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Neuroendocrine regulation of the transcription factor SKN-1/Nrf2 in oxidative stress response
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Neuroendocrine regulation of the transcription factor SKN-1/Nrf2 in oxidative stress response
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Content
Neuroendocrine regulation of the transcription factor
SKN-1/Nrf2 in oxidative stress response
by
Qi Jia
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2015
Copyright 2015 Qi Jia
i
ACKNOWLEDGEMENT
First of all I want to express my sincere appreciation and thanks to my mentor Dr.
Derek Sieburth. I really appreciate that he offered me the opportunity to do research in
his lad and work on this wonderful project. His intellectual knowledge and enthusiasm
for scientific research has really been inspiring me, and he always gave me instructive
advice, helping me build up more scientific research spirit and pursue my degree. I also
want to express my gratitude to him for being supportive and patient throughout my
research, which really guide and help me to finish my experiment and to write my thesis.
Further I want to express my thanks to my labmates, who share all the joy and tears
throughout my two years study and lab research. Also I want to express my thanks to Dr.
Zoltan Tokes, who is the chair of my committee. He has been really kind and
encouraging throughout two years of master, and helped us to pursuit academic
achievement.
Finally, I want to thank my parents and my friends, who share all the joy and tears
with me through these two years and support me to succeed in pursuing my master
degree.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENT i
LIST OF FIGURES iv
ABBREVIATIONS v
ABSTRACT vi
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: MATERIALS AND METHODS 5
2.1 C. elegans strains 5
2.2 Molecular biology 5
2.3 Transgenic lines 6
2.4 Microscopy and analysis 6
2.5 SKN-1::GFP Translocation assay 7
2.6 Toxicity assay 7
2.7 Statistical analysis 8
CHAPTER 3: RESULTS 9
3.1 Neuroendocrine signals function in organismal survival 9
3.2 Neuropeptides function in organismal survival 11
3.3 EGL-3/PC2 regulates SKN-1 translocation in the intestine 16
3.4 EGL-3/PC2 in the nervous system promotes organismal survival 20
iii
3.5 The neuropeptide, FLP-1, promote organismal survival 24
3.6 GPCR candidates in the intestine function in the SKN-1 activation pathway 28
3.7 The GPCR, FSHR-1, promotes organismal survival through SKN-1 activation. 30
CHAPTER 4: DISCUSSION 33
REFERENCE 37
iv
LIST OF FIGURES
Figure 1. Regulation of SKN-1 activity in the intestine.
Figure 2. Toxicity assay on DCV release mutants.
Figure 3. Neuropeptides are processed in the DCV.
Figure 4. Expression of egl-3 and egl-21 are regulated by SKN-1 in the nervous system.
Figure 5. Toxicity assay on neuropeptide processing mutants.
Figure 6. SKN-1 translocation into the nucleus is blocked in egl-3 mutant.
Figure 7. SKN-1 abundance in the intestine decreases egl-3 mutant.
Figure 8. Expression of SKN-1 downstream targets decreases in egl-3 mutants
Figure 9. EGL-3/PC2 promotes organismal survival under stressed condition.
Figure 10. Neuronal expression of egl-3 rescues its sensitivity phenotype.
Figure 11. Specific expression of egl-3 in motor neurons did not rescue its phenotype.
Figure 12. FLP-1 is regulated by SKN-1 in the nervous system.
Figure 13. FLP-1 promotes organismal survival under stressed condition.
Figure 14. SKN-1 activation and translocation is blocked in egl-3 and flp-1 mutants.
Figure 15. Toxicity assay on putative FLP-1 receptor mutants.
Figure 16. Toxicity assay on GPCRs mutants.
Figure 17. fshr-1 functions in the oxidative stress response.
Figure 18. SKN-1 translocation and activation is blocked in fshr-1 mutant.
Figure 19. egl-3 mutants reduce the resistance in daf-2 mutants.
v
ABBREVIATIONS
Nrf: nuclear factor E2-related factor
ARE: antioxidant response element
GPCR: G-protein coupled receptor
PLC: phospholipase C
DAG: diacylglcerol
MAPK: mitogen-activated protein kinase
SV: synaptic vesicle
DCV: dense core vesicle
PKCE: protein kinase C epsilon
WT: wild type
PC: propeotein convertase
ROS: reactive oxygen species
ARE: antioxidant response element
gf: gain of function
BDM: 2,3-butanedione monoxime
GABA: gamma-aminobutyric acid
vi
ABSTRACT
The Nrf2 transcription factor plays a critical role in mediating adaptive responses to
cellular stress and defending against neurodegeneration, aging, and cancer. Nrf2 activity
is regulated both by oxidative stress and by endogenous signaling pathways that control
its translocation into the nucleus where it directs the transcription of a variety of
antioxidant genes. However the molecular mechanisms underlying the regulation of Nrf2
activity in the context of multicellular organisms are not well understood. Here, we report
a novel role for inter-tissue neuroendocrine signaling in regulating the activity of the
Caenorhabditis elegans Nrf homolog SKN-1 during the oxidative stress response. We
show that mutants with impaired neuropeptide processing or release show decreased
survival following treatment with toxins that increase oxidative stress, whereas mutants
with increased neuropeptide release are resistant to the toxic effects of oxidative stress.
We find that the proprotein convertase, egl-3/PC2, which is a key enzyme involved in
neuropeptide maturation, functions in the nervous system to promote survival following
treatment with the oxidant juglone. Strikingly, egl-3 mutations block stress-induced
SKN-1 nuclear translocation and expression of the SKN-1 target
gst-4/glutathione-S-transferase in the intestine. We identify a neuropeptide, FLP-1,
expressed in the nervous system, that is critical for survival in the presence of stress, and
we are investigating its involvement in SKN-1 activation in the intestine. Together these
results identify the nervous system as a critical regulator of SKN-1 activity in distal
tissues and suggest that neuroendocrine signaling may be a novel mechanism by which
vii
Nrf2 is regulated in vivo.
1
CHAPTER 1
INTRODUCTION
Organismal oxidative stress is produced due to the imbalance between the production
of reactive oxygen species (ROS) and the endogenous defense against ROS in cells. Such
imbalance may cause damage to cellular components including lipids, proteins and DNA
molecules, resulting in detrimental effects on cellular metabolism. (Finkel, et, al, 2000)
The high metabolic demands and low regenerative ability in the nervous system
contribute to the prediction that increased oxidative stress level in organisms is
particularly harmful to nervous system. Several neurodegenerative diseases, including
Parkinson’s disease and Alzheimer’s disease, have been shown to be significantly related
to increased oxidative stress level. (Halliwell, 2006; Abou-Sleiman, et, al, 2006)
Organisms have evolved mechanisms that act against oxidative stress. The Nrf
(nuclear factor E2-related factor) family of transcription factors primarily participates in
the oxidative stress response and protects tissues from detrimental damages. (Itoch, et, al,
1997; Kobayashi, et, al, 2006) In normal conditions, Nrf2 is degraded in the cytoplasm by
the kelch-domain containing protein Keap1, and when faced with oxidative stress, Nrf2
gets released from Keap1 sequestering and translocates into the nucleus, activating the
transcription of oxidative stress response genes by binding to the antioxidant response
element (ARE). (Lee, et, al, 2005; Itoh, et, al, 1999)
2
Figure 1. Regulation of SKN-1 in the intestine. In normal condition SKN-1 is
down-regulated by WDR-23, and SKN-1 can also be regulated in parallel by DAF-2 and
GPCRs pathways. GPCRs act through protein kinase cascade to activate SKN-1, while
DAF-2 acts through serine/threonine protein kinase complex to down-regulate SKN-1
activation.
3
In Caenorhabditis elegans, SKN-1, homologous to Nrf2 in mammalian cells,
participates in the response to elevated organismal oxidative stress level. Under normal
conditions, SKN-1 is negatively regulated by the WD40 repeat protein, WDR-23, in the
cytoplasm. However, SKN-1 gets stabilized and released from WDR-23 under stressed
conditions and translocates into the nucleus, activating the transcription of downstream
targets. (Chow, et, al, 2009; Angers, et, al, 2006; Higa, et, al, 2006) WDR-23 belongs to
the DDB1-CUL4 ubiquitin ligase family and regulates protein degradation as a substrate
specificity subunit. (Angers, et, al, 2006) SKN-1 can also be regulated in parallel by
insulin signaling pathway and G protein-coupled receptors (GPCRs) pathway. GPCRs in
the intestine have been predicted to activate phospholipase C (PLC), which produces
diacylglycerol (DAG) to activate PMK-1, which is homologous to MAPK
(mitogen-activated protein kinase) in mammalian cells, through the serine/threonine
kinase SEK-1, which is homologous to MAPK kinase in mammalian. (Zugasti, et, al,
2104) On the other hand, DAF-2, which is the sole receptor for insulin signals in C.
elegans, has been to shown to inactivate SKN-1 through a serine/threonine protein kinase
complex of AGE-1, SGK-1 and AKT-1. (Tullet, et, al, 2008) (Figure 1) SKN-1 has also
been shown to function in a pair of neuron cells to regulate longevity in C. elegans.
(Bishop, et, al, 2007)
However, the regulation of SKN-1 participation in the oxidative stress response has
not been defined. In this study, we identified that the proprotein convertase, EGL-3/PC2,
which participates in neuropeptide processing, functions in the nervous system to
4
promote organismal survival under stressed conditions. Furthermore, we identify a
neuropeptide, FLP-1, which is processed by EGL-3/PC2 in the nervous system, that may
function as a neuroendocrine signal to regulate SKN-1 activation in the intestine,
promoting organismal-wide survival in response to oxidative stress.
5
CHAPTER 2
MATERIALS AND METHODS
2.1 C. elegans strains – All the strains were maintained under standard condition and
young hermaphrodite adults were used to perform all the experiment unless otherwise
indicated. The following strains were provided by the Caenorhabditis Genetics Center,
which is funded by NIH National Center for Research Resources (NCRR): egl-3(nr2090),
egl-21(n476), sbt-1(rb907), flp-1(ok2811), pkc-1(nu444), unc-13(s69);nuIs152,
unc-17(e113), dgk-1(nu62), egl-30(js126gf), npr-11(ok594), npr-5(ok1583),
ckr-2(tm3082), npr-4(tm1782), npr-22(ok1598), gnrr-1(ok238), tkr-3(ok381),
nmur-4(ok1381), ntr-1(ok2780), fshr-1(ok778), daf-2(e1370), skn-1(lax188),
wdr-23(tm1817), bwIs2[Pflp-1::gfp], idIs7[Pskn-1::skn-1 b/c::gfp];glo-1,
CL2166[Pgst-4::gfp], idIs3[Pgcs-1::gfp]. The wild type reference strain was N2 Bristol.
2.2 Molecular biology – Promoter regions were amplified from lysate genomic DNA.
cDNA concentrated from C. elegans was used to clone genes into vector pPD49.26 using
standard molecular biological techniques and the following plasmids were constructed:
pDS437[Pegl-3::gfp], pDS436[Pegl-21::gfp], pJQ03[Pegl-3::egl-3::gfp],
pJQ04[Pflp-1::flp-1], pJQ06[Punc-47::egl-3::gfp], pJQ07[Pges-1::egl-3::gfp],
pSK1[Prab-3::egl-3::gfp], pTS227[Punc-17::egl-3::venus]
Oligo Sequences(5’--3’) Gene/Promoter
oJQ11 CCCCCCGGATCCATGCTTCTCGAGTCTTACCG Pflp-1::flp-1
6
oJQ12 CCCCCCACTAGTTGCGACCAAATTTACTATGGG Pflp-1::flp-1
2.3 Transgenic lines – Transgenic strains were generated by injecting the expression
plasmids (25ng/µL) together with the co-injection marker KP#708 (Pttx-3::rfp) (40ng/µL)
into N2 strain or corresponding mutants as indicated. Microinjection was performed
using standard techniques. At least three lines were generated and examined
representatively. The following arrays were generated: vjEx800[pDS436],
vjEx801[pDS437], vjEx824[pTS227], vjEx827[pJQ04], vjEx828[pJQ03],
vjEx843[pJQ06], vjEx848[pJQ07].
2.4 Microscopy and analysis – To image C. elegans, L4 stage or young adult worms
were paralyzed by using 2,3-butanedione monoxime (BDM, 30mg/mL) and mounted on
2% agarose pads for imaging. Images were captured with the Nikon eclipse 90i
microscope equipped with a Nikon PlanApo 60x or 100x objective (NA=1.4) and a
PhotometricsCoolsnap ES2 camera. For fluorescence imaging flp-1 expression plasmid,
images were captured from the ventral nerve cord near the posterior gonadal bend of the
worm and for fluorescence imaging egl-3 and egl-21 reporter plasmids, images were
captured from the dorsal nerve cord near the posterior gonadal bend of the worm.
Metamorph 7.0 software (Universal Imaging/Molecular Devices) was used to capture
serial image stacks and the line scans of the maximum intensity projection image was
used for analysis of the dorsal nerve cord. Puncta 6.0 software was used to quantify the
fluorescence intensity values. For all the imaging, fluorescence values were normalized
to the values of 0.5 µm FluoSphere beads (Invitrogen) captured during each imaging
7
session.
2.5 SKN-1::GFP Translocation assay – 50mM of sodium azide (NaN3) solution was
freshly made in M9 buffer during each assay session. L4 stage worms were treated with
50mM sodium azide solution for 10 min, and then were paralyzed with 2,3-butanedione
monoxime (BDM, 30mg/mL) and immediately analyzed for SKN-1 translocation in the
intestine. Same stage worms were treated with M9 buffer and paralyzed with BDM for
analysis as control. The numbers of fluorescent nuclei were counted under the Nikon
eclipse 90i microscope equipped with a Nikon PlanApo 60x or 100x objective (NA=1.4)
and a PhotometricsCoolsnap ES2 camera. Numbers between 1 and 10 were counted as
“Low”, numbers between 11 and 20 would be counted as “Medium” and numbers above
21 would be counted as “High”. For quantification of nuclear fluorescence intensity,
Metamorph 7.0 software (Universal Imaging/Molecular Devices) was used to capture
serial image stacks for each half of the intestine, and the circle scans of the maximum
intensity projection image was used for analysis of each nucleus. Puncta 6.0 software was
used to quantify the fluorescence intensity values.
2.6 Toxicity assay – 300 µM Juglone plates were made by dissolving 50mM juglone
stock solution into standard NGM worm plates and juglone stock solution was made
freshly during each toxicity session. And OP50 bacteria were immediately added to
juglone plates and all the plates were kept in dark all the time except during evaluation.
L4 stage worms of each strain were separated onto separate standard NGM worm plates
in the same day evening and placed on the juglone plates on the next day. 4M fructose
8
ring was applied around the bacteria to restrain the movement of worms. Survival level
was evaluated after 12 hours treatment on the juglone plates with 3 hours interval
between every two time points.
2.7 Statistical analysis – A student’s t test was used to determine significance when
comparing fluorescence level of flp-1 expression plasmid between different genetic
backgrounds and toxicity resistance between different mutants.
9
CHAPTER 3
RESULTS
3.1 Neuroendocrine signals function in organismal survival
To address the question that whether the nervous system participates in the oxidative
stress response, we first test organismal survival to oxidative stress in C. elegans strains
with altered neurotransmitter secretion. Neurons secrete two types of transmitters, fast
transmitters, such as acetylcholine and glutamate and slow transmitters such as
neuropeptides. Fast transmitters are released from synaptic vesicles (SVs) and slow
transmitters are released from dense core vesicles (DCVs). pkc-1, which encodes protein
kinase C epsilon (PKCE) (Monje, et, al, 2011), positively regulates the release of
neuropeptides from DCVs. unc-13, which encodes a C2 and phorbol ester-binding
domain synaptic protein that binds syntaxin, promotes the release of both SV and DCVs.
(Kohn, et al, 2000; Chan, et, al, 2012) The heterotrimeric G protein egl-30, positively
regulates DVC release whereas dgk-1/DAGK, negatively regulate DCV release. (Monje,
et, al, 2011) To test the impacts of SV and DCV secretion on oxidative stress response,
we determined the impacts of mutants defective in these proteins on survival following
juglone treatment.
Juglone is derived from the bark of plants in the Juglandaceae family (Khalafy, et, al,
2002), and it has been shown to inhibit enzymes that are needed for normal metabolic
functions in cells. (Strustad, 2012; Dama, et, al, 1997) Juglone treatment causes toxicity
to C. elegans, and adult animals grown on agar plates that contain juglone die within 24
10
hours. (Khalafy, et, al, 2002; Cao, et, al, 2012; Harding, et, al, 2003)
Figure 2. Toxicity assay on DVC release mutants. Mutants that have either decreased
or increased DCV release were treated with oxidative drug, juglone, including pkc-1,
dgk-1 and egl-30(gf). unc-13, which is shown to affect both SV and DCV release and
unc-17, which is shown to specifically affect SV release were set as control groups. (*
denotes p < 0.01, ** denotes p < 0.001, *** denotes p < 0.0001)
We observed that pkc-1 mutant that is specifically defective in DCV release showed
increased sensitivity to juglone, compared to wild type (WT), (Chan, et, al, 2012;
Stigloher, et, al, 2011) and on the contrary, dgk-1 and egl-30 gain of function (gf)
mutants, which have increased DCV release, showed increased resistance to juglone,
consistent with previous functional studies. (Chan, et, al, 2012; Allen, et, al, 2011; Myers,
0%
20%
40%
60%
80%
100%
Percentage alive
(300uM juglone)
The effect of DCV release mutants
on survival
WT pkc-1/
PKC
unc-13/
Munc-13
Unc-17/
VAchT
dgk-1/
DAGK
egl-30(gf)/
Gαq
DCV
↓
DCV+
SV↓
SV
↓
DCV
↑
***
**
*
*
11
2012) (Figure 2) However, unc-17 mutant, which is specifically defective in the
generation of acetylcholine, did not give sensitive phenotype, and decreased both DCV
and SV release in unc-13 mutant induced sensitiveness, indicating that neuropeptides,
which are packaged in DCV, instead of neurotransmitters, which are packaged in SV,
function as the signals to promote organismal survival under stressed condition.
3.2 Neuropeptides function in organismal survival
Figure 3. Neuropeptides are processed in the DCV. EGL-3/PC and EGL-21/CPE are
the two major enzymes that participate in the neuropeptide processing. SBT-1 encodes
for the chaperone for EGL-3/PC2.
In a previous study, we identified several neuropeptide-related genes, including egl-3,
egl-21 and sbt-1, that are regulated by SKN-1 in the nervous system by using RNAseq.
12
(Staab, et, al, 2014) egl-3 encodes for the proprotein convertase (PC), which participates
in neuropeptide processing, while sbt-1 encodes for the neuroendocrine chaperone 7B2,
which is functional in the maturation and activation of EGL-3/PC2 in neuropeptides
processing. EGL-3/PC2 recognizes and cuts at basic amino acids within the propeptides.
(Stawicki, et, al, 2013; Liu, et, al, 2007; Husson, et, al, 2007) egl-21 encodes for the
carboxypeptidase that is homologous to the convertase/carboxypeptidase E in
mammalian cells, and EGL-21/CPE will further cut off those basic amino acids at the
carboxyl terminal, and this is required for processing of some neuropeptides including
FMRFamid-like (FLP) and neuropeptide-like (NLP) peptides. (Liu, et, al, 2007; Husson,
et, al, 2007) (Figure 3) It is also shown that EGL-21 regulates acetylcholine release at the
neuromuscular junctions. (Jacob, et, al, 2003)
13
Figure 4. Expression of egl-3 and egl-21 are regulated by SKN-1 in the nervous
system. A, C. expression pattern of EGL-3 and EGL-21 in WT, skn-1(gf) and wdr-23
mutant. B, D. quantitative analysis of EGL-3 and EGL-21 expression. Expression of gfp
is driven by egl-3 and egl-21 promoters in WT, skn-1(gf) and wdr-23 mutant. GFP
fluorescence intensity increased in skn-1(gf) mutant, and even increased more in wdr-23
mutant. (** denotes p < 0.001, *** denotes p < 0.0001)
0
100
200
300
400
500
600
700
Arbitrary fluorescence,
normalized to 100
0
50
100
150
200
250
300
Arbitrary fluorescence,
normalized to 100
Pegl-3::gfp
Pegl-21::gfp
WT
skn-1(gf)
wdr-23
WT
skn-1(gf)
wdr-23
A B
WT skn-1(gf) wdr-23
WT skn-1(gf) wdr-23
D C
**
**
***
***
14
First we tested these neuropeptide-related SKN-1 targets in nervous system. We
constructed reporter plasmids for egl-3 and egl-21 by fusing promoter for either egl-3 or
egl-21 together with GFP and then injected these reporters together with dissecting
markers in WT, skn-1(gf) and wdr-23 mutants. skn-1(gf) mutant is hyperactive in skn-1
gene due to the T19E single amino acid mutation in axon 4 and wdr-23 mutant is
defective in the negative regulator of SKN-1, WDR-23, resulting in hyperactivation of
SKN-1. (Staab, et, al, 2013) As shown in Figure 3, our data indicated the expression
pattern of egl-3 and egl-21 in the dorsal nerve cord and by quantifying fluorescence
microscopy images, we showed that the expression level of EGL-3 and EGL-21 increase
by 2 fold in skn-1(gf) mutant compared to WT and it is even higher in wdr-23 mutants
with proximately 6 fold and 2.5 fold for EGL-3 and EGL-21 respectively. Our data
validates that the expression of these neuropeptide-related targets are regulated by SKN-1
in the nervous system.
15
Figure 5. Toxicity assay on neuropeptide processing mutants. Mutants that are
defective in neuropeptide processing showed significant sensitivity to juglone. (***
denotes p < 0.0001)
Next we tested juglone toxicity on mutants that are defective in neuropeptide
processing (Figure 5), including egl-3, egl-21 and sbt-1, and they all showed dramatically
increased sensitivity to oxidative stress that is induced by juglone with over 90% death
after 12 hours of juglone treatment. This finding supports the hypothesis that
neuropeptides may serve as neuroendocrine signals to promote organismal survival under
stressed conditions.
0%
20%
40%
60%
80%
100%
percentage alive
(300uM juglone)
Neuropeptide
processing mutants
egl-3 WT egl-21 sbt-1
***
***
***
16
3.3 EGL-3/PC2 regulates SKN-1 translocation in the intestine
Based on our findings that neuropeptides participate in the regulation of organismal
survival under stressed condition, we next wanted to identify the mechanism by which
the cellular and molecular mechanisms they do so. SKN-1 is the major transcription
factor that regulates oxidative stress response by binding to the AREs in the promoter
regions of downstream targets after being activated in the intestine. (Lee, et, al, 2005;
Itoh, et, al, 1999) To test the translocation of SKN-1 in the intestine and the regulation of
this process, we use a transgenic strain OJ1531(idis7[Pskn-1::skn-1 b/c::gfp;glo-1]) that
expresses skn-1::GFP under its own promoter in glo-1(vu391) mutation background.
glo-1 has been identified to participate in the gut granule formation and glo-1 mutation is
shown to reduce gut fluorescence under excitation for green fluorescent protein(GFP) in
the intestine. (Hermann, et, al, 2005) We used 50mM sodium azide (NaN
3
) as previously
described to induce oxidative stress in C. elegans and SKN-1 translocation in the
intestine. (An, et, al, 2003)
17
Figure 6. SKN-1 translocation into the nucleus is blocked in egl-3 mutant. A. SKN-1
translocation in the intestine. B. quantitative analysis of SKN-1 translocation. Activation
and translocation of SKN-1 is measured after NaN
3
treatment in both WT and egl-3
mutant by counting the number of fluorescent nucleus. Numbers between 1 and 10, 11
and 20, and above 21 were counted as Low, Medium and High respectively.
The intestine is an epithelium composed of ~25 cells. Under normal conditions
SKN-1::GFP can not be observed probably because it is degraded in the cytoplasm.
Following treatment, fluorescence can be observed in many intestinal nuclei. Compared
to M9 treatment, sodium azide dramatically increased SKN-1 translocation in the
intestine with over 50% high translocation level in OJ1531, indicating that SKN-1 is
activated in response to stressed condition and translates into the nucleus (Figure 6). We
crossed OJ1531 to egl-3 mutant, and our data showed that the defectiveness in
neuropeptide processing in egl-3 mutants blocked SKN-1 translocation with
A B
18
approximately 20% decrease in high translocation level and 40% increase in none
translocation level in the intestine, compared to OJ1531. (Figure 6) This indicates that
neuropeptides processed by EGL-3/PC2 may serve as positive signals to activate SKN-1.
Because egl-3 mutants did not completely block SKN-1 translocation in the intestine,
other signals may also participate in activation of SKN-1 in the intestine.
Figure 7. SKN-1 abundance in the intestine decreases egl-3 mutant. Along with
decreased number of fluorescent nucleus, the abundance of SKN-1 in the nucleus also
decreased in egl-3 mutant. (** denotes p < 0.001)
We also quantified SKN-1 translocation in the intestine by measuring fluorescence
intensity under fluorescence
microscopy. (Figure 7) Our data showed that in egl-3
mutants the intensity was reduced by 71.42% compared to WT controls. This further
0
100
200
300
400
Arbitrary fluorescence
(normalized to 100)
SKN-1::GFP nuclear
abundance
WT egl-3
**
19
supports the idea that egl-3 is required for stress induced SKN-1 nuclear translocation in
the intestine
To further address the defectiveness of SKN-1 activation egl-3 mutants, we tested
two downstream targets of SKN-1, gst-4/glutathione S transferase and
gcs-1/gamma-glutamine cysteine synthetase, which are also shown to mainly function in
antioxidative response. Our data showed that the expression of either gst-4 or gcs-1
decreases in egl-3 mutants, indicating that the defectiveness of SKN-1 translocation in
egl-3 mutants results in the defectiveness of SKN-1 activation in the intestine.
20
Figure 8. Expression of SKN-1 downstream targets decreases in egl-3 mutants. The
defectiveness of SKN-1 translocation in egl-3 mutants results in defectiveness of
activation of SKN-1 and its downstream targets. (* denotes p < 0.01, *** denotes p <
0.0001)
3.4 EGL-3/PC2 in the nervous system promotes organismal survival
EGL-3 is reported to be expressed exclusively in the nervous system, (Husson, et, al,
2006) suggesting that an egl-3 processed signal released from the nervous system
regulates skn-1 in the intestine. To directly test this idea, we directed expression of wild
type full length egl-3 cDNA specifically in the nervous system. We first constructed a
0
100
200
300
400
500
600
700
800
-‐
-‐
+
+
-‐
-‐
+
+
Arbitrary
fluorescence
(normalized
to
100)
Intes9nal
fluorescence
n
gcs-1::GFP
☐
gst-4::GFP
***
*
*
21
plasmid pJQ03[Pegl-3::egl-3::GFP] to express egl-3 under its own promoter and
determined whether this could rescue the juglone hypersensitivity of egl-3 mutants. Our
data showed that egl-3 expression under its own promoter in egl-3 mutant was able to
rescue egl-3 mutant phenotype back to WT when treated with juglone. (Figure 9) Next,
we expressed egl-3 under a heterologous promoter that is exclusively expressed in the
nervous system (Prab-3) or a gut specific promoter (Pges-1). Neuronal but not gut
expression was sufficient to rescue, confirming that egl-3 functions in the nervous system
for the stress response. (Figure 10) Furthermore, overexpression of egl-3 caused
resistance. (Figure 9) This suggests that levels of egl-3 are important for the stress
response.
22
Figure 9. EGL-3/PC2 promotes organismal survival under stressed condition. egl-3
expression under its own promoter was able to rescue egl-3 phenotype and induce
resistance in WT.
0%
20%
40%
60%
80%
100%
120%
14 15 16 17 18 19 20 21 22
Percentage alive
(300uM juglone)
Time(hours)
egl-3(XS) wild type egl-3(rescue) egl-3 pkc-1(nj3)
23
Figure 10. Neuronal expression of egl-3 rescues its sensitivity phenotype. Expression
of egl-3 driven by pan neuronal specific promoter recued egl-3 phenotype, but not by gut
specific promoter. (** denotes p < 0.001, *** denotes p < 0.0001)
The C. elegans nervous system is made up of 302 neurons, including 68 sensry, 28
inter- and 214 motor neurons. To further identify the subset of neurons where
EGL-3/PC2 functions, we constructed plasmids, pTS227[Punc-17::egl-3::VENUS],
pJQ06[Punc-47::egl-3::GFP] and to express egl-3 under rab-3, unc-17, unc-47 promoters.
(Lee, et, al, 2001; Ewald, et, al, 2012) unc-17 and unc-47 promoters drive expression
exclusively in cholinergic and GABAergic neurons respectively. (Mathews, et, al, 2012)
egl-3 expression driven by either unc-17 or unc-47 promoter, which are motor neuron
specific promoters, did not rescue egl-3 phenotype (Figure 11), indicating that SKN-1
***
**
24
activation may require egl-3 expression in the whole nervous system or in other parts of
the nervous system, including interneurons and sensory neurons.
Figure 11. Specific expression of egl-3 in motor neurons did not rescue its phenotype.
Expression of egl-3 under either cholinergic or GABAergic neuronal promoter failed to
rescue egl-3 phenotype.
3.5 The neuropeptide, FLP-1, promote organismal survival
Our previous study identified the neuropeptide gene, flp-1, to be regulated by SKN-1
in the nervous system (Staab, et, al, 2013). Flp-1 is an FMRFamide like peptide, one of
31 in the C. elegans genome. (Frooninckx, et, al, 2012) Flp-1 is reported to be expressed
in a subset of neurons. (Nelson, et, al, 1998) To test FLP-1 regulation in the oxidative
0%
20%
40%
60%
80%
100%
14
15
16
17
18
19
20
21
22
Percentage
alive
(300uM
juglone0
Time(hours)
WT
egl-‐3
Punc-‐47::egl-‐3(rescue)
Punc-‐17::egl-‐3(rescue)
Pges-‐1::egl-‐3(rescue)
Pegl-‐3::egl-‐3(rescue)
25
stress response, we first tested flp-1 expression with transgenic strain MU1085, which
expresses GFP under its flp-1 promoter and we crossed MU1085 into skn-1(gf) and
wdr-23 mutants. Our data showed the expression of Pflp-1::gfp in the ventral nerve cord
increased approximately 2 fold in skn-1(gf) or wdr-23 mutants. (Figure 12) This suggests
that that the neuropeptide gene, flp-1, is one of the SKN-1 targets in the nervous system
in C. elegans.
Figure 12. FLP-1 is regulated by SKN-1 in the nervous system. A. Expression pattern
of FLP-1 in the nervous system in WT, skn-1(gf), wdr-23 mutants. B. Expression of
Pflp-1::gfp is up-regulated in skn-1(gf) and wdr-23 mutant. (** denotes p < 0.001, ***
denotes p < 0.0001)
0
50
100
150
200
250
Arbitrary fluorescence
(normalized to 100)
WT
skn-1(gf)
wdr-23
Pflp-1::gfp
WT skn-1(gf) wdr-23
B A
**
***
26
Figure 13. FLP-1 promotes organismal survival under stressed condition. flp-1
mutation incuded sensitivity and flp-1 overexpression induced resistance, compared to
WT and egl-3 mutant.
We obtained a mutant containing a deletion of flp-1(ok2811). This mutant has an
250bp deletion in the flp-1 gene that removes exons two and part of exon one, and is
predicted to eliminate flp-1 gene activity. We found that flp-1 mutants are hypersensitive
to juglone (Figure 13). Further, we constructed plasmid pJQ04[Pflp-1::flp-1] to express
flp-1 under its own promoter and injected into WT and flp-1 mutant. As shown in figure
13, flp-1 overexpression in WT background induced resistance to juglone, compared to
flp-1 mutant and WT. Together, this indicates that the neuropeptide, FLP-1, serves as a
neuroendocrine signals processed in the nervous system to promote organismal survival
0%
20%
40%
60%
80%
100%
120%
11 16 21 26 31 36 41
Percentage alive
(300uM juglone)
Time(hours)
flp-1(4x) egl-3 WT flp-1(XS)
27
in C. elegans. To further address the regulation of FLP in oxidative stress response, we
crossed OJ153 into flp-1 mutation background and tested SKN-1 translocation in the
intestine by treating with sodium azide (NaN
3
). Our date showed that flp-1 mutation
blocked SKN-1 translocation in the intestine, compared to idIs7;glo-1.(figure 14)
However, this blockage of SKN-1 translocation in flp-1 mutant was not severe as in egl-3
mutant, indicating that other neuropeptides processed by EGL-3/PC2 may also participate
in the activation of SKN-1 in the intestine.
Figure 14. SKN-1 activation and translocation is blocked in egl-3 and flp-1 mutants.
Activation and translocation of SKN-1 is measured after NaN
3
treatment in WT, egl-3
and flp-1 mutants by counting the number of fluorescent nucleus. Numbers between 1
and 10, 11 and 20, and above 21 were counted as Low, Medium and High respectively.
0%
20%
40%
60%
80%
100%
SKN-1::GFP nuclear fluorescence
High
Medium
Low
None
- +
WT
- +
egl-3
- +
flp-1
NaN3
28
3.6 GPCR candidates in the intestine function in the SKN-1 activation pathway
Figure 15. Toxicity assay on putative FLP-1 receptor mutants. Five potential GPCRs
for FLP-1 were tested with toxicity assay, but none of these mutants showed sensitive to
juglone treatment. (* denotes p < 0.01)
To further address SKN-1 activation through FLP-1 signaling pathway, we next
wanted to identify the G-protein coupled receptor (GPCR) for FLP-1 in the intestine. C.
elegans encodes over 1150 GPCRs, 125 of which are predicted to ligand with
neuropeptides. (Frooninckx, et, al, 2012) We first looked into those GPCRs that have
been genetically or biochemically linked to FLP-1. (Urano, et, al, 2002; Li, 2005)
However, our data showed mutants corresponding to each of these five GPCRs did not
show significant sensitivity to juglone, compared to WT. (Figure 15) We predict that
there must be other GPCRs that act downstream of FLP-1 and regulate SKN-1 activation
0%
20%
40%
60%
80%
100%
120%
Percentage alive
(300uM juglone)
Putative FLP-1 receptor mutants
WT npr-2 npr-11 npr-5 npr-4 ckr-2
*
*
*
*
29
in oxidative stress response and another possibility would be that one or more of these
GPCRs may function redundantly in oxidative stress response.
Figure 16. Toxicity assay on GPCRs mutants. Some accessible GPCRs mutants were
tested with toxicity assay, and three mutants including tkr-3, ntr-1 and fshr-1 gave
sensitive phenotype. (* denotes p < 0.01, ** denotes p < 0.001, *** denotes p < 0.0001)
We next examined other available mutants encoding additional GPCRs. (Figure 16)
We found that, tkr-3, ntr-1 and fshr-1 mutants showed significant sensitivity to juglone
compared to wild type controls in survival assays. tkr-3 is predicted to encode for
neuropeptide Y receptor based on protein domain analysis, which is orthologous to
human prokeneticin receptor 1 (HGNC:PROKR1), but its corresponding ligands and
regulatory pathways have not been studied or identified yet. (Updike, et, al, 2009; Styer,
et, al, 208) Our subsequent RNAi screening showed that tkr-3 knockdown in the intestine
caused sensitivity to juglone in C. elegans. ntr-1 is predicted to be orthologous to human
0%
20%
40%
60%
80%
100%
120%
Percentage alive
(300uM juglone)
GPCRs mutants
*
*
*
**
***
***
30
vasopressin receptor type II based on protein domain analysis, but its ligands and
regulatory pathways have not been identified yet. (Beets, et, al, 2012) Our results identify
these GPCRs candidates that regulate organismal survival under stressed condition, and
further research will be done to clarify the participation of these GPCRs in FLP-1
signaling pathway and in regulation and activation of SKN-1 in the intestine.
3.7 The GPCR, FSHR-1, promotes organismal survival through SKN-1 activation.
fshr-‐1
is
predicted
to
encode
for
human
follicle-‐stimulating
hormone
receptor
and
luteinizing
hormone
receptor
orthologs
and
previous
study
have
shown
that
FSHR-‐1
participates
in
the
innate
immune
response.
(Powell,
et,
al,
2009)
However,
whether
this
GPCR
functions
in
the
regulation
of
oxidative
stress
response
has
not
been
identified.
We
obtained
this
outcrossed
version
of
fshr-‐1
mutant,
which
gets
rid
of
some
background
mutation
and
solidifies
the
fshr-‐1
phenotype.
As
shown
in
Figure
17,
fshr-‐1
mutant
showed
similar
sensitivity
to
oxidative
drug
as
flp-‐1
mutant,
indicating
its
function
in
oxidative
stress
response.
31
Figure
17.
fshr-‐1
functions
in
the
oxidative
stress
response.
fshr-‐1
mutant
induced
sensitivity
to
juglone
treatment.
To
further
address
the
question
that
how
fshr-‐1
functions
in
the
oxidative
stress
response,
we
tested
SKN-‐1::GFP
translocation
in
fshr-‐1
mutants.
Our
data
showed
that,
similar
as
egl-‐3
mutants,
fshr-‐1
mutants
can
also
block
SKN-‐1
translocation
and
activation
in
the
intestine
(Figure
18),
but
this
blockage
was
not
severe
as
in
egl-‐3
mutants,
indicating
that
other
GPCRs
may
also
be
activated
by
the
neuropeptides
processed
by
EGL-‐3/PC2.
0%
20%
40%
60%
80%
100%
12
17
22
27
32
37
Percent
alive
(300uM
juglone)
Time(hours)
N2
egl-‐3
fshr-‐1
^lp-‐1(ok2811)6X
32
Figure
18.
SKN-‐1
translocation
and
activation
is
blocked
in
fshr-‐1
mutant.
fshr-‐1
mutant
can
also
block
SKN-‐1
translocation
in
the
intestine,
comparing
to
WT
and
egl-‐3
mutant.
0%#
20%#
40%#
60%#
80%#
100%#
idIs7;glo21# egl23;idIs7;glo21# flp21;idIs7;glo21# fshr21;idIs7;glo21#
SKN$1::GFP*Nuclear*Fluorescence None# Low# Medium# High#
33
CHAPTER 4
DISCUSSION
Oxidative stress that is produced during metabolism is predicted to be associated to
many neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease.
(Finkel, et, al, 2000) Nrf-2, which is a homologue of SKN-1 in C. elegans, is the major
transcription factor that is shown to participate in the oxidative stress response in
mammalian cells. We found that neuroendocrine signals that are processed by
EGL-3/PC2 regulate oxidative stress response and organismal survival through SKN-1 in
the intestine. Mutants that are defective in neuropeptide release show increased
sensitivity to juglone treatment, which is functional as an oxidative stress inducer, and
increased neuropeptide release caused increased resistance to juglone treatment. We also
showed that impaired neuropeptide processing in egl-3 mutants induced sensitivity to
oxidative stress that is induced by juglone treatment. On the other hand, we showed that
expression egl-3 either under its own promoter or under pan-neuronal promoter, Prab-3,
can rescue egl-3 phenotype of being sensitive to oxidative drugs back to wild type, and
increased expression of egl-3, which leads to increased neuropeptide signaling, promotes
organismal resistance to oxidative drugs. Together we predicted that neuropeptides
processed by EGL-3/PC2 serve as neuroendocrine signals to promoter organismal
survival under stressed condition. We also showed that egl-3 mutations could block
SKN-1 translocation in intestinal nuclei, indicating that neuropeptide signals participate
in the regulation of SKN-1 in the oxidative stress response through inter-tissue signaling.
34
However, translocation of SKN-1 in response to oxidative stress was not completely
blocked in egl-3 mutants, and corresponding to previous study that the upper regulator of
SKN-1, SEK-1, can also be activated by other factors and SKN-1 can also be activated in
parallel by insulin signaling pathway (Zugasti, et, al, 2014; Tullet, et ,al, 2008), this
indicates that in the absence of egl-3, SKN-1 translocation can be activated by other
minor factors. Our data also showed that egl-3 mutants could reduce the resistance to
oxidative drugs in daf-2 mutants (Figure 19), indicating that neuropeptide signaling and
insulin signaling may function in parallel to regulate SKN-1 in the intestine, or their
functional pathway partially overlap.
Figure 19. egl-3 mutants reduce the resistance in daf-2 mutants. egl-3;daf-2 double
mutants showed intermediate sensitivity comparing to egl-3 and daf-2 single mutants.
35
Next, we did neuropeptide mutants screening and identified the neuropeptide, flp-1,
potentially serves as the neuroendocrine signal to regulate SKN-1 activation and
organismal oxidative stress response. We showed that flp-1 mutant was sensitive to
oxidative stress, which was induced by juglone, similar to egl-3 mutant and
overexpression of flp-1 caused resistance to oxidative drugs in WT. And flp-1 mutant also
was shown to block SKN-1 activation and translocation into the nucleus in intestine.
However, this blockage in flp-1 mutants was slightly weaker than that in egl-3 mutants,
indicating that there might be other neuroendocrine signals processed by EGL-3/PC2 that
activates SKN-1 in the intestine.
To identify potential GPCRs for FLP-1 in the intestine, we first tested the five
GPCRs mutants that were genetically or biochemically linked to FLP-1, however none of
these GPCRs mutants showed sensitivity to oxidative drugs. We predicted that there
would be other GPCRs that act downstream of FLP-1 to regulate SKN-1 in the intestine,
or one or more of these GPCRs function redundantly in the FLP-1 regulatory pathway to
activate SKN-1. We further identified some GPCRs candidates including tkr-3, ntr-1 and
fshr-1 that showed increased sensitivity to oxidative drugs in their corresponding mutants,
but the pathways or the ligands of these GPCRs have not been clearly identified yet. To
genetically show whether these GPCRs would be receptors for FLP-1, we further would
look into double mutants of each of these GPCRs mutants and flp-1, we predict that the
doubles mutants would show similar sensitivity to oxidative drugs as single
corresponding mutant if a GPCR functions as the receptor for FLP-1. Also for the GPCR,
36
FSHR-1, our data showed that fshr-1 mutants can also induce sensitivity to oxidative
drugs and block SKN-1 translocation and activation, indicating FSHR-1 also function in
the oxidative stress response through SKN-1 pathway. Further experiment will be needed
to show whether FSHR-1 functions downstream of FLP-1. We also identified some
GPCRs mutant that showed increased resistance to oxidative drugs, indicating that
GPCRs may both positively and negatively regulate downstream pathways, and the
integrated signaling regulates SKN-1 activity. Our future work would focus on screening
of GPCR candidates with RNAi, and identify corresponding pathways and sites of action
of GPCR candidates.
37
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Abstract (if available)
Abstract
The Nrf2 transcription factor plays a critical role in mediating adaptive responses to cellular stress and defending against neurodegeneration, aging, and cancer. Nrf2 activity is regulated both by oxidative stress and by endogenous signaling pathways that control its translocation into the nucleus where it directs the transcription of a variety of antioxidant genes. However the molecular mechanisms underlying the regulation of Nrf2 activity in the context of multicellular organisms are not well understood. Here, we report a novel role for inter-tissue neuroendocrine signaling in regulating the activity of the Caenorhabditis elegans Nrf homolog SKN-1 during the oxidative stress response. We show that mutants with impaired neuropeptide processing or release show decreased survival following treatment with toxins that increase oxidative stress, whereas mutants with increased neuropeptide release are resistant to the toxic effects of oxidative stress. We find that the proprotein convertase, egl-3/PC2, which is a key enzyme involved in neuropeptide maturation, functions in the nervous system to promote survival following treatment with the oxidant juglone. Strikingly, egl-3 mutations block stress-induced SKN-1 nuclear translocation and expression of the SKN-1 target gst-4/glutathione-S-transferase in the intestine. We identify a neuropeptide, FLP-1, expressed in the nervous system, that is critical for survival in the presence of stress, and we are investigating its involvement in SKN-1 activation in the intestine. Together these results identify the nervous system as a critical regulator of SKN-1 activity in distal tissues and suggest that neuroendocrine signaling may be a novel mechanism by which Nrf2 is regulated in vivo.
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Creator
Jia, Qi
(author)
Core Title
Neuroendocrine regulation of the transcription factor SKN-1/Nrf2 in oxidative stress response
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
07/09/2015
Defense Date
03/19/2015
Publisher
University of Southern California
(original),
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Tag
C. elegans,FLP-1,neuropeptide,OAI-PMH Harvest,oxidative stress,SKN-1
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Language
English
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Tokes, Zoltan A. (
committee chair
), Langen, Ralf (
committee member
), Sieburth, Derek (
committee member
)
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jqjackey@gmail.com,qij@usc.edu
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https://doi.org/10.25549/usctheses-c3-589898
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
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Tags
C. elegans
FLP-1
neuropeptide
oxidative stress
SKN-1