)

STAR Methods

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to,

and will be fulfilled by, John Labbadia: j.labbadia@ucl.ac.uk

Materials availability

This study did not generate new, unique reagents.

Data and code availability

RNA-seq data have been deposited at GEO and are publicly available as of the date

of publication. Accession numbers are listed in the key resources table. All data

contained in this paper will be shared by the lead author upon request.

The paper does not report original code.

Any additional information required to re-analyze the data reported in this paper is

available from the lead contact upon request.

Experimental model and study participant details

C. elegans strains, culture conditions and fasting

All strains were maintained at 20



C on NGM plates seeded with

Escherichia coli

OP50

using standard husbandry techniques

. Strains used in this study were: N2 Bristol

(wild type laboratory strain), AD1116

eat-2(ad1116)

, CF1903

glp-1(e2144ts)

, MQ989

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isp-1(qm150);ctb-1(qm189)

, AM738 (rmIs297 [

vha-6

p::Q(44)::YFP,

rol-6(su1006)

]),

AM140 (rmIs132 [

unc-54

p::Q(35)::YFP]), GMC101 dvIs100[unc-54p::Abeta-1-

42::unc-

54 3’ UTR + mtl-2p::GFP], CL2122 dvIs15[pPD30.38) unc-54(vector) +

(pCL26) mtl-2p::gfp], PS3551

hsf-1(sy441),

CF1038

daf-16(mu86),

RB711

pqm-

1(ok485)

, TM1153

atf-6(tm1153)

, SJ17

xbp-1(zc12)

, RB545

pek-1(ok275)

, TM4525

atfs-1(tm4525)

, JIN1375

hlh-30(tm1978)

, BR4006

pink-1(tm1779)

, VC1024

pdr-

1(gk448)

, TM2648

sir-2.2(tm2648)

, RB654

sir-2.3(ok444)

, WBM321 (wbmIs321[acs-

2p::gfp + rol-6(su1006)]), STE68

nhr-49(nr2041)

, VC936

jmjd-3.1(gk384)

. SJ4103

zcls14

[myo-3::GFP(mit)], TH188 ddIs105[sir-2.2::TY1::EGFP::3XFLAG(92C12) +

unc-119(+)], MAH215 sqIs11[lgg-1p::mCherry::GFP::lgg-1 + rol-6(su1006)]

PP563

hhIs64[unc-119p(+); sur-5p::UbV::gfp] and PP556 hhIs57[unc-119(+); sur-5p::gfp].

Worms were fasted by moving mid-stage L4s to plates without food for indicated

lengths of time (4, 8, 12, 16, 20, or 24 hours). Following fasting, worms were moved

back to plates containing food for subsequent stress resistance or lifespan assays. To

ensure efficient removal of food from roller strains (those carrying a

rol-6

selection

marker), mid-L4 stage worms were sequentially moved to plates without food three

times, with 10-minute periods of incubation/crawling allowed between each transfer.

Mouse breeding and maintenance

All procedures were performed in accordance with the Animals (Scientific Procedures)

Act 1986, complied with ARRIVE guidelines and were approved by the University

College London Ethical Review Process Committee. Animals used for fasting and

QuantiGene

analysis

experiments

were

(CBA/Ca × C57Bl/6J)F1

mice

(B6CBAF1/OlaHsd, Envigo). Male mice at 11 weeks of age were used for all treatment

groups. Mouse husbandry was performed with up to five mice housed per cage. Mice

were housed in individually ventilated cages with Aspen Chips 4 Premium bedding

(Datesand) and with environmental enrichment which included chew sticks and a play

tunnel (Datesand). Mice had unrestricted access to food (Teklad global 18% protein

diet, Envigo) and water. The temperature was regulated at 21

°C ± 1 °C and animals

were kept on a 12 h light/dark cycle. The animal facility was barrier-maintained and

quarterly non-sacrificial FELASA screens found no evidence of pathogens.

NVP-HSP990 formulation and dosing of fed and fasted mice

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NVP-HSP990 (2-amino-7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one) was obtained

from Novartis Pharma AG. NVP-HSP990 was formulated as a suspension in 2%

methylcellulose (Sigma), diluted in 0.9% saline solution (Severn Biotech) and

sonicated twice at high frequency in an ultrasonic bath. Both vehicle and NVP-HSP990

solutions were freshly prepared for the dosing experiment. Thorough mixing was

carried out between doses to maintain NVP-HSP990 as an even suspension. Male

mice at 11 weeks of age were fasted by complete removal of food for 16 hours. At this

point, animals were dosed with vehicle (n = 6 fed; n = 5 fasted) or 12 mg / kg NVP-

HSP990 (n = 9 fed; n = 9 fasted) by oral gavage and sacrificed 2 hours later (total of

18 hours without food for fasted group). Sample sizes were based on previous

experimental results

. For this dosing experiment, male wild-type mice were always

randomised with respect to litter of origin and age matched at 11 weeks of age. 2 hours

after treatment with NVP-HSP990 or vehicle, mice were sacrificed by a schedule 1

procedure, dissected, tissues were snap-frozen in liquid nitrogen and stored at

− 80 °C.

Method details

Tissue homogenisation and QuantiGene gene expression assays

QuantiGene experiments were performed as previously described

. Tissue samples

were homogenised using a Polytron homogeniser for brain regions or liquid nitrogen

and pre-chilled pestle and mortar for muscle, using QuantiGene reagents from Thermo

Fisher Scientific, following the manufacturer’s recommendations. Housekeeping

genes used for normalization in cerebrum (brain) tissue were

Canx, Atp5b, Eif4a2,

Sdha, Gapdh

and

Rpl13a

. For quadricep (muscle) tissue,

Canx, Atp5b, Eif4a2,

and

Sdha

were used. The tissue lysate dilutions used for this study are listed in

Supplemental Table 2. Information pertaining to probe regions and accession

numbers for the QuantiGene multiplex assays used in this study can be found in

Supplemental Table 3. The median fluorescent intensity (MFI) was read in a Magpix

(Luminex) using the xPonent software.

RNA interference

All clones were sequenced verified before use and were obtained from the Ahringer

RNAi library. RNAi was performed by growing bacteria for 16 hours at 37



C in LB

containing 100 µg/ml ampicillin, with shaking (220 rpm). Cultures were then induced

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with 5 mM IPTG and allowed to grow at 37



C for a further 3 hours. Following induction,

bacteria were allowed to cool at room temperature and were then seeded onto NGM

plates containing 100 µg/ml ampicillin and 1 mM IPTG. Seeded plates were allowed

to dry at room temperature before use.

Lifespan and stress resistance assays

In all lifespan and stress resistance assays, survival was scored by gently touching

worms with a platinum pick at indicated time points. Worms were scored as dead in

the absence of touch response and absence of pharyngeal pumping. In lifespan

assays, worms exhibiting intestinal prolapse through the vulva (rupturing) or internal

hatching of progeny (bagging) were censored. For thermorecovery assays, worms

were heat shocked on seeded NGM plates at 35



C for 4 hours and allowed to recover

at 20



C. For tunicamycin or paraquat treatment, worms were transferred to seeded

NGM plates containing tunicamycin (50 µg/ml) or paraquat (10 mM) and maintained

at 20



C until dead.

Proteostasis sensor assays

Polyglutamine aggregation was scored in muscle and intestinal proteostasis sensors

at indicated time points under a Nikon SMZ1270 fluorescence dissecting

stereomicroscope. Aggregates were determined to be any discrete foci exhibiting

fluorescence signal above the background diffuse signal. Muscle function was

assessed in polyglutamine expressing animals by scoring paralysis at different days

of adulthood. Animals were scored as paralysed when they were unable to move

forwards or backwards at least one body length in response to touch with a platinum

pick. For Abeta (GMC101) and control (CL2122) animals, muscle health was assessed

by measuring thrashing ability in M9 buffer (number of body bends per minute) under

a Leica M80 stereo dissection microscope. Worms were shifted to 25



C at the L4 stage

to induce paralysis/thrashing defects. All scoring was performed blind to treatment

groups.

Autophagy reporter

Animals were synchronised by egg lay for 1 hour at 20



C. Once animals reached the

L4 stage, they were transferred onto NGM plates with or without food for 24 hours.

After this period, worms were moved back to plates containing food for 24 hours, at

which point, fed and fasted animals were heat-shocked in a water bath for 4 hours at

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

C, or kept at 20



C for an equivalent period of time. Immediately following heat

shock, worms were mounted onto slides for image acquisition. Worms were mounted

on 2% agarose pads in 5 mM levamisole and imaged immediately. For image

acquisition, a Zeiss Imager.Z2 microscope with a Hamamatsu C13440 ORCA-

Flash4.0 V3 digital camera, Apotome.2 for Z-stack images and ZenBlue software were

used. Z-stacks were acquired at 0.6 µm slice intervals using 100x objective and

processed as maximum intensity projections. Based on previous studies

, a Z-

position was selected where the nucleus could be seen in the intestine and posterior

pharyngeal bulb in the pharynx. The area of the first two intestinal cells was used for

the quantification of visible puncta in the intestine, whereas the area of the posterior

pharyngeal bulb was used for the quantification of visible puncta in the pharynx. Green

or yellow puncta were scored as autophagosomes while red puncta were scored as

autolysosomes.

UPS reporter

Animals were synchronised by egg lay for 1 hour at 20



C. Once animals reached the

L4 stage, they were transferred onto NGM plates with or without food for 24 hours.

After this period, worms were moved back to plates containing food for 24 hours, at

which point, fed and fasted animals were heat-shocked in a water bath for 4 hours at



C, or kept at 20



C for an equivalent period of time. Immediately following heat

shock, worms were mounted onto slides for image acquisition. Worms were mounted

on 2% agarose pads in 3 mM levamisole and imaged immediately. For image

acquisition, a Nikon SMZ1270 fluorescence dissecting stereomicroscope with a DS-

Fi3 5.9 MP colour camera was used. Images were processed using ImageJ

RNA extraction, cDNA synthesis and RTqPCR

Approximately 100-200 adult animals per treatment group were lysed in 250



l of Trizol

by vortexing for 20 minutes at 4



C. RNA was purified using an RNeasy extraction kit

as per

manufacturer’s instructions. cDNA was generated using 1 µg of total RNA and

an iScript cDNA synthesis kit. Real-time quantitative PCR was performed using a

Biorad CFX96 Real-time PCR detection system and BioRad SsoAdvanced SYBR

green super mix. Expression of genes of interest was calculated relative to the

housekeeping genes

rpb-2

and

cdc-42

using the standard curve method. Sequences

for all primer pairs used in this study can be found in Supplemental Table 3. For

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quantification of mRNA levels following heat shock, animals were harvested

immediately after the heat shock conditions specified (35



C, 30 minutes).

RNA-sequencing and analysis

RNA integrity was assessed using an RNA Nano 6000 assay kit and an Agilent

Bioanalyzer 2100 system. Following this, mRNA was purified from 1



g of total RNA

using poly-dT magnetic beads and cDNA libraries were generated using random

hexamers and M-MuLV reverse transcriptase for first strand synthesis, followed by

second strand synthesis using DNA polymerase I and RNase H. Fragments were

blunt-ended and adapters were ligated before PCR was performed using Phusion high

fidelity DNA polymerase. PCR products were purified using an AMPure XP system

and library quality was checked using an Agilent Bioanalyzer 2100. Libraries were

sequenced using an Illumina Novaseq 6000 platform to generate paired-end 150 base

pair reads at a depth of 20 million reads per sample. Following sequencing, raw data

was checked and reads containing adapter sequences, poly-N reads or poor-quality

sequences were removed. Reads were then aligned to the

C. elegans

reference

genome using Hisat2 v2.0.5 and featureCounts v1.5.0-p3 was used to quantify the

number of reads mapped to each gene. Differential expression testing was carried out

using DESeq2 and p-values were adjusted using Benjamini-Hochberg. Z-scores were

calculated for each gene from log FPKM expression data for all differentially expressed

genes (> 1.5-fold, adj p < 0.05). Up or down regulated genes were analysed using the

g:profiler tool to identify KEGG processes/pathways and Gene Ontology categories

that were enriched in either group. Raw and processed data can be found at the NCBI

Gene Expression Omnibus using accession number GSE236616

For quantification

of mRNA levels following heat shock, animals were harvested immediately after the

heat shock conditions specified (35



C, 30 minutes).

Protein extraction and western blotting

To extract protein for western blotting, worms (approximately 500

– 1000) were

collected in M9, pelleted, and then resuspended in RIPA buffer supplemented with a

protease inhibitor cocktail tablet. Worm pellets were then flash frozen in liquid nitrogen

and ground in microcentrifuge tubes using a plastic dounce homogenizer. Freezing

and grinding were performed twice, and effectiveness of lysing was confirmed by

checking a sample of the lysate under a dissecting microscope. Lysates were then

Journal Pre-proof

centrifuged at 15,000 x

at 4



C for 15 minutes and the supernatant was collected.

Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes

before probing with primary antibodies. Blots were incubated with primary antibodies

for 1 hour at room temperature (Histone H3 - 1:5000, tubulin - 1:10,000) or overnight

at 4



C (anti-H3K27me3 (1:1000), anti-H3K27ac (1:1000)), washed three times with

PBS-0.2% Tween, incubated with secondary antibodies for 1 hour at room

temperature (mouse-HRP

– 1:5000, rabbit-HRP – 1:5000), washed a further three

times with PBS-0.2% Tween, and then developed using ECL detection reagents and

an Amersham ImageQuant800 detection system. Densitometry of protein bands was

performed using ImageJ gel analysis tools.

Chromatin immunoprecipitation

Worms (20,000 per treatment group) were harvested in M9, pelleted and re-

suspended in 1% formaldehyde-PBS to promote cross-linking. Worms were then fixed

for 30 minutes at room temperature, washed three times in PBS and resuspended in

FA buffer. Worms were then dounce homogenized on ice before being subjected to

sonication using a Diagenode Bioruptor sonicator (15 rounds of 30 s on 1 min off).

Samples were then centrifuged at 4



C for 15 minutes and lysates were subjected to 5

more rounds of sonication to shear chromatin to approximately 500 bp in size.

Pulldowns were performed by incubating 2 mg of pre-cleared chromatin with 20 µl of

washed and pre-blocked Protein G Dynabeads and 2 µg antibody (anti-GFP) in 1 ml

of FA buffer, overnight at 4



C. Following incubation, beads were washed twice with

FA buffer, once with low salt wash buffer, once with high salt wash buffer, and once

with TEL buffer before being eluted from beads using 100 µl of 1% SDS. Cross-linking

was reversed by incubating samples at 65



C overnight in the presence of 20 mM NaCl,

before protein and RNA were removed by RNAse A (30 min at 37



C) and Proteinase

K (1 hour at 55



C) treatment. Samples were boiled at 95



C and DNA was purified

using a Qiagen PCR purification kit, as per

manufacturer’s instructions.

Mitochondrial morphology assays

The mitochondria morphology in muscle of the

zcls14[myo-3::GFP(mit)]

animals was

examined. 2% of molten agarose in water was used to make glass slides. Animals

were immobilized on agarose pads in 3 mM levamisole with a glass cover slip on the

top. To acquire images, 63x/1.40 oil objective lens of the Zeiss Imager.Z2 microscope

Journal Pre-proof

was used. Images were processed using ImageJ. Qualitative analysis of the

mitochondria morphology in each muscle section was performed. The largest regions

of clear body wall muscle were identified between the pharynx and the vulva or

between the vulva and tail. No significant difference was observed between these two

segments. Adjacent regions to the tail, pharynx and vulva were excluded due to the

disrupted mitochondria morphology which naturally occurs there. Images were scored

as fused, tubular, intermediate and fragmented based on the mitochondria structure

organization.

Quantification and statistical analysis

All statistical tests (Mantel Cox Log-rank, one-way ANOVA, two-way ANOVA and

Student’s t-test) were carried out as stated within each figure legend using GraphPad

Prism 9. The statistical details of all experiments can be found within the

accompanying figure legends or in Table S1.

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Key resources table

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit monoclonal anti Histone H3 - Nuclear Marker

ABCAM

Cat# ab176842,
RRID:AB_2493104

Rabbit monoclonal anti Histone H3 (tri methyl K27)

ABCAM

Cat# ab192985,
RRID:AB_2650559

Rabbit monoclonal anti Histone H3 (acetyl K27)

ABCAM

Cat# ab177178,
RRID:AB_2828007

Mouse monoclonal anti-alpha tubulin

SIGMA

Cat# T5168, B512;
RRID: AB_477579

Bacterial and virus strains

E. coli

OP50

CGC

WB OP50; RRID:
WB-STRAIN:OP50

E. coli

HT115

CGC

WB HT115; RRID:
WB-STRAIN:HT115

Biological samples

Chemicals, peptides, and recombinant proteins

Paraquat

SIGMA

Cat# 856177

Tunicamycin

Cambridge
Bioscience

Cat# 11445

Critical commercial assays

RNeasy extraction kit

QIAGEN

Cat# 74104

iScript cDNA synthesis kit

BIO-RAD

Cat# 1708890

Qiagen PCR purification kit

QIAGEN

Cat# 28104

Deposited data

All raw and processed RNA-sequencing data

Gene

Expression

Omnibus

GSE236616

Experimental models: Cell lines

Experimental models: Organisms/strains

C. elegans

: STRAIN N2 Bristol: (wild type laboratory strain)

CGC

WB Strain: N2

C. elegans

: STRAIN DA1116:

eat-2(ad1116)

CGC

WB Strain:

DA1116

C. elegans

: STRAIN CF1903:

glp-1(e2144ts)

CGC

WB Strain:

CF1903

C. elegans

: STRAIN MQ989:

isp-1(qm150);ctb-1(qm189)

CGC

WB Strain:

MQ989

C. elegans

: STRAIN AM738: rmIs297 [

vha-6

p::Q(44)::YFP,

rol-6(su1006)

]

Gift from

Morimoto Lab

WB Strain: AM738

C. elegans

: STRAIN AM140: rmIs132 [

unc-54

p::Q(35)::YFP]

Gift from

Morimoto Lab

WB Strain: AM140

C. elegans

: STRAIN GMC101: dvIs100[unc-54p::Abeta-1-42::unc-

54 3’

UTR + mtl-2p::GFP]

CGC

WB Strain:

GMC101

C. elegans

: STRAIN CL2122: dvIs15[pPD30.38) unc-54(vector) + (pCL26)

mtl-2p::gfp]

CGC

WB Strain:

CL2122

C. elegans

: STRAIN PS3551:

hsf-1(sy441)

CGC

WB Strain:

PS3551

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C. elegans

: STRAIN CF1038:

daf-16(mu86)

CGC

WB Strain:

CF1038

C. elegans

: STRAIN RB711:

pqm-1(ok485)

CGC

WB Strain:

RB711

C. elegans

: STRAIN TM1153:

atf-6(tm1153)

NBRP

WB Strain:

TM1153

C. elegans

: STRAIN SJ17:

xbp-1(zc12);hsp-4::GFP

CGC

WB Strain:

SJ17

C. elegans

: STRAIN RB545:

pek-1(ok275)

CGC

WB Strain:

RB545

C. elegans

: STRAIN TM4525:

atfs-1(tm4525)

NBRP

WB Strain:

TM4525

C. elegans

: STRAIN JIN1375:

hlh-30(tm1978)

CGC

WB Strain:

JIN1375

C. elegans

: STRAIN BR4006:

pink-1(tm1779)

CGC

WB Strain:

BR4006

C. elegans

: STRAIN VC1024:

pdr-1(gk448)

CGC

WB Strain:

VC1024

C. elegans

: STRAIN TM2648:

sir-2.2(tm2648)

NBRP

WB Strain:

TM2648

C. elegans

: STRAIN RB654:

sir-2.3(ok444)

CGC

WB Strain:

RB654

C. elegans

: STRAIN WBM321: (wbmIs321[acs-2p::gfp + rol-6(su1006)]),

Gift from Mair

Lab

WB Strain:

WBM321

C. elegans

: STRAIN VC936:

jmjd-3.1(gk384)

CGC

WB Strain:

VC936

C. elegans

: STRAIN SJ4103:

zcls14

[myo-3::GFP(mit)]

CGC

WB Strain:

SJ4103

C. elegans

: STRAIN TH188: ddIs105[sir-2.2::TY1::EGFP::3XFLAG(92C12)

+ unc-119(+)]

CGC

WB Strain: TH188

C. elegans

: STRAIN MAH215: sqIs11[lgg-1p::mCherry::GFP::lgg-1 + rol-

6(su1006)]

CGC

WB Strain:

MAH215

C. elegans

: STRAIN PP563: hhIs64[unc-119p(+); sur-5p::UbV::gfp]

Gift from

Hoppe Lab

WB Strain: PP563

C. elegans

: STRAIN PP556: hhIs57[unc-119(+); sur-5p::gfp]

Gift from

Hoppe Lab

WB Strain: PP556

C. elegans

: STRAIN STE68:

nhr-49(nr2041)

CGC

WB Strain: STE68

Oligonucleotides

Please see Table S3 for a complete
list of probes used in the QuantiGene 16-plex assay

N/A

Please see Table S4 for a complete
list of primers used in this study

N/A

Recombinant DNA

Software and algorithms

ImageJ

Schneider et
al.

https://imagej.nih.g
ov/ij/

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