Available via license: CC BY
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TYPE Original Research
PUBLISHED 05 August 2022
DOI 10.3389/fnagi.2022.955113
OPEN ACCESS
EDITED BY
Michael Wink,
Heidelberg University, Germany
REVIEWED BY
Chatrawee Duangjan,
University of Southern California,
United States
Shaoxiong Zhang,
Fujian Agriculture and Forestry
University, China
*CORRESPONDENCE
Xiangwei Song
songxiangwei@ccsfu.edu.cn
Xueli Wang
xueliwang101@hotmail.com
SPECIALTY SECTION
This article was submitted to
Alzheimer’s Disease and Related
Dementias,
a section of the journal
Frontiers in Aging Neuroscience
RECEIVED 28 May 2022
ACCEPTED 11 July 2022
PUBLISHED 05 August 2022
CITATION
Song X, Sun Y, Wang Z, Su Y, Wang Y
and Wang X (2022) Exendin-4
alleviates β-Amyloid peptide toxicity
via DAF-16 in a Caenorhabditis
elegans model of Alzheimer’s disease.
Front. Aging Neurosci. 14:955113.
doi: 10.3389/fnagi.2022.955113
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©2022 Song, Sun, Wang, Su, Wang
and Wang. This is an open-access
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author(s) and the copyright owner(s)
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reproduction is permitted which does
not comply with these terms.
Exendin-4 alleviates β-Amyloid
peptide toxicity via DAF-16 in a
Caenorhabditis elegans model
of Alzheimer’s disease
Xiangwei Song1*, Yingqi Sun1, Zhun Wang2, Yingying Su1,
Yangkun Wang1and Xueli Wang3*
1School of Life Sciences, Changchun Normal University, Changchun, China, 2Plant Inspection and
Quarantine Laboratory, Changchun Customs Technical Center, Changchun, China, 3School of
Grain, Jilin Business and Technology College, Changchun, China
Epidemiological analyses indicate that type 2 diabetes mellitus (T2DM) is a risk
factor for Alzheimer’s disease (AD). They share common pathophysiological
mechanisms. Thus, it has been increasingly suggested that several anti-T2DM
drugs may have therapeutic potential in AD. Exendin-4, as a glucagon-like
peptide-1 (GLP-1) receptor agonist, is an approved drug used to treat T2DM. In
this research, the neuroprotective eect of Exendin-4 was investigated for the
first time using transgenic Caenorhabditis elegans. Our results demonstrated
that Exendin-4 attenuated the amyloid-β(1-42) (Aβ1-42) toxicity via multiple
mechanisms, such as depressing its expression on protein and mRNA and
reducing Aβ(1-42) accumulation. Exendin-4 at 0.5 mg/ml had been shown
to extend life by 34.39% in CL4176 and delay the onset of paralysis in
CL4176 and CL2006 which were increased by 8.18 and 8.02%, respectively.
With the treatment of Exendin-4, the nuclear translocation of DAF-16 in the
transgenic nematode TJ356 was enhanced. Superoxide dismutase-3 (SOD-3),
as a downstream target gene regulated by DAF-16, was upregulated on mRNA
level and activity. The reactive oxygen species (ROS) level was decreased.
In contrast, we observed that the ability of Exendin-4 to regulate SOD was
decreased in CL4176 worms with the DAF-16 gene silenced. The activity of
SOD and the mRNA level of sod-3 were downregulated by 30.45 and 43.13%,
respectively. Taken together, Exendin-4 attenuated Aβ(1-42) toxicity in the
C. elegans model of AD via decreasing the expression and the accumulation of
Aβ(1-42). Exendin-4 exhibited the ability of antioxidant stress through DAF-16.
With continuous research, Exendin-4 would become a potential therapeutic
strategy for treating AD.
KEYWORDS
Alzheimer’s disease, Exendin-4, Caenorhabditis elegans, DAF-16, Amyloid-βpeptide,
neuroprotection
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Song et al. 10.3389/fnagi.2022.955113
Introduction
As a chronic neurodegenerative disease, Alzheimer’s disease
(AD) poses a serious threat to individuals’ health. Despite
years of massive investigation, the pathogenic mechanism of
AD is still in the stage of hypothesis theories, such as the
hyperphosphorylated tau protein hypothesis, amyloid toxicity
hypothesis, oxidative stress hypothesis, and acetylcholine
hypothesis. However, the amyloid toxicity hypothesis is widely
accepted. Amyloid-βpeptides (Aβ1-40 and Aβ1-42) are
produced due to aberrant cleavage of the amyloid precursor
protein (APP), which accumulates on the neuron. Furthermore,
Aβ(1-42), as a mediator of oxidative stress, has been proposed
to play a central role in the pathogenesis of AD (Butterfield and
Boyd-Kimball, 2004), such as decreasing the levels of superoxide
dismutase (SOD) and increasing the levels of MDA and reactive
oxygen species (ROS) (Wang et al., 2021). As a target, amyloid is
often applied to drug screening and clinical diagnosis.
The data of epidemiological investigation suggest that the
correlation between type 2 diabetes mellitus (T2DM) and AD is
highly significant. Diabetic patients are twice as likely to suffer
from AD as the normal population. AD and T2DM always
share common pathophysiological symptoms (Caberlotto et al.,
2019), such as hyperglycemia, hypercholesterolemia, and insulin
signaling dysfunction.
Thus, it has been strongly suggested that some drugs treated
with T2DM may have therapeutic potential in AD (Sebastiao
et al., 2014). Exendin-4, as a glucagon-like peptide-1 (GLP-1)
receptor agonist, has been approved for the treatment of T2DM.
However, the strong preclinical evidence suggests that Exendin-
4 is neuroprotective in AD (Mullins et al., 2019). For instance,
Exendin-4 appears to prevent the hyperphosphorylation of
AD-associated tau protein due to the increased insulin signaling
pathway in the brain (Xu et al., 2015). Exendin-4 significantly
attenuated Aβ-induced memory deficits in the Morris water
maze and Y-maze test (Garabadu and Verma, 2019).
Many kinds of transgenic Caenorhabditis elegans models
(Wu and Luo, 2005) of AD had been used to research the
pathogenesis of AD (Link, 2006) due to their short lifetime
and progressive paralysis phenotype. Moreover, the transgenic
C. elegans, such as CL4176 (Wu and Luo, 2005; Zhang et al.,
2016) and CL2006 (Smith and Luo, 2003), promotes the onset
of the ROS, and it is similar to the onset of ROS observed in
patients with AD, which is consistent with the amyloid cascade
hypothesis on oxidative stress. Oxidative stress is extensive in the
AD brain. Aβ(1-42) has been shown to induce oxidative stress
and neurotoxicity in vitro and in vivo. Therefore, transgenic
C. elegans strains accelerate the pace of understanding the
mechanisms of Aβ(1-42) toxicity in biological systems. At the
same time, they can be used for anti-AD drug screening in vivo
(Ewald and Li, 2010; Wolozin et al., 2011).
Herewith, we mainly employed transgenic C. elegans
as the model to investigate the neuroprotective effects of
Exendin-4. Our conclusion presents that Exendin-4 develops
its neuroprotective effect by reducing the accumulation and
expression of Aβ(1-42) directly. Exendin-4 also showed the
excellent performance of antioxidant stress via the transcription
factors DAF-16.
Materials and methods
C. Elegans strains and maintenance
The wild-type N2 worms and the transgenic worms
CL4176 {smg-1ts [myo-3/Aβ1–42 long 3′-untranslated
region (UTR)]} were obtained from Caenorhabditis Genetics
Center (University of Minnesota, Minneapolis, MN). TJ356
[daf-16p::daf-16a/b::GFP +rol-6], CL2006 {dvIs2 [pCL12(unc-
54/human Aβ1–42 minigene) +pRF4]}, the transgenic
CF1553 {muIs84 [(pAD76) sod-3p::GFP +rol-6(su1006)]},
and Escherichia coli were kindly donated by Dr. Liping Wang.
CL4176 was maintained at 16 ◦C. The wild-type N2, CL2006,
CF1553, and TJ356 were maintained at 20 ◦C.
All worms were on a solid nematode growth medium
(NGM) seeded with live E. coli (OP50) as a food source.
Brood size and body length assays
The wild-type N2 worms were used in brood size and
body length assays. For the brood size assay, each worm
(L4 stage) was transferred onto NGM plates treated with
different concentrations of Exendin-4 (0.1, 0.3, 0.5, and 1.2
mg/ml). The worms were cultivated at 20 ◦C. The number of
all eggs that it hatched was recorded (Rangsinth et al., 2019).
For the body length assay, worms (L4 stage) were transferred
onto different NGM plates treated with different concentrations
of Exendin-4 (0.1, 0.3, 0.5, and 1.2 mg/ml) and cultured at 20 ◦C.
Adult day-1 worms were paralyzed using 0.1% sodium azide and
mounted on a glass slide. At least thirty worms per group were
imaged using light microscopes (Leica, DM3000 LED, Germany)
and measured using the Leica Application Suite version 4.12
software (Leica, Germany).
Paralysis assays
We synchronized the transgenic C. elegans strain CL4176 on
different concentrations of Exendin-4 (0 mg/ml, 0.02 mg/ml, 0.1
mg/ml, 0.3 mg/ml, 0.5 mg/ml, and 1.2 mg/ml) and caffeine (1.2
mg/ml) treated plates at 16 ◦C for 48 h. Then, worms (L2 stage)
were transferred from 16 ◦C to 23 ◦C. After cultivating 40 h at
23 ◦C, the paralyzed worms had been counted at 2 h intervals
until all the worms were paralyzed (Dostal and Link, 2010;
Takahashi et al., 2014). The principle of paralysis is stipulated
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that they moved their heads only or failed to move their bodies
by touching stimuli.
Life span assays
To obtain the synchronized worms, gravid adults (L1 stage)
of CL4176 were raised on different concentrations of Exendin-4
(0 mg/ml, 0.02 mg/ml, 0.1 mg/ml, 0.3 mg/ml, and 0.5 mg/ml)-
treated plates to lay eggs for 2–3 h. Generally, 280 worms
should be promised in one plate. After 48 h of synchronization,
Aβ(1-42) expression was induced by upshifting the temperature
to 23 ◦C. We continued to collect the data for 20 days. All life
span assays were preceded independently at least three times.
Worms that were missing, attaching to walls or worm bags, were
not included during the life statistics.
TABLE 1 Lists of primers.
Gene Forward primer Reverse primer
actin-1 5′AAGACCACGTCAT
CAAGG3′
5′TTCTCCATATCAT
CCCAGTT3′
daf-16 5′GCGAATCGGTTCCAGCAAT
TCCAA3′
5′ATCCACGGACACTGTT
CAACTCGT3′
sod-3 TATTAAGCGCGACTTCGG
TTCCCT
CGTGCTCCCAAACGTCAAT
TCCAA3
Aβ(1-42) CCGACATGACTCAGGATA
TGAAGT
CACCATGAGTCCAATGA
TTGCA3
Western blotting
Worms were treated with Exendin-4 (0.5 mg/ml) and
induced for 44 h at 23 ◦C. Worms (L4 stage) were harvested
and centrifuged at 12,000 rpm for 10 min. The pellets were
resuspended and sonicated after freeze-thawed as described
in the WormBook (http://www.wormbook.org). The gel was
transferred onto polyvinylidene fluoride (PVDF) membranes.
The monoclonal antibody (ab201060, ABCam, UK) was used to
detect Aβ(1-42). Experiments were repeated independently for
three times.
Staining of Aβ
Worms (L2 stage) were transferred from 16 ◦C to 23 ◦C
to cultivate for 40 h after synchronization. A total of 100
worms (L4 stage) were picked up and washed with M9
buffer. After centrifugation, worms were fixed with 150 µl of
4%paraformaldehyde/PBS buffer (pH =7.4) at 4 ◦C for 24 h.
Fixed worms were treated with 150 µl 5% β-mercaptoethanol,
1% Triton X-100, and 125 mM Tris pH 7.4, at 37 ◦C for 24 h.
Treated worms were washed with PBS buffer three times, and
200 µl of 0.125% thioflavin T (Sigma) in 50% ethanol was added
for 90 s and destained with 500 µl of 50% ethanol until the
suspension became transparent. Worms were resuspended with
100 µl M9 buffer. The animals were finally transferred to slides
using a drop of glycerol, and fluorescence images were acquired
by confocal laser scanning fluorescence microscopy (CarlZeiss
LSM710, Germany).
FIGURE 1
The eects of Exendin-4 on wild-type N2 Caenorhabditis elegans.(A) Body length assay and (B) brood size assay. There was no significant
dierence in the treatment groups on brood size and body length. Values are mean ±SEM of experiments repeated independently at least for
three times.
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FIGURE 2
Exendin-4 alleviated Aβ-induced paralysis in transgenic C. elegans strains CL4176 and CL2006. (A) In CL4176. (B) In CL2006. Curves show the
process of Aβ-induced paralysis in transgenic C. elegans treated with a vehicle control (H2O) or dierent concentrations of Exendin-4 (0.02
mg/ml, 0.1 mg/ml, 0.3 mg/ml, 0.5 mg/ml, and 1.2 mg/ml). Caeine (1.2 mg/ml) is positive control. The onset of paralysis was prolonged in
CL4176 and CL2006 worms in a dose-dependent manner (at least 40 worms were tested in each group, and the experiments were repeated
independently for three times).
FIGURE 3
The life span assay administrated Exendin-4 in transgenic
C. elegans strain CL4176. CL4176 was treated with a vehicle
control (H2O) or dierent concentrations of Exendin-4 (0.02
mg/ml, 0.1 mg/ml, 0.3 mg/ml, and 0.5 mg/ml). The median
survival of worms treated with 0.5 mg/ml Exendin-4 was 13
days, which had a significant expansion compared with 8 days
of control worms (p<0.001). The experiment was repeated
independently for three times.
ROS assays
Age-synchronized C. elegans (more than 30 eggs per
plate) were transferred to NGM plates containing vehicle
or 0.5 mg/ml Exendin-4 and incubated for 48 h at 16 ◦C.
After incubation at 23 ◦C for 44 h, ROS was determined
based on published studies (Wu et al., 2012). The examined
nematodes were transferred to 0.5 ml of M9 buffer containing
5µM CM-H2DCFDA (D6883, Sigma-Aldrich, USA) and
preincubated for 3 h at 20 ◦C. Later, nematodes were mounted
on 2 % agar pads and examined using a fluorescence
microscope (Nikon, SMZ 18, Japan) at 495 nm of excitation
wavelength and 537 nm of emission filter. More than 30
animals were counted for the statistical analysis. The relative
fluorescence intensities of the worm were semi-quantified
using the ImageJ software. The semi-quantified ROS was
expressed as relative fluorescence units (RFU). Three replicates
were performed.
SOD activity assays
The transgenic C. elegans strain CL4176 was synchronized
on 0.5 mg/ml Exendin-4 treated and untreated plates at 16
◦C for 48 h, respectively. Then, t he worms (L2 stage) were
cultivated for another 40 h at 23 ◦C. A total of 200 worms
(L4 stage) were collected and centrifuged with M9 buffer.
The pellets were disrupted according to the Western blotting
assay protocol. After protein quantification using bicinchoninic
acid (BCA) assay, the analysis of SOD activity was proceeded
using nitrotetrazolium blue chloride (NBT) method (Total
Superoxide Dismutase Assay Kit with NBT, #S0109, Beyotime
Biotechnology, China). Four replicates were performed.
Real-time PCR analysis
Worms were treated according to the Western blotting
protocol. The worms (L4 stage) were collected by washing the
plate with 1 ml M9 buffer. The pellets were washed three times by
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FIGURE 4
Exendin-4 attenuated the protein and mRNA levels of Aβ(1-42) in C. elegans CL4176. (A) Western blotting assays of experiments repeated
independently for three times are shown. Control groups were not treated with Exendin-4. The test groups were treated with 0.5 mg/ml of
Exendin-4. (B) The relative intensity of Aβ(1-42) expression was analyzed using the ImageJ software. After treatment with 0.5 mg/ml Exendin-4,
the protein expression level was reduced by 39.78%. p<0.01 (**).(C) The mRNA levels of Aβ(1-42) were analyzed by real-time PCR. mRNA levels
were reduced by 29.67%. p<0.001 (***).
centrifugation at 25,000 rpm for 5 min. The worms were freeze-
thawed and transferred directly into 1 ml of TRIzol reagent
(Thermo Fisher Scientific, Shanghai, China). The total nematode
RNA was extracted first and measured using a Nanophotometer
(IMPLEN N60, German). The cDNAs were synthesized using
the Transcription Kit (Promega, A5000, USA). The expression
of genes was determined by real-time PCR performed on 7500
Real-Time PCR System (ABI) with SYBR Green real-time PCR
kit (Roche). According to the instruction of the real-time PCR
kit, the cycling conditions were as follows: (1) holding stage at 50
◦C for 2 min; (2) holding stage at 95 ◦C for 10 min; (3) cycling
stage at 95 ◦C for 15 s, at 55 ◦C for 1 min; the number of cycling
for 40 cycles; and (4) melt curve stage at 95 ◦C for 10 s, at
60 ◦C for 1 min, at 95 ◦C for 10 s. The relative levels of gene
expression were calculated using the 2 −11CT method using
the gene actin-1 as the internal control. The experiment was
repeated in triplicate. Primers are listed in Table 1.
DAF-16 nuclear translocation analysis
Age-synchronized nematodes of the transgenic strain TJ356
stably expressing a DAF-16::GFP fusion protein were treated
with Exendin-4 (0.5 mg/ml) and kept at 20 ◦C for 48 h. Later,
the worms (L4 stage) were anesthetized with 0.1% sodium and
transferred to slides using a drop of glycerol. The subcellular
DAF-16 distribution among twenty worms per group was
analyzed using a Nikon SMZ 1500 fluorescence microscope. The
experiments were performed more than 3 times. The amount of
DAF-16::GFP was analyzed using the ImageJ software.
The C. Elegans RNA interference assay
Worms (L1 stage) were treated with 0.5 mg/ml Exendin-4.
DAF-16 was knocked down by feeding the C. elegans CL4176
with E. coli strain R13H8.1 bacteria carrying daf-16 dsRNA.
Worms fed with R13H8.1 bacteria with the empty vector L4440
were used as negative controls. Notably, 1 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG) was used on NGM plates. CL4176
was maintained at 16 ◦C after synchronization. After 48 h, more
than 50 worms (L2 stage) were cultured for another 40 h at
23 ◦C. Then, the process of paralysis assays was carried out. The
experiments were performed more than 3 times.
Statistical analysis
Statistical analyses of differences between groups in the
paralysis assays and life span assay were performed using
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FIGURE 5
Aβ(1-42) accumulation assay with thioflavin T staining in transgenic C. elegans CL4176. (A) Control groups were not treated with Exendin-4. Aβ
(1-42) deposits stained with thioflavin-T show abundant fluorescence patches near the pharynx in transgenic worm CL4176. (B–D) Transgenic
worms fed with 0.3 mg/ml, 0.6 mg/ml, and 1.2 mg/ml Exendin-4. They show a significant reduction in the deposition of Aβ(1-42) in a
dose-dependent way. The yellow arrows show the sites of Aβ(1-42). (E) The number of Aβ(1-42) patches in dierent groups. p<0.01 (**) and
p<0.001 (***).
the log-rank test. Data other than paralysis and life span
were analyzed using Student’s t-test. Results were expressed
as the mean ±standard deviation of experiments repeated
independently for three times. The GraphPad Prism software 5.0
(GraphPad, La Jolla, CA, United States) was used for statistical
analyses. The difference in statistical data is shown with p-value;
p<0.05 (∗), p<0.01 (∗∗), and p<0.001 (∗∗∗ ) were regarded
as significant.
Results
Eects of exendin-4 on the development
and reproduction of C. Elegans
To investigate the toxicity of Exendin-4, we performed body
length and brood size assays to monitor the development and
fertility rate of wild-type N2 worms, respectively. The data
showed that Exendin-4 with 0.1, 0.3, 0.5, and 1.2 mg/ml has no
toxic effects on nematodes in terms of spawning and body length
(Figures 1A,B).
Exendin-4 delays the progression of
paralysis in AD worms
To investigate the potential influence of Exendin-4 on the
progression of paralysis induced by A β(1-42), we treated
CL4176 and CL2006, respectively. The onset of paralysis
was dramatically delayed in a dose-dependent manner after
treatment of Exendin-4 (Figures 2A,B). The low dose of
Exendin-4 (0.02 mg/ml) cannot slow down the process
of paralysis significantly. Notably, 1.2 mg/ml of Exendin-
4 displayed the best effect among all groups. The mean
paralysis time was increased by 8.18% (CL4176) and 8.02%
(CL2006), respectively. In CL4176, the duration of paralysis
in the 1.2 mg/ml Exendin-4 group was prolonged by 3.67 h.
The average paralysis time was 0.7 h longer than that of the
positive control (Supplementary Table 1). Exendin-4 showed
better performance than the positive control. In CL2006, the
duration of paralysis in the 1.2 mg/ml Exendin-4 group was
prolonged by 3.58 h. The average paralysis time was 0.11 h
longer than that of the positive control (Supplementary Table 2).
Combined with the experimental results in the two mutant
nematodes, these observations suggested that Exendin-4 may
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FIGURE 6
The eect of antioxidant stress of Exendin-4 in C. elegans CL4176 and CL2006. ROS was analyzed using CM-H2DCFDA. Photographs were
taken using a fluorescence microscope at 495 nm of excitation wavelength and 537 nm of emission filter. (A,D) The untreated CL4176 and
CL2006. (B,E) The treated CL4176 and CL2006 with 0.5 mg/ml Exendin-4. (C,F) The fluorescence intensity analyzed using the ImageJ software.
(G,H) The mRNA levels of superoxide dismutase-3 (sod-3). (I,J) The activity of sod-3. All the experiments were repeated independently for three
times. p<0.05 (*), p<0.01 (**).
have the potential to relieve the paralysis caused by Aβ(1-
42).
Exendin-4 increases life span in AD
worms
The life span assay was examined under different Exendin-
4 concentrations (0.02, 0.1, 0.3, and 0.5 mg/ml). We collected
the data for 20 days. Exendin-4 treatment showed a significant
increase in the life span in a dose-dependent manner (Figure 3).
Median survival time was prolonged in each treated group.
Notably, 0.3 mg/ml and 0.5 mg/ml of Exendin-4 displayed
remarkable performance in extending life span. They almost
increased the median survival time to 13 days. Compared
with the control group, lifetime was increased by 32.05 and
34.39%, respectively (Supplementary Table 3). Results implied
that Exendin-4 treatment attenuated the harmful effect of
longevity induced by Aβ(1-42) toxicity in worms.
Exendin-4 reduced the levels of Aβ(1-42)
in AD worms
To clarify why Exendin-4 can relieve paralysis in CL4176,
we monitored the Aβ(1-42) levels on protein and mRNA.
All worms were collected after being induced to paralysis
for 44 h, and parallel populations were processed for real-
time PCR and the Western blotting assay. The group treated
with 0.5 mg/ml Exendin-4 resulted in a 39.78% decrease in
the protein expression of Aβ(1-42) (Figures 4A,B) and a
significant reduction by 29.67% in the mRNA level of Aβ(1-42)
(Figure 4C). Thus, the ability of Exendin-4 to delay paralysis
is directly demonstrated by downregulating the protein and
mRNA levels of Aβ(1-42). We detected the change of Aβ(1-
42) accumulation with a thioflavin T staining assay. We focused
on the area near the pharynx of C. elegans CL4176 to count
the number of deposits. Lots of Aβ(1-42) deposits appeared
after being induced (Figures 5A–D). Instead, worms treated with
Exendin-4 displayed less Aβ(1-42) deposits in a dose-dependent
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FIGURE 7
Exendin-4 lost the ability to alleviate Aβ-induced paralysis when the daf-16 gene is knockout in transgenic C. elegans strain CL4176. DAF-16
expression was knocked down by feeding the C. elegans CL4176 with Escherichia coli strain R13H8.1 bacteria carrying daf-16 dsRNA. Curves
show that the eect of Exendin-4 (0.5 mg/ml) on inhibiting the paralysis rate of C. elegans vanished after daf-16 gene knockout. (At least 50
worms were tested in each group, and the experiments were repeated independently for three times.).
manner (Figure 5E). Exendin-4 possessed the positive effect to
inhibit the accumulation of Aβ(1-42).
Exendin-4 increases oxidative stress
resistance in AD worms
The ROS will be increased by triggering the expression of Aβ
(1-42). The increased production of ROS associated with age and
neurotransmission in neurons leads to cognitive dysfunction
in AD.
To test the antioxidant effect of Exendin-4, two kinds of
strains (CL2006 and CL4176) were treated with 0.5 mg/ml
of Exendin-4.
Reactive oxygen species was measured using the
CM-H2-DCFDA method. The worms treated with Exendin-4
showed lower production of ROS in CL2006 and CL4176
(Figures 6A,B,D,E). The fluorescence intensity was analyzed
using the ImageJ software. A volume of 0.5 mg/ml Exendin-4
decreased ROS level by 13.57% (p<0.01) and 17.22% (p<0.05)
in CL4176 and CL2006, respectively (Figures 6C,F). A volume
of 0.5 mg/ml Exendin-4 improved the mRNA levels of sod-3
by 37.33% (p<0.01) and 63.33% (p<0.05) in CL4176 and
CL2006, respectively (Figures 6G,H). A volume of 0.5 mg/ml
Exendin-4 improved the activity of sod-3 by 36.04% (p<0.05)
and 27.11% (p<0.05) in CL4176 and CL2006, respectively
(Figures 6I,J).
We have a primary judgment that Exendin-4 is provided
with delaying the process of AD induced by Aβtoxicity through
its antioxidative stress activity.
DAF-16 was essential to the
neuroprotective eect of exendin-4
DAF-16, which is a major regulator in the insulin/insulin-
like growth factor 1 signaling (IIS) pathway, integrates
signals from upstream pathways to elicit transcriptional
changes in many genes involved in aging, development,
stress, metabolism, and immunity. Exendin-4 has a profound
effect on the insulin signaling pathway to prevent the
process of AD (Xu et al., 2015; Yang et al., 2016). Hence,
we investigated whether Exendin-4 can delay the process
of paralysis in worms when daf-16 was knocked down
using RNA interference (RNAi) (Supplementary Figure 2).
Exendin-4 at 0.5 mg/ml lost its ability to delay Aβ-induced
paralysis significantly when daf-16 of the worms was knockout
(Figure 7).
Meanwhile, we examined whether Exendin-4 affected the
translocation of DAF-16. The transgenic strain TJ356 can
stably express a DAF-16::GFP fusion protein. The indication of
fluorescence showed that Exendin-4 enhanced the translocation
of DAF-16 from the cytoplasm to nuclei. The subcellular
distribution includes “cytosol,” “intermediate,” and “nucleus”
(Figures 8A–C). The amount of DAF-16 in nuclei was improved
obviously by estimating the fluorescence particles using the
ImageJ software (Figure 8D). The ratio of nucleus location was
increased from 15 to 45% compared with control worms.
However, Exendin-4 was still able to improve the mRNA
expression levels of the daf-16 gene (Figure 8E). These findings
indicated that DAF-16 was required for the protective effects of
Exendin-4 on Aβ(1-42) toxicity.
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FIGURE 8
The eect of Exendin-4 on DAF-16. The subcellular distribution of DAF-16 was examined in approximately 20 worms for each group by
fluorescence microscopy. (A) Cytosol, (B) intermediate, (C) nucleus, and (D) the treated worms with 0.5 mg/ml Exendin-4 significantly increase
the ratio of nucleus translocation. The histogram represents the ratio of each subcellular distribution in the whole population in each group.
(E) The mRNA levels of daf-16 were analyzed by real-time PCR. mRNA levels was increased by 69.00% (p<0.001).
Exendin-4 regulates SOD via DAF-16
SOD-3 is a typical downstream target regulated by
DAF-16. To explore whether Exendin-4 regulates SOD via
DAF-16, SOD was detected in the activity and mRNA
levels after knocking down the gene of DAF-16 by RNAi.
We observed that the ability of Exendin-4 to regulate
SOD decreased in CL4176 worms with the DAF-16 gene
silenced. The activity of SOD and mRNA level of sod-
3 was downregulated by 30.45 and 43.13%, respectively
(Figures 9A,B). The CL4176 worms with empty vector L4440
were administrated with or without Exendin-4, and the same
trends as the above results in Figure 6 were displayed due to
the existence of DAF-16. All results indicated that Exendin-
4 regulated the SOD at least partially through a DAF-16-
based mechanism.
Discussion
Epidemiological studies showed that diabetes is a high-
risk factor for Alzheimer’s disease. Common pathophysiological
features exist between T2DM and AD, including oxidative stress
and insulin resistance. It suggests that effective drugs for T2DM
could provide an effective treatment option for AD. Exendin-4 is
a GLP-1R agonist approved to treat T2DM. Recently, Exendin-
4 has shown neuroprotective effects in vitro and in vivo model
systems (Wang et al., 2015, 2016; Qiu et al., 2016; Rocha-Ferreira
et al., 2018). However, the neuroprotective effect of Exendin-4
on AD has never been reported in the C. elegans models.
In this research, we investigated the neuroprotective effects
of Exendin-4 in C. elegans model of AD for the first
time. We found that Exendin-4 alleviated Aβ(1-42) toxicity
via DAF-16.
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Song et al. 10.3389/fnagi.2022.955113
FIGURE 9
Exendin-4 regulates SOD via DAF-16. CL4176 worms with empty vector L4440 as control were treated with or without 0.5 mg/ml Exendin-4.
CL4176 worms were knocked down the daf-16 gene by RNAi and treated with 0.5 mg/ml Exendin-4. (A) The total SOD activity in worms was
investigated using the NBT method. Compared with the control group treated with Exendin-4, the SOD activity was decreased by 30.45% after
knocking down the daf-16 gene (p<0.01). (B) Compared with the control group treated with Exendin-4, the mRNA levels of sod-3 were
decreased by 43.13% after knocking down the daf-16 gene (p<0.05).
We utilized transgenic C. elegans strains CL4176 and
CL2006 induced expressing Aβ(1-42) to imitate the onset
of paralysis. Our results indicated that Exendin-4 attenuated
the process of paralysis and enhanced life span in a dose-
dependent manner.
So far, it has not been reported that Exendin-4 can inhibit
the onset of paralysis. In this research, we clarified that Exendin-
4 possesses the effect of inhibiting paralysis in C. elegans model
of AD for the first time.
However, it has also been reported that Exendin-4 possesses
the ability to increase life span in Huntington’s disease mice
(Martin et al., 2012). We have proved again that Exendin-
4 has the effect of prolonging life span in C. elegans
model of AD.
We hypothesized that exendin-4 can inhibit paralysis rates
or prolong the life of C. elegans due to its ability to attenuate Aβ
(1-42) toxicity. The content of Aβ(1-42) is the critical reason for
its toxicity.
In previous studies, Exendin-4 mainly focuses on the study
of protecting against cascading damage caused by Aβ(1-42),
such as inhibiting cell apoptosis (Qiu et al., 2016) and relieving
oxidative damage (An et al., 2019). Few studies were focused
on how Exendin-4 affects the change of Aβ(1-42) directly. Our
results of Western blotting and qPCR testified that Exendin-
4 attenuated the content of Aβ(1-42) on protein and mRNA
levels directly. Subsequently, Aβ(1-42) aggregation deposits
were reduced.
As we all know, ROS played a negative role in longevity
(Sanz, 2016; Dilberger et al., 2019; Huang et al., 2021) and
paralysis (Tang et al., 2022), and the aggregation of Aβ(1-42)
will lead to the accumulation of ROS, which will affect the onset
of paralysis and life span (Ewald, 2018; Kalmankar et al., 2021).
Our results showed that exendin-4 reduced ROS, which was
beneficial to prolong life and improve paralysis.
We traced the effect of SOD. We found that the SOD was
improved in its activity and mRNA level. It was a positive effect
on decreasing ROS content. This kind of effect is consistent with
previous studies that Exendin-4 upregulated the SOD on the
activity (Zhou et al., 2016) and gene expression in neuronal cells
(Hu et al., 2013; Yasuda et al., 2016).
The antioxidant effect of Exendin-4 has been described in
many oxidative stress models induced by different drugs in
vivo and in vitro. Exendin-4 may inhibit lipotoxicity-induced
oxidative stress in β-cells (Shen et al., 2020). Exendin-4 alleviates
oxidative stress by activating Nrf2/HO-1 in streptozotocin-
induced diabetic mice (Fang et al., 2019). This is the first study
to report that Exendin-4 alleviates the oxidative stress induced
by Aβ(1-42) in C. elegans.
As a known nuclear transcription factor, DAF-16 can induce
transcriptional regulation of many genes involved in aging,
development, and stress. SOD-3, as a downstream target gene,
is regulated by DAF-16. To study whether Exendin-4 regulates
SOD through DAF-16/FOXO, the nuclear translocation of
DAF-16 was detected in transgenic C. elegans TJ356 expressed
DAF-16::GFP. We found that Exendin-4 promoted the nuclear
translocation of DAF-16. The nuclear translocation of DAF-16
contributed to the upregulation of SOD-3, leading to the
antioxidant effect of Exendin-4. In contrast, worms knocked
down daf-16 gene were treated with Exendin-4. We found that
the ability of Exendin-4 to upregulate SOD was reduced.
Meanwhile, Exendin-4 lost the ability to alleviate the process
of paralysis after silencing the daf-16 gene. These results
Frontiers in Aging Neuroscience 10 frontiersin.org
Song et al. 10.3389/fnagi.2022.955113
indicated that DAF-16 played a pivotal role in alleviating Aβ
(1-42) toxicity when Exendin-4 exerted its neuroprotective
effects. It has been reported that Exendin-4 upregulated FoxO
1 expression both in vitro and in vivo (Wang et al., 2017), or
regulated the phosphorylation of FoxO 1 in cardiomyocytes (Eid
et al., 2021). So our results about the important roles of DAF-
16 in mediating the function of exendin-4 are consistent with
previous studies. We can draw a conclusion from the present
data that Exendin-4 has a positive effect on the FoxO family.
Conclusion
Although Exendin-4 was reported that possesses
neuroprotective effects in various AD models, we reported
for the first time that Exendin-4 exhibits the ability to delay
the development of paralysis and prolong the life span in the
C. elegans model of AD. In our research, we elucidated some
neuroprotective mechanisms of Exendin-4 on oxidative stress
and Aβ(1-42) toxicity. The DAF-16 is required for Exendin-4
to display the antioxidant effects and neuroprotective effects.
Taken together, our study testified that Exendin-4 as a marketed
diabetes drug has the potential to be a kind of drug for AD.
Data availability statement
The original contributions presented in the study are
included in the article/Supplementary material, further inquiries
can be directed to the corresponding authors.
Author contributions
XS and XW designed this study. YSun, ZW, and YSu
performed the experiments. YSu and ZW collected and analyzed
the data. XS wrote the manuscript. XW contributed to the
manuscript review. All authors contributed to the article and
approved the submitted version.
Funding
This study was supported by the Scientific and Technological
Developing Scheme of Jilin Province (20190304051YY) and
the Scientific Research Project of Jilin Provincial Department
of Education (JJKH20210879KJ) and the Natural Science
Foundation of Changchun Normal University 2018 (012).
Acknowledgments
We would like to thank Dr. Liping Wang and Dr. Shunwen
Guan for the C. elegans strains.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those
of the authors and do not necessarily represent those of
their affiliated organizations, or those of the publisher,
the editors and the reviewers. Any product that may
be evaluated in this article, or claim that may be made
by its manufacturer, is not guaranteed or endorsed
by the publisher.
Supplementary material
The Supplementary Material for this article can be
found online at: https://www.frontiersin.org/articles/10.3389/
fnagi.2022.955113/full#supplementary-material
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