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Endogenous H2O2 signaling positively regulates the release of neuropeptides during a neuron-gut axis mediated oxidative stress response in caenorhabditis elegans

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Endogenous H2O2 signaling positively regulates the release of neuropeptides during a neuron-gut axis mediated oxidative stress response in caenorhabditis elegans

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Content ENDOGENOUS H2O2 SIGNALING POSITIVELY REGULATES THE RELEASE OF
NEUROPEPTIDES DURING A NEURON-GUT AXIS MEDIATED OXIDATIVE STRESS
RESPONSE IN CAENORHABDITIS ELEGANS
by
Qi Jia
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(DEVELOPMENT, STEM CELLS AND REGENERATIVE MEDICINE)
May 2024
copyright 2024 Qi Jia

ii
Acknowledgements
First of all, I would like to thank my mentor Dr. Derek Sieburth and I really appreciate the
opportunity I was given to work on this project. Your enthusiasm about scientific research and
ability to attack a project from multiple angles has been inspiring me and helping me pursue my
degree. I also thank you for your knowledge and support throughout my research, making it a
successful and insightful project. I also would like to thank Mingxi Hu and other current and
previous labmates, who share all the joy and tears over the years, for your help throughout my
research.
And I thank my committee members, Dr. Karen Chang and Dr. Dion Dickman, for your
time and participation in my project. I appreciate your guide and encouragement, both have
contributed to the success of my project. I also would like to acknowledge DSR and PIBBS faculty,
and ZNI faculty for all the help I received over the years.
Finally I want to thank my friends and family for supporting and believing in me during
the good and the hard times. I appreciate all the love and help that have motivated and supported
me to pursue my degree.

iii
Table of Contents
Acknowledgements ....................................................................................................................... ii
List of Tables................................................................................................................................. v
List of Figures............................................................................................................................... vi
Abstract........................................................................................................................................ vii
Chapter 1 ....................................................................................................................................... 1
Introduction................................................................................................................................... 1
1.1 Reactive Oxygen Species...................................................................................................................1
1.1.1 Superoxide................................................................................................................................................... 2
1.1.2 Hydrogen Peroxide...................................................................................................................................... 2
1.1.3 Physiological role of ROS........................................................................................................................... 2
1.2 Antioxidant Defense Systems ...........................................................................................................3
1.2.1 Antioxidants ................................................................................................................................................ 3
1.2.2 The Nrf (nuclear factor erythroid-derived 2 related factors) transcription factor family............................ 4
1.3 Neuropeptide and Synaptic Transmission ......................................................................................5
1.3.1 Neuropeptide ............................................................................................................................................... 5
1.3.2 Exocytosis of DCVs.................................................................................................................................... 6
1.3.3 Neuropeptide Signaling............................................................................................................................... 6
1.4 Gut-Brain Axis...................................................................................................................................7
1.5 Caenorhabditis elegans.....................................................................................................................8
1.5.1 C. elegans simple yet complex nervous system .......................................................................................... 9
1.5.2 C. elegans intestine and epithelial cells....................................................................................................... 9
Chapter 2 ..................................................................................................................................... 11
Mitochondrial hydrogen peroxide positively regulates neuropeptide secretion during dietinduced activation of the oxidative stress response ................................................................. 11
2.1 Summary ..........................................................................................................................................11
2.2 Introduction .....................................................................................................................................11
2.3 Results...............................................................................................................................................13
2.3.1 Neuropeptide signaling activates the intestinal oxidative stress response ................................................ 13
2.3.2 The FLP-1 neuropeptide activates the intestinal oxidative stress response .............................................. 15
2.3.3 FLP-1 secretion from the AIY interneuron activates the oxidative stress response.................................. 16
2.3.4 FLP-1 antioxidant signaling is mediated by the NPR-4 GPCR in the intestine........................................ 18
2.3.5 FLP-1 secretion is positively regulated by mtH2O2................................................................................... 19
2.3.6 Mitochondrial calcium influx is required for ROS-induced FLP-1 secretion........................................... 21
2.3.7 TRX-2/thioredoxin and PRDX-3/peroxiredoxin regulate AIY mtH2O2 levels and FLP-1 secretion........ 22
2.3.8 Localized H2O2 production by axonal mitochondria regulates FLP-1 secretion....................................... 23
2.3.9 PKC-1/PKC mediates the effects of H2O2 on FLP-1 secretion................................................................. 24
2.3.10 PKC-1(C524) is necessary for juglone-induced FLP-1 secretion ........................................................... 25
2.3.11 Bacterial food sources alter H2O2 levels, FLP-1 secretion and oxidative stress response ...................... 26
2.4 Discussion.........................................................................................................................................28
2.5 Acknowledgements..........................................................................................................................34
2.6 Materials and Methods...................................................................................................................34
2.6.1 Strains........................................................................................................................................................ 34

iv
2.6.2 Toxicity Assays......................................................................................................................................... 35
2.6.3 Cell ablation and ROS production by miniSOG ....................................................................................... 35
2.6.4 Channelrhodopsin activation..................................................................................................................... 36
2.6.5 Microscopy and Analysis.......................................................................................................................... 36
2.6.6 RNA interference....................................................................................................................................... 37
2.6.7 Statistical analysis ..................................................................................................................................... 38
Chapter 3 ..................................................................................................................................... 67
Endogenous hydrogen peroxide positively regulates secretion of a gut-derived peptide in
neuroendocrine potentiation of the oxidative stress response in C. elegans.......................... 67
3.1 Summary ..........................................................................................................................................67
3.2 Introduction .....................................................................................................................................67
3.3 Results...............................................................................................................................................69
3.3.1 Neuronal FLP-1 secretion is regulated by neuropeptide signaling from the intestine. ............................. 69
3.3.2 FLP-2 signaling from the intestine potentiates neuronal FLP-1 secretion and the oxidative stress
response. ............................................................................................................................................................. 70
3.3.3 FLP-2 secretion from the intestine is H2O2-regulated............................................................................... 72
3.3.4 SOD-1 and SOD-3 superoxide dismutases regulate FLP-2 release. ......................................................... 73
3.3.5 SOD-1 and SOD-3 regulate intestinal mitochondrial H2O2 levels............................................................ 74
3.3.6 The peroxiredoxin-thioredoxin system regulates endogenous H2O2 levels and FLP-2 secretion............. 75
3.3.7 PKC-2/PKCα/β mediates H2O2 induced FLP-2 secretion from the intestine............................................ 77
3.3.8 DAG positively regulates FLP-2 secretion................................................................................................ 78
3.4 Discussion.........................................................................................................................................78
3.4.1 A new function for flp-2 signaling in the antioxidant response. ............................................................... 79
3.4.2 AIY as a target for flp-2 signaling............................................................................................................. 80
3.4.3 A role for endogenous H2O2 in regulated neuropeptide secretion............................................................. 81
3.4.4 Regulation of FLP-2 exocytosis by PKC-2/PKCα/β and AEX-4/SNAP25. ............................................. 82
3.4.5 Similar molecular mechanisms regulating FLP-1 and FLP-2 release. ...................................................... 83
3.5 Acknowledgement ...........................................................................................................................83
3.6 Materials and Methods...................................................................................................................84
3.6.1 Strains and transgenic lines....................................................................................................................... 84
3.6.2 Molecular Biology..................................................................................................................................... 84
3.6.3 Toxicity Assay........................................................................................................................................... 85
3.6.4 RNAi Interference ..................................................................................................................................... 85
3.6.6 Microscopy and Fluorescence Imaging..................................................................................................... 85
3.6.7 CRISPR/Cas9 Editing ............................................................................................................................... 86
3.6.8 Statistics..................................................................................................................................................... 87
chapter 4 .................................................................................................................................... 103
Summary and future directions............................................................................................... 103
4.1 Neuropeptides regulate stress-induced SKN-1 activation. ........................................................103
4.2 Conserved PKC dependent mechanism mediates endogenous H2O2 signaling.......................104
4.3 Bacterial diet modulates synaptic transmission. ........................................................................104
References.................................................................................................................................. 106

v
List of Tables
Table 2.1 Neuropeptide Screening for Juglone Toxicity………………...…….……..………………52
Table 2.2 GPCR Screening for Juglone Toxicity……………...………………………………………54

vi
List of Figures
Figure 1.1 Cellular Sources of Endogenous ROS Production…………………………………………1
Figure 1.2 Regulation of Nrf2/SKN-1 Activity in Mammals and C. elegans………………………...3
Figure 1.3 DCVs Mediate Neuropeptide Signaling and Undergo SNARE Mediated Exocytosis.....4
Figure 2.1 Neuronal FLP-1 Signaling Promotes the SKN-1-mediated Intestinal
Antioxidant Response..………………………………………………………………………………….27
Figure 2.2 FLP-1 Released from AIY Interneurons Promotes the Antioxidant Response…………30
Figure 2.3 FLP-1 Activates the Antioxidant Response through NPR-4/GPCR in the Intestine…...32
Figure 2.4 FLP-1 Secretion from AIY Interneurons Relies on Mitochondria-generated H2O2……34
Figure 2.5 Regulation of FLP-1 Secretion by Mitochondrial Calcium and the
Thioredoxin-Peroxiredoxin System……………………………………………………………….…...36
Figure 2.6 Trafficking of Mitochondria and DCVs to AIY Axons is Required for JugloneInduced FLP-1 Secretion……….……………………………………………………………………….38
Figure 2.7 mtH2O2-induced FLP-1 Secretion Requires C524 of PKC-1……………………………39
Figure 2.8 Bacterial Food Sources Alter FLP-1 Secretion and the Antioxidant Response………...41
Supplementary Figure 2.1 EGL-3 and FLP-1 Signaling Regulate Specific Stress Responses…….43
Supplementary Figure 2.2 mito-miniSOG-induced AIY Ablation and Effects of ATP
Generating Mutants on FLP-1 Secretion.….….……………………………………………………….45
Supplementary Figure 2.3 Specificity of Juglone-induced FLP-1 Secretion from AIY…………...46
Supplementary Figure 2.4 The Peroxiredoxin-Thioredoxin System Negatively Regulates
FLP-1::Venus Secretion from AIY………………………………………………………………..…...48
Supplementary Figure 2.5 TOMM-7::UNC-116 Fusion Protein Schematic………………………..49
Supplementary Figure 2.6 Localization of PKC-1 in AIY and Juglone-induced
INS-22 Secretion………………………………………………………………………….….…...….….50
Supplementary Figure 2.7 Sensory Input or Starvation do not Impact FLP-1 Secretion…………...52
Figure 3.1 Peptidergic Gut-to-Neuron FLP-2 Signaling Potentiates the Oxidative
Stress Response…………………………………………………….………………………….…70
Figure 3.2 FLP-2 Secretion from the Intestine is Stress Regulated…………………………………..72
Figure 3.3 SOD-1/SOD-3 Mediates Endogenous H2O2 Regulates FLP-2 Release from
the Intestine……………………………………………………………………...…….….……………...73
Figure 3.4. PRDX-2/PRDX and TRX-3/TRX Regulate Endogenous H2O2 and FLP-2 Secretion..75
Figure 3.5. PKC-2/PKCα/β Activation by H2O2 Promotes FLP-2 Secretion from the Intestine….76
Figure 3.6. DAG Promotes PKC-2 Mediated FLP-2 Secretion from the Intestine…………………77
Supplementary Figure 3.1. The Effect of Intestinal DCV Secretion Mutations on FLP-1
Release from AIY…………………………………………………….….….……………………79
Supplementary Figure 3.2. Specificity of Juglone on Intestinal Peptide Secretion, and FLP-2
and NLP-40 Localization in the Intestine……………………………………….….………………….80
Supplementary Figure 3.3. SODs Function in Juglone Induced FLP-2 Release from the
Intestine and Mitochondrial mCherry Control………………………………………………………...81
Supplementary Figure 3.4. PRDX-2 Intestinal Rescue and Mediates SOD-3 Dependent
Regulation of FLP-2 Release…………………………………………………………………………...82
Supplementary Figure 3.5. Juglone Promotes FLP-2 Release in pkc-1 Mutants and
Expulsion Analysis………………………………………………….….………………………………..83

vii
Abstract
Reactive oxygen species (ROS) production/exposure at a physiological level has been
shown to modulate synaptic functions and activities, and endogenous hydrogen peroxide (H2O2)
has been implicated in regulating synaptic vesicle (SV) and dense core vesicle (DCV) release.
Neuropeptides packaged into DCVs play an important role in mediating intercellular and intertissue communication and promoting organismal homeostasis in response to various
environmental cues. Using Caenorhabditis elegans as the model system, the goal of this
dissertation is to determine 1) how neuropeptide signaling promotes organismal protection against
oxidative stress-induced toxicity, 2) how the secretion of neuropeptide is regulated by reactive
oxygen species (ROS), 3) how endogenous H2O2 functions as signaling molecules in promoting
neuropeptide mediated inter-tissue regulation of antioxidative stress response.
The mammalian nuclear factor erythroid-derived 2 related factors (Nrf) family and its C.
elegans homolog, SKN-1, regulate the expression of detoxifying genes and promote organismal
protection against oxidative stress. Through genetic analysis and in vivo fluorescence imaging, I
identified the neuropeptide-like proteins FLP-1 and FLP-2 as essential components of a gut-brain
axis that coordinates SKN-1 activation and activity in the intestine. Functional characterization of
flp-1 signaling revealed that FLP-1 released from AIY interneurons is required to promote
intestinal SKN-1 activation in response mitochondrially generated H2O2. I showed that
pharmacological or genetic manipulations that lead to acute or chronic increases in mitochondrial
H2O2 levels, respectively, are sufficient to selectively increase FLP-1 secretion from DCVs at
release sites. The ability of acute H2O2 increases to increase FLP-1 secretion is dependent upon
the mitochondrial superoxide dismutase (SOD), SOD-2/SOD2, and H2O2 generation and FLP-1
secretion is cell-autonomously negatively regulated by thioredoxin, TRX-2 and peroxiredoxin,
PRDX-3. H2O2 induces FLP-1 release by activating protein kinase C (PKC), PKC-1/PKCε/η,
possibly through direct cysteine modification. Finally, I showed that gut bacteria potentiate
endogenous H2O2 production, FLP-1 release and SKN-1 activity, establishing that intestinederived signaling initiated by diet is responsible for regulating neuronal function.
Further genetic screening led to the identification of a second neuropeptide-like protein,
FLP-2, that I showed functions from the intestine to positively regulate the release of FLP-1
response from AIY, and the subsequent activation of SKN-1 in the intestine. I showed that FLP-2
release from the intestine is also positively regulated by endogenously generated H2O2 in the

viii
intestine by both mitochondrial SOD-3 and cytoplasmic SOD-1. Conversely, PRDX2/peroxiredoxin negatively regulates SOD-1 and SOD-3 dependent H2O2 production, and FLP-2
release from the intestine. Endogenous H2O2 promotes FLP-2 secretion from the intestine in a
similar manner as FLP-1 secretion: by a mechanism that is dependent upon the protein kinase c
family member PKC-2/PKCα/β. Moreover, H2O2 -induced FLP-2 secretion relies on DCV
exocytosis mediated by a number of SNARE proteins including AEX-4/SNAP25. My results
reveal a fundamental role for endogenous H2O2 signaling in regulating neuropeptide secretion in
an inter-tissue stress-response circuit.

1
Chapter 1
Introduction
1.1 Reactive Oxygen Species
Reactive oxygen species (ROS) are the byproducts generated through metabolic processes
of oxygen, and to maintain physiological concentrations of cellular ROS, abundant mechanisms
of ROS production, regulation and detoxification exist. Aside from generating ATP, mitochondria
function as a major internal source of ROS and continuously produce ROS under normal
physiological conditions (Indo et al., 2007; Venditti et al., 2013). ROS are also produced in
response to external factors. Phagocytotic cells including macrophages have been shown to
generate superoxide (O2
·–
) and nitric oxide (NO·) radicals as part of primary immune response (S.
Y. Kim et al., 2017; L. Liu et al., 2018; Zheng et al., 2022). Other cellular organelles, including
endoplasmic reticulum (ER) and peroxisomes, also contribute to cellular ROS production (del Rio
& Lopez-Huertas, 2016; Zorov et al., 2014). However, carrying unpaired electrons enables ROS
to oxidize biomolecules, such as lipids and proteins, altering their functions, and excessive ROS
production has been associated with the onset and/or progression of many neurodegenerative
diseases and cancer (W. Ahmad et al., 2017; Lan et al., 2016; Parkash et al., 2006; SarmientoSalinas et al., 2021).
Figure 1.1 Cellular Sources of Endogenous ROS Production.
Schematic showing cellular sources of superoxide (O2
·–
) and hydrogen peroxide (H2O2) production,
and in detail showing ETC generated O2
·– is converted into H2O2 by superoxide dismutase (SOD).

2
1.1.1 Superoxide
Mitochondria-generated ROS (mtROS) are mainly generated through electron transport
chain (ETC)-mediated oxidative phosphorylation, during which electrons leak and reduce
molecular oxygen to superoxide (O2
·–
) and/or hydrogen peroxide (H2O2). Mitochondrial complex
I, in which flavin mononucleotide (FMN) mediates electron transport from nicotine adenine
dinucleotide (NADH), and complex III, in which ubiquinone (Q) cycle carries electrons, are the
primary sites for superoxide production (Figure 1.1 (D. Han et al., 2001; Hirst et al., 2008)). Flavin
adenine dinucleotide (FAD) in mitochondrial complex II also contribute to superoxide production
to a lesser extent (Quinlan et al., 2012).
Other cellular components also generate superoxide independent of mitochondria. NADPH
oxidase (NOX), including NOX1-5, and dual oxidase (DUOX), including DUOX1 and DUOX2,
also produce superoxide through trans-membrane electron transport to molecular oxygen (Figure
1.1 (Morand et al., 2009; Sumimoto, 2008)).
1.1.2 Hydrogen Peroxide
Superoxide dismutase (SOD) family next catalyze the dismutation of O2
·– by manganese
dependent SOD (Mn-SOD) in the matrix and copper and zinc dependent SOD (Cu/Zn-SOD) in
the intermembrane spaces to produce H2O2 (Brand, 2016; Fridovich, 1995). Besides mitochondria
being the major source of H2O2, peroxisomes and endoplasmic reticulum (ER) also contribute to
H2O2 production (del Rio & Lopez-Huertas, 2016; Margittai et al., 2015). H2O2 can be transported
across cellular membranes through aquaporin (AQP) channels to other cellular comartments.
AQP8 and AQP11, for example, mediate H2O2 transport from mitochondria and ER respectively
to cytosol (Bestetti et al., 2020; Calamita et al., 2005).
1.1.3 Physiological role of ROS
Despite imbalanced ROS accumulation being the underlying causes for oxidative damages,
ROS on a physiological level have also been established to play messenger roles in regulation of
various biological processes and environment-induced responses. H2O2 generated by DUOX
system, for example, has been shown to act as endogenous messenger at plasma membrane in the
regulation of SKN-1 (NFE2-related factor 2 (Nrf2) in mammals) mediated longevity in C. elegans
(S.-K. Park et al., 2009). O2
·– generated in response to angiotensin II (ANG II) regulates gene

3
expression through activation of MAPK cascades pathway in endothelial cells (Brandes, 2003; K.-
Y. Yu et al., 2014). ANG II also increases O2
·– production in the central nervous system in
regulation of vasopressin secretion (Zimmerman et al., 2002). Altered cellular ROS concentration
induces significant promotion and/or inhibition of cellular processes. Mitochondria generated ROS
(mtROS) has been implicated in inducing hypoxia and autophagy response, and regulating cellular
differentiation and aging (Chandel et al., 1998; Y. Chen et al., 2009; Dai et al., 2014; Hamanaka
et al., 2013).
Reversible modifications of amino acid provide a prominent molecular mechanism by
which ROS induce site specific regulation of protein function, converting oxidant signal into
biological response. The thiol side chain of cysteine has the ability to adopt various oxidation states,
for example oxidation of the sulfhydryl group (-SH) of cysteine leads to sulfenic acid (Cys-SOH),
which can be subject to other modification (C. E. Paulsen & Carroll, 2013). Reaction of sulfenic
acid with another thiol side chain or glutathione (GSH) produce intermolecular or intramolecular
disulfide bond. It’s also been proposed that sulfenic acid can be transformed into sulfonamide with
nitrogen (Barford et al., 2003; Jhoti et al., 2003). Chronic oxidation of sulfenic acid further yields
sulfinic acid (Cys-SO2H) and sulfonic acid (Cys-SO3H) (C. E. Paulsen & Carroll, 2013).
1.2 Antioxidant Defense Systems
1.2.1 Antioxidants
Small antioxidant molecules, such as vitamin C, vitamin E and glutathione, convey nonenzymatic detoxification by quenching ROS (Anderson, 1998; KC et al., 2005; Niki, 2014).
Antioxidative enzymes including SODs, catalase (CAT), thioredoxin peroxidase (Trx-Ps) and
glutathione peroxidase (GSH-Ps) play a central role in enzymatic detoxification of cellular ROS.
While CAT directly converts H2O2 into H2O, thioredoxin peroxidase and glutathione peroxidase
utilize thioredoxin and glutathione as substrates respectively to reduce H2O2 to H2O (Kudin et al.,
2012; Miranda-Vizuete et al., 2000). Interestingly, nuclear DNA encodes for all the mitochondria
localized antioxidant enzymes, which are transported into mitochondria after protein translation
(X. Li et al., 2013).

4
Figure 1.2 Regulation of Nrf2/SKN-1 Activity in Mammals and C. elegans.
Schematic showing Nrf2/SKN-1 is downregulated by Keap1/WDR-23 and activated by oxidative
stress and kinases in mammals (Left) and C. elegans (Right).
1.2.2 The Nrf (nuclear factor erythroid-derived 2 related factors) transcription factor family
The major function of the mammalian Nrf family is to regulate resistance against oxidative
stress through expression of antioxidants. Nrf2, a member of basic region leucine zipper (bZip)
transcription factors, consists of seven Nrf2-Ech homology (Neh) domains, in which bZip
containing Neh1 domain recognizes the antioxidant response elements (ARE) for activation of
gene expression and Neh2 domain interacts with Kelch-like Ech associated protein 1 (Keap1) for
ubiquitination and degradation (Deshmukh et al., 2017; W. Li & Kong, 2009; Nguyen et al., 2003).
In addition to Keap1 mediated degradation, multiple kinases such as phosphoinositide-3 kinase
(PI3K), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) have been proposed
to promote Nrf2 activation in many studies (Bloom & Jaiswal, 2003; Sotolongo et al., 2020; L.
Wang et al., 2008; R. Yu et al., 2000). It has been proposed that Nrf2 activity and Nrf2 regulated
gene expression decreases with age, and loss of Nrf2 function has been characterized in
neurodegenerative disorders. In C. elegans, the Nrf2 homolog, SKN-1, is targeted by WD40 repeat
protein WDR-23 for degradation under normal condition (Choe et al., 2009). Oxidative stress and
PMK-1/MAPK for example have been shown to regulate SKN-1 activation by disrupting its
association with WDR-23 and promoting nuclear translocation in a similar manner (Figure 1.2,
(Hoeven et al., 2011; Kell et al., 2007; S.-K. Park et al., 2009)). SKN-1 in C. elegans has been
well established for its function in regulating expression of phase II detoxificant genes and
promoting resistance against ROS induced toxicity, skn-1 mutant are highly sensitive to oxidative

5
stress (An, 2003; Hoeven et al., 2011; Inoue et al., 2005; Oliveira et al., 2009). Besides its major
expression and function in defense against oxidative stress in the intestine, SKN-1 expressed in a
pair of sensory neurons ASI has been shown to modulate synaptic transmission and function in
response to oxidative stressor (Staab et al., 2013), and regulate dietary restriction (DR) induced
increase in longevity. (Bishop & Guarente, 2007).
Figure 1.3 DCVs Mediate Neuropeptide Signaling and Undergo SNARE Mediated
Exocytosis.
(Left) Schematic showing prepropeptide (precursor) processed by prohormone convertase 2 (PC2)
and carboxypeptidase E (CPE). (Right) Schematic showing dense core vesicle (DCV) going
through SNRAE and calcium dependent exocytosis.
1.3 Neuropeptide and Synaptic Transmission
1.3.1 Neuropeptide
Neuropeptides are small chains of amino acid that are typically synthesized and released
by neurons, and act through G protein-coupled receptors (GPCRs). Neuropeptide synthesis starts
from prepropeptides, which contain a signal peptide, cleavage sites and precursor peptide
sequences (Elphick et al., 2018). Signal peptide targets prepropeptides to the ER, where it’s
removed by signal peptidase to generate propeptides, and propeptides travel along to the Golgi
apparatus, where propeptides are processed proteolytically into bioactive peptides by prohormone
convertase PC1 and PC2 (Creemers et al., 1995). Peptides are then packed into dense core vesicles
(DCVs), in which further cleavage and modification such as C-terminal amidation finish synthesis
(Figure 1.2), and DCVs are transported to release sites at the plasma membrane. In neurons, DCVs

6
can be trafficked by kinesin dependent motors along microtubules to release sites in axons or the
soma. In axons, DCVs can be released at the synaptic terminals as well as at extra-synaptic sites
along the axon (Kwinter et al., 2009). Synaptic vesicles (SVs), on the other hand, contain fast
neurotransmitters, such as glutamate and acetylcholine, often co-exist with DCVs at synapses, and
their membrane and protein components are recycled locally at synaptic terminals for reuse.
1.3.2 Exocytosis of DCVs
Both SVs and DCVs are released through Soluble N-ethylmaleimide-sensitive fusion
protein (NSF) attachment protein receptors- (SNAREs-) and calcium-mediated exocytosis (Figure
1.2). Core SNARE components consist of synaptobrevin, which is an α-helix inserted into the
vesicle membrane, syntaxin and SNAP-25, which is an α-helixes inserted into the cell plasma
membrane. SNARE complex dock DCVS to the cell plasma membrane, which also initiates
membrane fusion through interactions between the transmembrane domains of synaptobrevin and
syntaxin; calcium (Ca2+) goes on stimulating exocytosis by activating synaptotagmin, allowing
DCVs to fuse with presynaptic membrane (Goda, 1997). It is well documented that exocytosis of
SVs and DCVs are regulated independently. While SVs are fused to cellular membrane at
specialized active zones, DCVs accumulate and release at synaptic terminal as well as cell body
and dendrite (Y. Park & Kim, 2009). DCVs also host a distinct membrane protein population,
offering molecular basis for selective exocytosis. It is been shown that DCV exocytosis is mediated
by different synaptotagmins comparing to SV associated synaptotagmins (Z. Zhang et al., 2011).
CAPS (Ca2+-dependent activator proteins for secretion) protein, which contains a SNARE binding
domain, is also reported to regulate DCV exocytosis but not SV exocytosis (Speese et al., 2007;
van Keimpema et al., 2017).
1.3.3 Neuropeptide Signaling
Released neuropeptides act through G-protein coupled receptors (GPCRs) on the plasma
membrane of target cells. Neuropeptides generally function at longer distances compared to
classical transmitters, They can function in an autocrine or paracrine manner on nearby targets, or
they can function in an endocrine manner on distant targets. Neuropeptides mediate intracellular
signaling not only in the nervous system but also between the nervous and other, “distal” tissues,
such as the intestine. Growing research have suggested that neuropeptide signaling regulates

7
various developmental and physiological pathways. Glucose-induced neuropeptide 26RFa
secretion from the small intestine regulates glucose homeostasis in mice by activating insulin
activity and secretion (Prévost et al., 2015). In Drosophila, germline stem cell proliferation, which
is regulated by gut secreted neuropeptide F, can also be modified by neuronal neuropeptide
signaling under parasitic infection (Ameku et al., 2018; Sadanandappa et al., 2021).
In addition, neuropeptide signaling also regulates stress activated responses, and impaired
neuropeptide signaling has been reported to attenuate neuroprotection in animal model of
neurodegenerative diseases. Neuropeptide Y, for example, was shown to be secreted by chromaffin
cells to regulate stress induced neuronal plasticity in mice (Q. Wang et al., 2013). Increasing the
level of neuropeptide Y, as well as acylated ghrelin and other neuropeptides, enhanced
neuroprotection in mouse model of Parkinson’s disease (PD), on the other hand, neurotoxin
induced PD mouse model showed reduced neuropeptide signaling (J. A. Bayliss et al., 2016; Choi
et al., 2021; Zheng et al., 2021). Given the characteristics of neuropeptide signaling, they’ve
emerged to be targeted for drug development in pharmacological and clinical research. Recent
study with neuropeptide-based drugs demonstrated potential ability to break down amyloid-β (Aβ)
aggregates and improve neural cognitive function (Mocanu et al., 2022)
1.4 Gut-Brain Axis
Aside from neuropeptide-controlled neuronal regulation on the gastrointestinal functions,
neuropeptides also mediate communication from the gut to the nervous system, and gut microbiota,
specifically, has been suggested to regulate neuronal activity and plasticity not only through direct
interaction with the gastrointestinal cells, but also through microbial metabolites, and gut secreted
neuropeptides and hormones. A study in Drosophila showed that a gut released peptide suppresses
arousability through dopaminergic neurons during sleep (Titos et al., 2023a). Gut secreted peptides
are also shown to modulate the neurocircuits in regulation of feeding behavior and glucose
metabolism (Borgmann et al., 2021; Hayashi et al., 2023). Peptidergic signaling along the gutbrain axis not only plays a major role in maintaining physiological balance, but likely regulates
stress induced responses and promotes organism-wide protection. Many intestinal peptides have
been demonstrated to promote inflammatory responses against pathogenic bacteria (CamposSalinas et al., 2014; Magliano et al., 2018). Altered gut-brain axis equilibrium has been associated
with progression of many pathological and neurodegenerative diseases (Carabotti et al., 2015;

8
Grenham et al., 2011; Mayer et al., 2022). Studies with microbiota transplantation demonstrated
that changes in gut microbiota was sufficient to alter induce or reverse disease associated
behavioral abnormality (M.-S. Kim et al., 2020; Zhu et al., 2020).
The gut microbiota community can produce a wide range of metabolites with various
bioactivities on human health. For example, the human gut bacteria derived carbohydrate active
enzymes (CAZymes) play an essential role in dietary fiber breakdown (El Kaoutari et al., 2013;
Tasse et al., 2010). An important class of bioproducts from carbohydrate fermentation is short
chain fatty acids (SCFAs), which have been implicated in maintaining glucose homeostasis and
obesity (Kimura et al., 2013), anti-inflammatory protection (W. Yang et al., 2020). Gut microbiota
has also been shown to produce multiple essential vitamins, which humans lack the biosynthetic
enzymes for besides dietary vitamin supply (LeBlanc et al., 2013). While influencing local
intestinal modulation with the host, gut bacterial metabolites also play significant physiological
role in the regulation of distal neuronal function and activity. Gut bacteria generated vitamin B12,
for instance, reduces cholinergic signaling from the nervous system through modulation of choline
availability in C. elegans, and changing gut bacteria with different vitamin B12 production was
sufficient to modulate cholinergic signaling and behavior (Kang et al., 2024).
1.5 Caenorhabditis elegans
Since first discovered by Sydney Brenner, C. elegans has been widely used in research in
developmental biology and neurobiology as an outstanding model system. C. elegans homologs
overlap with 60-80% of human genes (Kaletta & Hengartner, 2006). Several features including
rapid life cycle, small size and easy cultivation, and fully sequenced genome enable C. elegans to
be studied as genetic experimental system in various behavior, development and disease models
both at the whole organismal level and at the specifically identified cellular level.
C. elegans is free living nematode with 0.25 millimeter in length for newly hatched larvae
and 1 millimeter in length for adult, and exists majorly as hermaphrodites and rarely arisen males.
Development and growth from egg to egg-laying adult takes 3 days at 25°C going through 4 larval
stages (L1-L4) with inactive period of lethargus and molting in between each stage.
Hermaphrodites can produce around 300 self-fertilized progenies (Kaletta & Hengartner, 2006).

9
1.5.1 C. elegans simple yet complex nervous system
Adult hermaphrodite has 302 neurons whose axons and dendrites majorly synapse to each
other in the nerve ring encircling around the pharynx, the ventral nerve cord, the dorsal nerve cord
and the tail ganglia (J. G. White et al., 1986). C. elegans nervous system share several general
characteristics as the mammalian neurons. Consisting of sensory neurons, interneurons and motor
neurons, C. elegans produce most common neurotransmitters including acetylcholine (Ach),
glutamate (Glu), γ-amino butyric acid (GABA), dopamine and serotonin, and make more than
7,000 chemical synapses and neuromuscular junctions (J. G. White et al., 1986). Exocytosis of
neurotransmitters and neuropeptides are mediated through SNARE and calcium dependent
mechanism.
Compared to the mammalian neurons, C. elegans neurons express various potassium and
other ion channels except voltage-gated sodium channels (Bargmann, 1998). Individual neurons
in C. elegans normally express multiple G-protein coupled receptors (GPCRs), rather than a single
receptor, enabling multifunctionality that would be achieved by multiple neurons in mammals. A
pair of olfactory neurons AWC, for example, are structural and functional similar but have
asymmetric diversity of GPCR expression and discrimination towards odor attractiveness
(Bargmann & Wes, 2001). Olfactory sensory neurons AWC have been shown to synapse to first
layer interneurons AIB and AIY, which is under the control of thermosensory neurons AFD
(Bargmann et al., 2007; Kimata et al., 2012). Another pair of interneuron AVA are under the
peptidergic influence of both O2-sensing neurons and osmosensory neurons (Busch et al., 2012;
Laurent et al., 2015) The multifunctionality of C. elegans neurons compress complex nervous
system and neuronal network into small number of neurons. Research have revealed a variety of
neural circuits in chemosensory, thermosensory and mechanosensory signaling transduction.
1.5.2 C. elegans intestine and epithelial cells
C. elegans intestine consisting of semi-circular intestinal epithelial cells attached to each
other takes up majority of its somatic mass and is responsible for food digest, and synthesis and
metabolism of many macromolecules, as well as a major site of response and defense against
environmental toxins and stressors, and pothogens (Leung et al., 1999; Mullaney & Ashrafi, 2009;
Pukkila-Worley & Ausubel, 2012). Many key features of mammalian epithelial cells are conserved
in C. elegans. C. elegans epithelial cell also has a polarized structure: the apical domain with

10
microvilli surface between intestinal lumen and the basolateral domain with basement membrane
facing the pseudocoelom. A number of genes have been identified to be expressed exclusively in
one or the other domain, as well as nonuniformly between the anterior and posterior regions
(Fukushige et al., 2005; Göbel et al., 2004; Labouesse et al., 2000; Schroeder & McGhee, 1998;
Wu et al., 1998). Like in mammalian epithelial cells, nutrients and other molecules are taken up
by endocytosis or, through transporters and channels (Balamurugan et al., 2007; C. C.-H. Chen et
al., 2006; Kitaoka et al., 2013; Patel & Soto, 2013; Spanier, 2014), and many conserved homologs
of mammalian contents for exocytosis have been identified in C. elegans (Lin-Moore et al., 2021;
Mahoney et al., 2008; Sato et al., 2011), servicing intestine a great model for real time analysis of
synaptic trafficking on both single cell level and whole animal level with a variety of microscopic
techniques.

11
Chapter 2
Mitochondrial hydrogen peroxide positively regulates neuropeptide secretion
during diet-induced activation of the oxidative stress response
Qi Jia and Derek Sieburth
2.1 Summary
Mitochondria play a pivotal role in the generation of signals coupling metabolism with
neurotransmitter release, but a role for mitochondrial-produced ROS in regulating neurosecretion
has not been described. Here we show that endogenously produced hydrogen peroxide originating
from axonal mitochondria (mtH2O2) functions as a signaling cue to selectively regulate the
secretion of a FMRFamide-related neuropeptide (FLP-1) from a pair of interneurons (AIY) in C.
elegans. We show that pharmacological or genetic manipulations that increase mtH2O2 levels lead
to increased FLP-1 secretion that is dependent upon ROS dismutation, mitochondrial calcium
influx, and cysteine sulfenylation of the calcium independent PKC family member PKC-1.
mtH2O2-induced FLP-1 secretion activates the oxidative stress response transcription factor SKN1/Nrf2 in distal tissues and protects animals from ROS-mediated toxicity. mtH2O2 levels in AIY
neurons, FLP-1 secretion and SKN-1 activity are rapidly and reversibly regulated by exposing
animals to different bacterial food sources. These results reveal a previously unreported role for
mtH2O2 in linking diet-induced changes in mitochondrial homeostasis with neuropeptide secretion.
2.2 Introduction
Mitochondria are complex organelles with roles in energy metabolism, cell signaling, and
calcium homeostasis. In neurons and neuroendocrine cells, mitochondria are highly enriched at
neurotransmitter release sites where they play a critical role in coupling metabolic demands with
neurotransmitter and hormone secretion. Mitochondria supply a majority of cellular ATP, which
is critical for fueling vesicle transport, release, and recycling (Harris et al., 2012). Mitochondria
are also important for regulating synaptic strength through the production of ATP locally at
synapses (Rangaraju et al., 2014), and for regulating vesicle exocytosis through cytoplasmic

12
calcium buffering (Ly & Verstreken, 2006), and the production of metabolites (Kibbey et al., 2007;
Pozzan et al., 1997). Mitochondria are the primary cellular source of reactive oxygen species (ROS)
that arise as byproducts of mitochondrial oxidative phosphorylation. At high levels, ROS can cause
detrimental effects by oxidizing cellular components such as proteins, lipids and nucleic acids, and
prolonged ROS exposure is linked to neuropathological conditions including ageing and
neurodegenerative diseases (Massaad & Klann, 2011). However, at physiological levels, ROS can
function as intracellular signals to regulate normal neuronal functions including neuronal
homeostasis (Veal et al., 2007), plasticity (Accardi et al., 2014; Eric Klann, 1998; Knapp & Klann,
2002b; Kwan Yeop Lee et al., 2010), and activity (Avshalumov et al., 2005; Bhatla & Horvitz,
2015; G. Li et al., 2016; Ostrowski et al., 2014; Pouokam et al., 2009).
Hydrogen peroxide (H2O2) is an endogenous ROS produced by mitochondria through the
dismutation of superoxide (O2
.-
) by mitochondrial superoxide dismutase (SOD2), and also outside
the mitochondria by cytoplasmic oxidases (Dale E. Edmondson, 2014; Ma et al., 2017). H2O2 is
relatively stable and freely diffusible, and in neurons mitochondrial H2O2 levels are tightly
regulated by synaptic activity and by cellular antioxidant systems such as the
peroxiredoxin/thioredoxin system (Bell & Hardingham, 2011; Dan Dunn et al., 2015). H2O2
signaling has been implicated in regulating synaptic plasticity (Kamsler & Segal, 2003, 2004;
Ohashi et al., 2016; Takahashi et al., 2007) and neurotransmitter release (A R Giniatullin & R A
Giniatullin, 2003; Avshalumov et al., 2003; B. T. Chen et al., 2001a; Giniatullin et al., 2019;
Oswald et al., 2018), and altering H2O2 levels by over-expression of superoxide dismutase causes
defects in hippocampal LTP and learning paradigms in mice (Gahtan et al., 2001; Levin et al.,
1998; Thiels et al., 2000). Studies of insulin secretion in pancreatic beta cells have shown that
glucose-stimulated insulin secretion, which is positively regulated by ATP, is inhibited by the
removal of H2O2 (Corinne Leloup et al., 2009; Pi et al., 2007). Despite evidence for roles for H2O2
signaling in regulating DCV secretion from neuroendocrine cells, the subcellular source(s) and
mechanism of action of H2O2 in regulating transmitter secretion are poorly understood.
Neuropeptides are conserved modulators of behavior, physiology and homeostasis. Studies
in C. elegans have established important roles for neuropeptides release from neurons in activating
stress responses in distal tissues (Apfeld & Kenyon, 1998; Li-Wa Shao Rong Niu Ying Liu, 2016;
Q. Zhang et al., 2018). For example, neuropeptide secretion from specific neurons regulates the
mitochondrial unfolded protein response (UPRmt) and the heat shock response in the intestine

13
through an inter-tissue signaling mechanism (S. Kim & Sieburth, 2018a; Li-Wa Shao Rong Niu
Ying Liu, 2016; Veena Prahlad & Richard I. Morimoto, 2011). In addition, a number of
neuropeptide signaling pathways are linked with sensory regulation of fat metabolism (Cohen et
al., 2009; Palamiuc et al., 2017), and longevity (Maier et al., 2010). Neuropeptides are packaged
into dense core vesicles (DCVs) in neuronal somas, and DCVs are subsequently trafficked to
release sites where they undergo soluble NSF attachment protein receptor (SNARE) and calciumdependent exocytosis. The release properties of DCVs and synaptic vesicles (SVs) differ in several
important respects including differences in release sites, calcium dependence, and release kinetics
(Sugita, 2008). Unlike SVs, the mechanisms underlying the regulation of DCV release are not well
defined.
Here we identify a role for endogenously produced H2O2 in promoting the secretion of the
FMRFamide-related neuropeptide, FLP-1, from a pair of interneurons (AIY), to activate the
antioxidant response in distal tissues in C. elegans. H2O2 originating from axonal mitochondria
selectively regulates FLP-1 release, and the mitochondrial peroxiredoxin-thioredoxin antioxidant
system in AIY functions to negatively regulate FLP-1 release. Changes in the bacterial diet of C.
elegans lead to rapid changes in endogenous H2O2 levels in AIY interneurons, FLP-1 secretion,
and antioxidant response activation in distal tissues. Finally, the effects of H2O2 on neuropeptide
secretion rely on a putative reactive cysteine residue in the calcium-independent protein kinase C
family member, PKC-1. Our results suggest that H2O2-induced neuropeptide secretion is a
mechanism by which organisms can rapidly and reversibly control antioxidant activity in response
to changing environmental inputs.
2.3 Results
2.3.1 Neuropeptide signaling activates the intestinal oxidative stress response
Nrf2 and its C. elegans ortholog, SKN-1, are evolutionarily conserved transcription factors
that are master regulators of the antioxidant response that control the expression of antioxidant
enzymes in response to oxidative stress. Nrf2 and SKN-1 are activated by ROS that originate cell
autonomously (Ewald et al., 2017; Hourihan et al., 2016), and can also be activated cellnonautonomously by ROS originating from neighboring neurons undergoing oxidative stress
(Baxter & Hardingham, 2016; Chew et al., 2015; McCallum et al., 2016). To determine the

14
mechanisms underlying the neuronal control of SKN-1 activation, we first sought to identify the
neuronal signal(s) that promotes the antioxidant response. Juglone is a naturally occurring
mitochondrial toxin that generates superoxide anion radicals (T. Ahmad & Suzuki, 2019; M. T.
Paulsen & Ljungman, 2005) and juglone treatment of C. elegans increases ROS levels (de Castro
et al., 2004), and activates the SKN-1-dependent antioxidant response (Przybysz et al., 2009).
Acute treatment with juglone for four hours leads to mild toxicity in adults 24 hours later, and loss
of skn-1 significantly increases the toxicity of juglone treatment (Figure 2.1A and (Hartwig et al.,
2009; S.-K. Park et al., 2009)). We found that mutants with impaired biosynthesis or secretion of
the classical neurotransmitter acetylcholine (unc-17/VChAT), GABA (unc-25/GAD), or glutamate
(eat-4/VGAT), or the biogenic amine serotonin (tph-1/TPH), dopamine (cat-2/TH), or octopamine
(tdc-1/DDC or tbh-1/DBH) exhibited similar survival following juglone treatment as wild type
controls. However, mutants with impaired neuropeptide biogenesis (egl-3/PC2, egl-21/CPE, or
sbt-1/7B2), or secretion (pkc-1/PKC) exhibited significantly decreased survival following juglone
treatment (Figure 2.1A). egl-3 encodes prohormone convertase 2, which performs the first
cleavage step in the maturation of neuropeptide precursors into bioactive peptides in DCVs
(Husson et al., 2006; Jacob & Kaplan, 2003). egl-3 mutants cultured in media containing juglone
exhibited significantly decreased survival compared to wild type controls over all time points
examined (Figure 2.1B). egl-3 mutants also exhibited decreased survival following exposure to
the SKN-1-activating oxidants thimerosal or sodium arsenite, but were not sensitive to toxicity
cause by heat stress (Supplementary Figure 2.1A-C, (Oliveira et al., 2009; Sharpe et al., 2012; The
PLOS Genetics Staff, 2014)).
Upon activation, SKN-1 translocates into the nucleus of intestinal cells where it regulates
the expression of antioxidant genes, including gst-4/glutathione-S-transferase (An, 2003). egl-3
mutants were defective in juglone-induced SKN-1::GFP nuclear translocation and jugloneinduced increases in intestinal expression of the Pgst-4::gfp reporter (Figures 2.1C and D). egl-3
mutants were not defective in the juglone-induced activation of the lifespan extension transcription
factor DAF-16 (Hartwig et al., 2009) or the unfolded protein response reporter Phsp-4::gfp
(Supplementary Figures 2.1E and F (Calfon et al., 2002)). Restoring egl-3 cDNA expression
selectively in a subset of neurons (under the flp-1 promoter, see below) fully rescued the juglone
survival and juglone-induced Pgst-4::gfp expression defects of egl-3 mutants, whereas expressing
egl-3 cDNA in the intestine failed to rescue juglone survival (Figures 2.1D and E). Together these

15
results reveal a specific role for neuropeptide signaling originating from the nervous system in
activating SKN-1 in the intestine and the antioxidant response.
2.3.2 The FLP-1 neuropeptide activates the intestinal oxidative stress response
To identify the neuropeptide(s) involved in SKN-1 activation, we first conducted a pilot
RNA interference (RNAi) screen (in an eri-1; lin-15b background to enhance RNAi knockdown
of neuronal genes (Sieburth et al., 2005; D. Wang et al., 2005)) of 88 candidates among the 111
neuropeptide-like genes encoded by C. elegans for altered sensitivity to toxicity by juglone (Table
2.1). Knockdown of six neuropeptide genes significantly increased sensitivity to juglone toxicity
compared to empty vector controls (Table 2.1). Among these, the FMRFamide-related peptide flp1 emerged as a strong candidate because putative null flp-1(ok2811) mutants exhibited
significantly increased sensitivity to juglone-mediated toxicity compared to wild type controls
(Figures 2.1B and E). flp-1 mutants were also significantly more sensitive to toxicity caused by
the oxidants thimerosal and sodium arsenite, but were not sensitive to toxicity caused by heat stress
or activation of the unfolded protein response (Supplementary Figures 2.1A-D). flp-1 mutations
significantly attenuated juglone-induced SKN-1::GFP nuclear translocation and Pgst-4::gfp
expression, without detectably altering baseline SKN-1 activity in the absence of stress (Figures
2.1C and D). The defects in survival and SKN-1 activation of flp-1 mutants were fully rescued by
expression of flp-1 genomic DNA under control of its endogenous promoter fragment (Pflp-1::flp1, Figure 2.1E). In addition, the rescue of the juglone-induced Pgst-4::gfp expression defects of
egl-3 mutants by egl-3 transgenes was completely blocked by flp-1 mutations (Figure 2.1D). To
further confirm that flp-1 is an activator of the oxidative stress response, we generated a flp-1
overexpression strain (under its own promoter) and found that these animals were significantly
more resistant to juglone toxicity than non-transgenic controls (Figure 2.1F). flp-1 overexpression
also increased juglone-induced intestinal Pgst-4::gfp expression without altering Pgst-4::gfp
expression in the absence of stress (Supplementary Figure 2.1G). Together these results reveal that
FLP-1 functions in the nervous system to activate the oxidative stress response by specifically
regulating stress-induced SKN-1 activation in the intestine. Because the defects in juglone
responsiveness of flp-1 mutants were significantly less severe than those observed in egl-3 mutants
(Figures 2.1B-E), additional neuropeptides processed by EGL-3 are likely to contribute to the
antioxidant response.

16
FLP-1 is predicted to be processed into nine mature peptides that share a common Cterminal seven amino acid consensus motif (Figure 1g, (Li & Kim, 2008)). Truncated flp-1
transgenes that encode just one peptide (FLP-1(P1)), two peptides (FLP-1(P1+P2)) or, at least
three peptides (FLP-1(P1+P2+P3), partially rescued, nearly completely rescued, or fully rescued
the sensitivity to juglone-mediated toxicity of flp-1 mutants, respectively (Figure 2.1G). These
results indicate that the mature FLP-1 peptides may be functionally equivalent, suggesting that
FLP-1 peptide levels rather than their specific sequences may determine the extent to which FLP1 confers stress protection.
2.3.3 FLP-1 secretion from the AIY interneuron activates the oxidative stress response
flp-1 is expressed in a subset of head interneurons, and has been reported to regulate lipid
homeostasis, reproduction, and behavioral plasticity in response to changes in a number of
environmental cues (Buntschuh et al., 2018; Jeong & Paik, 2017; Mutlu et al., 2020; L. S. Nelson
et al., 1998). We investigated in which neuron(s) flp-1 functions to activate the oxidative stress
response. FLP-1 reporters are expressed in six pairs of interneurons (AVK, AVA, AVE, RIG, AIY
and AIA), and two pairs of motoneurons (RMG and M5) (K. Kim & Li, 2004). Several results
indicate that FLP-1 secretion from the AIY interneurons (hereafter referred to as AIY) positively
regulates the oxidative stress response. First, we used mito-miniSOG, which is a mitochondrially
targeted light-activated singlet oxygen generator (Yingchuan B. Qi et al., 2012), to genetically
ablate flp-1 expressing cells. We found that AIY ablation using four different promoters that each
drive expression in AIY decreased survival following juglone treatment to a similar extent as flp1 mutations, whereas ablation of other flp-1-expressing cells had no effect on survival (Figure
2.2A). Specifically, animals expressing mito-miniSOG selectively in AIY (using the ttx-3
promoter, (Wenick & Hobert, 2004), which we hereafter used to drive AIY expression), exhibited
normal sensitivity to juglone toxicity in the absence of blue light, but blue light illumination of
adults for 30 minutes, which resulted in efficient AIY ablation (Supplementary Figure 2.2A), led
to a significant increase in sensitivity to juglone toxicity 24 hours later (Figure 2.2A). Second,
expression of tetanus toxin (TeTx), which disrupts SNARE-mediated exocytosis (Schiavo et al.,
1992), in AIY conferred significant sensitivity to juglone toxicity compared to non-transgenic
controls (Figure 2.2B). Third, the juglone sensitivity of egl-3 mutants was rescued by expressing
egl-3 selectively in AIY, and rescue was abolished by flp-1 mutations (Figure 2.2C). Finally,

17
expressing flp-1 selectively in AIY fully rescued the sensitivity to juglone toxicity of flp-1 mutants
(Figure 2.2D). AIY has been shown to integrate thermal and chemical cues from sensory neurons
to regulate behavioral and metabolic plasticity (Bono & Villu Maricq, 2005). Our results reveal a
previously undescribed function for AIY in regulating the oxidative stress response in peripheral
tissues.
To determine whether FLP-1 is secreted from AIY, we generated transgenic animals
expressing FLP-1::Venus fusion proteins in AIY. AIY is a unipolar neuron located in the head that
extends a single axon anteriorly from the soma, where it forms cholinergic synapses to multiple
targets in the nerve ring. FLP-1::Venus adopted a punctate pattern of fluorescence in the AIY soma
and in the axon. In axons, puncta were sparsely distributed and were most prominent at the axonal
bend and tip (Figure 2.2E), where cholinergic presynaptic terminals are located (Colon-Ramos et
al., 2007; J. G. White et al., 1986). FLP-1::Venus is secreted from AIY because fluorescence was
also observed in coelomocytes (marked with mCherry, Figures 2.2E and F), which are scavenger
cells that take up material released into the pseudocoelom into endocytic compartments by bulk
endocytosis. Changes in steady-state fluorescence intensity in coelomocytes is widely used as a
measure of efficacy of neuropeptide secretion (Ailion et al., 2014; Ch’ng et al., 2008; Sieburth et
al., 2006). Venus fluorescence intensity in coelomocytes of FLP-1::Venus expressing animals was
significantly reduced by unc-31/CAPS or by unc-2/voltage gated calcium channel mutations,
which impair calcium-dependent DCV exocytosis (Figure 2.2F, (Ch’ng et al., 2008; Speese et al.,
2007)). On the other hand, AIY-specific activation of the light-gated cation channel,
Channelrhodopsin2 (ChR2, which increases transmitter release (Nagel et al., 2005)) significantly
increased FLP-1::Venus coelomocyte intensity (Figure 2.2G). FLP-1::Venus coelomocyte
fluorescence was significantly reduced by disruption of electron transport chain function
specifically in AIY (gas-1 or mev-1 mutations (Ishii et al., 1998; Kayser et al., 2001)) or glycolysis
(gpd-3 mutations (Huang et al., 1989)) (Supplementary Figures 2.2B and C), in agreement with
reports that ATP generated by oxidative phosphorylation and glycolysis are critical for DCV
secretion (Maechler & Wollheim, 1998; Zhao et al., 2018). Together these results show that FLP1::Venus is secreted from AIY via calcium- and ATP-dependent exocytosis of DCVs.

18
2.3.4 FLP-1 antioxidant signaling is mediated by the NPR-4 GPCR in the intestine
Neuropeptides exert their biological effects by binding to G-protein coupled receptors
(GPCRs) on target cells. We reasoned that we could identify the FLP-1 receptor by screening for
GPCRs that cause sensitivity to juglone toxicity when knocked down. frpr-7 and npr-6 are
neuropeptide GPCRs reported to function downstream of FLP-1 to regulate movement (Oranth et
al., 2018). We found that null frpr-7 or npr-6 mutants exhibited normal sensitivity to juglone
toxicity (Table 2.2), suggesting that FLP-1-mediated activation of the oxidative stress response
occurs through a different GPCR. We conducted an RNAi screen of an additional 21 GPCRs that
are reported to be expressed in the intestine for altered juglone sensitivity (Table 2.2), and we
subsequently confirmed that null mutations in npr-4/GPCR, significantly decreased survival
following juglone-treatment compared to wild type controls (Figure 2.3A). npr-4 was previously
implicated in regulating foraging behavior, food preference, and fat homeostasis (Bhardwaj et al.,
2018; McCallum et al., 2016; Mutlu et al., 2020; Y. Yu et al., 2016), and npr-4 mediates the effects
of flp-1 in fat regulation (Mutlu et al., 2020). The increased sensitivity to juglone-induced toxicity
of npr-4 mutants was similar to that of flp-1 mutants, and flp-1; npr-4 double mutants exhibited
similar sensitivity to juglone as flp-1 or npr-4 single mutants (Figure 2.3A and Table 2.2). The
resistance to juglone-induced toxicity by flp-1 overexpression in AIY was abolished by npr-4
mutations (Figure 2.3B). These results suggest that NPR-4 functions downstream of FLP-1 in a
common genetic pathway to promote survival.
npr-4 is reported to be expressed in the nervous system, coelomocytes, and the intestine
(Y. Yu et al., 2016). Transgenic expression of npr-4 cDNA in the intestine (using the ges-1
promoter) fully restored protection against juglone-induced toxicity, whereas expression of npr-4
cDNA in either the coelomocytes (using the ofm-1 promoter) or nervous system (using the rab-3
promoter) did not rescue (Figure 2.3C). npr-4 mutants had defects in juglone-induced Pgst-4::gfp
intestinal expression that were similar to those of flp-1 mutants, and were not enhanced by flp-1
mutations. The gst-4 expression defects of npr-4 mutants were fully rescued by intestinal npr-4
cDNA expression (Figure 2.3D). Finally, npr-4 mutations also blocked the enhanced jugloneinduced Pgst-4::gfp expression resulting from AIY flp-1 overexpression (Figure 2.3D). Together
these data support a model whereby neuronal FLP-1 secretion regulates SKN-1 activation and the
oxidative stress response through the activation of NPR-4 in the intestine.

19
2.3.5 FLP-1 secretion is positively regulated by mtH2O2
We hypothesized that if FLP-1 functions to activate the antioxidant response, FLP-1
secretion from AIY might be regulated by mtROS themselves (Figure 2.4A). To monitor ROS
levels in AIY, we used the H2O2 sensor HyPer, which is a dual ratio-metric sensor that increases
fluorescence in response to levels of H2O2 as low as 0.1uM, (Back et al., 2012; Belousov et al.,
2006). HyPer targeted to mitochondria in AIY (Pttx-3::mito-HyPer) adopted a punctate pattern of
fluorescence in axons that colocalized with the mitochondrial marker TOMM-20::mCherry
(Supplemenraty Figure 2.3A). Juglone treatment for ten minutes increased mito-HyPer punctal
fluorescence intensity 2.5-fold, without altering TOMM-20::mCherry intensity (Figure 2.4B and
Supplementary Figure 2.3B). sod-2 encodes the neuronal mitochondrial superoxide dismutase2
(SOD2), which converts superoxide anions into H2O2 (Y. Wang et al., 2018). sod-2 mutants
exhibited similar mito-HyPer punctal fluorescence intensity as wild type controls. However,
juglone treatment failed to increase mito-HyPer fluorescence intensity in sod-2 mutants (Figure
2.4B). These results confirm that mito-HyPer is a specific sensor for mtH2O2, and they indicate
that juglone treatment increases mitochondrial H2O2 levels in AIY axons through the dismutation
of superoxide by SOD-2.
We found that juglone treatment caused a dose-dependent increase in coelomocyte
fluorescence in FLP-1::Venus-expressing animals that was maximal at 300uM (Supplementary
Figure 2.3C), the same concentration used for toxicity assays. A time course revealed that juglone
treatment for as little as two minutes caused a significant increase in coelomocyte fluorescence in
FLP-1::Venus expressing animals and 10 minute treatment resulted in a maximal response
(Supplementary Figure 2.3D). Similarly, a 10 minute treatment with sodium arsenite significantly
increased FLP-1::Venus secretion compared to untreated controls (Supplementary Figure 2.3E).
Juglone treatment did not detectably alter the distribution FLP-1::Venus fluorescence in AIY
axons or somas (Supplementary Figure 2.3F). sod-2 mutants, which exhibited slightly reduced
FLP-1::Venus secretion, exhibited no increase in FLP-1::Venus secretion following juglone
treatment (Figure 2.4C). Transgenes expressing wild type sod-2 cDNA specifically in AIY fully
restored juglone-induced FLP-1 secretion to sod-2 mutants, whereas transgenes expressing sod-2
variants lacking their mitochondrial localization signal, which disrupted the mitochondrial
localization of SOD-2::GFP fusion proteins (Supplementary Figure 2.3G), failed to restore juglone
responsiveness to sod-2 mutants (Figure 2.4C). unc-31/CAPS mutations, which blocked the

20
juglone-induced increase in FLP-1::Venus secretion (Figure 2.2G), did not block juglone-induced
increases in mitochondrial H2O2 levels in AIY (Figure 2.4B). The rapid coelomocyte fluorescence
increase caused by juglone treatment eliminated the possibility that it arose through increased flp1::Venus transgene expression or protein synthesis. Juglone treatment did not impact constitutive
secretion of signal sequence-tagged Venus (Pttx-3::ss-Venus) from AIY, (Supplementary Figure
2.3H, (Hummer et al., 2017)), suggesting that juglone does not generally boost secretion, nor does
it alter bulk endocytosis of coelomocytes. We conclude that juglone-induced ROS production leads
to a rapid increase in FLP-1 secretion from DCVs in AIY.
To address the specificity of juglone on DCV secretion, we first examined secretion of
FLP-18, which is a FMRF-like neuropeptide protein whose release from AIY regulates foraging
behavior and fat metabolism (Cohen et al., 2009). Unlike flp-1 mutants, flp-18 mutants were not
sensitive to juglone toxicity (Supplementary Figure 2.3I). FLP-18::mCherry fusion proteins
adopted a punctate pattern of localization in AIY axons that was similar to that of FLP-1::Venus
(Supplementary Figure 2.3J), and FLP-18::Venus secretion from AIY was dependent upon unc31/CAPS (Supplementary Figure 2.3K). However, FLP-18::Venus secretion from AIY was not
significantly altered by juglone treatment (Supplementary Figure 2.3K). Second, we examined
FLP-1 secretion from the AVK interneuron, which secretes FLP-1 to regulate locomotion (Oranth
et al., 2018). Animals expressing FLP-1::Venus selectively in AVK (under the Pflp-1(513bp)
promoter (Oranth et al., 2018)), exhibited a punctate pattern of fluorescence along AVK axons,
and FLP-1::Venus secretion from AVK was dependent upon unc-31/CAPS and increased after
food withdrawal (Supplementary Figures 2.3L-N), as reported. FLP-1::Venus secretion from AVK
was not significantly different in animals treated with juglone compared to untreated controls
(Supplementary Figure 2.3M). Finally, we examined secretion of the insulin-like protein INS22::Venus, which is released from motor neurons in an unc-31/CAPS dependent manner (Sieburth
et al., 2006). Juglone treatment had no detectable effect on coelomocyte fluorescence intensity of
INS-22::Venus-expressing animals (Supplementary Figure 2.3O). Together these results indicate
that the effect of juglone on DCV secretion is dictated by cellular context as well as the identity of
DCV cargo, and that juglone may exhibit specificity in promoting FLP-1 secretion from AIY.
We next considered whether ROS production cell-autonomously in AIY could promote
FLP-1 secretion. To test this, we activated mito-miniSOG with blue light for short times, which
elicits ROS production without cell death (Wojtovich & Foster, 2014). As expected, a one minute

21
light exposure of animals expressing mito-miniSOG selectively in AIY resulted in a significant
elevation of mitochondrial H2O2 levels in AIY ten minutes later (Figure 2.4B), without altering
mitochondrial mass (Supplementary Figure 2.3B). We detected a corresponding ~2-fold increase
in FLP-1::Venus secretion following mito-miniSOG activation, similar to the increase caused by
juglone or H2O2 (Figure 2.4D). Mito-miniSOG activation failed to induce FLP-1 secretion in the
absence of sod-2 or unc-31 (Figure 2.4D). These results indicate that ROS produced by
mitochondria in AIY itself can promote FLP-1 secretion.
To determine whether H2O2 can elicit FLP-1 secretion, we treated animals with H2O2,
which has been shown to reach micromolar levels in C. elegans tissues within minutes following
exposure (Back et al., 2012). We found that H2O2 treatment for 10 minutes robustly induced FLP1 secretion to a similar extent as juglone treatment, and the increase was completely blocked by
unc-31 mutations (Figure 2.4E). Unlike juglone treatment, H2O2 increased FLP-1 secretion in the
absence of sod-2 (Figure 2.4E). Thus, exogenous H2O2 can bypass the requirement for
mitochondrial H2O2 production to rapidly induce FLP-1 secretion from AIY.
2.3.6 Mitochondrial calcium influx is required for ROS-induced FLP-1 secretion
Mitochondrial calcium uptake regulates neurotransmitter and neuropeptide secretion in a
variety of systems (Kennedy & Wollheim, 1998; Ly & Verstreken, 2006; Pozzan et al., 1997). We
found that juglone treatment significantly increased mitochondrial calcium levels (as measured by
mito-GCaMP3) in wild type animals, sod-2/SOD2 mutants, and unc-2/VGCC mutants (Figure
2.5A). mcu-1 encodes the C. elegans ortholog of the mitochondrial calcium uniporter (Rizzuto et
al., 2012), and mcu-1 mediates uptake of calcium and ROS production in mitochondria of C.
elegans skin in response to injury (Xu & Chisholm, 2014). In the absence of juglone, mcu-1 null
mutants had normal mitochondrial calcium levels (Figure 2.5A), mtH2O2 levels (Figure 2.5B) and
FLP-1::Venus secretion (Figure 2.5C). However, juglone treatment failed to increase
mitochondrial calcium levels, mtH2O2 levels, or FLP-1::Venus secretion in mcu-1 mutants. The
FLP-1::Venus secretion defects of mcu-1 mutants were fully rescued by expressing mcu-1 cDNA
selectively in AIY (Figure 2.5D). micu-1 encodes the ortholog of MICU1 (mitochondrial calcium
uptake 1), a regulatory subunit of MCU-1 that is proposed to promote the retention of accumulated
calcium inside the mitochondrial matrix (Dong et al., 2017; Madreiter-Sokolowski et al., 2016).
As expected, disruption of micu-1 blocked juglone-induced FLP-1::Venus secretion

22
(Supplementary Figure 2.4A). vdac-1 encodes the voltage dependent anion channel that transports
ATP and other small metabolites across the outer mitochondrial membrane (Camara et al., 2017).
vdac-1 disruption did not significantly alter juglone-induced FLP-1::Venus secretion
(Supplementary Figure 2.4A). These results suggest that mitochondrial calcium entry in AIY is
critical for juglone-induced increases in mtH2O2 levels and FLP-1 secretion.
2.3.7 TRX-2/thioredoxin and PRDX-3/peroxiredoxin regulate AIY mtH2O2 levels and FLP-1
secretion
To determine whether H2O2 produced by mitochondria under normal physiological
conditions contributes to FLP-1 secretion, we sought to genetically increase mtH2O2 levels in AIY
without experimentally inducing mitochondrial stress. The peroxiredoxin-thioredoxin system is an
evolutionarily conserved antioxidant system that specifically removes H2O2, thereby antagonizing
H2O2 action. Peroxiredoxin converts H2O2 to water by catalyzing the transfer of oxidizing
equivalents from H2O2 to a reactive cysteine residue at the peroxiredoxin active site. Thioredoxins,
in turn, reduce oxidized peroxiredoxins for reuse to consume additional H2O2 molecules (MirandaVizuete et al., 2000; Netto & Antunes, 2016). Once oxidized, thioredoxins themselves are reduced
and thereby recycled by thioredoxin reductases (TrxR), utilizing NADPH as a cofactor (Figure
2.5E, (Arnér & Holmgren, 2000)). C. elegans encodes one ortholog each of mitochondrial
peroxiredoxin, PRDX-3 (Ranjan et al., 2013), mitochondrial thioredoxin, TRX-2 (Cacho-Valadez
et al., 2012), and mitochondrial thioredoxin reductase, TRXR-2 (Cacho-Valadez et al., 2012;
Lacey & Hondal, 2006). Null mutants in prdx-3, trx-2 or trxr-2 exhibited significantly increased
FLP-1::Venus secretion compared to wild type controls (Figure 2.5F), whereas mutants of the
cytoplasmic peroxiredoxin, prdx-2 exhibited wild type FLP-1 secretion (Figures 2.5G,
Supplementary Figure 2.4B and C). The increase in FLP-1 secretion of trx-2 mutants was blocked
by sod-2 mutations but was not further increased by juglone treatment (Figure 2.5G). trx-2 or prdx3 mutants also exhibited increased mito-HyPer punctal fluorescence intensity in AIY compared to
wild type controls (Figure 2.4B). These results suggest that the peroxiredoxin-thioredoxin system
negatively regulates FLP-1 secretion from AIY by reducing mtH2O2 levels.
trx-2 is expressed in a small number of neurons and is highly expressed in AIY (CachoValadez et al., 2012). Expression of full length trx-2 cDNA selectively in AIY fully restored wild
type FLP-1::Venus secretion to trx-2 mutants (Figure 2.5G). TRX-2 contains a mitochondrial

23
localization signal (MLS) on its N-terminus, and a conserved thiol-disulfide active site that
contains reactive cysteines used for peroxiredoxin reduction (Supplementary Figure 2.4D).
Deletion of the TRX-2 MLS disrupted the mitochondrial localization of TRX-2::GFP fusion
proteins in AIY axons (Supplementary Figure 2.4E), and trx-2(∆MLS) transgenes failed to rescue
the increased FLP-1::Venus secretion defects of trx-2 mutants (Figure 2.5G). Similarly, trx-2
transgenes with mutations in the active site predicted to impair catalytic activity (TRX-2(∆CAT),
(Cacho-Valadez et al., 2012)) failed to rescue the FLP-1::Venus secretion defects of trx-2 mutants
(Figure 2.5G). These results reveal that TRX-2 functions in AIY mitochondria to catalyze the
removal of mtH2O2 and inhibit FLP-1 secretion, and they point to a critical role of endogenously
produced mtH2O2 in AIY as a signaling cue that positively regulates FLP-1 secretion.
2.3.8 Localized H2O2 production by axonal mitochondria regulates FLP-1 secretion
Since H2O2 is readily neutralized by cellular antioxidant mechanisms in the cytosol, we
hypothesized that if mtH2O2 promotes FLP-1 secretion, then mitochondria should be positioned in
close proximity to DCV release sites in AIY axons. Consistent with this, we found that TOMM20::mCherry puncta overlapped with FLP-1::Venus puncta along the entire length of AIY axons
(Figure 2.6A). To determine whether proximity of mitochondria and DCVs is important for FLP1 secretion, we examined mutants in which axonal mitochondria and DCVs had been genetically
separated. ric-7 encodes an adapter protein that is selectively required for the anterograde transport
of mitochondria to axons from the soma (Rawson et al., 2014). ric-7 mutations nearly eliminated
axonal TOMM-20::mCherry puncta without detectibly altering axonal FLP-1::Venus puncta,
implying a specific disruption of mitochondrial but not DCV trafficking in AIY axons (Figure
2.6A). ric-7 mutations significantly reduced baseline FLP-1::Venus secretion from AIY compared
to wild type controls, consistent with its reported role in positively regulating DCV secretion (Hao
et al., 2012). In addition, ric-7 mutations abolished juglone-induced FLP-1::Venus secretion
(Figure 2.6B). Expression of a chimeric protein (UNC-116::TOM-7, aka mito-truck), which
functions as an adapter between mitochondria and microtubules that can restore mitochondrial
trafficking to ric-7 mutants (Supplementary Figure 2.5, (Rawson et al., 2014; Zhao et al., 2018)),
rescued both the mitochondrial trafficking defects and juglone-induced FLP-1::Venus secretion
defects of ric-7 mutants (Figures 2.6A and B).

24
unc-104/KIF1A encodes a kinesin motor protein that mediates anterograde transport of
DCVs from the soma, where they are generated, into axons (Zahn et al., 2004), and unc-104 is
required for DCV secretion (Laurent et al., 2018). unc-104 mutations reduced axonal FLP1::Venus puncta without detectibly altering TOMM-20::mCherry puncta, indicative of a specific
disruption of DCV trafficking from the soma (Figure 2.6A). We found that unc-104 mutations
significantly reduced baseline FLP-1::Venus secretion from AIY compared to wild type controls,
and abolished juglone-induced FLP-1::Venus secretion. Double mutants lacking both ric-7 and
unc-104, which were largely devoid of both axonal mitochondria and DCVs (Figure 2.6A), had
defects in baseline and juglone-induced FLP-1::Venus secretion that were no more severe than
single mutants (Figure 2.6B). Together, these results suggest that the proximity of mitochondria
and FLP-1-containing DCVs in axons is critical for juglone-induced FLP-1 secretion. Importantly,
we found that the increase in FLP-1::Venus secretion caused by mito-miniSOG activation was
blocked by ric-7 mutations (Figure 2.4D). However, ric-7 mutations failed to block H2O2-induced
FLP-1::Venus secretion (Figure 2.6C), indicating that exogenous H2O2 can bypass the need for
axonal mitochondria. These results further support the idea that mtH2O2 generated locally at DCV
release sites in axons is essential for FLP-1 release.
2.3.9 PKC-1/PKC mediates the effects of H2O2 on FLP-1 secretion
What are the molecular mechanisms by which H2O2 promotes FLP-1 secretion and the
antioxidant response? H2O2 modifies reactive cysteine residues on target proteins, converting the
thiol groups (-SH) into sulfenic acid (-SOH), in a process known as sulfenylation (Figure 2.7A).
Sulfenylation is reversible and typically changes the conformation and/or activity of proteins. We
hypothesized that H2O2 might sulfenylate a target protein that regulates DCV exocytosis. PKCs
are a family of serine/threonine kinases that are implicated in the regulation of secretory vesicle
exocytosis and synaptic plasticity (Gillis et al., 1996; Nagy et al., 2002; Naoto Saitoh et al., 2001;
Scepek et al., 1998; Stevens & Sullivan, 1998; Yan Yang et al., 2002; Yu A. Chen et al., 1999),
and PKC family members are important intracellular targets for the effects of H2O2 on neuronal
function (Knapp & Klann, 2002a, 2002b; Servitja et al., 2000; Suzuki et al., 1997). pkc-1/ PKCε/η
encodes the sole member of the calcium-independent PKC subfamily. pkc-1 positively regulates
neuro-secretion from a number of neuron subtypes (Kitazono et al., 2017; Macosko et al., 2009;
Sieburth et al., 2006; Y. Yu et al., 2018), and pkc-1 has been implicated in mtROS signaling (H.

25
Chen et al., 2017; Zubovych et al., 2010). We found that pkc-1 null mutations significantly reduced
FLP-1::Venus secretion from AIY (Figure 2.7B) without altering FLP-18::Venus secretion
(Supplementary Figure 2.3K). pkc-1 mutations blocked the increased FLP-1::Venus secretion
induced by juglone, H2O2, mito-miniSOG activation, or trx-2 mutation (Figure 2.7B). The block
in H2O2-induced FLP-1 secretion by pkc-1 mutations cannot be explained by impaired mtH2O2
production (or transgene expression) because pkc-1 mutants exhibited similar baseline and
juglone-induced increases in AIY mito-HyPer fluorescence intensity as wild type controls (Figure
2.4B). Expressing pkc-1 cDNA selectively in AIY fully restored juglone-induced FLP-1::Venus
secretion to pkc-1 mutants (Figure 2.7C).
pkc-1 mutants were hypersensitive to toxicity caused by juglone (Figure 2.1A), and
exhibited normal baseline SKN-1 activity, but had significantly decreased juglone-induced SKN1::GFP nuclear translocation and Pgst-4::gfp expression compared to wild type controls (Figures
2.7D and E). Thus, PKC-1 functions cell-autonomously in AIY to selectively promote mtH2O2-
induced FLP-1 secretion and subsequent SKN-1 activation in the intestine.
2.3.10 PKC-1(C524) is necessary for juglone-induced FLP-1 secretion
PKC-1 contains a putative redox active cysteine residue (C524) in a conserved motif that
shares strikingly similarity to the redox-sensitive region of IRE-1, a protein kinase whose
sulfenylation by H2O2 on the corresponding reactive cysteine regulates responses to ER stress
(Figure 2.7F, (Hourihan et al., 2016)). C524 is conserved in mammalian calcium-independent PKC,
and is located adjacent to a conserved basic amino acid predicted to stabilize the sulfenyl
modification (Poole, 2015), and the DFG motif, which lies within the activation loop of the PKC1 kinase domain and is critical for kinase activity (R. Bayliss et al., 2012). Biochemical studies
have shown that mammalian PKCα becomes robustly sulfenylated by H2O2 in cell culture
(Hourihan et al., 2016; Lin & Takemoto, 2005). To test whether H2O2-induced FLP-1 secretion
relies on PKC-1 sulfenylation, we substituted C524 with serine, which replaces the thiol group (-
SH) with a hydroxyl group (-OH) rendering it unable to be sulfenylated by H2O2 (Figures 2.7A
and F, (Hourihan et al., 2016)). The C524S substitution did not appear to alter PKC-1 stability,
distribution, or kinase activity since PKC-1(C524S)::GFP fusion proteins adopted similar
fluorescence intensities and localization patterns in AIY axons as PKC-1(+)::GFP controls
(Supplementary Figure 2.6A), and pkc-1(C524S) transgenes fully rescued the pkc-1 mutant’s FLP-

26
1::Venus secretion defects when expressed in AIY, and the INS-22::Venus secretion defects when
expressed in motor neurons (under the unc-129 promoter) in the absence of juglone (Figures 2.7F
and Supplementary Figure 2.6B). However, pkc-1(C524S) transgenes failed to restore jugloneinduced FLP-1::Venus secretion from AIY to pkc-1 mutants (Figure 2.7F). These results suggest
that sulfenylation of C524 of PKC-1 plays a specific and critical role in mediating the effects of
H2O2 on FLP-1 secretion without altering PKC-1 kinase activity.
Further oxidation by H2O2 converts sulfenic acid into sulfinic acid (-SO2H) and
subsequently to sulfonic acid (-SO3H) (Figure 2.7A, (Luo et al., 2005). To test whether further
oxidation of C524 can promote secretion, we next generated the PKC-1(C524D) substitution,
which is predicted to mimic sulfonylated cysteine (Figures 2.7A and F). PKC-1(C524D) variants
were expressed at similar levels and distribution in AIY as PKC-1(+) controls (Supplementary
Figure 2.6A), and rescued the baseline FLP-1::Venus secretion of pkc-1 mutants, but failed to
restore juglone-induced FLP-1 secretion to pkc-1 mutants (Figure 2.7F). This suggests that
sulfonic acid modified C524 is not likely to contribute to FLP-1::Venus secretion in response to
stress. We conclude that H2O2-mediated sulfenylation or sulfinylation but not sulfonylation of
PKC-1 C524 promotes DCV exocytosis.
2.3.11 Bacterial food sources alter H2O2 levels, FLP-1 secretion and oxidative stress response
Finally, we investigated whether the antioxidant response activated by FLP-1 signaling is
regulated by environmental cues. AIY receives direct synaptic input from several olfactory and
gustatory sensory neurons (Ohnishi et al., 2011). Therefore, we tested whether sensory input
regulates FLP-1 secretion from AIY. osm-6 encodes a homolog of mammalian intraflagellar
transport 52 protein that is necessary for cilium biogenesis in sensory neurons that provide synaptic
input to AIY, and osm-6 mutants have defects in both olfactory and gustatory chemosensation
(Collet et al., 1998; Starich et al., 1995). We found that osm-6 mutants did not exhibit altered FLP1 secretion either in the absence or presence of juglone (Supplementary Figure 2.7A). Thus, the
H2O2-induced FLP-1 secretion is unlikely to require synaptic input to AIY from the sensory system.
C. elegans is an obligate bacterial eater, and the types and abundance of bacteria that C.
elegans consumes has dramatic effects on mitochondrial function and metabolism. We found that
food restriction does not impact FLP-1 secretion because mutations in eat-2, which cause
decreased feeding rates and are a classic model for caloric restriction (Avery, 1993; Bernard

27
Lakowski & Siegfried Hekimi, 1998; Maier et al., 2010), had no effect on baseline or jugloneinduced coelomocyte fluorescence in animals expressing FLP-1::Venus (Supplementary Figure
2.7A). Similarly, we found that food deprivation by starvation, which increased FLP-1 secretion
from AVK (Supplementary Figure 2.4M), did not alter FLP-1::Venus secretion from AIY
(Supplementary Figure 2.7B).
To determine whether differences in food source impact FLP-1 secretion we compared
animals reared on the standard E. coli OP50 strain (which was used throughout this study) to
animals reared on HT115. C. elegans reared on OP50 have a number of differences in
mitochondrial function and metabolism compared to those reared on HT115. Notably, animals
grown on OP50 exhibit greater sensitivity to killing by oxidants, including H2O2 and juglone
compared to animals grown on HT115 (Revtovich et al., 2019), suggesting a reduced antioxidant
capacity of OP50-fed animals. We found that animals reared on HT115 had similar mitochondrial
distribution and abundance in AIY as animals reared on OP50 culture plates (Supplementary
Figure 2.7C). However, HT115-reared animals had 2-fold greater AIY mtH2O2 levels and 2.5-fold
greater FLP-1::Venus secretion from AIY than animals grown on OP50 (Figures 2.8A and B). The
increase in FLP-1 secretion of HT115-reared animals was reduced to OP50 levels by sod-2
mutations (Figure 2.8B). HT115-reared animals exhibited 3-fold greater intestinal Pgst-4::gfp
expression compared to animals grown on OP50 (Figure 2.8C). Heat-killed HT115 were as
effective at increasing FLP-1 secretion as live HT115 (Figure 2.8D), indicating that the signal that
increases FLP-1 secretion is likely to be a bacterial metabolic product present in HT115 prior to
its ingestion. These results indicate that the type of food source plays a critical role in setting the
strength of both H2O2-regulated flp-1 signaling from AIY and antioxidant activity.
When animals grown on OP50 culture plates were switched to plates containing HT115,
we observed a 2-fold increase in AIY H2O2 levels within 10 minutes and a 2.5-fold increase in
FLP-1::Venus coelomocyte fluorescence within 30 minutes compared to non-switched animals
(Figures 2.8A and E). In the converse experiment, animals that were maintained on HT115 and
switched to OP50 exhibited significantly reduced AIY mtH2O2 levels beginning about 60 minutes
after being switched, with reductions in FLP-1::Venus coelomocyte fluorescence beginning about
120 minutes after the switch, and reaching levels seen in OP50-reared animals after 180 minutes
(Figures 2.8A and E). The lags between the changes in mtH2O2 levels and coelomocyte
fluorescence likely reflect the time it takes for coelomocytes to uptake and degrade FLP-1::Venus

28
following H2O2-regulated increases and decreases in FLP-1::Venus secretion, respectively.
Together, these results indicate that the ingestion of (or exposure to) HT115 leads to the rapid and
reversible increase in mtH2O2 in AIY and the subsequent increase in FLP-1 secretion. Importantly,
the observation that changes in FLP-1 secretion are reversible within minutes upon switching food
sources reinforces the idea that H2O2 performs a signaling function rather than functioning as a
damaging oxidant, in which case changes would be expected to be slower or irreversible
Animals grown on either OP50 or HT115 showed similar juglone-induced increases in
FLP-1 secretion and intestinal Pgst-4::gfp expression (Figures 2.8B and C), as well as increased
sensitivity to juglone-mediated toxicity in the absence of flp-1 (Figure 2.8F). However, OP50-
reared animals exhibited significantly reduced Pgst-4::gfp expression compared to HT115-reared
animals following exposure to lower juglone concentrations that do not elicit maximal responses
(Figure 2.8C). These results suggest that OP50-reared animals have a less robust oxidative stress
response than HT115 animals, likely due to lower baseline flp-1 signaling on this food source.
2.4 Discussion
By analyzing the mechanisms by which the nervous system activates the antioxidant
response, we have discovered a physiological role for H2O2 originating from mitochondria in
regulating the secretion of a neuropeptide, FLP-1, that functions as a neuroendocrine stress signal
to activate the oxidative stress response in distal tissues. We showed that FLP-1 secretion from the
AIY interneurons is necessary and sufficient to promote activation of the antioxidant response in
the intestine and organism-wide protection against oxidative stress. Exposure of animals to
different types of bacterial food sources leads to rapid local changes in H2O2 levels in axonal
mitochondria in AIY and to corresponding changes in FLP-1 secretion that depend on the
mitochondrial superoxide dismutase, sod-2. We identified a role in AIY for the antioxidant
peroxiredoxin-thioredoxin system in reducing mtH2O2 levels in axons and inhibiting FLP-1
secretion. The effects of H2O2 on FLP-1 secretion rely on a putative redox active cysteine residue
in the serine threonine kinase, PKC-1.
We propose a function for AIY as a “stress sensing” neuron that responds to bacteria or
other toxins that are ingested and signal to AIY from the alimentary system. When fed OP50
bacteria, mtH2O2 levels and flp-1 signaling in AIY are low. Upon changing food source to HT115,
or exposure to oxidative stressors, levels of mtH2O2 are rapidly increased in axonal mitochondria

29
in the vicinity of FLP-1 release sites. After exiting mitochondria, H2O2 functions as a signaling
cue where it sulfenylates PKC-1 on C524, which in turn positively regulates exocytosis of FLP-1
containing DCVs. Once secreted, FLP-1 functions as a neuroendocrine stress signal that tunes the
antioxidant response in distal tissues by activating NPR-4/GPCR and positively regulating SKN1 signaling in the intestine. Under conditions when mtH2O2 levels are low in AIY or in the absence
of flp-1, pkc-1, or npr-4, SKN-1 activation is compromised, and worms are more susceptible to
the deleterious effects of oxidative stress arising from environmental insults. Our data support a
model whereby the regulated secretion of FLP-1 from AIY by mtH2O2 is a mechanism by which
animals maintain antioxidant homeostasis as they adapt to changes in the composition of diverse
food sources to ensure survival (Figure 2.8G).
Neurons are among the most metabolically active cells, yet in mammals, mature neurons
possess a limited capacity to neutralize ROS since Nrf2 activity is weak. Instead, neurons
undergoing stress can take up antioxidant precursors that are secreted by neighboring astrocytes.
The release of “stress signals” from neurons undergoing stress is proposed to lead to Nrf2
activation in astrocytes and the subsequent secretion of antioxidant precursors (Agata Habas et al.,
2013; Baxter & Hardingham, 2016; Jimenez-Blasco et al., 2015). Although neuronal glutamate
release has been suggested to activate Nrf2 in astrocytes, additional signals, possibly neuropeptides,
are likely to contribute to Nrf2 activation (Agata Habas et al., 2013). Interestingly, SKN-1
activation in the worm intestine not only activates the antioxidant response, but also leads to the
secretion of diffusible signals that regulate neuronal function (S. Kim & Sieburth, 2018b; Staab et
al., 2013). Thus, the activation of SKN-1/Nrf2-in distal tissues by neuronal activity, and the
subsequent feedback signaling to neurons may be an evolutionary conserved mechanism by which
neurons maintain redox homeostasis in response to oxidative stress.
Here we identified a previously undescribed function for the AIY interneuron pair in
relaying changes in levels of mitochondrially generated H2O2 to the rest of the organism through
FLP-1 secretion. The AIY pair is a first layer amphid interneuron that receives glutamatergic
synaptic input from a number amphid sensory neurons to regulate food, odor, and thermal-evoked
behaviors through cholinergic synaptic transmission (H. Liu et al., 2018). AIY also plays a critical
role in regulating development and metabolism in response to environmental signals through the
secretion of FLP-1 and FLP-18 (Cohen et al., 2009; Jeong & Paik, 2017; Mutlu et al., 2020). Our
observation that FLP-1 but not FLP-18 secretion from AIY is increased by juglone and by pkc-1

30
suggests that FLP-1 and FLP-18 secretion may be differentially regulated, raising the possibility
that these peptides are packaged into distinct DCV pools that may differ in their proximity to
mitochondria, or their ability to be regulated by PKC-1 signaling. Consistent with this, differential
release of DCV pools from single cells has been observed in a number of neuroendocrine cell types
(Mcneilly et al., 2003; Rao et al., 2017; Z. Zhang et al., 2011). Thus, AIY appears to be unique
among neurons we examined in its ability to specifically regulate FLP-1 secretion through mtH2O2
signaling, revealing a specialized function for AIY in the oxidative stress response. Interestingly,
we found that flp-1 mutants are sensitive to juglone-induced toxicity, whereas flp-18 mutants are
resistant to juglone-induced toxicity, and flp-1; flp-18 double mutants exhibit an intermediate
juglone response (Supplementary Figure 2.3I), suggesting a role for flp-18 in inhibiting the
oxidative stress response by antagonizing the protective effects of flp-1 signaling.
How might endogenous H2O2 levels in AIY be regulated to calibrate the strength of FLP1 secretion? First, H2O2 levels could be regulated by controlling ROS production by mitochondria.
Mitochondrial ROS production has been shown to be controlled by numerous signals including by
glucose in pancreatic beta cells, synaptic activity in neurons, and peptide hormones in smooth
muscle cells (Ahmed Alfar et al., 2017; Papadia et al., 2008; Roma & Jonas, 2019; K.-Y. Yu et al.,
2014). In C. elegans, mitochondrial activity is also highly dynamic and can be influenced by
intrinsic metabolic processes driven by changes in diet and the gut microbiota (Jones et al., 2012;
Revtovich et al., 2019). Second, mtH2O2 levels in AIY could be regulated by controlling the rate
of H2O2 consumption by the peroxiredoxin-thioredoxin system (Netto & Antunes, 2016). In
mammals, the activity of the mitochondrial peroxiredoxin-thioredoxin system can be regulated by
cAMP arising from extracellular signals in the adrenal cortex (Kil et al., 2012), and by synaptic
activity in cortical neurons (Papadia et al., 2008). Third, H2O2 levels could be regulated by
controlling calcium influx into mitochondria. We found that juglone treatment leads to increased
mitochondrial calcium levels, and that mitochondria calcium influx is required for mtH2O2
production and for juglone-induced mitochondrial calcium increases. Thus, juglone treatment may
increase calcium levels in the cytosol that then enters the mitochondrial through MCU1.
Alternatively, juglone may directly regulate calcium levels in the mitochondria for example by
regulating MCU channel activity. The activity of the MCU channel is proposed to be modulated
by a number of post-transcriptional modifications, including by phosphorylation, and by ROS
mediated S-glutathionylation (Mammucari et al., 2017). The mechanism by which increased

31
mitochondrial calcium levels facilitate the generation of H2O2 is less clear. Calcium is an activator
the ETC, and sustained ETC activation can lead to mtROS production (Williams et al., 2015).
Finally, FLP-1 secretion could be controlled by regulating the rate of mtH2O2 exit from
mitochondria possibly through aquaporins (Tamma et al., 2018), or the extent of H2O2 buffering
in the cytoplasm by cytosolic antioxidant enzymes (Winterbourn & Hampton, 2008), or the
proximity of mitochondria to DCVs (Griesche et al., 2019; Zhao et al., 2018). Regulation of any
one or more of these steps by stress signals could modulate FLP-1 secretion from AIY.
Our studies reveal a central role for PKC-1/PKCε/η in mediating the effects of H2O2 on
FLP-1 secretion. PKC has been implicated in a number of redox signaling events in excitable cells
(Barnett et al., 2007; Rayudu Gopalakrishna et al., 2008; Steinberg, 2015). In hippocampal neuron
cultures, PKC mediates the effects of ROS on long term plasticity (Knapp & Klann, 2002b). In
Aplysia, ROS activates novel PKC, which is important for the establishment of changes in synaptic
strength (Zabouri & Sossin, 2002). PKCε protects neurons and cardiac myocytes in models of
ischemia/reperfusion injury and is activated by ROS produced by mitochondria during hypoxia
(Alexandre D. T. Costa et al., 2006). In C. elegans, pkc-1 is required for the protective effects of
mitochondrial ROS generated by mutants with impaired mitochondrial respiration to an antimitotic
toxin (Iryna Zubovych et al., 2006; Zubovych et al., 2010). In addition, pkc-1 has been shown to
function in AIY to mediate the effects of the ROS generator graphene oxide on oxidative stress
the intestine (H. Chen et al., 2017). Whether pkc-1 regulates FLP-1 secretion or the secretion of
other signals during these responses remains to be determined.
Our genetic studies point to sulfenylation of the conserved PKC-1 C524 residue in
promoting H2O2 -induced FLP-1 secretion. Cysteine sulfenylation is a rapid enzyme-independent
reversible modification (Luo et al., 2005), making this an attractive mechanism by which PKC-1
could rapidly and reversibly regulate FLP-1 secretion. H2O2 signaling can lead to cysteine
sulfenylation either directly by oxidization of reactive cysteines on target proteins, or indirectly by
oxidation of a “relay” protein such as peroxiredoxin, which then transfers oxidizing equivalents to
the target protein (Giniatullin et al., 2019). We do not favor the idea that H2O2 leads to PKC-1
sulfenylation indirectly through PDRX-3, since we found that prdx-3 mutations do not decrease
FLP-1 release, which would be expected if PRDX-3 positively links H2O2 to PKC-1 activation.
Instead, we propose that PKC-1 is either directly sulfenylated by H2O2 or is sulfenylated by an
alternative redox-active “relay” protein.

32
TRX-2 is nearly exclusively expressed at high levels in AIY suggesting a unique capacity
of AIY to remove mtH2O2 through the peroxiredoxin-thioredoxin system. How then could H2O2
in AIY reach levels high enough to promote FLP-1 secretion? One explanation, termed the
“floodgate hypothesis”, proposes that when H2O2 levels increase beyond a certain threshold,
peroxiredoxins become hyperoxidized and are inactivated allowing H2O2 to build up sufficiently
to perform its signaling functions (Woo, 2003; K.-S. Yang et al., 2002). In this scenario, PRDX-3
would remove H2O2 efficiently under normal conditions, but an increase in mtH2O2, arising from
stress signals, would lead to PRDX-3 hyperoxidation resulting in further mtH2O2 accumulation to
levels that would promote FLP-1 secretion. Interestingly, the function we found for pdrx-3 in
inhibiting H2O2 signaling contrasts with that of the cytoplasmic peroxiredoxin, prdx-2, which
promotes H2O2 signaling by functioning as a redox-active “relay” protein for H2O2 in sensory
neurons during sensory transduction (Bhatla & Horvitz, 2015; G. Li et al., 2016), revealing distinct
mechanisms by which cytoplasmic vs. mitochondrial peroxiredoxins impact H2O2 signaling in C.
elegans.
Peroxiredoxins are efficient in H2O2 sensing and scavenging (Netto & Antunes, 2016) and
mammalian peroxiredoxin 3 is estimated to account for up to 90% of cellular H2O2 detoxification
(Cox et al., 2009; Wozniak et al., 2014), suggesting that it may play a major role in mitochondrial
redox signaling. Mouse peroxiredoxin 3 knockouts appear largely healthy, but have increased
mtH2O2 levels, and have defects in mitochondrial and metabolic homeostasis in a number of tissues
(Y. J. Lee, 2020), whereas overexpression of peroxiredoxin 3 results in decreased ROS levels (L.
Chen et al., 2008). Peroxiredoxin 3 and thioredoxin 2 are expressed broadly in the nervous system,
where they have well documented roles in neuroprotection in response to oxidative damage in
hippocampal and cortical neurons (Hattori et al., 2003; Rothman & Olney, 1995; Silva-Adaya et
al., 2014). Studies in insulinoma cells have shown that glucose-stimulated insulin release is
enhanced upon peroxiredoxin 3 knockdown and blocked upon peroxiredoxin 3 overexpression
(Huh et al., 2012), and peroxiredoxin 3 knockouts have elevated fasting insulin plasma levels and
are insulin resistant (Gabriele Wolf et al., 2010), , suggesting a potentially conserved function for
prdx-3 and mammalian peroxiredoxin 3 in inhibiting DCV secretion by the removal of mtH2O2. It
will be interesting to determine whether the peroxiredoxin-thioredoxin system regulates
neurotransmitter or neuropeptide secretion from mammalian neurons.

33
We found that cysteine sulfenylation of PKC-1 does not appear to affect kinase activity
since the PKC-1(C524S) mutant transgenes fully supported neuropeptide secretion in the absence
of stress. PKCs are cytosolic proteins whose activities are primarily regulated by targeting to
cellular membranes through their calcium and/or lipid binding C1 and C2 domains. In their role in
promoting secretory granule secretion, the recruitment of PKC to release sites promotes a late step
in vesicle exocytosis (Y.-S. Park et al., 2006). H2O2 treatment induces membrane translocation of
PKCγ, which is involved in ischemia (Lin & Takemoto, 2005). Therefore, we propose that H2O2-
induced C524 sulfenylation promotes membrane recruitment of PKC-1 to DCV release sites,
where it facilitates calcium-dependent FLP-1 release. PKC-1 may promote FLP-1 secretion
through phosphorylation of known PKC target proteins involved in secretory granule exocytosis
(Barclay et al., 2003; Hilfiker et al., 1999; Nagy et al., 2002; Shoji‐Kasai et al., 2002; Zeidman et
al., 2002). Alternatively, PKC-1 could promote DCV secretion by regulating mitochondrial
function. Calcium-independent PKCs interact with a number of mitochondrial proteins (Barnett et
al., 2007), and have been proposed to promote insulin secretion by regulating mitochondrial ATP
production in pancreatic beta cells (Santo-Domingo et al., 2017, 2019). Finally, PKC-1 may
regulate DCV secretion through a less direct mechanism. For example, pkc-1 is reported to regulate
the expression of the MAP kinase cascade activator lin-45/Raf in AIY following exposure to
oxidative stress (H. Chen et al., 2017).
Our results suggest that diet plays a critical role in setting the baseline antioxidant response
through the regulation AIY H2O2 levels and FLP-1 secretion. OP50, which is a B strain and HT115,
which is a K-12 strain, differ in both the amount and composition of nutrients and metabolites,
including specific lipids, mitochondrial byproducts and amino acids (Brooks et al., 2009; Reinke
et al., 2010), and some of these differences are thought to underly differences in mitochondrial
function and stress responses in animals that consume each bacterial type. Because of the relatively
fast rates at which changing diet leads to changes in FLP-1 secretion, we propose that a signal or
metabolite from consumed bacteria is sensed either directly or indirectly by AIY, leading to
changes in H2O2 levels in AIY. C. elegans also can ingest oxidants directly from food or from the
environment. For example, some pathogenic bacteria consumed by C. elegans produce levels of
H2O2 as high as 2 mM (Maike Bolm et al., 2004; W. T. M. Jansen et al., 2002). Similarly, juglone
is a naturally occurring oxidant exuded into the soil by the roots of black walnut trees (von Kiparski
et al., 2007), where it may be consumed by C. elegans at physiologically relevant concentrations

34
to impact ROS production in AIY. Determining how bacterial-derived signals from diet regulate
H2O2-dependent neurosecretion from AIY may have general relevance for understanding
mechanisms underlying the crosstalk between the nervous system and the gut microbiome.
2.5 Acknowledgements
C. elegans strains used in this work were provided by the Caenorhabditis Genetics Centre
(Univ. of Minnesota), which is funded by the NIH National Center for Research Resources
(NCRR). Thanks to Trisha Staab for the initial observation that egl-3 mutants are hypersensitive
to juglone toxicity, to the Lillian Schoofs lab for testing the interaction between FLP-1 and NPR4, to Karen Chang for providing the HyPer construct, and to Eric Jorgensen for providing the UNC116::TOM-7 constructs. Thanks to members of the Sieburth lab for discussions and critical reading
of the manuscript. This work was supported by the Southern California Environmental Health
Sciences Center, grant #P30ES007048 and National Institutes of Health (NIH), grant #NS099414
to D.S..
2.6 Materials and Methods
2.6.1 Strains
C. elegans Strains were maintained on standard nematode growth medium (NGM) plates
seeded with OP50 Escherichia coli bacteria as food source, unless otherwise indicated, and
cultured in a dark 20°C incubator. All strains were synchronized by picking mid L4 stage animals
and either analyzed immediately (for coelomocyte imaging) or as young adults 24 hours later (for
AIY imaging or toxicity assays). The wild type reference strain was Bristol N2. Mutants used in
this study were outcrossed at least two times and are listed in the Supplemental Note.
Transgenic animals were generated by micro-injecting plasmid mixes into the gonads of
young adult N2 hermaphrodites following standard techniques as previously described (Mello et
al., 1991). All microinjection mixes were prepared using expression constructs injected at 10ng/ul,
plus the co-injection markers KP#708 (Pttx-3-rfp, 40 ng/µL), KP#1106 (Pmyo-2-gfp, 10 ng/µL),
pJQ70 (Pofm-1-rfp, 25 ng/µL) or pMH163 (Podr-1-mCherry, 40 ng/µL) to a final concentration
of 100ng/ul. At least three transgenic lines for each transgene were examined. Integration of arrays
was performed by radiating 100 transgenic L4 worms on unseeded NGM plates with the lid

35
removed in a UV cross-linker. 200 F1 transgenic worms were singled onto separate NGM plates,
and F2 transgenic worms with high level of fluorescence were selected for candidates for 100%
homozygous transgenic F3 worms (Mariol et al., 2013). Heat killing bacteria was performed by
placing bacteria containing conical in a 100°C water bath for ~30 min, verified by failure to form
colonies on LB plates, as described (Kaeberlein et al., 2006).
2.6.2 Toxicity Assays
Stock solutions of 50mM juglone in DMSO, 20mM thimerosal in water, and 4mM paraquat
in water (each freshly made before each assay), 0.5% w/v sodium arsenite in water, and 30% (9.8M)
H2O2 solution were used for toxicity assays. High concentrations for treatment were required for
some drugs since the C. elegans cuticle is not permeable to most drugs (A. R. Burns et al., 2010).
For liquid toxicity assays, about 50-80 synchronized adults were transferred into a 1.5 mL
Eppendorf tube with M9 buffer, and washed three times. Oxidants were added to washed worms
at final concentrations of 100-300 µM (juglone), 50 µM (thimerosal), 2 mM (sodium arsenite),
unless indicated otherwise, and incubated for 4 hours on rotating mixer. Animals were then washed
once and transferred to fresh plates seeded with OP50 or HT115 to recover in dark at 20°C for 16
hours. Survival was assayed by counting the number of alive and dead animals. For plate toxicity
assays, plates containing 4M fructose rings around the bacteria were freshly prepared to restrain
the movement of worms. Toxicity assays were performed in triplicate.
2.6.3 Cell ablation and ROS production by miniSOG
For miniSOG-induced cell ablation, an EXFO mercury arc lamp was used as the blue light
source. 30-40 L4 animals were transferred onto fresh NGM plates with OP50 and animals were
exposed to continuous 50mW/cM2 blue light for 30min and recovered at 20°C in dark for 16 hours
before toxicity assays. Blue light illumination was performed in triplicate. For miniSOG-induced
ROS generation, LED light with pre-built MSOG0001 filter module (TriTech Research) was used
as the blue light source. 30-40 L4 stage worms were transferred onto fresh NGM plates with OP50
and animals were exposed to continuous 100mW/cM2 blue light for 10min and recovered at 20°C
in dark for 10min before taking images. Plates were placed without covers on the stage with blue
light passing through without objectives.

36
2.6.4 Channelrhodopsin activation
Blue light induced channelrhodopsin 2 (ChR2) activation was performed as previously
described (Nagel et al., 2005). 100mW/cm2 blue LED light source was used for light illumination.
Briefly, 30-40 animals expressing ChR2::gfp transgene were transferred onto NGM plates with
OP50 spread supplemented with 500µM all-trans retinal (Sigma), and recovered at 20°C in dark
for 4 hours before light illumination. Plates were placed without covers on the stage with blue light
passing through without objectives. For light illumination, animals were exposed to continuous
blue light for 1min and recovered at 20°C in dark for 10min before taking images.
2.6.5 Microscopy and Analysis
Fluorescence microscopy experiments to quantify AIY axonal and coelomocyte
fluorescence were performed as previously described (Ch’ng et al., 2008). Briefly, 30-40 animals
were paralyzed with 30mg/ml 2,3-butanedione monoxime (BDM) in M9 buffer and mounted on
2% agarose pads. Images were captured with the Nikon eclipse 90i microscope equipped with a
Nikon PlanApo 40x or 100x objective (NA=1.4) and a Hamamatsu Orca Flash LT+ CMOS camera,
and Metamorph 7.0 software (Universal Imaging/Molecular Devices) was used to capture serial
image stacks and the maximum intensity projection image was used for analysis.
For transcriptional reporter imaging, 30-40 young adults were transferred into M9 solution,
washed three times and then exposed to oxidant for 1 hour on a rotating mixer. Animals were then
washed three times and transferred onto fresh OP50-seeded NGM plates and allowed to recover
in dark at 20°C for 4 hours before imaging. The posterior end of the intestine at the gonad bend
was imaged using a 60x objective for each reporter. For quantification of Pgst-4::gfp expression,
a 16-pixel diameter circle was drawn in the posterior intestine (ROI) and the average fluorescence
intensity within the area of the circle was calculated using MetaMorph. Background fluorescence
was measured as the average intensity within a same-sized circle positioned next to the animal
(coverslip fluorescence) and this value was subtracted from the ROI fluorescence to generate the
fluorescence intensity value. For SKN-1::GFP and DAF-16::GFP reporter imaging, 40-50 L4
animals were exposed to the working concentration of oxidant for 10 minutes in liquid, prior to
imaging. Z-stacks of the intestine (20 nuclei/image) were captured using a 40x objective and the
total number of GFP+ nuclei in the intestine were counted. Fluorescent nuclei between 1 and 10,
11 and 20, and above 20 were binned into Low, Medium and High categories, respectively.

37
For imaging fluorescently tagged fusion proteins in AIY, 30-40 synchronized young adults
were exposed to either oxidant or M9 buffer for the indicated times (usually 10 minutes) and then
paralyzed in BDM for 10 minutes prior to imaging. Only animals oriented such that the left AIY
neuron was facing the objective were imaged. Z stacks of the axons were captured, and the
fluorescence intensity values were then quantified by obtaining linescans in Metamorph and using
custom Puncta 6.0 software written with Igor Pro (Wavemetrics) to illustrate fluorescence
distribution or to quantify punctal fluorescence, as previously described (A. R. Burns et al., 2010;
Ch’ng et al., 2008; Mariol et al., 2013). Z stacks were obtained using GFP (excitation/emission:
450nm/520nm) and CFP (excitation/emission: 420/480nm) filter sets sequentially. mito-HyPer
fluorescence amplitude was quantified as the ratio of GFP to CFP punctal fluorescence intensity
changes with respect to the baseline [(Ft-F0)/F0].
For coelomocyte imaging, 30-40 L4 stage animals were washed three times with M9 and
then exposed to oxidants for 10min in M9 unless indicated otherwise. Animals were then paralyzed
in BDM in M9 buffer before taking images of the posterior most coelomocytes at the posterior
gonad bend. Z stacks were quantified by measuring average fluorescence/pixel of FLP-1::Venus
from 2-4 endocytic in the posterior coelomocyte of each animal in Metamorph. All FLP-1::Venus
secretion assays from AIY neurons were performed using the vjIs150 transgene, which is
integrated on LG III, except for trxr-2 mutants, which were analyzed using vjIs152, which is
integrated on LG I.
2.6.6 RNA interference
Feeding RNAi was performed as described (Jeremy S. Dittman & Joshua M. Kaplan, 2006).
Briefly, 20-25 gravid adult animals were placed on RNAi plates seeded with HT115(DE3) bacteria
transformed with L4440 vector containing the insert of the gene targeted for knockdown or empty
L4440 vector as a control, and eggs were collected for 4 hours to obtain age-matched synchronized
worm populations. Young adult animals were used for all RNAi assays. RNAi clones were from
the Ahringer RNAi library (Kamath, 2003), and clones not represented in the library were made
from genomic DNA and confirmed by sequencing. See Supplemental Notes (STAR table) for a
list of plasmids made.

38
2.6.7 Statistical analysis
Statistical analysis was performed on GraphPad Prism 8. Unpaired t-test (two tails) was
used to determine the statistical significance between DMSO (control) and juglone treatment,
nested one way-ANOVA was used to determine the statistical significance between wild type and
mutant strains. P values are indicated with asterisks * (p < 0.05), **(p < 0.01), ***(p < 0.001), or
number symbols # (p < 0.05), ## (p < 0.01), ### (p < 0.001). Error bars in the figures indicate the
standard error of the mean (±SEM). Bar graph and Box-and-whisker plots were generated on
GraphPad Prism 8. Sample sizes for coelomocyte assays were 20-30 animals per condition, for
toxicity assays were 100-200 animals, for mito-HyPer and GCaMP imaging were 20-30.

39

40
Figure 2.1 Neuronal FLP-1 Signaling Promotes the SKN-1-mediated Intestinal Antioxidant
Response.
A Percentage of surviving animals of the indicated genotypes 16 h following 4 h treatment of
young adults with juglone. VAChT vesicular acetylcholine transporter, GAD glutamate
decarboxylase, VGluT vesicular glutamate transporter, TPH glutamate decarboxylase, TH tyrosine
hydroxylase, TDC tyrosine decarboxylase, TBH tyramine-beta hydroxylase, PC2 prohormone
convertase, SPE carboxypeptidase, 7B2 secretogranin V, PKC protein kinase C. Unlined ns and
*** denote statistical analysis compared to “wild type”. n =129, 64, 164, 108, 139, 91, 158, 165,
153, 149, 114, 63, 109 independent animals over three independent experiments. B Percentage of
surviving animals of the indicated genotypes following chronic exposure of young adults to culture
plates containing juglone for the indicated times. n = 111, 142, 121 independent animals over three
independent experiments. C Representative fluorescent images and quantification of the number
of intestinal nuclei with SKN-1::GFP in adult animals following 10 min of M9 or juglone treatment.
Neuronal flp-1 denotes transgenes expressing flp-1 gDNA under control of the endogenous flp-1
promoter. Dotted lines demarcate intestinal regions. Nuclear translocation of SKN-1::GFP was
measured by counting the number of fluorescent nuclei in the intestine. Fewer than 10, between
11 and 20, and above 20 fluorescent nuclei are denoted Low, Medium, and High, respectively.
Unlined *** denotes statistical analysis compared to “wild type”; unlined ## denotes statistical
analysis compared to M9 control group of the same mutant strain. n = 63, 51, 52, 67, 45, 81, 36,
55 independent animals. Scale bar: 100 μm. D Representative images and quantification of the
posterior regions of transgenic worms expressing the oxidative stress reporter Pgst-4::gfp after 1
h of M9 or juglone treatment and 4 h of recovery. Asterisks mark the intestinal region used for
quantitation. Pgst-4::gfp expression in body wall muscles, which appears as fluorescence on the
edge of animals in some images, was not quantified. Neuronal egl-3 and neuronal flp-1 denote
expression of egl-3a cDNA and flp-1 genomic DNA under the flp-1 promoter respectively.
Unlined ns and *** denote statistical analysis compared to “wild type”; a and b denote statistical
analysis compared to “wild type+juglone”, P < 0.001 and P < 0.001; c denotes statistical analysis
compared to “neuronal flp-1;flp-1+juglone”, P < 0.001; d denotes statistical analysis compared to
“neuronal egl-3;egl-3+juglone”, P < 0.001. n = 20, 25, 30, 31, 20, 20, 31, 30, 17, 33, 19, 25
independent animals. Scale bar: 50 μm. E Percentage of surviving animals of the indicated
genotypes 16 h following 4 h juglone treatment of young adults. Intestinal egl-3 denotes expression
of egl-3a cDNA under the ges-1 promoter. Unlined ns denotes statistical analysis compared to
“egl-3”; unlined *** denotes statistical analysis compared to “wild type”. n =89, 94, 83, 119, 129,
118 independent animals over three independent experiments. F Percentage of surviving animals
of the indicated genotypes 16 h following treatment of young adults with juglone for 4 h. flp-1(OE)
denotes expression of flp-1 gDNA under control of the flp-1 promoter in wild type animals.
Unlined *** denotes statistical analysis compared to “wild type”. n =152, 186 independent animals
over three independent experiments. G (Top) Protein structure of FLP-1B with the predicted EGL3 cleavage sites (K = lysine, R= arginine) marked above each mature peptide (numbered P1-P9).
(Bottom) (Left) Percentage of surviving animals of the indicated genotypes 16 h following
treatment of young adults with juglone for 4 h. Neuronal flp-1(SS) denotes expression of signal
peptide sequence of flp-1 under the ges-1 promoter; neuronal flp-1(P1), flp-1(P1+P2), flp1(P1+P2+P3), flp-1(P1+P2+P3+P4) denotes expression of truncated flp-1 variants under the
ges-1 promoter; neuronal flp-1(+) denote expression of flp-1 gDNA under the ges-1 promoter.
(Bottom) (Right) Amino acid sequence alignment of each mature FLP-1 peptide. Conserved amino
acids are highlighted in gray and invariant amino acids are marked in bold. Unlined *** denotes

41
statistical analysis compared to “wild type”; unlined ns, #, ## and ### denote statistical analysis
compared to “flp-1”. n = 160, 160, 159, 123, 154, 147, 90, 99 independent animals over three
independent experiments. Data are mean values ± s.e.m. ns not significant, # P < 0.05, ## P <
0.01, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple
comparisons test.

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Figure 2.2 FLP-1 Released from AIY Interneurons Promotes the Antioxidant Response.
A Juglone-induced toxicity of wild-type animals expressing mito-miniSOG under the control of
the indicated promoters, with and without 50 mW/cm2 blue light illumination for 30 min. The
neurons in which each promoter drives mito-miniSOG expression are indicated below. The ttx-3
promoter is reported to be expressed weakly in AIA neurons (Hobert et al., 1997), indicated by
parenthesis. Unlined ns denotes statistical analysis compared to “wild type”. n = 102, 96, 98, 70,
146, 67, 77, 88, 84, 70, 85, 78, 134 independent animals over three independent experiments. B
Percentage of surviving animals expressing tetanus toxin under the ttx-3 promoter (AIY TeTx) 16
h following 4 h juglone treatment of young adults. n = 203, 303 independent animals over three
independent experiments. C Percentage of surviving animals of the indicated genotypes 16 h
following 4 h treatment of young adults with juglone. AIY egl-3 denotes expression of egl-3a
cDNA under the ttx-3 promoter. n= 156, 190, 162, 156 independent animals over three independent
experiments. D Percentage of surviving animals of the indicated genotypes 16 h following 4 h
treatment of young adults with juglone. AIY flp-1 denotes expression of flp-1 gDNA under the ttx3 promoter. Unlined *** denotes statistical analysis compared to “wild type”. n = 189, 174, 148
independent animals over three independent experiments. E Schematic showing the positions of

43
AIY, intestine, and coelomocytes. Transgenic animals used to measure FLP-1 secretion co-express
FLP-1::Venus in AIY and mCherry in coelomocytes. An image of AIY shows the distribution of
FLP-1::Venus fusion proteins in puncta in an AIY axon and soma. Scale bar: 10 μm. FLP-1::Venus
secreted from AIY accumulates in the pseudocoelom and is taken up by coelomocytes. An image
of the posterior-most coelomocyte that has taken up Venus into endocytic compartments is shown.
DCV dense core vesicle. Scale bar: 5 μm. F Representative images and quantification of average
coelomocyte fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in
AIY following M9 or juglone treatment for 10 min. CAPS calcium-dependent activator protein for
secretion, VGCC voltage-gated calcium channel. Unlined * and *** denotes statistical analysis
compared to “wild type”. n =30, 30, 30, 30 independent animals. Scale bar: 5 μm. G Quantification
of average coelomocyte fluorescence of animals co-expressing FLP-1::Venus fusion proteins and
channelrhodopsin (ChR2) in AIY with and without 1 min blue light exposure or 10 min juglone
treatment. Unlined *** denotes statistical analysis compared to “wild type”. n =30, 30, 30, 30
independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns not
significant, * and # P < 0.05, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with
Dunnett’s T3 multiple comparisons test.

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Figure 2.3 FLP-1 Activates the Antioxidant Response through NPR-4/GPCR in the Intestine.
A Percentage of surviving animals of the indicated genotypes 16 h following 4 h juglone treatment
of young adults. Unlined *** denotes statistical analysis compared to “wild type”. n = 323, 253,
284, 301 independent animals over three independent experiments. B Percentage of surviving
animals of the indicated genotypes 16 h following treatment of young adults with juglone for 4 h.
AIY flp-1(OE) denotes expression of flp-1 gDNA under the ttx-3 promoter in wild type animals.
Unlined *** denotes statistical analysis compared to “wild type”. n = 399, 184, 211 independent
animals over three independent experiments. C Percentage of surviving animals of the indicated
genotypes 16 h following treatment of young adults with juglone for 4 h. Coelomocyte npr-4
denotes expression of npr-4a cDNA under the ofm-1 promoter, neuronal npr-4 denotes expression
of npr-4a cDNA under the rab-3 promoter, intestinal npr-4 denotes expression of npr-4a cDNA
under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type, unlined
### denotes statistical analysis compared to “npr-4”. n =144, 215, 178, 188, 212 independent
animals over three independent experiments. D Representative images and quantification of the
posterior regions of transgenic worms expressing Pgst-4::gfp after 1 h of M9 or juglone treatment
and 4 h recovery. Asterisks mark the intestinal region used for quantitative analysis. Pgst-4::gfp
expression in body wall muscles, which appears as fluorescence on the edge of animals in some
images, was not quantified. Unlined *** ”; a, b, c and d denote statistical analysis compared to
“wild type+juglone”, P < 0.01, P < 0,01, P < 0.01, P < 0.01 respectively. n =20, 28, 25, 32, 19,

45
29, 17, 32, 22, 31, 15, 17, 19, 21 independent animals. Scale bar: 50 μm. Data are mean values ±
s.e.m normalized to wild type controls. ns not significant, *** and ### P < 0.001 by BrownForsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

46
Figure 2.4 FLP-1 Secretion from AIY Interneurons Relies on Mitochondria-generated H2O2.
A Schematic representation of the generation of mitochondrial hydrogen peroxide (mtH2O2) by
dismutation of superoxide (O2
−) by mitochondrial SOD-2/superoxide dismutase, and its
consumption by the mitochondrial TRX-2/thioredoxin PRDX-3/peroxiredoxin system, which
converts mtH2O2 to H2O. B Representative images and quantification of fluorescence in AIY
axons of the indicated transgenic animals expressing mitochondrial-targeted HyPer (mito-HyPer)
driven by ttx-3 promoter. Arrowheads indicate mitochondrial fluorescence in AIY axons. The 520
nm/480 nm (GFP/CFP) ratio of HyPer was used to measure mtH2O2 levels. Unlined *** and ###
denote statistical analysis compared to “wild type”. n =20, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20
independent animals. Scale bar: 10 μm. C Quantification of average coelomocyte fluorescence of
the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following M9 or juglone
treatment for 10 min. AIY sod-2 denotes expression of sod-2 cDNA under the ttx-3 promoter, AIY
sod-2(ΔMLS) denotes expression of sod-2(ΔMLS) variants under the ttx-3 promoter.Unlined ***
denotes statistical analysis compared to “wild type”, unlined ### denotes statistical analysis
compared to “sod-2+juglone”. n = 30, 30, 30, 30, 30, 30 independent animals. D Quantification of
average coelomocyte fluorescence of the indicated mutants coexpressing FLP-1::Venus and mitominiSOG in AIY following 1 min illumination with 50 mW/cm2 blue light and 10 min recovery.
Unlined *** denotes statistical analysis compared to “wild type”. n =30, 30, 30, 30 biologically
independent animals. E Quantification of average coelomocyte fluorescence of the indicated
mutants expressing FLP-1::Venus fusion proteins in AIY following 10 min M9 or H2O2 treatment.
Unlined *** denotes statistical analysis compared to “wild type”. n = 30, 30, 30, 30 independent
animals. Data are mean values ± s.e.m normalized to wild type controls. ns not significant, ***

47
and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple
comparisons test.

48
Figure 2.5 Regulation of FLP-1 Secretion by Mitochondrial Calcium and the ThioredoxinPeroxiredoxin System.
A Representative images and punctal fluorescence quantification of AIY axons from adults
expressing mitochondrial-targeted GCaMP3 (mito-GCaMP) driven by the ttx-3 promoter after 10
min M9 or juglone treatment. Unlined *** denotes statistical analysis compared to “wild type”. n
=20, 20, 20, 20, 20, 20, 20, 20 independent animals. Scale bar: 10 μm. B Representative images
and punctal fluorescence quantification of AIY axons from adults expressing mitochondrialtargeted HyPer (mito-HyPer) driven by the ttx-3 promoter after 10 min M9 or juglone treatment.
The 520 nm/480 nm (GFP/CFP) ratio of HyPer was used to measure H2O2 levels. Unlined ***
denotes statistical analysis compared to “wild type”. n = 20, 20, 20, 20, 20, 20 independent animals.
Scale bar: 10 μm. C Quantification of average coelomocyte fluorescence intensity following
exposure of the indicated mutants expressing FLP-1::Venus in AIY to juglone for 10 min. Unlined

49
*** denotes statistical analysis compared to “wild type”. n = 30, 30, 30, 30, 30, 30 independent
animals. D Quantification of average coelomocyte fluorescence intensity following exposure of
indicated mutants expressing FLP-1::Venus in AIY to juglone for 10 min. AIY mcu-1 denotes
expression of mcu-1 cDNA under the ttx-3 promoter. Unlined *** denotes statistical analysis
compared to “wild type”. n =30, 30, 30, 30 independent animals. E Schematic showing the redox
cycle used by peroxiredoxin, thioredoxin, and thioredoxin reductase to consume H2O2. F
Quantification of average coelomocyte fluorescence intensity following exposure of indicated
mutants expressing FLP-1::Venus in AIY to juglone for 10 min. Unlined *** and ### denote
statistical analysis compared to “wild type”. n =30, 30, 30, 30 independent animals. G
Quantification of average coelomocyte fluorescence intensity following exposure of indicated
mutants expressing FLP-1::Venus in AIY to juglone for 10 min. AIY trx-2 denotes expression of
trx-2 cDNA under the ttx-3 promoter, AIY trx-2(ΔMLS) denotes expression of trx-2(ΔMLS)
variants under the ttx-3 promoter, AIY trx-2(ΔCAT) denotes expression of trx-2(ΔCAT) variants
under the ttx-3 promoter. Unlined *** denotes statistical analysis compared to “wild type”, unlined
### denotes statistical analysis compared to “trx-2/TXN2”. n = 30, 30, 30, 30, 30, 30, 30, 30
independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns not
significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3
multiple comparisons test.

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Figure 2.6 Trafficking of Mitochondria and DCVs to AIY Axons is Required for JugloneInduced FLP-1 Secretion.
A Representative images and linescans of fluorescence distribution in AIY axons of animals coexpressing FLP-1::Venus (to mark DCVs) and TOMM-20::mCherry (to mark mitochondria) in the
indicated mutants. Arrowheads mark fluorescent puncta in AIY axons and arrows mark AIY somas.
Scale bar: 10 μm. B Quantification of average coelomocyte fluorescence intensity of the indicated
mutants expressing FLP-1::Venus in AIY following 10 min juglone treatment. Unlined ns, * and
*** denotes statistical analysis compared to “wild type”, unlined ## denotes statistical analysis
compared to “AIY mito-truck;ric-7”, a denotes statistical analysis compared to “ric-7+juglone”, P
< 0.001. n =30, 30, 30, 30, 30, 30, 30, 30, 30, 30 independent animals. C Quantification of average
coelomocyte fluorescence intensity of the indicated mutants expressing FLP-1::Venus in AIY
following 10 min H2O2 treatment or 1 min mito-miniSOG activation. Unlined *** denotes
statistical analysis compared to “wild type”. n = 30, 30, 30, 30, 30 independent animals. Data are
mean values ± s.e.m normalized to wild type controls. ns not significant, * P < 0.05, ## P < 0.01,
*** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple
comparisons test.

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Figure 2.7 mtH2O2-induced FLP-1 Secretion Requires C524 of PKC-1.
A Schematic representation of progressive oxidation of the sulfhydryl groups (-SH) of cysteine
(Cys) residues by H2O2. The cysteine to serine (Ser) substitution mimics non-oxidizable cysteine,
and the cysteine to aspartic acid (Asp) substitution mimics sulfonylated cysteine. B Quantification
of average coelomocyte fluorescence intensity of the indicated mutants expressing FLP-1::Venus
in AIY following 10 min treatment with the indicated oxidants. Unlined ns denotes statistical
analysis compared to “pkc-1”; unlined * and *** denote statistical analysis compared to “wild
type”; a denotes statistical analysis compared to “wild type+H2O2”, P < 0.001; b denotes statistical
analysis compared to “wild type+mito-miniSOG”, P < 0.001. n = 30, 30, 30, 30, 30, 30, 30, 30,
30, 30 independent animals. C Quantification of average coelomocyte fluorescence intensity of
indicated mutants expressing FLP-1::Venus in AIY following 10 min juglone treatment. AIY pkc-

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1 denotes expression of pkc-1a cDNA under the ttx-3 promoter. Unlined *** denotes statistical
analysis compared to “wild type”. n =30, 30, 30, 30 independent animals. D Representative images
and quantification of the number of intestinal nuclei with SKN-1::GFP in adult animals before and
after 10 min of juglone treatment. Dotted lines demarcate intestinal regions. Nuclear translocation
of SKN-1::GFP was measured by counting the number of fluorescent nuclei in the intestine. Fewer
than 10, between 11 and 20, and above 20 fluorescent nuclei are denoted Low, Medium, and High,
respectively. Unlined *** denotes statistical analysis compared to “wild type”, unlined # denotes
statistical analysis compared to “pkc-1”. n = 36, 55, 49, 47 independent animals. Scale bar: 100
μm. E Representative images and quantification of the posterior regions of transgenic worms
expressing the oxidative stress reporter Pgst-4::gfp after 1 h of juglone treatment and 4 h of
recovery. Asterisks mark the intestinal region used for quantitative analysis. Pgst-4::gfp expression
in body wall muscles, which appears as fluorescence on the edge of animals in some images, was
not quantified. Unlined *** denotes statistical analysis compared to “wild type”; unlined ###
denotes statistical analysis compared to “pkc-1”; a denotes statistical analysis compared to “wild
type+juglpne”, P < 0.001. n =20, 29, 31, 31 independent animals. Scale bar: 50 μm. F (Top)
Schematic representation of the protein structure of PKC-1, showing the conserved domains (C1-
C4) the pseudosubstrate domain (PS) and the putative redox active region containing cysteine 524,
which is conserved in human PKCε and C. elegans IRE-1. (Bottom) Quantification of average
coelomocyte fluorescence intensity of the indicated mutants expressing FLP-1::Venus in AIY
following 10 min treatment with juglone. AIY pkc-1(C524S) and pkc-1(C524D) denote expression
of pkc-1a(C524S) and pkc-1a(C524D) variants under the ttx-3 promoter. Unlined * and *** denote
statistical analysis compared to “wild type”, unlined # denotes statistical analysis compared to
“pkc-1”. n = 30, 30, 30, 30, 30, 30, 30 independent animals. Data are mean values ± s.e.m
normalized to wild type controls. ns not significant, * and # P < 0.05, *** and ### P < 0.001 by
Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Figure 2.8 Bacterial Food Sources Alter FLP-1 Secretion and the Antioxidant Response.
A Representative images and punctal fluorescence quantification of AIY axons from adults
expressing mitochondrial-targeted HyPer (mito-HyPer) driven by the ttx-3 promoter cultured on
OP50 or HT115 bacteria and switched to the other food source for the indicated times. The 520
nm/480 nm (GFP/CFP) ratio of HyPer was used to measure H2O2 production. Arrowheads mark
fluorescent puncta in AIY axons. (Top) n = 20, 20, 20, 20, 20, 20, 20 independent animals, (Bottom)
n = 20, 20, 20, 20, 20, 20 independent animals. Scale bar: 10 μm. B Quantification of average
coelomocyte fluorescence intensity of animals expressing FLP-1::Venus in AIY cultured with
either OP50 or HT115 bacteria with or without 10 min juglone treatment. Unlined *** denotes
statistical analysis compared to “wild type” on the same bacterial diet; unlined ### denotes

54
statistical analysis compared to “wild type+juglone” on the same bacterial diet; a denotes statistical
analysis compared to “wild type+juglone” on OP50 diet, P < 0.01; b denotes statistical analysis
compared to “sod-2+juglone” on OP50 diet, P > 0.00. n = 30, 30, 30, 30, 30, 30 independent
animals. C Representative images and quantification of average Pgst-4::gfp expression in the
posterior intestine of adult animals (asterisks) cultured on OP50 or HT115 following exposure to
the indicated concentrations of juglone for 1 and 4 h recovery. Pgst-4::gfp expression in body wall
muscles, which appears as fluorescence on the edge of animals in some images, was not quantified.
Unlined *, ** and *** denote statistical analysis compared to “wild type” on the same bacterial
diet. n =20, 20, 20, 20, 20, 20, 20, 20, 20, 20 independent animals. Scale bar: 50 μm. D
Quantification of average coelomocyte fluorescence intensity of animals expressing FLP-1::Venus
in AIY cultured on live or heat-killed HT115 bacteria. Unlined *** denotes statistical analysis
compared to “OP50”. n = 30, 30, 30 independent animals. E Quantification of average
coelomocyte fluorescence intensity of animals expressing FLP-1::Venus in AIY cultured on OP50
or HT115 bacteria and switched to the other food source for the indicated times. (Top) n = 30, 30,
30, 30, 30, 30, 30, 30 independent animals. (Bottom) n = 30, 30, 30, 30, 30, 30, 30, 30 independent
animals. F Percentage of surviving animals of the indicated genotypes 16 h following treatment
of young adults with juglone for 4 h. Survival of flp-1 mutants was normalized to wild-type animals
raised on the same bacterial strain. Unlined *** denotes statistical analysis compared to “wild type”
on the same bacterial diet; a denotes statistical analysis compared to “flp-1” on OP50 diet. n = 447,
426, 426, 423 independent animals over three independent experiments. G A schematic model in
which cues originating from ingested bacteria (or other stressors) rapidly regulate mitochondrial
H2O2 levels in AIY, leading to PKC-1 C524 sulfenylation and FLP-1 secretion from AIY axons.
Once secreted, FLP-1 activates intestinal SKN-1 signaling and the antioxidant response through
the GPCR NPR-4. The regulation of FLP-1 secretion by H2O2 may represent a way in which the
nervous system can rapidly adjust antioxidant defenses in distal tissues in order to adapt to
constantly changing food sources. Data are mean values ± s.e.m normalized to wild type controls.
ns not significant, * and # P < 0.05, ** P < 0.01, *** and ### P < 0.001 by Brown-Forsythe and
Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 2.1 EGL-3 and FLP-1 Signaling Regulate Specific Stress Responses.
A-D Percentage of surviving animals of the indicated strains following treatment of young adults
with the indicated stressors and 16 hour recovery. Unlined ns, ** and *** denote statistical analysis
compared to “wild type”. (A) n = 116, 57, 78; (B) n = 147, 96, 108; (C) n = 154, 90, 126; (D) n =
70, 49, 78 independent animals over 3 independent experiments. E Representative images and
quantification of nuclear fluorescence the posterior regions of transgenic animals expressing DAF16::GFP after 10min of M9 or juglone treatment. Fewer than 10, between 11 and 20, and above
20 fluorescent nuclei are denoted Low, Medium, and High, respectively. Unlined *** denotes
statistical analysis compared to “wild type”, unlined ### denotes statistical analysis compared to
“egl-3”. n = 25, 35, 22, 24 independent animals. Scale bar: 100μm. F Representative images and
quantification of the posterior regions of transgenic animals expressing the ER stress marker Phsp4::gfp after 1hour of M9 or juglone treatment and 4 hours recovery. Asterisks mark the intestinal

56
region used for quantitative analysis. Unlined *** denotes statistical analysis compared to “wild
type”. n = 20, 20, 20, 20 independent animals. Scale bar: 50μm. G Representative images and
quantification of average Pgst-4::gfp expression in the posterior intestine of adult animals
(asterisks) following treatment with the indicated concentrations of juglone or M9. Neuronal flp1(OE) denotes expression of flp-1 gDNA under the flp-1 promoter. Pgst-4::gfp expression in body
wall muscles, which appears as fluorescence on the edge of animals in some images, was not
quantified. Unlined *, ** and *** denotes statistical analysis compard to “wild type”; unlined ##
and ### denote statistical analysis compared to “neuronal flp-1(OE)”. n = 20, 20, 21, 17, 21, 20,
23, 21, 21, 21 independent animals. Scale bar: 50 μm. Data are mean values ± s.e.m normalized to
wild type controls. ns not significant, * and # P < 0.05, ** and ## P < 0.01, *** and ### P < 0.001
by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 2.2 mito-miniSOG-induced AIY Ablation and Effects of ATP
Generating Mutants on FLP-1 Secretion.
A Representative images and quantification of mCherry marked AIY neurons from transgenic
animals expressing mito-miniSOG under the ttx-3 promoter after 30min of 50mW/cm2 blue light
illumination and overnight recovery on NGM plates. Efficiency of mito-miniSOG induced cell
ablation was measured by counting the number of visible mCherry-marked AIY neurons. Arrows
indicate somas, asterisks indicate somas of the second AIY and arrowheads indicate axons. n = 50,
50 independent animals. Scale bar: 10μm. B Quantification of average coelomocyte fluorescence
of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following M9 or juglone
treatment for 10 minutes. AIY gas-1 denotes expression of gas-1 cDNA under the ttx-3 promoter.
AIY mev-1 denotes expression of mev-1 cDNA under the ttx-3 promoter. NDUFS2,
NADH:ubiquinone oxidoreductase core subunit S2; SDHC, succinate dehydrogenase complex
subunit C. Unlined * and *** denote statistical analysis compared to “wild type”. n = 30, 30, 30,
30, 30, 30, 30, 30, 30, 30 independent animals. C Quantification of average coelomocyte
fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following
M9 or juglone treatment for 10 minutes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Unlined * and *** denote statistical analysis compared to “wild type”. n = 30, 30, 30, 30
independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns not
significant, * P < 0.05, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3
multiple comparisons test.

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Supplementary Figure 2.3 Specificity of Juglone-induced FLP-1 Secretion from AIY.

59
A Representative images showing co-localization of mito-HyPer puncta in the YFP or CFP
channels with the mitochondrial marker TOMM-20::mCherry in AIY axons. Scale bar: 10μm. B
Representative images and quantification of mCherry fluorescence from the indicated mutants coexpressing mito-HyPer and TOMM-20::mCherry in AIY axons with or without 10 minutes of the
indicated oxidative stress treatments. Unlined ns denotes statistical analysis compared to “wild
type”. n = 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, 20 independent animals. Scale bar: 10μm. C
Quantification of average coelomocyte fluorescence intensity following exposure of FLP1::Venus-expressing animals to the indicated juglone concentrations for 10 minutes. Unlined *, **
and *** denote statistical analysis compared to “M9”. n = 30, 30, 30, 30, 30 independent animals.
D Quantification of average coelomocyte fluorescence following exposure of FLP-1::Venusexpressing animals to 300uM juglone for the indicated number of minutes. Unlined ns, *, ** and
*** denote statistical analysis compared to “0 min”. n = 30, 30, 30, 30, 30, 30, 30 independent
animals. E Quantification of average coelomocyte fluorescence intensity following exposure of
FLP-1::Venus-expressing animals to the indicated sodium arsenite concentrations for 10 minutes.
Unlined * and *** denote statistical analysis compared to “M9”. n = 20, 20, 20, 20, 20 independent
animals. F Representative image of AIY in adult animals expressing FLP-1::Venus in AIY
following 10 minutes of M9 or juglone treatment. Scale bar: 10μm. G Representative images
showing that SOD-2::GFP puncta co-localized with TOMM-20::mCherry puncta in AIY axons. In
contrast, SOD-2(∆MLS)::GFP fusion proteins adopted a diffuse pattern of localization in AIY
axons. Scale bar: 10μm. H Representative images and quantification of average coelomocyte
fluorescence following exposure of animals expressing constitutively secreted Venus in AIY (Pttx3::ss-Venus) to juglone for 10 minutes. n = 20, 20 independent animals. Scale bar: 5μm. I
Percentage of surviving animals of the indicated strains 16 hours following treatment of young
adults with juglone for 4 hours. Unlined ns and *** denote statistical analysis compared to “wild
type”. n = 89, 74, 123, 88 independent animals over 3 independent experiments. J Representative
images of AIY axons of adults co-expressing FLP-18::Venus and FLP-1::mCherry. Arrowheads
denote axonal puncta and arrows denote the AIY soma. Scale bar: 5μm. K Representative images
and quantification of average coelomocyte fluorescence following exposure of animals expressing
FLP-18::Venus in AIY to juglone. Unlined ns and *** denote statistical analysis compared to
“wild type”. n = 30, 30, 30, 30 independent animals. Scale bar: 5μm. L Representative image of
an adult expressing FLP-1::Venus in AVK using the flp-1(513bp) promoter. Arrowheads denote
FLP-1::Venus puncta in the AVK axon and arrows denote AVK soma. Scale bar: 30μm. M
Quantification of average coelomocyte fluorescence following exposure of animals expressing
FLP-1::Venus in AVK to juglone. Unlined ns and *** denote statistical analysis compared to “wild
type”. n = 25, 25, 30 independent animals. N Representative images and quantification of average
coelomocyte fluorescence following exposure of animals expressing FLP-1::Venus in AVK to
starvation for 1 hour. n = 25, 25 independent animals. Scale bar: 5μm. O Representative images
and quantification of average coelomocyte fluorescence following exposure of animals expressing
INS-22::Venus in motor neurons (under the unc-129 promoter) to juglone. Unlined ns denotes
statistical analysis compared to “wild type”. n = 20, 20 independent animals. Data are mean values
± s.e.m normalized to wild type controls. ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001
by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 2.4 The Peroxiredoxin-Thioredoxin System Negatively Regulates
FLP-1::Venus Secretion from AIY.
A Quantification of average coelomocyte fluorescence intensity in animals subject to RNAimediated knockdown of the indicated genes. Unlined ns and ** denote statistical analysis
compared to “empty vector”. n = 20, 20, 20, 20 independent animals. B Quantification of average
coelomocyte fluorescence intensity of indicated mutants expressing FLP-1::Venus in AIY.
Unlined ns denotes statistical analysis compared to “wild type”. n = 30, 30 independent animals.
C Quantification of average coelomocyte fluorescence intensity of indicated mutants expressing
FLP-1::Venus in AIY. n = 30, 30 independent animals. D Amino acid sequence alignment of
human TRX2 and C. elegans TRX-2 showing the mitochondrial localization sequence that was
truncated (arrow) and the positions of the conserved cysteines (bold) that were mutated to alanine
to generate catalytically inactive TRX-2 (C68A, C71A). E Representative images of AIY in
animals co-expressing the mitochondrial marker TOMM-20::mCherry and either wild type TRX2::GFP or TRX-2(∆MLS)::GFP, which lacks the TRX-2 mitochondrial localization sequence.
Scale bar: 10μm. Data are mean values ± s.e.m normalized to wild type controls. ns not significant,
** P < 0.01, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple
comparisons test.

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Supplementary Figure 2.5 TOMM-7::UNC-116 Fusion Protein Schematic.
Schematic showing how TOMM-7::UNC-116 fusion proteins can restore anterograde
mitochondrial trafficking to ric-7 mutants, adapted from (Rawson et al., 2014).

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Supplementary Figure 2.6 Localization of PKC-1 in AIY and Juglone-induced INS-22
Secretion.
A Representative images of AIY axons from animals co-expressing the indicated PKC-1::GFP
fusion proteins and TOMM-20::mCherry under the ttx-3 promoter. Scale bar: 10μm. B
Representative images and quantification of average coelomocyte fluorescence intensity of the
indicated mutants expressing INS-22::Venus in DA/DB motor neurons (under the unc-129
promoter) following 10 minute treatment with juglone. Unlined *** denotes statistical analysis
compared to “wild type”, unlined ### denotes statistical analysis compared to “pkc-1”. n = 20, 20,
20, 20 independent animals. Scale bar: 5μm. Data are mean values ± s.e.m normalized to wild type
controls. *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3
multiple comparisons test.

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Supplementary Figure 2.7 Sensory Input or Starvation do not Impact FLP-1 Secretion.
A Quantification of average coelomocyte fluorescence intensity of the indicated mutants
expressing FLP-1::Venus in AIY following juglone treatment. Unlined *** denotes statistical
analysis compared to “wild type”. n = 30, 30, 30, 30, 30, 30 independent animals. B Quantification
of average coelomocyte fluorescence intensity of the indicated mutants expressing FLP-1::Venus
in AIY following 1 hour starvation. n = 30, 30 independent animals. C Representative images and
quantification of TOMM-20:mCherry punctal fluorescence in AIY axons of animals grown on
OP50 or HT115 bacteria. Unlined ns denotes statistical analysis compared to “OP50”. n = 20, 20
independent animals. Scale bar: 10μm. Data are mean values ± s.e.m normalized to wild type
controls. ns not significant, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s
T3 multiple comparisons test.

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Table 2.1 neuropeptide screening for juglone toxicity
RNAi Juglone Survival** RNAi Juglone Survival**
egl-3 20%* nlp-14 91%
flp-1 63%* nlp-15 88%
flp-2 72%* nlp-16 81%
flp-3 85% nlp-17 86%
flp-4 96% nlp-18 96%
flp-5 102% nlp-19 91%
flp-6 124%* nlp-20 90%
flp-7 83% nlp-21 122%*
flp-8 103% nlp-22 99%
flp-9 93% nlp-23 101%
flp-10 94% nlp-24 93%
flp-11 85% nlp-25 84%
flp-12 72%* nlp-26 105%
flp-13 89% nlp-27 96%
flp-14 89% nlp-28 96%
flp-15 94% nlp-29 99%
flp-16 88% nlp-30 99%
flp-17 98% nlp-31 95%
flp-18 109% nlp-32 98%
flp-19 101% nlp-33 97%
flp-20 95% nlp-34 100%
flp-21 102% nlp-35 102%
flp-22 78% nlp-36 104%
flp-23 102% nlp-37 83%
flp-24 86% nlp-38 95%
flp-25 113% nlp-39 96%
flp-26 101% nlp-40 90%
flp-27 100% nlp-41 93%
flp-28 98% nlp-42 104%
flp-32 83% nlp-43 95%
flp-33 98% nlp-44 92%
flp-34 90% nlp-45 108%
nlp-1 94% nlp-46 100%
nlp-2 89% nlp-47 101%
nlp-3 67%* nlp-48 124%*
nlp-4 108% nlp-49 97%
nlp-5 103% nlp-50 97%
nlp-6 95% nlp-51 92%
nlp-7 95% nlp-52 94%
nlp-8 99% nlp-53 98%
nlp-9 88% nlp-54 114%
nlp-10 76%* nlp-55 60%*

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nlp-11 91% nlp-56 109%
nlp-12 93% nlp-57 103%
nlp-13 99%
* denotes p < 0.05, compared to empty vector controls
** denotes percentage of surviving animals normalized to empty vector
controls
animals were fed with bacteria expressing dsRNA corresponding to the
indicated genes

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Table 2.2 GPCR screening for juglone toxicity
RNAi Juglone
Survival** mutants Juglone
Survival** reference
GPCR expressed in the intestine
npr-20 96% (Cao et al., 2017)
npr-28 104% (Cao et al., 2017)
nmur-4 84% ok1381 98% (Cao et al., 2017)
npr-23 95% (Cao et al., 2017;
Kaletsky et al., 2018)
npr-26 101% (Cao et al., 2017)
npr-8 94% (Cao et al., 2017)
npr-4 68%* tm1782 25% (Cao et al., 2017;
Kaletsky et al., 2018)
npr-12 94% (Cao et al., 2017)
frpr-19 91% (Cao et al., 2017)
npr-30 92% (Cao et al., 2017)
W10C4.1 59%* (Kaletsky et al., 2018)
gnrr-2 64%* (Kaletsky et al., 2018)
M04G7.3 107% (Kaletsky et al., 2018)
lat-1 101% (Kaletsky et al., 2018)
pdfr-1 99% (Kaletsky et al., 2018)
lat-2 104% (Kaletsky et al., 2018)
B0334.6 99% (Kaletsky et al., 2018)
F32D8.10 70%* (Kaletsky et al., 2018)
dmsr-1 148%* (Kaletsky et al., 2018)
known FLP-1 GPCRs
frpr-7 84% vj290 102% (Oranth et al., 2018)
npr-6 74%* vj288 102% (Oranth et al., 2018)
GPCR that function in the intestine for other pathways
npr-22 82% ok1598 98% (Palamiuc et al., 2017)
npr-5 89% ok1583 89% (Bhardwaj et al., 2020)
* denotes p < 0.05, compared to empty vector controls
** denotes percentage of surviving animals normalized to empty vector
controls
animals were fed with bacteria expressing dsRNA corresponding to the
indicated genes

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Chapter 3
Endogenous hydrogen peroxide positively regulates secretion of a gut-derived
peptide in neuroendocrine potentiation of the oxidative stress response in C.
elegans
Qi Jia, Drew Young and Derek Sieburth
3.1 Summary
The gut-brain axis mediates bidirectional signaling between the intestine and the nervous
system and is critical for organism-wide homeostasis. Here we report the identification of a
peptidergic endocrine circuit in which bidirectional signaling between neurons and the intestine
potentiates the activation of the antioxidant response in C. elegans. We identify a FMRF-amidelike peptide, FLP-2, whose release from the intestine is necessary and sufficient to activate the
intestinal oxidative stress response by promoting the release of the antioxidant FLP-1 neuropeptide
from neurons. FLP-2 secretion from the intestine is positively regulated by endogenous hydrogen
peroxide (H2O2) produced in the mitochondrial matrix by sod-3/superoxide dismutase, and is
negatively regulated by prdx-2/peroxiredoxin, which depletes H2O2 in both the mitochondria and
cytosol. H2O2 promotes FLP-2 secretion through the DAG and calcium-dependent protein kinase
C family member pkc-2 and by the SNAP25 family member aex-4 in the intestine. Together, our
data demonstrate a role for intestinal H2O2 in promoting inter-tissue antioxidant signaling through
regulated neuropeptide-like protein exocytosis in a gut-brain axis to activate the oxidative stress
response.
3.2 Introduction
The gut-brain axis is critical for communication between the intestine and the nervous
system to regulate behavior and maintain homeostasis, and altered gut-brain signaling is associated
with neurodegeneration, obesity and tumor proliferation (Carabotti et al., 2015; Grenham et al.,
2011; Mayer et al., 2022; Mehrian-Shai et al., 2019; Vitali et al., 2022). Over the last decade the
importance of peptides that function as signals in gut-brain signaling has gained recognition.

68
Numerous gut peptides are distributed throughout the gastrointestinal (GI) tract with regional
specificity (Haber et al., 2017), and gut-secreted peptides can modulate neurocircuits in regulation
of feeding behavior and glucose metabolism (Batterham & Bloom, 2003; W. Han et al., 2018;
Song et al., 2019), can promote inflammatory responses against pathogenic bacteria (CamposSalinas et al., 2014; H. B. Yu et al., 2021), and can regulate food intake and meal size (Batterham
et al., 2002; Chelikani et al., 2005; Gibbs et al., 1973; Lutz et al., 1994, 1995; West et al., 1984).
A gut released peptide suppresses arousal through dopaminergic neurons during sleep in
Drosophila (Titos et al., 2023b). In C. elegans, gut released peptides regulate rhythmic behavior
and behavioral responses to pathogenic bacteria (K. Lee & Mylonakis, 2017; Singh & Aballay,
2019; H. Wang et al., 2013). Conversely, peptides released from the nervous system regulate many
aspects of intestinal function including gut mobility, inflammation and immune defense (Browning
& Travagli, 2014; Furness et al., 2014; Lai et al., 2017). In C. elegans, the secretion of peptides
from various neurons regulates the mitochondrial unfolded protein response (UPRmt), the heat
shock response, and the antioxidant response in the intestine (Jia & Sieburth, 2021; Maman et al.,
2013; Prahlad et al., 2008; Shao et al., 2016). In spite of the many roles of peptides in the gut-brain
axis, the mechanisms underlying the regulation of intestinal peptide secretion and signaling remain
to be fully defined.
Hydrogen peroxide (H2O2) is emerging as an important signaling molecule that regulates
intracellular signaling pathways by modifying specific reactive residues on target proteins. For
example, H2O2 induced tyrosine and serine phosphorylation activate the inhibitor of nuclear factor
κB (NF-κB) kinases (IKKs), leading to the activation of NF-κB during development, inflammation
and immune response. (Kamata et al., 2002; Oliveira-Marques et al., 2009; Takada et al., 2003).
H2O2 induced tyrosine and cysteine modifications contribute to redox regulation of c-Jun Nterminal kinase 2 (JNK2), Src family kinase, extracellular signal-regulated kinases 1 and 2
(ERK1/2), protein kinase C (PKC) and other protein kinases (Kemble & Sun, 2009; Konishi et al.,
1997; Y.-J. Lee et al., 2003; K. J. Nelson et al., 2018). H2O2 signaling has been implicated in
regulating neurotransmission and transmitter secretion. H2O2 at low concentration increases
neurotransmission at neuromuscular junctions without influencing lipid oxidation (A R Giniatullin
& R A Giniatullin, 2003; Giniatullin et al., 2019; Shakirzyanova et al., 2009). Studies with rat
brain tissue demonstrated that enhanced endogenous H2O2 generation regulates dopamine release
(Avshalumov et al., 2005; Avshalumov & Rice, 2003; Bao et al., 2005; B. T. Chen et al., 2001b,

69
2002). Acute H2O2 treatment increases exocytosis of ATP-containing vesicles in astrocytes (Z. Li
et al., 2019). Finally, mitochondrially-derived H2O2 regulates neuropeptide release from neurons
in C. elegans (Jia & Sieburth, 2021). Cellular H2O2 levels are tightly controlled through the
regulation of its production from superoxide by superoxide dismutases (SODs) and cytoplasmic
oxidases (Fridovich, 1995, 1997; Messner & Imlay, 2002; Zelko et al., 2002), and through its
degradation by catalases and peroxidases (Chance et al., 1979; Marinho et al., 2014). In the
intestine, endogenously produced H2O2 plays important roles as an antibacterial agent in the lumen,
and in activating the ER unfolded protein response (UPRER) through protein sulfenylation in C.
elegans (Botteaux et al., 2009; Corcionivoschi et al., 2012; Hourihan et al., 2016; Miller et al.,
2020).
Here we demonstrate a role of endogenous H2O2 signaling in the intestine in regulating the
release of the intestinal FMRFamide-like peptide, FLP-2, to modulate a neurocircuit that activates
the anti-oxidant response in the intestine in C. elegans. Intestinal FLP-2 signaling functions by
potentiating the release of the antioxidant neuropeptide-like protein FLP-1 from AIY interneurons,
which in turn activates the antioxidant response in the intestine. FLP-2 secretion from the intestine
is rapidly and positively regulated by H2O2. Intestinal H2O2 levels and FLP-2 secretion are
positively regulated by superoxide dismutases in the mitochondrial matrix and cytosol, and are
negatively regulated by the peroxiredoxin-thioredoxin system in the cytosol. Intestinal FLP-2
release is mediated by aex-4/SNAP25-dependent exocytosis of dense core vesicles and H2O2-
induced FLP-2 secretion is dependent upon the production of intestinal diacylglycerol and on pkc2/PKCα/β kinase activity.
3.3 Results
3.3.1 Neuronal FLP-1 secretion is regulated by neuropeptide signaling from the intestine.
We previously showed that 10 minute treatment with the mitochondrial toxin juglone leads
to a rapid, reversible and specific increase in FLP-1 secretion from AIY, as measured by a twofold increase in coelomocyte fluorescence in animals expressing FLP-1::Venus fusion proteins in
AIY (Fig. 1A and B, (Jia & Sieburth, 2021)). Coelomocytes take up secreted neuropeptides by
bulk endocytosis (Fares & Greenwald, 2001) and the fluorescence intensity of Venus in their
endocytic vacuoles is used as a measure of regulated neuropeptide secretion efficacy (Ailion et al.,

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2014; Ch’ng et al., 2008; Sieburth et al., 2006). To determine the role of the intestine in regulating
FLP-1 secretion, we first examined aex-5 mutants. aex-5 encodes an intestinal subtilisin/kexin type
5 prohormone convertase (PCSK5), that functions to proteolytically process peptide precursors
into mature peptide fragments in dense core vesicles (DCVs) (S. L. Edwards et al., 2019; Thacker
& Rose, 2000), and aex-5 mutants are defective in peptide signaling from the intestine (Mahoney
et al., 2008). We found that aex-5 mutants expressing FLP-1::Venus in AIY exhibited no
significant difference in coelomocyte fluoresce compared to wild type controls in the absence of
juglone (Fig. 1B). However, coelomocyte fluorescence did not significantly increase in aex-5
mutants treated with juglone. Expression of aex-5 cDNA selectively in the intestine (under the
ges-1 promoter) fully restored normal responses to juglone to aex-5 mutants, whereas aex-5 cDNA
expression in the nervous system (under the rab-3 promoter) failed to rescue (Fig. 1B). Thus,
peptide processing in intestinal DCVs is necessary for juglone-induced FLP-1 secretion from AIY.
Next, we examined a number of mutants with impaired SNARE-mediated vesicle release
in the intestine including aex-1/UNC13, aex-3/MADD, aex-4/SNAP25b, and aex-6/Rab27 (Fig.
S1A (Iwasaki et al., 1997; Mahoney et al., 2006; Thacker & Rose, 2000; Thomas, 1990; Wang et
al., 2013)), and they each exhibited no increases in FLP-1 secretion following juglone treatment
above levels observed in untreated controls (Fig. S1B). NLP-40 is a neuropeptide-like protein
whose release from the intestine is presumed to be controlled by aex-1, aex-3, aex-4 and aex-6
(Lin-Moore et al., 2021; Mahoney et al., 2008; Shi et al., 2022; H. Wang et al., 2013). Null mutants
in nlp-40 or its receptor, aex-2 (H. Wang et al., 2013), exhibited normal juglone-induced FLP-1
secretion (Fig. S1C). These results establish a gut-to-neuron signaling pathway that regulates FLP1 secretion from AIY that is likely to be controlled by peptidergic signaling distinct from NLP-40.
3.3.2 FLP-2 signaling from the intestine potentiates neuronal FLP-1 secretion and the oxidative
stress response.
flp-1 protects animals form the toxic effects of juglone (Jia & Sieburth, 2021). We reasoned
that the intestinal signal that regulates FLP-1 secretion should also protect animals from jugloneinduced toxicity. We identified the FMRF-amide neuropeptide-like protein, flp-2, in an RNA
interference (RNAi) screen for neuropeptides that confer hypersensitivity to juglone toxicity upon
knockdown (Jia & Sieburth, 2021). flp-2 signaling has been implicated in regulating lifespan,
reproductive development, locomotion during lethargus, and the mitochondrial unfolded protein

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response (UPRmt) (Chai et al., 2022; D. Chen et al., 2016; Kageyama et al., 2022; Shao et al., 2016).
Putative flp-2(ok3351) null mutants, which eliminate most of the flp-2 coding region, are
superficially as healthy as wild type animals, but they exhibited significantly reduced survival in
the presence of juglone compared to wild type controls (Fig. 1C). The reduced survival rate of flp2 mutants was similar to that of flp-1 mutants, and flp-1; flp-2 double mutants exhibited survival
rates that were not more severe than those of single mutants (Fig. 1C), suggesting that flp-1 and
flp-2 may function in a common genetic pathway.
To determine whether flp-2 signaling regulates FLP-1 secretion from AIY, we examined
FLP-1::Venus secretion. flp-2 mutants exhibited normal levels of FLP-1 secretion in the absence
of stress, but FLP-1 secretion failed to significantly increase following juglone treatment of flp-2
mutants (Fig. 1D). flp-2 is expressed in a subset of neurons as well as the intestine (Chai et al.,
2022), and flp-2 functions from the nervous system for its roles in development and the UPRmt
(Chai et al., 2022; D. Chen et al., 2016; Kageyama et al., 2022; Shao et al., 2016). Expressing flp2 genomic DNA (gDNA) in the nervous system failed to rescue the FLP-1::Venus defects of flp2 mutants, whereas expressing flp-2 selectively in the intestine fully restored juglone-induced FLP1::Venus secretion to flp-2 mutants (Fig. 1D). Intestinal overexpression of flp-2 had no effect on
FLP-1 secretion in the absence of stress, but significantly enhanced the ability of juglone to
increase FLP-1 secretion (Fig. 1D). These results indicate that although flp-2 signaling has minimal
impacts on FLP-1 secretion under normal conditions, flp-2 originating from the intestine is
necessary and sufficient to positively regulate FLP-1 release from AIY in the presence of juglone
to promote oxidative stress resistance.
Previously we showed that FLP-1 signaling from AIY positively regulates the activation
of the antioxidant transcription factor SKN-1/Nrf2 in the intestine. Specifically, flp-1 mutations
impair the juglone-induced expression of the SKN-1 reporter transgene Pgst-4::gfp (Fig. 1E and
(Jia & Sieburth, 2021)). We found that mutations in flp-2 caused a similar reduction in jugloneinduced Pgst-4::gfp expression as flp-1 mutants, and that flp-1; flp-2 double mutants exhibited
similar impairments in juglone-induced Pgst-4::gfp expression as flp-1 or flp-2 single mutants (Fig.
1E). Conversely, overexpression of flp-2 selectively in the intestine elevated juglone-induced Pgst4::gfp expression, without altering baseline Pgst-4::gfp expression, and the elevated Pgst-4::gfp
expression in juglone-treated animals overexpressing flp-2 was entirely dependent upon flp-1 (Fig.
1F). It’s noteworthy that overexpressing flp-2 in the intestine did not enhance FLP-1::Venus

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release or Pgst-4::gfp expression in the absence of stress, indicating that the regulation of the FLP1 mediated anti-oxidant pathway by flp-2 is stress activated. Together this data indicates that flp2 signaling originating in the intestine positively regulates the stress-induced secretion of FLP-1
from AIY, as well as the subsequent activation of anti-oxidant response genes in the intestine. We
propose that FLP-1 and FLP-2 define a bidirectional gut-neuron signaling axis, whereby during
periods of oxidative stress, FLP-2 released from the intestine positively regulates FLP-1 secretion
from AIY, and FLP-1, in turn, potentiates the antioxidant response in the intestine (Fig. 1A).
3.3.3 FLP-2 secretion from the intestine is H2O2-regulated.
To directly investigate the mechanisms underlying the regulation of FLP-2 secretion, we
examined FLP-2::Venus fusion proteins expressed in the intestine under various conditions (Fig.
2A). FLP-2::Venus fusion proteins adopted a punctate pattern of fluorescence throughout the
cytoplasm of intestinal cells and at the plasma membrane (Fig. 2A), and FLP-2::Venus puncta colocalized with the DCV cargo protein AEX-5/PCSK5 tagged to mTurquoise2 (AEX-5::mTur2,
Fig. 2B). FLP-2::Venus fluorescence was also observed in the coelomocytes (marked by mCherry)
(Fig. 2A, C), indicating that FLP-2 is released from the intestine. SNAP25 forms a component of
the core SNARE complex, which drives vesicular membrane fusion and transmitter release (Y. A.
Chen & Scheller, 2001; Goda, 1997; Jahn & Scheller, 2006). aex-4 encodes the C. elegans
homolog of SNAP25, and mutations in aex-4 disrupt the secretion of neuropeptides from the
intestine (Lin-Moore et al., 2021; Mahoney et al., 2008; H. Wang et al., 2013). We found that aex4 null mutations significantly reduced coelomocyte fluorescence in FLP-2::Venus expressing
animals, and expression of aex-4 cDNA selectively in the intestine fully restored FLP-2 secretion
to aex-4 mutants (Fig. 2C). Together these results suggest that intestinal FLP-2 can be packaged
into DCVs that undergo release via SNARE-dependent exocytosis.
To test whether intestinal FLP-2 secretion is regulated by oxidative stress, we examined
coelomocyte fluorescence in FLP-2::Venus-expressing animals that had been exposed to a number
of different commonly used oxidative stressors. We found that 10 minute exposure to juglone,
thimerosal, or paraquat, which promote mitochondria targeted toxicity (Castello et al., 2007;
Elferink, 1999; Sharpe et al., 2012), each significantly increased Venus fluorescence intensity in
the coelomocytes compared to untreated controls (Fig. 2C and D). We conducted four controls for
specificity: First, juglone treatment did not significantly alter fluorescence intensity of mCherry

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expressed in coelomocytes (Fig. S2A). Second, impairing intestinal DCV secretion (by either aex4/SNAP25 or aex-6/Rab27 mutations (Lin-Moore et al., 2021; Mahoney et al., 2006, 2008;
Thomas, 1990), blocked the juglone-induced increase in coelomocyte fluorescence in FLP2::Venus expressing animals (Fig. 2C). Third, nlp-40 and nlp-27 encode neuropeptide-like proteins
that are released from the intestine (J. Liu et al., 2023; Taylor et al., 2021; H. Wang et al., 2013),
and juglone treatment had no detectable effects on coelomocyte fluorescence in animals expressing
intestinal NLP-40::Venus or NLP-27::Venus fusion proteins (Fig S2B and C), and NLP-40::mTur2
puncta did not overlap with FLP-2::Venus puncta in the intestine (Fig. S2D). Finally, flp-1 mutants
exhibited wild type levels of FLP-2 secretion both in the absence and presence of juglone (Fig.
2E). Together, these results indicate that acute oxidative stress selectively increases the exocytosis
of FLP-2-containing DCVs from the intestine, upstream of flp-1 signaling.
3.3.4 SOD-1 and SOD-3 superoxide dismutases regulate FLP-2 release.
Juglone treatment promotes the production of mitochondrial superoxide, which can then
be rapidly converted into H2O2 by superoxide dismutase. To determine whether H2O2 impacts
FLP-2 secretion, we first examined superoxide dismutase mutants. C. elegans encodes five
superoxide dismutase genes (sod-1 through sod-5). sod-1 or sod-3 null mutations blocked jugloneinduced FLP-2 secretion without altering baseline FLP-2 secretion, whereas sod-2, sod-4, or sod5 mutations had no effect on FLP-2 secretion in the presence of juglone (Fig. 3A and B, S3A).
sod-1; sod-3 double mutants exhibited juglone-induced FLP-2 secretion defects that was similar
to single mutants, without significantly altering FLP-2 secretion in the absence of stress (Fig. 3C).
sod-1 encodes the ortholog of mammalian SOD1, which is a cytoplasmic SOD implicated in the
development of amyotrophic lateral sclerosis (ALS) and cancer (Giglio et al., 1994; Papa et al.,
2014; X. Wang et al., 2021; F. Zhang et al., 2007). SOD-1::fusion proteins adopted a diffuse
pattern of fluorescence in intestinal cells, consistent with a cytoplasmic localization (Fig. 3D).
Transgenes expressing the sod-1 cDNA selectively in the intestine fully rescued the jugloneinduced FLP-2::Venus secretion defects of sod-1 mutants (Fig. 3A). sod-3 encodes a homolog of
mammalian SOD2, which is a mitochondrial matrix SOD implicated in protection against
oxidative stress induced neuronal cell death (Fukui & Zhu, 2010; Vincent et al., 2007). Intestinal
SOD-3::GFP fusion proteins localized to round structures that were surrounded by the outer
membrane mitochondrial marker TOMM-20::mCherry (Ahier et al., 2018), consistent with a

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mitochondrial matrix localization (Fig. 3E). Expression of sod-3 cDNA in the intestine fully
restored juglone induced FLP-2 release to sod-3 mutants (Fig. 3B). sod-3 variants lacking the
mitochondrial localization sequence (sod-3(DMLS)), were no longer localized to mitochondria
(Fig. 3F) and failed to restore normal responsiveness to juglone to sod-3 mutants (Fig. 3B). Thus,
the generation of H2O2 by either SOD-1 in the cytoplasm or by SOD-3 in the mitochondrial matrix
is necessary for juglone to increase FLP-2 secretion.
Next, to determine if H2O2 can regulate FLP-2 secretion, we examined the effects of acute
exogenous H2O2 exposure on coelomocyte fluorescence of FLP-2::Venus-expressing animals. We
found that 10 minute treatment with H2O2 increased FLP-2::Venus secretion to a similar extent as
juglone treatment. aex-4/SNAP25, or aex-6/Rab27 mutants exhibited no increase in FLP-2
secretion in response to H2O2 treatment compared to untreated controls (Fig. 3G). In contrast, sod1 or sod-3 mutants (or sod-1; sod-3 double mutants) exhibited an increase in FLP-2 secretion in
response to H2O2 that was similar to that of wild type controls (Fig. 3H), suggesting that exogenous
H2O2 can bypass the requirement of SODs but not SNAREs to promote FLP-2 secretion. Together
these results suggest that H2O2 generated by SODs can positively regulate intestinal FLP-2
exocytosis form DCVs (Fig. 3I).
3.3.5 SOD-1 and SOD-3 regulate intestinal mitochondrial H2O2 levels.
To directly monitor H2O2 levels in the intestine, we generated transgenic animals
expressing HyPer7, which is a pH stable genetically encoded H2O2 sensor in which the ratio of
GFP/CFP fluorescence intensity increases in the presence of H2O2 (Pak et al., 2020). We targeted
HyPer7 to either the mitochondrial matrix (matrix-HyPer7) by generating fusion proteins with the
cytochrome c MLS, or to the cytosolic face of the outer mitochondrial membrane (OMM-HyPer7)
by generating fusion proteins with TOMM-20. When co-expressed in the intestine with the OMM
marker TOMM-20::mCherry, matrix-HyPer7 formed round structures throughout the cytoplasm
that were surrounded by the OMM, and OMM-HyPer7 formed ring-like structures throughout the
cytoplasm that co-localized with the OMM marker (Fig. 3J). Ten minutes treatment with H2O2
significantly increased the fluorescence intensity by about two-fold of both matrix-HyPer7 and
OMM-HyPer7 without altering mitochondrial morphology or abundance (Fig. 3J, S3B and C),
validating the utility of HyPer7 as a sensor for acute changes in H2O2 levels in and around intestinal
mitochondria.

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To determine whether juglone treatment impacts H2O2 levels in the intestine, we first
treated matrix-HyPer7 expressing animals with juglone for 10 minutes. Juglone treatment led to a
similar two-fold increase in matrix-HyPer7 fluorescence as H2O2 treatment (Fig. 3J). sod-3
mutations did not alter baseline H2O2 levels in the matrix, but they completely blocked jugloneinduced increases H2O2 levels, whereas sod-1 mutations had no effect on either baseline or
juglone-induced increases H2O2 levels (Fig. 3J). These results indicate that superoxide produced
by juglone treatment is likely to be converted into H2O2 by SOD-3 in the matrix (Fig. 3I).
Next, we examined H2O2 levels on the outer surface of mitochondria using OMM-HyPer7
and we found that juglone treatment led to a two-fold increase in OMM-HyPer7 fluorescence,
similar to H2O2 treatment (Fig. 3J). sod-3 or sod-1 mutations did not alter baseline H2O2 levels on
the OMM, but sod-1 single mutations attenuated, juglone-induced increases in OMM H2O2 levels,
while sod-3 mutations had no effect (Fig. 3J). In sod-1; sod-3 double mutants, the juglone-induced
increase in OMM H2O2 levels was completely blocked, whereas baseline H2O2 levels in the
absence of stress were unchanged (Fig. 3J). These results suggest that sod-3 and sod-1 are
exclusively required for H2O2 production by juglone and that both mitochondrial SOD-3 and
cytosolic SOD-1 contribute to H2O2 levels in the cytosol. One model that could explain these
results is that juglone-generated superoxide is converted into H2O2 both by SOD-3 in the matrix,
and by SOD-1 in the cytosol, and that the H2O2 generated in the matrix can exit the mitochondria
to contribute to cytosolic H2O2 levels needed to drive FLP-2 secretion (Fig. 3I).
3.3.6 The peroxiredoxin-thioredoxin system regulates endogenous H2O2 levels and FLP-2
secretion.
To determine whether endogenous H2O2 regulates FLP-2 secretion, we examined
mutations in the peroxiredoxin-thioredoxin system. Peroxiredoxins and thioredoxins detoxify
excessive H2O2 by converting it into water and they play a critical role in maintaining cellular
redox homeostasis (Netto & Antunes, 2016) (Fig. 4A). C. elegans encodes two peroxiredoxin
family members, prdx-2 and prdx-3, that are expressed at high levels in the intestine (Taylor et
al., 2021). Null mutations in prdx-2 significantly increased FLP-2::Venus secretion compared to
wild type animals in the absence of stress (Fig. 4B), whereas null mutations in prdx-3 had no effect
on FLP-2 secretion (Fig. S4A). We observed a corresponding increase in both matrix-HyPer7 and
OMM-HyPer7 fluorescence intensity in prdx-2 mutants (Fig. 4C and D), demonstrating that

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endogenous H2O2 is neutralized by peroxiredoxin and establishing a correlation between
endogenous H2O2 levels and FLP-2 secretion. The increase in FLP-2 secretion in prdx-2 mutants
was not further increased by juglone treatment (Fig. 4B). These results suggest that H2O2 generated
under either normal conditions or by juglone-treatment can positively regulate FLP-2 secretion.
There are three isoforms of prdx-2 that arise by the use of alternative transcriptional start
sites (Fig. 4A). Expressing the prdx-2b isoform selectively in the intestine fully rescued the
elevated FLP-2::Venus secretion defects of prdx-2 mutants, whereas expressing prdx-2a or prdx2c isoforms failed to rescue (Fig. 4B, S4B and C). To independently verify the role of prdx-2b
function in FLP-2 release, we generated a prdx-2b-specific knockout mutant by introducing an in
frame stop codon within the prdx-2b-specific exon 1 using CRISPR/Cas9 (prdx-2b(vj380) Fig.
4A). prdx-2b(vj380) mutants exhibited increased H2O2 levels in the mitochondrial matrix and
OMM (Fig. 4C and D), as well as increased FLP-2::Venus secretion compared to wild type
controls that were indistinguishable from prdx-2 null mutants (Fig. 4B). prdx-2b mutations could
no longer increase FLP-2 secretion when either sod-1 or sod-3 activity was impaired (Fig. 4E and
Fig. S4D). Thus, the prdx-2b isoform normally inhibits FLP-2 secretion likely by promoting the
consumption of H2O2 in the mitochondrial matrix and/or cytosol.
Once oxidized, peroxiredoxins are reduced by thioredoxins (TRXs) for reuse [refs]. TRX3 is an intestine-specific thioredoxin promoting protection against specific pathogen infections
(Jiménez-Hidalgo et al., 2014; Miranda-Vizuete et al., 2000; Netto & Antunes, 2016). Mutations
in trx-3 elevated FLP-2::Venus release in the absence of juglone and expressing trx-3 transgenes
in the intestine restored wild type FLP-2 release to trx-3 mutants (Fig. 4B). Juglone treatment
failed to further enhance FLP-2::Venus release in trx-3 mutants (Fig. 4B). Mutations in
cytoplasmic sod-1 but not in mitochondrial sod-3 reduced the elevated FLP-2::Venus release in
trx-3 mutants to wild type levels (Fig. 4B). Mutations in trx-3 increased H2O2 levels in the OMM
but had no effect on matrix H2O2 levels (Fig. 4C and D). Thus TRX-3 likely functions in the
cytosol but not in the matrix to neutralize H2O2, and elevated H2O2 levels in the cytosol are
sufficient to drive FLP-2 secretion without SOD-3-mediated H2O2 generation in the matrix.
Finally, to investigate the physiological significance of elevated endogenous H2O2 levels
on the oxidative stress response we examined the effects of prdx-2b mutations on expression of
gst-4. prdx-2b mutants had significantly increased Pgst-4::gfp expression in the intestine compared
to wild type controls (Fig. 4F). The increased Pgst-4::gfp expression in prdx-2b mutants was

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completely dependent upon flp-2 signaling, since gst-4 expression was reduced to wild type levels
in prdx-2b; flp-2 double mutants (Fig. 4F). Together our data suggest that prdx-2b functions in the
intestine to maintain redox homeostasis following SOD-1/SOD-3 mediated H2O2 production by
regulating the secretion of FLP-2 (Fig. 3I).
3.3.7 PKC-2/PKCα/β mediates H2O2 induced FLP-2 secretion from the intestine.
H2O2 functions as a cellular signaling molecule by oxidizing reactive cysteines to sulfenic
acid, and this modification on target proteins can regulate intracellular signaling pathways (García
Santamarina et al., 2014). One of the validated targets of H2O2 signaling is the protein kinase C
(PKC) family of serine threonine kinases (Jia & Sieburth, 2021; Konishi et al., 1997, 2001; Min et
al., 1998). C. elegans encodes four PKC family members including pkc-1 and pkc-2, which are
expressed at highest levels in the intestine (Islas-Trejo et al., 1997; Taylor et al., 2021). pkc-1 null
mutants had no effect on baseline or juglone-induced FLP-2 secretion (Fig. S5A). pkc-2 null
mutations did not alter baseline intestinal FLP-2 secretion, but they eliminated juglone-induced
FLP-2 secretion (Fig. 5A). pkc-2 encodes a calcium and diacylglycerol (DAG) stimulated PKCα/β
protein kinase C that regulates thermosensory behavior by promoting transmitter secretion (M. R.
Edwards et al., 2012; Land & Rubin, 2017). Expressing pkc-2 cDNA selectively in the intestine
fully restored juglone-induced FLP-2 secretion to pkc-2 mutants (Fig. 5A), whereas expressing a
catalytically inactive pkc-2(K375R) variant (Van et al., 2021) failed to rescue (Fig. 5A). The
intestinal site of action of pkc-2 is in line with prior studies showing that pkc-2 can function in the
intestine to regulate thermosensory behavior (Land & Rubin, 2017). pkc-2 mutants exhibited wild
type H2O2 levels in the mitochondrial matrix and OMM of the intestine in both the presence and
absence of juglone (Fig. 5B and C). Increasing H2O2 levels by either acute H2O2 treatment or by
prdx-2 mutation failed to increase FLP-2 secretion in pkc-2 mutants (Fig. 5D and E). To determine
whether pkc-2 can regulate the secretion of other peptides from the intestine, we examined
expulsion frequency, which is a measure of NLP-40 secretion (Mahoney et al., 2008; H. Wang et
al., 2013). pkc-2 mutants showed wild type expulsion frequency (Fig. S5B), indicating that
intestinal NLP-40 release is largely unaffected. Together, these results show that pkc-2 is not a
general regulator of intestinal peptide secretion, and that PKC-2 functions in the intestine
downstream or in parallel to H2O2 to promote FLP-2 secretion by a mechanism that involves
phosphorylation of target proteins.

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3.3.8 DAG positively regulates FLP-2 secretion.
PKCα/β family members contain two N terminal C1 domains (C1A and C1B) whose
binding to DAG promotes PKC recruitment to the plasma membrane (D. J. Burns & Bell, 1991;
Darby et al., 2017; Johnson et al., 2000; K. M. Kim et al., 2016; Ono et al., 1989; Yanase et al.,
2011). To address the role of DAG in promoting FLP-2 secretion by PKC-2, we examined mutants
that are predicted to have altered DAG levels. Phosphatidylinositol phospholipase C beta (PLCβ)
converts phosphatidyl inositol phosphate (PIP2) to DAG and inositol triphosphate (IP3, Fig. 6A),
and impairing PLC activity leads to reduced cellular DAG levels (Nebigil, 1997). C. elegans
encodes two PLC family members whose expression is enriched in the intestine, plc-2/ PLCβ and
egl-8/ PLCβ (Taylor et al., 2021). plc-2 null mutants exhibited baseline and juglone-included FLP2 secretion that were similar to wild type controls (Fig. S6). egl-8 loss-of-function mutants
exhibited wild type baseline FLP-2 secretion, but juglone-induced FLP-2 secretion was completely
blocked (Fig. 6B). H2O2 levels in egl-8 mutants were similar to wild type controls, both in the
presence and absence of juglone (Fig. 6C and D). Thus, egl-8/PLCb functions downstream of or
in parallel to H2O2 production to promote FLP-2 secretion.
DAG kinase converts DAG into phosphatidic acid (PA), and is therefore a negative
regulator or DAG levels (Fig. 6A) (Topham, 2006; van Blitterswijk & Houssa, 2000). In C. elegans,
dgk-2/DGKε is the highest expressing DAG kinase in the intestine. Mutations in dgk-2 elevated
FLP-2::Venus secretion (Fig. 6E) without altering H2O2 levels in the intestinal mitochondrial
matrix or OMM (Fig. 6F and G). Expressing dgk-2 transgenes selectively in the intestine restored
normal FLP-2::Venus secretion to dgk-2 mutants (Fig. 6E). Finally, the increase in FLP-2 secretion
in dgk-2 mutants was not further increased by juglone treatment, but it was completely blocked by
pkc-2 mutations or aex-4/SNAP25 mutations (Fig. 6E and H). These results show that FLP-2
secretion can be regulated bidirectionally by DAG, and they suggest that DAG and H2O2 function
in a common genetic pathway upstream of pkc-2 to promote FLP-2 secretion (Fig. 6A).
3.4 Discussion
By screening for intercellular regulators of FLP-1 signaling from the nervous system in
promoting the anti-oxidant response, we have uncovered a function for peptidergic signaling in
mediating gut-to-neuron regulation of the anti-oxidant response in C. elegans. We identified the

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neuropeptide-like protein FLP-2 as an inter-tissue signal originating in the intestine to potentiate
stress-induced FLP-1 release from AIY neurons and the subsequent activation of SKN-1 in the
intestine. We found that H2O2 generated endogenously in the intestine or exogenously by acute
oxidant exposure increases FLP-2 secretion from intestinal DCVs. H2O2 promotes FLP-2
exocytosis through PKC-2, and AEX-4/SNAP25. The use of oxidant-regulated peptide secretion
exemplifies a mechanism that can allow the gut and the nervous system to efficiently and rapidly
communicate through endocrine signaling to promote organism-wide protection in the
3.4.1 A new function for flp-2 signaling in the antioxidant response.
Previous studies have identified roles for flp-2 signaling in development and in stress
responses. flp-2 promotes locomotion during molting (D. Chen et al., 2016) promotes entry into
reproductive growth (Chai et al., 2022), regulates longevity (Kageyama et al., 2022), and activates
the UPRmt cell-non autonomously during mitochondrial stress (Shao et al., 2016). The function we
identified for flp-2 in the antioxidant response has some notable similarities with flp-2’s other
functions. First, flp-2 mediates its effects at least in part by regulating signaling by other peptides.
flp-2 signaling increases the secretion of the neuropeptide like protein PDF-1 during lethargus (D.
Chen et al., 2016) and INS-35/insulin-like peptide for its roles in reproductive growth choice and
longevity (Kageyama et al., 2022), in addition to regulating AIY FLP-1 secretion (Fig. 5G). In
mammals, release of the RF-amide neuropeptide kisspeptin from the anteroventral periventricular
nucleus (AVPV) regulates reproduction by inducing the release of gonadotropins via its
stimulatory action on GnRH neurons (S.-K. Han et al., 2005). Second, the secretion of FLP-2 is
dynamic. FLP-2 secretion decreases during lethargus (D. Chen et al., 2016) and increases under
conditions that do not favor reproductive growth (Kageyama et al., 2022), as well increasing in
response to oxidants (Fig. 2C and D). However, in some instances, the regulation of FLP-2
secretion may occur at the level of flp-2 expression (Kageyama et al., 2022), rather than at the
level of exocytosis (Fig. 2C). Finally, genetic analysis of flp-2 has revealed that under normal
conditions, flp-2 signaling may be relatively low, since flp-2 mutants show no defects in
reproductive growth choice when animals are well fed (Chai et al., 2022), show only mild defects
in locomotion during molting in non-sensitized genetic backgrounds (D. Chen et al., 2016), and
do not have altered baseline FLP-1 secretion or antioxidant gene expression in the absence of
exogenous oxidants (Fig. 1D and E). It is notable that increased ROS levels are associated with

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molting (Back et al., 2012; Knoefler et al., 2012), ageing (Back et al., 2012; Van Raamsdonk &
Hekimi, 2010), starvation (Tao et al., 2017), and mitochondrial dysfunction (Dingley et al., 2010),
raising the possibility that flp-2 may be used in specific contexts associated with high ROS levels
to affect global changes in physiology, behavior and development.
One major difference we found for flp-2 signaling in our study is that intestinal, but not
neuronal flp-2 activates the oxidative stress response, whereas flp-2 originates from neurons for its
reported roles in development and the UPRmt. The intestine is uniquely poised to relay information
about diet to the rest of the animal, and secretion of a number of neuropeptide-like proteins from
the intestine (e.g, INS-11, PDF-2 and INS-7) is proposed to regulate responses to different bacterial
food sources (K. Lee & Mylonakis, 2017; Murphy et al., 2007; O’Donnell et al., 2018). Since
bacterial diet can impact ROS levels in the intestine (Pang & Curran, 2014), secretion of FLP-2
from the intestine could function to relay information about bacterial diet to distal tissues to
regulate redox homeostasis. In addition, the regulation of intestinal FLP-2 release by oxidants may
meet a unique spatial, temporal or concentration requirement for activating the antioxidant
response that cannot be met by its release from the nervous system.
3.4.2 AIY as a target for flp-2 signaling.
AIY interneurons receive sensory information from several neurons primarily as
glutamatergic inputs to regulate behavior (Bargmann et al., 2007; Clark et al., 2006; Satoh et al.,
2014). Our study reveals a previously undescribed mechanism by which AIY is activated through
endocrine signaling originating from FLP-2 secretion from the intestine. FLP-2 could act directly
on AIY, or it may function indirectly through upstream neurons that relay FLP-2 signals to AIY.
frpr-18 encodes an orexin-like GPCR that can be activated by FLP-2-derived peptides in
transfected mammalian cells (Larsen et al., 2013; Mertens et al., 2005), and frpr-18 functions
downstream of flp-2 in the locomotion arousal circuit (D. Chen et al., 2016). frpr-18 is expressed
broadly in the nervous system including in AIY (D. Chen et al., 2016), and loss-of-function frpr18 mutations lead to hypersensitivity to certain oxidants (Ouaakki et al., 2023). FRPR-18 is
coupled to the heterotrimeric G protein Gaq (Larsen et al., 2013; Mertens et al., 2005), raising the
possibility that FLP-2 may promote FLP-1 secretion from AIY by directly activating FRPR-18 in
AIY. However, flp-2 functions independently of frpr-18 in the reproductive growth circuit, and
instead functions in a genetic pathway with the GPCR npr-30 (Chai et al., 2022). In addition, FLP-

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2-derived peptides can bind to the GPCRs DMSR-1, or DMSR-18 in transfected cells (Beets et al.,
2023). Identifying the FLP-2 receptor and its site of action will help to define the circuit used by
intestinal flp-2 to promote FLP-1 release from AIY.
FLP-1 release from AIY is positively regulated by H2O2 levels generated from
mitochondria (Jia & Sieburth, 2021). Here we showed that FLP-1 release is also regulated by
intestinal FLP-2 signaling. Interestingly, H2O2 treatment is not sufficient to promote FLP-1
secretion in the absence of flp-2, and intestinal-derived FLP-2 is not sufficient to promote FLP-1
secretion in the absence of H2O2 (Fig. 1D). These results point to a model whereby AIY must
integrate at least two stress signals: increased mt H2O2 levels and increased flp-2 signaling input,
and only when both conditions are met will FLP-1 secretion increase. AIY shows a sporadic Ca2+
response regardless of the presence of explicit stimulation (Ashida et al., 2019; Bargmann et al.,
2007; Clark et al., 2006), and FLP-1 secretion from AIY is calcium dependent (Jia & Sieburth,
2021). How mitochondrial H2O2 levels are established in AIY by intrinsic or extracellular inputs,
and how AIY integrates H2O2 and flp-2 signaling to control FLP-1 secretion remain to be defined.
3.4.3 A role for endogenous H2O2 in regulated neuropeptide secretion.
Using HyPer7, we showed that acute juglone exposure results in a rapid elevation of
endogenous H2O2 levels inside and outside intestinal mitochondria and a corresponding increase
of FLP-2 release from the intestine that depends on the cytoplasmic superoxide dismutase sod-1,
and mitochondrial sod-3. We favor a model whereby superoxide generated by juglone in the
mitochondria is converted to H2O2 by SOD-3 in the matrix and by SOD-1 in the cytosol. In this
case, both the superoxide generated by juglone and the H2O2 generated by SOD-3 would have to
be able to exit the mitochondria and enter the cytosol. Superoxide and H2O2 can be transported
across mitochondrial membranes through anion channels and aquaporin channels, respectively
(Bienert & Chaumont, 2014; Ferri et al., 2003; D. Han et al., 2003; Kontos et al., 1985). The
observation that both SOD-1 and SOD-3 activity are necessary to drive FLP-2 release suggests
that H2O2 levels much reach a certain threshold in the cytoplasm to promote FLP-2 release, and
this threshold requires the generation of H2O2 by both SOD-1 and SOD-3.
We identified a role for the antioxidant peroxiredoxin-thioredoxin system, encoded by
prdx-2 and trx-3, in maintaining low endogenous H2O2 levels in the intestine and in negatively
regulating FLP-2 secretion. We showed that the prdx-2b isoform functions to inhibit FLP-2

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secretion and to lower H2O2 levels in both the mitochondrial matrix and on the cytosolic side of
mitochondria. These observations are consistent with a subcellular site of action for PRDX-2B in
either the matrix only or in both the matrix and cytosol. In contrast, trx-3 mutations do not alter
mitochondrial H2O2 levels, suggesting that TRX-3 functions exclusively in the cytosol. Thus, the
PRDX-2B-TRX-3 combination may function in the cytosol, and PRDX-2B may function with a
different TRX family member in the matrix. There are several thioredoxin-domain containing
proteins in addition to trx-3 in the C. elegans genome that could be candidates for this role.
Alternatively, prdx-2 may function alone or with other redox proteins. PRDX-2 may function
without thioredoxins in its roles in light sensing and stress response in worms (G. Li et al., 2016;
Oláhová et al., 2008; Oláhová & Veal, 2015). PRDX-2B contains a unique N terminal domain that
is distinct from the catalytic domain and is not found on the other PRDX-2 isoforms. This domain
may be important for targeting PRDX-2B to specific subcellular location(s) where it can regulate
FLP-2 secretion.
3.4.4 Regulation of FLP-2 exocytosis by PKC-2/PKCα/β and AEX-4/SNAP25.
We demonstrated that pkc-2 mediates the effects of H2O2 on intestinal FLP-2 secretion,
and H2O2- and DAG-mediated PKC-2 activation are likely to function in a common genetic
pathway to promote FLP-2 secretion. Our observations that DAG is required for the effects of
juglone (Fig. 6B), are consistent with a two-step activation model for PKC-2, in which H2O2 could
first modify PKC-2 in the cytosol, facilitating subsequent PKC-2 recruitment to the membrane by
DAG. Alternatively, DAG could first recruit PKC-2 to membranes, where it is then modified by
H2O2. We favor a model whereby H2O2 modification occurs in the cytosol, since H2O2 produced
locally by mitochondria would have access to cytosolic pools of PKC-2 prior to its membrane
translocation.
We defined a role for aex-4/SNAP25 in the fusion step of FLP-2 containing DCVs from
the intestine under normal conditions as well as during oxidative stress. In neuroendocrine cells,
phosphorylation of SNAP25 on Ser187 potentiates DCV recruitment into releasable pools (Nagy
et al., 2002; Shu et al., 2008; Y. Yang et al., 2007), and exocytosis stimulated by the DAG analog
phorbol ester (Gao et al., 2016; Shu et al., 2008), without altering baseline SNAP25 function.
Interestingly, the residue corresponding to Ser187 is conserved in AEX-4, raising the possibility
that PKC-2 potentiates FLP-2 secretion by phosphorylating AEX-4. Since SNAP25

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phosphorylation on Ser187 has been shown to increase its interaction with syntaxin and promote
SNARE complex assembly in vitro (Gao et al., 2016; Y. Yang et al., 2007), it is possible that
elevated H2O2 levels could promote FLP-2 secretion by positively regulating SNARE-mediated
DCV fusion at intestinal release sites on the basolateral membrane through AEX-4/SNAP25
phosphorylation by PKC-2. Prior studies have shown that PKC-2 phosphorylates the SNAREassociated protein UNC-18 in neurons to regulate thermosensory behavior (M. R. Edwards et al.,
2012; Land & Rubin, 2017). Thus, PKC-2 may have multiple targets in vivo and target selection
may be dictated by cell type and/or the redox status of the cell.
3.4.5 Similar molecular mechanisms regulating FLP-1 and FLP-2 release.
The molecular mechanisms we identified that regulate FLP-2 secretion from the intestine
are similar in several respects to those regulating FLP-1 secretion from AIY. First, the secretion
of both peptides is positively regulated by H2O2 originating from mitochondria. Second, in both
cases, H2O2 promotes exocytosis of neuropeptide-containing DCVs by a mechanism that depend
upon the kinase activity of protein kinase C. Finally, the secretion of both peptides is controlled
through the regulation of H2O2 levels by superoxide dismutases and by the peroxiredoxinthioredoxin system. H2O2-regulated FLP-1 and FLP-2 secretion differ in the identity of the family
members of some of the genes involved. prdx-3-trx-2 and sod-2 family members regulate H2O2
levels in AIY, whereas prdx-2-trx-3 and sod-1/sod-3 family members regulate H2O2 levels in the
intestine. In addition, pkc-1 promotes H2O2 induced FLP-1 secretion from AIY whereas pkc-2
promotes H2O2 -induced FLP-2 secretion from the intestine. Nonetheless, it is noteworthy that two
different cell types utilize largely similar pathways for the H2O2-mediated regulation of
neuropeptide release, raising the possibility that similar mechanisms may be utilized in other cell
types and/or organisms to regulate DCV secretion.
3.5 Acknowledgement
C. elegans strains used in this study were provided by the Caenorhabditis Genetics Centre
(CGC), which is funded by the NIH National Center for Research Resources (NCRR). We thank
members of the Sieburth lab for critical reading and discussion of the manuscript. This work was
supported by grants from National Institute of Health NINDS R01NS071085 and R01NS110730
to D.S.

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3.6 Materials and Methods
3.6.1 Strains and transgenic lines
C. elegans strains were maintained at 20°C in the dark on standard nematode growth
medium (NGM) plates seeded with OP50 Escherichia coli as food source, unless otherwise
indicated. All strains were synchronized by picking mid L4 stage animal either immediately before
treatment (for coelomocyte imaging and intestine imaging) or 24h before treatment (for Pgst4::gfp imaging). The wild type strain was Bristol N2. Mutants used in this study were out-crossed
at least four times.
Transgenic lines were generated by microinjecting plasmid mixes into the gonads of young
adult animals following standard techniques (Mello et al., 1991). Microinjection mixes were
prepared by mixing expression constructs with the co-injection markers pJQ70 (Pofm-1::rfp,
25ng/μL), pMH163 (Podr-1::mCherry, 40ng/μL), pMH164 (Podr-1::gfp, 40ng/μL) or pDS806
(Pmyo-3::mCherry, 20ng/μL) to a final concentration of 100ng/μL. For tissue-specific expression,
a 1.5kb rab-3 promoter was used for pan-neuronal expression (Nonet et al., 1997), a 2.0kb ges-1
or a 3.5kb nlp-40 promoter was used for intestinal expression (Egan et al., 1995; H. Wang et al.,
2013). At least three transgenic lines were examined for each transgene, and one representative
line was used for quantification. Strains and transgenic lines used in this study are listed in the
Supplementary Table.
3.6.2 Molecular Biology
All gene expression vectors were constructed with the backbone of pPD49.26. Promoter
fragments including Prab-3, and Pges-1 were amplified from genomic DNA; genes of interest,
including cDNA fragments (aex-5, snt-5, sod-1b, sod-3, isp-1, prdx-2a, prdx-2b, prdx-2c, trx-3,
pkc-2b, dgk-2a, aex-4) and genomic fragments (flp-2, flp-40, nlp-36, nlp-27) were amplified from
cDNA library and genomic DNA respectively using standard molecular biology protocols.
Expression plasmid of HyPer7 was designed based on reported mammalian expression plasmid
for HyPer7 (Pak et al., 2020) and was synthesized by Thermo Fisher Scientific with codon
optimization for gene expression in C. elegans. Plasmids and primers used in this study are listed
in the Supplementary Table.

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3.6.3 Toxicity Assay
Stock solution of 50mM juglone in DMSO was freshly made on the same day of liquid
toxicity assay, working solution of juglone in M9 buffer was prepared using stock solution before
treatment. Around 60-80 synchronized adult animals were transferred into a 1.5mL Eppendorf
tube with fresh M9 buffer and washed three times. Working solution of juglone was added to the
animals at indicated concentration. Animals were incubated in dark for 4h on rotating mixer before
being transferred onto fresh NGM plates seeded with OP50 to recover in dark at 20°C. Percentage
of survival was assayed by counting the number of alive and dead animals. Toxicity assays were
performed in triplicates.
3.6.4 RNAi Interference
Plates for feeding RNAi interference were prepare as described (Kamath, 2003). Around
20-25 gravid adult animals with indicated genotype were transferred onto the RNAi plates that
were seeded with HT115(DE3) bacteria transformed with L4440 vectors with targeted gene inserts
or empty L4440 vectors. Eggs were collected for 4h to obtain synchronized populations. L4 stage
animals were collected for further assays. RNAi clones were from Ahringer or Vidal RNAi library,
or made from genomic DNA. Details were listed in the Supplementary Table.
3.6.5 Behavioral assays
The defecation motor program was assayed as previously described (D. W. Liu & Thomas,
1994). Twenty to thirty L4 animals were transferred onto a fresh NGM plate seeded with OP50 E.
coli and were stored in a 20°C incubator for 24 hours. After 24 hours, ten consecutive defecation
cycles were observed from three independent animals and the mean and the standard error was
calculated for each genotype. The pBoc and aBoc steps were recorded using custom Etho software
(James Thomas Lab website:http://depts.washington.edu/jtlab/software/otherSoftware.html)
3.6.6 Microscopy and Fluorescence Imaging
Approximately 30-40 age matched animals were paralyzed with 30mg/mL 2,3-butanedione
monoxime (BDM) in M9 buffer and mounted on 2% agarose pads. Images were captured using
the Nikon eclipse 90i microscope equipped with Nikon Plan Apo 20x, 40x, 60x, and 100x oil

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objective (N.A.=1.40), and a Photometrics Coolsnap ES2 camera or a Hamamatsu Orca Flash
LT+CMOS camera. Metamorph 7.0 software (Universal Imaging/Molecular Devices) was used to
capture serial image stacks and to obtain the maximum intensity projection image for analysis.
For transcriptional reporter imaging, young adult animals with indicated genotype were
transferred into a 1.5mL Eppendorf tube with M9 buffer, washed three times and incubated in
working solution of juglone with indicated concentration for 4h in dark on rotating mixer before
recovering on fresh NGM plates with OP50 for 1h in dark at 20°C. The posterior end of the
intestine was imaged with the 60x objective and quantification for average fluorescence intensity
of a 16-pixel diameter circle in the posterior intestine was calculated using Metamorph.
For coelomocyte imaging, L4 stage animals were transferred in fresh M9 buffer on a cover
slide, washed six times with M9 before being exposed to juglone or H2O2 in M9 buffer. Animals
were then paralyzed in BDM and images of coelomocytes next to the posterior end of intestine
were taken using the 100x oil objective. Average fluorescence intensity of Venus from the
endocytic compartments in the posterior coelomocytes was measured in ImageJ.
For fusion protein fluorescence imaging, L4 stage animals were exposed to M9 buffer or
indicated oxidants for 10min before being paralyzed in BDM and taken images of the posterior
end of the intestine using 100x oil objective. For HyPer7 imaging, Z stacks were obtained using
GFP (excitation/emission: 500nm/520nm) and CFP (excitation/emission: 400nm/520nm) filter
sets sequentially, HyPer7 fluorescence signal was quantified as the ratio of GFP to CFP
fluorescence intensity changes with respect to the baseline [(Ft − F0)/F0].
3.6.7 CRISPR/Cas9 Editing
prdx-2b(vj380) knock-out mutants were generated using a co-CRISPR protocol (Arribere
et al., 2014). A sgRNA and a repair single stranded oligodeoxynucleotides (ssODN) targeting dpy10 were co-injected with a sgRNA for genes of interest and a ssODN that induces homologydirected repaire (HDR) to introduce Cas9 mediated mutagenesis. Fifteen young adult animals were
injected to produce around thirty singled F1 animals carrying Dpy or Rol phenotype. F2 animals
were genotyped for mutations based on PCR and enzyme digest. Homozygous mutants were
outcrossed with wild type animals at least four times before being used for assays.

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3.6.8 Statistics
Statistical analysis was performed on GraphPad Prism 9. Unpaired t-test with two tails was
used for two groups and one-way ANOVA with multiple comparison corrections was used to three
or more groups to determine the statistical significance. Statistical details and n are specified in
the figure legends. All comparisons are conducted based on wild-type controls unless indicated by
lines between genotypes. Bar graphs with plots were generated using GraphPad Prism 9.

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Figure 3.1 Peptidergic Gut-to-Neuron FLP-2 Signaling Potentiates the Oxidative Stress
Response.
A (Top) Schematic showing the positions of AIY, intestine and coelomocytes of transgenic
animals co-expressing FLP-1::Venus in the intestine and mCherry in coelomocytes.
Representative image of the posterior coelomocyte that has taken up Venus into the endocytic
compartment. Scale bar: 5μM. (Bottom) Schematic showing FLP-1 and FLP-2 peptides as intertissue signals in gut-intestine regulation of the antioxidant response. B Representative images and
quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP1::Venus fusion proteins in AIY following vehicle M9 or juglone treatment for 10min. Neuronal
aex-5 denotes expression of aex-5 cDNA under the rab-3 promoter; intestinal aex-5 denotes
expression of aex-5 cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis
compared to “wild type”; a denotes statistical analysis compared to “aex-5+juglone”, P < 0.001; b

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denotes statistical analysis compared to “intestinal aex-5; aex-5+juglone”, P < 0.01. n = 30, 30,
24, 30, 26, 30, 30 independent animals. Scale bar: 5μM. C Average percentage of surviving young
adult animals of the indicated genotypes after 16h recovery following 4h juglone treatment.
Unlined ** denotes statistical analysis compared to “wild type”, a denotes statistical analysis
compared to “flp-1”, P > 0.99; b denotes statistical analysis compared to “flp-2”, P > 0.99. n =
213, 156, 189, 195 independent biological samples over three independent experiments. D
Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP1::Venus fusion proteins in AIY following M9 or juglone treatment for 10min. Neuronal flp-2
denotes expression of flp-2 gDNA under the rab-3 promoter; intestinal flp-2 denotes expression of
flp-2 gDNA under the ges-1 promoter; intestinal flp-2(OE) denotes expression of flp-2 gDNA
under the ges-1 promoter in wild type animals. Unlined *** denotes statistical analysis compared
to “wild type”; a denotes statistical analysis compared to “flp-2+juglone”, P < 0.0001; b denotes
statistical analysis compared to “wild type”, P > 0.99; c denotes statistical analysis compared to
“wild type+juglone”, P < 0.01. n = 20, 20, 25, 20, 20, 20, 25, 22 independent animals. E
Representative images and quantification of average fluorescence in the posterior region of
transgenic animals expressing Pgst-4::gfp after 4h vehicle M9 or juglone exposure. Asterisks mark
the intestinal region used for quantification. Pgst-4::gfp expression in the body wall muscles,
which appears as fluorescence on the edge animals in some images, was not quantified. Unlined
*** and ns denote statistical analysis compared to “wild type”; a, b, and c denote statistical analysis
compared to “wild type+juglone”, P < 0.01, P < 0.001, P < 0.01 respectively; unlined ns denotes
statistical analysis compared to “wild type”. n = 25, 26, 25, 25, 25, 25, 25, 25 independent animals.
Scale bar: 10μM. F Representative images and quantification of average fluorescence in the
posterior region of transgenic animals expressing Pgst-4::gfp after 4h M9 or juglone exposure.
Asterisks mark the intestinal region for quantification. Pgst-4::gfp expression in the body wall
muscles, which appears as fluorescence on the edge animals in some images, was not quantified.
Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical
analysis compared to “wild type+juglone”; a denotes statistical analysis compared to “intestinal
flp-2(OE)”, P < 0.0001; b denotes statistical analysis compared to “intestinal flp-2(OE)+juglone”,
P < 0.0001. n = 23, 25, 25, 26, 24, 25 independent animals. Scale bar: 10μM. Data are mean values
± s.e.m normalized to wild type controls. ns. not significant, ** and ## P < 0.01, *** and ### P <
0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Figure 3.2 FLP-2 Secretion from the Intestine is Stress Regulated.
A Schematic showing the positions of intestine and coelomocytes of transgenic animals coexpressing FLP-2::Venus in the intestine and mCherry in coelomocytes. Representative images of
the posterior coelomocyte that have taken up Venus into the endocytic compartment (Scale bar:
5μM) and the posterior intestinal region showing the distribution of FLP-2::Venus in puncta in the
intestine are shown (Scale bar: 15μM). B Representative images of fluorescence distribution in the
posterior intestinal region of transgenic animals co-expressing FLP-2::Venus and AEX-5::mTur2
fusion proteins. Arrowheads denote puncta marked by both fusion proteins. Scale bar: 5μM. C
Representative images and quantification of average coelomocyte fluorescence of the indicated
mutants expressing FLP-2::Venus fusion proteins in the intestine following M9, juglone or H2O2
treatment for 10min. Unlined *** and ns denote statistical analysis compared to “wild type”. n =
29, 25, 24, 30, 23, 30, 25, 25, 25 independent animals. Scale bar: 5μM. D Quantification of average
coelomocyte fluorescence of transgenic animals expressing FLP-2::Venus fusion proteins in the
intestine following treatment of vehicle (DMSO) or the indicated stressors for 10min. Unlined ***
denotes statistical analysis compared to “M9”. n = 23, 25, 25 independent animals. E
Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined **
denotes statistical analysis compared to “wild type”; unlined ## denotes statistical analysis
compared to “flp-1”; a denotes statistical analysis compared to “wild type+juglone”, P > 0.90. n
= 30, 30, 30, 30 independent animals. Data are mean values ± s.e.m normalized to wild type
controls. ns. not significant, ** and ## P < 0.01, *** and ### P < 0.001 by Brown-Forsythe and
Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Figure 3.3 SOD-1/SOD-3 Mediates Endogenous H2O2 Regulates FLP-2 Release from the
Intestine.

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A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal
sod-1 denotes expression of sod-1b cDNA under the ges-1 promoter. Unlined *** denotes
statistical analysis compared to “wild type”. n = 25, 22, 24, 24, 25 independent animals. B
Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal
sod-3 and sod-3(ΔMLS) denote intestinal expression of sod-3 cDNA and sod-3(ΔMLS) variants,
which lacks the mitochondrial localization sequence, under the ges-1 promoter. Unlined ***
denotes statistical analysis compared to “wild type”. n = 25, 25, 25, 25, 25, 25 independent animals.
C Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined
*** denotes statistical analysis compared to “wild type”. n = 25, 25, 22, 25 independent animals.
D Representative images of fluorescence distribution in the posterior intestinal region of transgenic
animals expressing SOD-1b::GFP fusion proteins in contrast against auto-fluorescence of gut
granules. Scale bar: 10μM. E Representative images of fluorescence distribution in the posterior
intestinal region of transgenic animals co-expressing SOD-3::GFP and TOMM-20::mCherry (to
target mitochondria) fusion proteins. Scale bar: 15μM. F Representative images of fluorescence
distribution in the posterior intestinal region of transgenic animals co-expressing SOD3(ΔMLS)::GFP and TOMM-20::mCherry fusion proteins. Scale bar: 15μM. G Quantification of
average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion
proteins in the intestine following M9, juglone or H2O2 treatment for 10min. Unlined *** and ns
denote statistical analysis compared to “wild type”. n = 29, 30, 25, 25, 25, 24, 25 independent
animals. H Quantification of average coelomocyte fluorescence of the indicated mutants
expressing FLP-2::Venus fusion proteins in the intestine following M9 or H2O2 treatment for
10min. n = independent animals. I Schematic showing that SOD-1 and SOD-3 mediate jugloneinduced H2O2 production in promoting FLP-2 release, and the PRDX-2/TRX-3 system detoxifies
excessive H2O2. J Schematic, representative images and quantification of fluorescence in the
posterior region of the indicated transgenic animals co-expressing mitochondrial matrix targeted
HyPer7 (matrix-HyPer7) or mitochondrial outer membrane targeted HyPer7 (OMM-HyPer7) with
TOMM-20::mCherry following M9 juglone or H2O2 treatment. Unlined *** and ns denote
statistical analysis compared to “wild type; a and b denote statistical analysis compared to “wild
type+juglone”, P < 0.01, P > 0.19. (top) n = 20, 20, 18, 20, 19, 19, 20, 20 independent animals.
(bottom) n = 20, 20, 19, 20, 20, 20, 20, 20 independent animals. Scale bar: 5μM. Data are mean
values ± s.e.m normalized to wild type controls. ns. not significant, * P < 0.05, *** P < 0.001 by
Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Figure 3.4. PRDX-2/PRDX and TRX-3/TRX Regulate Endogenous H2O2 and FLP-2
Secretion.
A (Top) Schematic showing the PRDX/TRX system in H2O2 detoxification. (Bottom) Schematic
showing the three isoforms of prdx-2 transcripts and vj380 allele of prdx-2b knockout. B
Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal
prdx-2b denotes expression of prdx-2b cDNA under the ges-1 promoter. Intestinal trx-3 denotes
expression of trx-3 cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis
compared to “wild type”; a denotes statistical analysis compared to “prdx-2”, P < 0.0001; b, c and
d denote statistical analysis compared to “trx-3”, P < 0.001, P < 0.0001, P > 0.99 respectively.
n= 25, 23, 25, 25, 25, 25, 25, 25, 25, 25, 25 independent animals. C and D Quantification of
fluorescence in the posterior region of the indicated transgenic animals co-expressing matrixHyPer7 (C) or OMM-HyPer7 (D) with TOMM-20::mCherry following M9 or juglone treatment.
Unlined *** and ns denote statistical analysis compared to “wild type. (C) n = 20, 20, 20, 20, 20
independent animals. (D) n = 20, 20, 20, 20, 20 independent animals. E Quantification of average
coelomocyte FLP-2::Venus fluorescence of transgenic animals fed with RNAi bacteria targeting
the indicated genes following M9 treatment for 10min. Unlined *** denotes statistical analysis
compared to “empty vector”. n = 25, 23, 24 independent animals. F Representative images and
quantification of average fluorescence in the posterior region of transgenic animals expressing
Pgst-4::gfp after 4h M9 or juglone exposure. Asterisks mark the intestinal region for quantification.
Pgst-4::gfp expression in the body wall muscles, which appears as fluorescence on the edge
animals in some images, was not quantified. Unlined ** denotes statistical analysis compared to
“wild type”, unlined ## denotes statistical analysis compared to “prdx-2b”. n = 25, 25, 25
independent animals. Scale bar: 10μM. Data are mean values ± s.e.m normalized to wild type
controls. n.s. not significant, ** and ## P < 0.01, *** P < 0.001 by Brown-Forsythe and Welch
ANOVA with Dunnett’s T3 multiple comparisons test.

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Figure 3.5. PKC-2/PKCα/β Activation by H2O2 Promotes FLP-2 Secretion from the Intestine.
A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal
pkc-2 denotes expression of pkc-2b cDNA under the ges-1 promoter. Intestinal pkc-2b(K375R)
denotes expression of pkc-2b(K375R) variants under the ges-1 promoter. Unlined *** and ns
denote statistical analysis compared to “wild type”; ### denote statistical analysis compared to
“intestinal pkc-2b;pkc-2+juglone”. n = 24, 24, 25, 25, 25, 25 independent animals. B and C
Quantification of fluorescence in the posterior region of the indicated transgenic animals coexpressing matrix-HyPer7 (B) or OMM-HyPer7 (C) with TOMM-20::mCherry following M9 or
juglone treatment. Unlined *** denotes statistical analysis compared to “wild type”; unlined ###
denotes statistical analysis compared to “pkc-2”. (B) n = 20, 20, 19, 20 independent animals, (C)
n = 20, 20, 20, 20 independent animals. D Quantification of average coelomocyte fluorescence of
the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or
H2O2 treatment for 10min. n = 23, 25, 25 independent animals. E Quantification of average
coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the
intestine following M9 treatment for 10min. Unlined *** denotes statistical analysis compared to
“wild type”; unlined ### denotes statistical analysis compared to “prdx-2”. n = 25, 25, 25, 25
independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not
significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3
multiple comparisons test.

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Figure 3.6. DAG Promotes PKC-2 Mediated FLP-2 Secretion from the Intestine.
A Schematic showing PLC and DGK mediates DAG metabolism and DAG functions in H2O2-
mediated FLP-2 signaling. B Quantification of average coelomocyte fluorescence of the indicated
mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone
treatment for 10min. n = 25, 25, 25, 25 independent animals. C and D Quantification of
fluorescence in the posterior region of the indicated transgenic animals co-expressing matrixHyPer7 (C) or OMM-HyPer7 (D) with TOMM-20::mCherry following M9 or juglone treatment.
Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical
analysis compared to “egl-8”. (C) n = 22, 20, 20, 21 independent animals, (D) n = 20, 20, 20, 20
independent animals. E Quantification of average coelomocyte fluorescence of the indicated
mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone
treatment for 10min. Intestinal dgk-2 denotes expression of dgk-2a cDNA under the ges-1
promoter. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes
statistical analysis compared to “dgk-2/DGKε”. n = 25, 25, 25, 25, 24 independent animals. F and
G Quantification of fluorescence in the posterior region of the indicated transgenic animals co-

96
expressing matrix-HyPer7 (F) or OMM-HyPer7 (G) with TOMM-20::mCherry following M9
treatment. (F) n = 20, 20 independent animals, (G) n = 20, 20 independent animals. H
Quantification of average coelomocyte fluorescence of the indicated transgenic animals fed with
RNAi bacteria targeting the indicated genes in the intestine following M9 treatment for 10min. n
= 25, 24, 25, 30 independent animals. I (Top) Schematic showing the position of intestine and AIY
neurons in FLP-1-FLP-2 mediated axis. (Bottom) Schematic showing endogenous H2O2 promotes
PKC-2/AEX-4 mediated FLP-2 release from the intestine in FLP-1-FLP-2 regulated inter-tissue
axis. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, ** P <
0.01, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple
comparisons test.

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Supplementary Figure 3.1. The Effect of Intestinal DCV Secretion Mutations on FLP-1
Release from AIY.
A Schematic showing the locations of AEX-1/UNC13, AEX-3/MADD, AEX-4/SNAP25 and
AEX-6/Rab27 relative to a DCV. B Quantification of average coelomocyte fluorescence of the
indicated mutants expressing FLP-1::Venus fusion proteins in AIY following vehicle (DMSO) or
juglone treatment for 10min. Unlined *** and ### denotes statistical analysis compared to “wild
type”. n = 30, 30, 29, 30, 30, 30 independent animals. C Quantification of average coelomocyte
fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following
vehicle (DMSO) or juglone treatment for 10min. Unlined *** and ns denote statistical analysis
compared to “wild type”. n = 24, 24, 25, 25, 30, 30 independent animals. Data are mean values ±
s.e.m normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by BrownForsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 3.2. Specificity of Juglone on Intestinal Peptide Secretion, and FLP-2
and NLP-40 Localization in the Intestine.
A Quantification of average coelomocyte fluorescence of the indicated mutants co-expressing
FLP-2::Venus in the intestine (under the ges-1 promoter) and mCherry in the coelomocytes (under
the ofm-1 promoter) following M9 or juglone treatment for 10min. n = 23, 19 independent animals.
B Quantification of average coelomocyte fluorescence of transgenic animals expressing NLP40::Venus fusion proteins in the intestine following M9 or juglone exposure for 10min. n = 25, 24
independent animals. C Quantification of average coelomocyte fluorescence of transgenic animals
expressing NLP-27::Venus fusion proteins in the intestine following M9 or juglone exposure for
10min. n = 23, 25 independent animals. D Representative images of fluorescence distribution in
the posterior intestinal region of transgenic animals co-expressing FLP-2::Venus fusion proteins
(marked by arrowheads) and NLP-40::mTur2 fusion proteins (marked by arrows). Scale bar: 5μM.
Data are mean values ± s.e.m normalized to wild type controls. ns. not significant by BrownForsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 3.3. SODs Function in Juglone Induced FLP-2 Release from the
Intestine and Mitochondrial mCherry Control.
A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined
***, ### and ns denote statistical analysis compared to “wild type+juglone”. n = 29, 27, 29, 27,
25, 26, 24 independent animals. B and C Representative images and quantification of average
fluorescence intensity of TOMM-20::mCherry proteins in transgenic animals co-expressing
matrix-HyPer7 (B) or OMM-HyPer7 (C) following M9 or H2O2 treatment for 10min. (B) Scale
bar: Scale bar: 5μM. n = 20, 20 independent animals. (C) Scale bar: Scale bar: 5μM. n = 20, 22
independent animals.

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Supplementary Figure 3.4. PRDX-2 Intestinal Rescue and Mediates SOD-3 Dependent
Regulation of FLP-2 Release.
A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 treatment for 10min. n = 30, 29 independent
animals. B Quantification of average coelomocyte fluorescence of the indicated mutants
expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min.
Intestinal prdx-2a denotes expression of prdx-2a cDNA under the ges-1 promoter. n = 30, 30, 25
independent animals. C Quantification of average coelomocyte fluorescence of the indicated
mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for
10min. Intestinal prdx-2c denotes expression of prdx-2c cDNA under the ges-1 promoter. n = 25,
25, 25 independent animals. D Quantification of average coelomocyte fluorescence of the
indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9
treatment for 10min. n = 25, 23, 22 independent animals. Data are mean values ± s.e.m normalized
to wild type controls. Data are mean values ± s.e.m normalized to wild type controls. ns. not
significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3
multiple comparisons test.

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Supplementary Figure 3.5. Juglone Promotes FLP-2 Release in pkc-1 Mutants and Expulsion
Analysis.
A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined ns
and ** denote statistical analysis compared to “wild type”. n = 24, 25, 20, 25 independent animals.
B Quantification of the number of expulsions (Exp) per defecation cycle in adult animals of the
indicated genotypes. n = 30, 30, 30, 30 in three independent animals. Data are mean values ± s.e.m
normalized to wild type controls. ns. not significant, ** P < 0.01, *** P < 0.001 by BrownForsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Supplementary Figure 3.6. Juglone Promotes FLP-2 Release in plc-2 Mutants.
Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined
*** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis
compared to “plc-2/PLCβ”. n = 25, 25, 23, 28 independent animals. Data are mean values ± s.e.m
normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by Brown-Forsythe
and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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chapter 4
Summary and future directions
Using C. elegans as the model system, I examined peptidergic signaling in an inter-tissue
axis mediated anti-oxidative stress response. SKN-1 mediated protection against juglone induced
toxicity is partially activated by FLP-1 signaling from AIY interneurons and FLP-2 signaling from
the intestine. Interestingly, FLP-2 released from the intestine potentiates the responsiveness of
FLP-1 signaling from AIY and FLP-1 dependent SKN-1 activation against juglone exposure,
suggesting a peptidergic signaling mediated inter-tissue pathway to promotes systematic and
whole-organismal activation of antioxidative protection. This work also revealed that the release
of both FLP-1 and FLP-2 are positively regulated by stress induced peroxide production through
protein kinase C (PKC) dependent exocytosis mechanisms.
4.1 Neuropeptides regulate stress-induced SKN-1 activation.
SKN-1 has been shown to be responsible for transcriptional expression of detoxificants
and enzymes largely implicated in response against environmental stress such as heavy metal
exposure, pathogenic infection and oxidative stress (An, 2003; Oliveira et al., 2009; S.-K. Park et
al., 2009; Przybysz et al., 2009). Through genetic screening, I showed in Chapter 2 and Chapter 3
that the neuron derived peptide FLP-1 and the intestine derived peptide FLP-2 function in a gutneuron signaling pathway responsible for partially promoting neuronal activation of intestinal
SKN-1 activity against juglone induced toxicity. In addition to juglone, I confirmed that neuronal
FLP-1 signaling is also regulated by another mitochondrial stressor, sodium arsenite, which has
minimal effects on FLP-2 signaling from the intestine; on the other hand, in Chapter 3 I
demonstrated that paraquat, another commonly used mitochondrial toxicant, promotes FLP-2
signaling from the intestine but not FLP-1 signaling from AIY, I hypothesize that while
overlapping in the same peptidergic pathway for defense against juglone induced toxicity, FLP-1
and FLP-2 signaling have distinct functions in response against different stressors. Consistently,
AIY derived FLP-1 has been demonstrated to be activated in response to starvation through the
glutamate receptor homolog, MGL-1 (Jeong & Paik, 2017). Investigating in detail how FLP-1
mediated and FLP-2 mediated activation of SKN-1 activity and expression of SKN-1 downstream

104
target genes in response to different stressors would help better understand the difference of FLP1 and FLP-2 mediated SKN-1 activation.
4.2 Conserved PKC dependent mechanism mediates endogenous H2O2 signaling.
Tightly regulated endogenous peroxide production has been demonstrated to signal
through cysteine modification in activation of a significant variety of redox sensitive proteins, in
Chapter 2 and Chapter 3 I studied the function of two conserved C. elegans homologs of
mammalian PKCε/η and PKCα/β, PKC-1 and PKC-2, in mediating peroxide dependent activation
of exocytosis; in detail I demonstrated that a well conserved cysteine residue in the kinase domain
of PKC-1 might be a direct peroxide target for redox modification. Such cysteine contained motif
has also be identified in the kinase IRE-1 in activation of UPRER response (Hourihan et al., 2016).
A recent mass spectrometry study that mapped oxidation sensitive cysteines in C. elegans redox
proteins revealed several conserved redox sensitive cysteine residues in PKC-1 and IRE-1 and
other kinases, as well as in PKC-2 (Meng et al., 2021). I hypothesize that cysteine dependent redox
modification is a conserved mechanism through which endogenous H2O2 promotes stress induced
activation of signaling cascades. Future research is needed to investigate in how redox cysteines
in PKC-2 signal in response to endogenous peroxide generation.
4.3 Bacterial diet modulates synaptic transmission.
In Chapter 2 I demonstrated that bacterial diet promotes acute activation of endogenous
peroxide production and FLP-1 release from AIY, possibly through bacterial metabolic products,
as I propose that FLP-1 signaling from AIY is modulated by FLP-2 originated from the intestine,
one logical explanation would be that gut bacteria and/or bacterial metabolites modulate neuronal
FLP-1 release through FLP-2 signaling from the intestine. A recent study demonstrated that
intestine derived INS-11/insulin negatively regulates neuronal function in aversive learning
behavior (K. Lee & Mylonakis, 2017). I propose another explanation that gut bacteria and/or
bacterial metabolites signal to the intestine independent of neuronal regulation of AIY activity.
One of the gut bacterial metabolites vitamin B12, for example, has been shown to reduce neuronal
cholinergic signaling (Kang et al., 2024). Gut bacteria has been proposed to influence sensory
behavior directly through bacteria generated neurotransmitters (O’Donnell et al., 2020), or through
gut derived hormones (Martin et al., 2019). The next question I ask if whether/how gut bacteria

105
and/or bacterial metabolites modulate FLP-2 signaling from the intestine. Taking into
consideration that FLP-2 functions in the same signaling pathway with FLP-1 signaling from AIY,
I propose that gut bacteria and/or bacterial metabolites may also modulate endogenous H2O2 level
and FLP-2 signaling.
There has been growing interest in the mechanism and signaling pathway underlying the
influence of gut microbiota on host neural function and neurological health, a recent study
demonstrated that pharmaceutical or genetic reduction of ROS generation in the gastrointestinal
(GI) tract inhibits probiotic regulation on synaptic transmission (Chandrasekharan et al., 2019),
suggesting endogenous ROS signaling may function as redox messenger in mediating bacterial
influence on the nervous system. Gut bacteria-derived metabolites play an important role in not
only modulating neuronal activity, many clinical studies reported that depending on the specific
bacterial metabolites, it can exert both beneficial and detrimental effects on neuronal functions and
it is associated with many neurodegenerative disorders (Swer et al., 2023; Vogt et al., 2018).
Investigating in whether/how microbiome produced metabolites affect FLP-2 signaling pathway
in the intestine would help understand better about the mechanism under which gut microbiome
neuronal activity through a intestine derived peptidergic signaling pathway.

106
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Abstract (if available)

Abstract Reactive oxygen species (ROS) production/exposure at a physiological level has been shown to modulate synaptic functions and activities, and endogenous hydrogen peroxide (H2O2) has been implicated in regulating synaptic transmission. Neuropeptides packaged into DCVs play an important role in mediating intercellular and inter-tissue communication and promoting organismal homeostasis in response to various environmental cues. The mammalian nuclear factor erythroid-derived 2 related factors (Nrf) family and its C. elegans homolog, SKN-1, regulate the expression of detoxifying genes and promote organismal protection against oxidative stress. Through genetic analysis and in vivo fluorescence imaging, I identified the neuropeptide-like proteins FLP-1 and FLP-2 as essential components of a gut-brain axis that coordinates SKN-1 activation and activity in the intestine. Functional characterization of flp-1 and flp-2 signaling revealed that FLP-1 released from AIY interneurons and FLP-2 from the intestine are activated by mitochondrially generated H2O2. Superoxide dismutases (SODs) modulate endogenous H2O2 level, FLP-1 and FLP-2 signaling and SKN-1 response. H2O2 induces neuorpeptide release by activating protein kinase C (PKC), possibly through direct cysteine modification. Moreover, H2O2 -induced FLP-2 secretion relies on DCV exocytosis mediated by a number of SNARE proteins including AEX-4/SNAP25. Finally, I showed that gut bacteria potentiate endogenous H2O2 production, FLP-1 release and SKN-1 activity, establishing that intestine-derived signaling initiated by diet is responsible for regulating neuronal function. My results reveal a fundamental role for endogenous H2O2 signaling in regulating neuropeptide secretion in an inter-tissue stress-response circuit.

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Creator Jia, Qi (author)

Core Title Endogenous H2O2 signaling positively regulates the release of neuropeptides during a neuron-gut axis mediated oxidative stress response in caenorhabditis elegans

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School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Degree Conferral Date 2024-05

Publication Date 04/17/2024

Defense Date 03/28/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag AEX-4/SNAP25,elegans,FLP-1,FLP-2,H2O2,mitochondria,neuropeptide,OAI-PMH Harvest,oxidative stress,PKC-1/PKC,PKC-2/PKC,PRDX-2/PRDX,PRDX-3/PRDX,SKN-1/Nrf2,SOD-1/SOD1,SOD-2/SOD2,SOD-3/SOD2,TRX-2/TRX,TRX-3/TRX

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Advisor Chang, Karen (committee member), Dickman, Dion (committee member), Sieburth, Derek (committee member)

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