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A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye

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Alzheimer's disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human Aβ42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer's like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Aβ42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.
Lunasin downregulates JNK signaling to block Aβ42 mediated neurodegeneration. (A) Schematic presentation of JNK signaling pathway. (B) Levels of phospho-JNK (pJNK) in a semi-quantitative Western Blot can provide the status of JNK signaling. The higher levels of JNK signaling in GMR > Aβ42 as compared to the wild-type background were significantly downregulated in GMR > Aβ42 + Lun background. The tubulin bands served as controls to normalize the levels of total protein loaded in all three conditions. The p-JNK band staining intensity was calculated by ImageJ. In comparison to the wild-type (C) eye imaginal discs and (D) adult eyes, activation of JNK signaling in GMR domain using (E,F) Djun aspv7 (GMR > jun) and (K,L) constitutively active hep Act (GMR > hep) result in strong neurodegenerative phenotype. Furthermore, activation of JNK signaling in GMR > Aβ42 background (G,H) GMR > Aβ42 + jun (M,N) GMR > Aβ42 + hep exhibits stronger neurodegenerative phenotype which are not rescued by misexpression of Lunasin (I,J) GMR > Aβ42 + Lun + jun and (O,P) GMR > Aβ42 + Lun + hep. (Q,R) Downregulation of JNK signaling by misexpression of puc, a dual phosphatase, results in near wild-type (Q) eye imaginal discs and (R) adult eyes. (S-V) Misexpression of puc in (S,T) GMR > Aβ42 (GMR > Aβ42 + puc) and (U,V) GMR > Aβ42 + Lun (GMR > Aβ42 + Lun + puc) results in significant rescue as seen in eye imaginal discs and the adult eyes. The puc-lacZ reporter is used as a functional read out of JNK signaling pathway. Expression of puc-lacZ reporter (Green) in (W,W') Wild-type, (X,X') GMR > Aβ42 and (Y,Y') GMR > Aβ42 + Lun eye imaginal discs. (W,W') Note that puc has weak expression in the developing photoreceptor neurons in the wild-type eye imaginal discs. However, (X,X') puc expression is dramatically upregulated in GMR > Aβ42 background. (Y,Y') Misexpression of Lunasin (Lun) along with Aβ42 (GMR > Aβ42 + Lun) can significantly downregulate puc expression in the developing third instar eye disc. Magnification of all eye discs is 20X.
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SCiENTifiC RePoRtS | (2018) 8:13545 | DOI:10.1038/s41598-018-31787-7
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A soy protein Lunasin can
ameliorate amyloid-beta 42
mediated neurodegeneration in
Drosophila eye
Ankita Sarkar1, Neha Gogia1, Neil Glenn2, Aditi Singh1, Gillian Jones6, Nathan Powers6,
Ajay Srivastava6, Madhuri Kango-Singh1,2,3,4 & Amit Singh
1,2,3,4,5
Alzheimer’s disease (AD), a fatal progressive neurodegenerative disorder, also results from
accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal
signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of
human Aβ42 in the dierentiating retinal neurons of the Drosophila eye, which results in the Alzheimer’s
like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a
possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer
eect and reduces stress and inammation. We show that misexpression of Lunasin by transgenic
approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and
results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates
the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can
prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective
function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and
is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin
can signicantly reduce mortality rate caused by misexpression of human Aβ42 in ies. Our studies
identied the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can
ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.
Alzheimer’s Disease (AD; OMIM: 104300), an irreversible, progressive neurodegenerative disorder, which results
in the loss of neurons in the hippocampus and cortex. AD manifests as the loss of memory, cognition func-
tions and eventually results in the death of patient14. e two major causes for AD are accumulation of amyloid
plaques and generation of neurobrillary tangles (NFTs) due to hyper-phosphorylation of microtubule binding
protein Tau1,47. e amyloid plaques are generated from improper cleavage of amyloid precursor protein (APP),
a transmembrane protein, by β- and then γ-secretase enzymes to form a 42 amino-acid long fragment, which
is referred to as amyloid-beta 42 (Aβ42)8. ese Aβ42 brils self-assemble into extracellular Aβ42 plaques2,9.
Generally, APP cleavage results in a forty amino acid long polypeptide (Aβ40). e two extra amino acids in
Aβ42 polypeptide makes it hydrophobic, which results in accumulation of the amyloid plaques47,10,11. ese
Aβ42 plaques cause membrane defects, disruption of neural networks, trigger aberrant signaling and disrupt
normal cellular processes resulting in neurodegeneration. us, the current consensus is that Aβ42 conver-
sion and self-assembly into oligomeric forms and plaques is responsible for neuronal death in AD by unknown
molecular-genetic mechanism(s)27,10,12. Various animal models of AD have been developed to understand the
molecular-genetic basis of AD13 as the genetic machinery is highly conserved across organisms including mouse,
C. elegans and fruit y4,1323.
1Department of Biology, University of Dayton, Dayton, OH, 45469, USA. 2Premedical Program, University of Dayton,
Dayton, OH, 45469, USA. 3Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton,
Dayton, OH, 45469, USA. 4The Integrative Science and Engineering Center, University of Dayton, Dayton, OH,
45469, USA. 5Center for Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN, USA. 6Department
of Biology and Biotechnology Center, Western Kentucky University, 1906 College Heights Boulevard, TCCW 351,
Bowling Green, KY, 42101, USA. Ankita Sarkar, Neha Gogia and Neil Glenn contributed equally. Correspondence and
requests for materials should be addressed to Amit Singh (email: asingh1@udayton.edu)
Received: 3 October 2017
Accepted: 24 August 2018
Published: xx xx xxxx
OPEN
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SCiENTifiC RePoRtS | (2018) 8:13545 | DOI:10.1038/s41598-018-31787-7
Drosophila melanogaster, fruit fly, is a highly versatile organism to model human disease13,14,16,22,23. The
Drosophila eye is used extensively to model human neurodegenerative disorders6,13,15,16,20,24,25 because the
important signaling pathways required for development and dierentiation of the y visual system are highly
conserved26. us, the information generated from the y model can help understand molecular-genetic under-
pinning of the human disease13,14,17,19,27. Drosophila, a holometabolous insect, has a blue print for its adult organs
housed inside the larva referred to as the imaginal discs28,29. e larval eye-antennal imaginal disc gives rise to the
adult compound eye, antenna and head upon dierentiation13,26,3032. In the developing Drosophila eye, the cell
fate specication and dierentiation is regulated by a group of genes like twin of eyeless (toy), eyeless (ey), eyegone
(eyg), twin of eyegone (toe), Optix (opt); eyes absent (eya), sine oculis (so), dachshund (dac) and optix (opt)3339,
which are called retinal determination (RD) genes4045.
e retinal precursor cells in the eye imaginal disc undergo dierentiation to form the photoreceptor neurons
in the adult eye26,4648. Eight photoreceptor neurons (PR1-8) and several support cells form a unit eye called as
the ommatidium. e axons from the photoreceptors (retinal neurons) fasciculate together to form an axonal
bundle, which traverses through the optic stalk and then innervate the dierent layers of the Drosophila brain49,50.
e axons from photoreceptors (PRs) 1–6 terminate in the lamina whereas PR7-PR8 end in a separate layer of
medulla aer passing through lamina. In the pupal retina, the excessive cells other than the dierentiated cells are
eliminated by programmed cell death (PCD)51. However, abnormal extracellular signaling due to inappropriate
levels of morphogens may trigger cell death in the larval eye imaginal disc52,53.
Previous work from our lab showed that evolutionarily conserved Wingless (Wg) and Jun-N terminal kinase
(JNK) signaling pathway are tightly regulated to allow dierentiation to occur and to prevent premature cell death
in the developing elds54. Wg, a member of highly conserved Wnt/Wg, is responsible for regulating early growth,
restricting eye fate and later Wg plays a role in triggering programmed cell death (PCD)55,56 in the pupal retina.
Wg also plays a role in developmental cell death during larval eye development53. Another highly conserved TGF
beta (TGFβ) signaling pathway, referred to as Decapentaplegic (Dpp) signaling in Drosophila5759, collaborates
with Hedgehog (Hh) signaling to promote retinal dierentiation in the developing eye as well as antagonize Wg
signaling60.
Activation of JNK signaling or stress activated kinase proteins of the mitogen-activated protein kinase
(MAPK) superfamily trigger cell death27,6163. JNK signaling is activated through a cascade of phosphorylation by
MAP Kinases to regulate cell homeostasis61,6466. JNK signaling acts downstream of the Tumor Necrosis Factor
(TNF) homolog Eiger (Egr) and its receptor Wengen (Wgn) by Tak1 (TGF-β- activating kinase 1), a JNK kinase
kinase (JNKKK), Hemipterous (Hep); a JNK Kinase, Basket (Bsk; Jun kinase) and Jun6365,67. It is known that
activation of JNK signaling leads to induction of cell death to eliminate developmentally aberrant cells63,67. e
functional read out for the activation of JNK signaling is puckered (puc), which encodes a dual phosphatase, and
acts via a negative feedback loop to downregulate the JNK activity27,63,66.
We developed a transgenic model system in Drosophila eye where we misexpress high levels of human
amyloid-beta (Aβ42) in the dierentiating retinal neurons of the developing y retina27 using a Glass Multiple
Repeat (GMR) Gal4 driver68. e transgenic ies with GMR-Gal4 driven UAS-Aβ42 have been abbreviated as
GMR > Aβ4227. Targeted misexpression of human Aβ42 (GMR > Aβ42) in the dierentiating photoreceptors
(retinal neurons) of the developing Drosophila eye69, exhibit progressive neurodegenerative phenotypes that
mimic the neuropathology of AD patients27. e frequency of this GMR > Aβ42 phenotype is 100%, which makes
this Drosophila eye model a highly reliable tool for identifying the genetic modiers of the GMR > Aβ42 mediated
neurodegeneration27,70,71. We used our AD model to test plant-based protein Lunasin for its role in blocking Aβ42
mediated neurodegeneration. Lunasin, a soy (glycine max) derived peptide, has multiple roles72. Lunasin protein
has four functional domains7274. It has an N terminal region of unknown function, followed by a chromatin
binding helical region, a carboxy terminal RGD cell adhesion motif, and an eight aspartic acid (poly-D) tail.
e poly-D tail and RGD motif have been shown to be essential for the bioactivity of Lunasin. Lunasin is known
to have anti-cancer eects74,75 and reduces stress and inammation. e odds of manifestation of AD increases
with chronic low-grade inammation like stress, depression, and obesity76. Here we present identication of
a plant protein Lunasin that can rescue Aβ42 mediated neurodegeneration. is neuroprotective function of
Lunasin is achieved by downregulating JNK signaling dependent cell death in the developing retinal neurons
of the Drosophila eye. Furthermore, gain-of-function of Lunasin can also reduce the mortality rate of the ies
expressing Aβ42 in the nervous system.
Materials and Methods
Fly Stocks. All y stocks used in this study are listed and described in Flybase (http://ybase.bio.indiana.edu).
e y stocks used in this study were Canton-S (Wild-type), GMR-Gal468, elav-Gal4 (BL#485)77, UAS-Aβ4227,
UAS-puc, pucE69, where lacZ reporter express under the control of puc regulatory element, and acts as the func-
tional read out of JNK signaling pathway66. Other stocks used were UAS-Djunaspv7 78, UAS-hepAct, wg-lacZ79,
dpp-lacZ80, where lacZ reporter81 express under the control of wg and dpp regulatory element. e UAS- Aβ42
transgenic ies were generated by microinjecting a UAS-construct where two tandem copies of human amyloid -
β1-42 (Aβ42) fused to signal peptide for secretion were cloned10,25,27. e rationale of bi-cistronic construct was
to mimic APP duplications associated with early onset of familial AD and to express high levels of Aβ42 to induce
strong eye phenotype25,82.
Generation of EGFP-Lunasin Transgenic flies. We employed gene tagging approach83 to generate
EGFP-Lunasin transgenic ies. e sequence for EGFP (Enhanced Green Fluorescent Protein) was fused to the 5
end of Lunasin sequence84. It has been shown that Lunasin tagged with EGFP show no observable dierences in
bioactivity73. e sequence of EGFP-Lunasin with start, stop codons and the restriction sites was synthesized in
vitro, sequence veried and cloned into pUAST vector. e GFP reporter can provide spatio-temporal localization
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SCiENTifiC RePoRtS | (2018) 8:13545 | DOI:10.1038/s41598-018-31787-7
of Lunasin transgene. e clones were sequence veried and microinjected in Drosophila embryos and the trans-
genic ies were generated. ese ies were balanced and used for genetic crosses.
Genetic Crosses. We employed a Gal4/UAS system for targeted misexpression studies69. All Gal4/UAS
crosses were maintained at 18 °C, 25 °C and 29 °C, unless specied, to sample dierent induction levels. e
adult ies were maintained at 25 °C, while the cultures aer egg laying (progeny) were transferred to 29 °C for
further growth. Misexpression of Aβ42 in the dierentiating retina (GMR-Gal4 > UAS-Aβ42) exhibits a stronger
neurodegenerative phenotype at 29 °C with no penetrance27,68. All the targeted misexpression experiments were
conducted using the Glass Multiple Repeat driver line (GMR-Gal4)68 or embryonic lethal abnormal visual system
(elav-GAL4) line77. GMR- Gal4 directs expression of transgenes in the dierentiating retinal precursor cells of the
developing eye imaginal disc and pupal retina68. e elav-Gal4 drives expression in the neurons77.
Immunohistochemistry. Eye-antennal discs from wandering third instar larvae were dissected, and xed
in 4% paraformaldehyde in Phosphate Buered Saline (PBS), and stained following the protocol8587. e pri-
mary antibodies used were rabbit anti-Dlg (1:200; a gi from K. Cho), mouse anti-Wg [1:50,Developmental
Studies Hybridoma Bank,(DSHB)], rat anti-Elav (1:50; DSHB), mouse anti-Dlg (1:100; DSHB), mouse anti-
22C10 (1:100; DSHB), mouse anti-Chaoptin (MAb24B10) (1:100; DSHB88), mouse anti-Ey (1:100, DSHB),
mouse anti-Eya (1:100, DSHB), mouse anti-Dac (1:100, DSHB), mouse anti-β-galactosidase (1:100; DSHB),
rabbit anti-β-galactosidase (1:200) (Cappel), and mouse anti-GFP (1:100, GFP-G1, DSHB). Secondary anti-
bodies (Jackson Laboratories) used consisted of donkey anti-rabbit IgG conjugated with FITC (1:200), donkey
anti-mouse IgG conjugated with Cy3 (1:250), and goat anti-rat IgG conjugated with Cy5 (1:250). e tissues
were mounted in Vectashield (Vector labs) and all immunouorescence images were captured using the Laser
Scanning Confocal Microscopy89 (Olympus Fluoview 1000). All images were taken at 20X magnication unless
stated otherwise. e nal images and gures were prepared using Adobe Photoshop CS6 soware.
Detection of Cell Death. Cell death was detected using TUNEL assays27,53,54,90,91. TUNEL assays were
used to identify the cells undergoing cell death where the cleavage of double and single stranded DNA is
labeled by a Fluorescent tag (TMR Red). e uorescently labeled nucleotides are added to 3 OH ends in a
template-independent manner by Terminal Deoxynucleotidyl Transferase (TdT). e uorescent label tagged
fragmented DNA within a dying cell can be detected by uorescence or confocal microscopy89. Eye-antennal
discs after secondary antibody staining were blocked in 10% normal donkey serum in phosphate buffered
saline with 0.2% Triton X-100 (PBT) and labeled for TUNEL assays using a cell death detection kit from Roche
Diagnostics(In Situ Cell Death Detection Kit, TMR red,12156792210).
e TUNEL positive nuclei were counted to determine the dying cell population from ve sets of imagi-
nal discs and were used for statistical analysis using Microso Excel 2013. e P-values were calculated using
two-tailed t-test and the error bars represent Standard Deviation from Mean27,70,71,92.
Adult Eye Imaging. Adult ies were prepared for imaging by freezing at 20 °C for approximately 2 hours
followed by mounting the y on a dissection needle54,86. e needle with the y was aligned horizontally over a
glass slide using molding putty. Images were captured on an MrC5 color camera mounted on an Axioimager.Z1
Zeiss Apotome using Z-sectioning approach. Final images were generated by compiling the individual stacks
from the Z-sectioning approach using the extended depth of focus function of Axiovision soware version 4.6.3.
Western Blot. Protein sample were prepared from third instar eye imaginal disc from Wild type,
GMR > Aβ42, GMR > Aβ42 + Lun larvae following the standardized protocol27,93. e Phospho SAPK/JNK
(Cell Signaling r183/Tyr185) (81E11) Rabbit antibody was used at 1:1000 dilution. Signal was detected using
Horse Radish Peroxidase(HRP) conjugated goat anti–rabbit IgG using supersignal chemiluminescence substrate
(Pierce). Images were captured using the BioSpectrum® 500 Imaging System.
Eclosure Assay. Eclosure assays are desirable assays to screen the eect of genetic backgrounds on eclosion of
ies77. We collected eggs on a grape plate from elav-Gal4 (control), elav-Gal4 drive UAS- Aβ42 (elav-Gal4 > Aβ42)
and elav-Gal4 drive UAS-Aβ42 + UAS-Lunasin (elav-Gal4 > Ab42 + Lun) ies. e eclosion assay was carried out
in four sets of 50 larvae each. We seeded rst instar larvae (50 in each set) from a synchronous culture in each vial.
We counted 200 larvae (4 sets of 50 larvae) for each cross. e larvae were allowed to develop and hatched/eclosed
adults were counted. All unhatched pupae were also counted. All four sets of y larvae each for every genotype
were seeded from the same grape plate to maintain consistency and accuracy in larval staging.
Results
Lunasin can rescue Aβ42 mediated neurodegeneration. In comparison to the wild-type eye imaginal
discs (Fig.1A) that develop into adult compound eyes (Fig.1F), targeted misexpression of Aβ42 in the develop-
ing Drosophila eye using the GMR-Gal4 enhancer results in a strong neurodegenerative phenotype in the eye
imaginal disc (Fig.1D), and the adult eye (Fig.1I)27. e neurodegenerative phenotype in the GMR > Aβ42 eye
imaginal disc (Fig.1D) worsens in the adult eye (Fig.1I). e adult eyes are highly reduced with glazed appear-
ance due to fusion of individual unit eyes and also exhibits some dark necrotic spots to mark neurodegeneration
(Fig.1I)27,70,71,92. e frequency of GMR > Aβ42 phenotype in the eye imaginal discs (Fig.1D, n = 72) as well as
adult eyes (Fig.1I, n = 151) is 100%. e controls GMR-Gal4 alone (n = 75, 100%, Fig.1B,G) and the transgene
stock UAS-Aβ42 alone (n = 75, 100%, Fig.1C,H) exhibits near wild-type eye phenotype in the eye imaginal
discs and the adult eyes. e gene tagging with standardized immune-epitopes or uorescent tags that permit
live imaging and do away with the requirement of generating antibodies against a protein is commonly used
approach83. We employed gene-tagging approach to generate UAS- based transgenic ies where the Soy plant
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SCiENTifiC RePoRtS | (2018) 8:13545 | DOI:10.1038/s41598-018-31787-7
based peptide Lunasin (Lun) is tagged with EGFP (UAS-Lun-EGFP). Misexpression of UAS-Lun-EGFP along
with Aβ42 (GMR > Aβ42 + Lun-EGFP) exhibits a strong rescue (n = 100, 70%) resulting in a near complete wild-
type eye (Fig.1E,J). ere were no necrotic spots observed in the adult eyes (Fig.1J). We conrmed that rescue
of GMR > Aβ42 neurodegenerative phenotype is due to misexpression of Lunasin based on EGFP transgene
expression in the GMR domain of the eye imaginal discs (Fig.1M) and the adult eyes (Fig.1N). A strong robust
GFP expression was detected by GFP antibody staining in the GMR domain of the GMR > Aβ42 + Lun-EGFP
eye imaginal discs (Fig.1M) and GFP reporter expression was seen in the adult eyes (Fig.1N). e GFP protein
was not detected in GMR > Aβ42 eye imaginal discs (Fig.1K), and, no GFP reporter expression was seen in the
adult eyes (Fig.1L). We also veried the data using a UAS-Lun construct which is not tagged with EGFP (data not
shown). ese data suggest that misexpression of Lunasin can rescue GMR > Aβ42 mediated neurodegeneration
likely by blocking cell death.
Lunasin can rescue Aβ42 mediated cell death in Drosophila eye. To test this, we employed the
TUNEL staining, which marks the dying cell nuclei by labelling the 5 end of the double and single stranded
DNA with a uorochrome. e uorochrome tagged TUNEL positive nuclei can be counted to quantify cell
death27,90,91,94. We performed TUNEL staining in the third instar eye-imaginal disc in the wild-type (Fig.2A,A),
GMR > Aβ42 (Fig.2B,B’) and GMR > Aβ42 + Lun (Fig.2C,C’) backgrounds. e TUNEL staining was per-
formed before the onset of developmentally controlled programmed cell death. e TUNEL positive cells were
counted from ve sets of imaginal discs from all three backgrounds, and the P values were calculated27,70,71,92. A
quantication (n = 5, p < 0.05) of dying cells from these genotypes further conrms that in comparison to the
wild-type eye disc, a 3–4 fold increase in cell death was observed in GMR > Aβ42 (Fig.2B,B’,D) background. In
Figure 1. Misexpression of the soy protein Lunasin can rescue Aβ42 mediated neurodegeneration. (A,F)
Wild-type (A) larval eye imaginal discs, which develop into (F) adult compound eyes comprising of nearly 800
unit eyes. Note that the eye imaginal disc is stained with membrane specic marker Disc large (Dlg: Green)
and pan neural marker Embryonic Lethal Abnormal Vision (Elav: Red), which marks nuclei of the retinal
neuron. In comparison to the wild-type eyes, (D,I) misexpression of Aβ42 (GMR > Aβ42) in the dierentiating
retinal neurons using GMR-Gal4 driver results in the induction of neuronal death as seen in (D) eye imaginal
discs and the (I) highly reduced adult eyes. Note that phenotype worsens from (D) larval eye imaginal disc
to the (I) the adult eye. e controls used are (B,G) GMR-Gal4 and (C,H) transgene stock UAS-Aβ42. (E,J)
Misexpression of soy polypeptide Lunasin (Lun) along with Aβ42 (GMR > Aβ42 + Lun) results in signicant
rescue of Aβ42 mediated neurodegeneration as seen in (E) the eye discs and (J) the adult eyes. Since Lunasin
is a plant protein, in order to determine if Lunasin (Lun) is actually expressed in our model system it has been
tagged with EGFP (as seen in E,J). Note that GFP reporter is detected in GMR domain of (E) eye imaginal discs
and (J) adult eyes. (K,M) Expression of Lunasin detected by GFP antibody staining in (K) control GMR > Aβ42
and (M) GMR > Aβ42 + Lun. Note that GFP antibody positively marks the GMR domain only in the (M)
GMR > Aβ42 + Lun eye disc. In adult eyes, GFP expression is detected by GFP reporter expression in control
(L) GMR > Aβ42 and (N) GMR > Aβ42 + Lun. e orientation of all imaginal discs in the gure is posterior to
le and dorsal up. Magnication of all eye discs is 20X.
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SCiENTifiC RePoRtS | (2018) 8:13545 | DOI:10.1038/s41598-018-31787-7
comparison to the GMR > Aβ42 (Fig.2B,B’,D) background, the number of TUNEL positive dying nuclei were
signicantly reduced in GMR > A β42 + Lun (Fig.2C,C’,D). It suggests that expression of Lunasin is capable of
inhibiting cell death as seen by the strong rescue phenotype in the adult eyes.
We wanted to test if Lunasin can rescue GMR > A β42 phenotype by preventing accumulation of amyloid
plaques or act downstream of amyloid plaque formation in the GMR > A β42 + Lun background. Using 6E10
antibody to mark Aβ42 plaques, we found that there is a little or no Aβ42 present in the wild-type (Fig.2E,H,
n = 50, 100%) eye discs. However, there is strong deposition of Aβ42 plaques in GMR > Aβ42 (Fig.2F,H, n = 52,
100%) and GMR > Aβ42 + Lun (Fig.2G,H, n = 46, 100%) eye discs. Furthermore, there is no signicant dier-
ence between Aβ42 plaque formation in GMR > Aβ42 and GMR > Aβ42 + Lun (Fig.2H) background. It suggests
that Lunasin misexpression does not aect Aβ42 plaques formation and acts downstream to GMR > Aβ42 plaque
formation.
Lunasin can restore axonal targeting defects seen in Aβ42 background. The neurodegen-
erative phenotype in AD encompasses disruption of axonal transport mechanism, which results in impaired
axonal targeting (incorrect axonal guidance)27,70,71,92. In order to understand, if these GMR > Aβ42 + Lun
imaginal discs where neurodegeneration phenotype is rescued have proper connection between retinal neu-
rons and brain, we employed 24B10 (Chaoptin, which marks photoreceptor neurons and their axons)88. In the
wild-type eye disc, R1-R6 axons of each ommatidium project to the lamina whereas R7 and R8 axons project
to the medulla, a separate layer of the optic lobe95 (Fig.3A) in (n = 51)100% eye imaginal discs. In comparison
to the wild-type eye imaginal discs, the GMR > Aβ42 eye discs show a severe disorganization in axonal target-
ing (Fig.3B) in (n = 47)100% larval eye imaginal discs. However, misexpression of Lunasin in GMR > Aβ42
(GMR > Aβ42 + Lun) background signicantly restore the axonal targeting (Fig.3C) in (n = 50) 70% of lar-
val eye imaginal discs. We also investigated the neurons and their axonal processes using 22C10 marker96. In
comparison to the wild-type expression of 22C10, that marks the retinal neurons and their processes (Fig.3D,
n = 50,100% eye discs), GMR > Aβ42 background show strong neurodegenerative phenotype (reduced neurons
Figure 2. Lunasin can block cell death to rescue Aβ42 mediated neurodegeneration. e dying cells nuclei
can be marked by TUNEL staining. TUNEL staining was carried out in (A,A’) Wild-type, (B,B’) GMR > Aβ42,
(C,C’) GMR > Aβ42 + Lun eye imaginal discs. e number of dying retinal neurons were counted in these
backgrounds (n = 5). Note that the number of dying cells increase nearly 3–4 fold in (B,D) GMR > Aβ42 as
compared to the (A,D) Wild-type eye discs. (C,D) Misexpression of Lunasin (Lun) along with GMR > Aβ42
(GMR > Aβ42 + Lun-GFP) results in signicant reduction in the dying retinal neurons. e number of
TUNEL positive nuclei were counted from ve eye imaginal discs for all three backgrounds. (D) A graph
comparing the number of dying nuclei of neurons validate that Lun misexpression along with GMR > Aβ42
(GMR > Aβ42 + Lun-GFP) rescues the GMR > Aβ42 neurodegeneration. ese numbers are signicant based
on the calculations of P-values using the two-tailed t- test using Microso Excel 2013. (EH) Accumulation
of amyloid plaque was detected using monoclonal antibody 6E10 in (E) Wild-type, (F) GMR > Aβ42 and (G)
GMR > Aβ42 + Lun eye imaginal discs. (H) e signal intensity of 6E10 staining was calculated from ve
(n = 5) eye discs of each background and plotted on a graph. Note that 6E10 levels are not signicantly dierent
between (F) GMR > Aβ42 and (G) GMR > Aβ42 + Lun background. e levels of amyloid plaques are barely
detected in Wild-type background. Magnication of all eye disc is 20X.
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and abnormality in their processes) (Fig.3E, n = 50, 100% eye discs). However, misexpression of Lunasin in
GMR > Aβ42 (GMR > Aβ42 + Lun) background signicantly restore the neurodegenerative phenotype (Fig.3F,
n = 50, 80%) as compared to the GMR > Aβ42 (Fig.3E, n = 50,100%) eye discs.
Neuroprotective function of Lunasin is independent of retinal dierentiation genes. Since our
experimental system is Drosophila eye, we need to rule out the possibility of Lunasin aecting the retinal dier-
entiation gene machinery rather than exhibiting the neuroprotective function. We investigated the role of RD
genes in neuroprotective function of Lunasin. e Pax-6 homolog eyeless (ey), eye fate selector gene, is expressed
in the early eye and later its expression is restricted anterior to the MF in the eye imaginal disc (Fig.4A, n = 50,
100%)30,44,85,97,98. A tyrosine phosphatase, eyes absent (eya), is expressed both in the dierentiated retinal neurons
as well as the retinal precursor cells anterior to MF30,31,33,70 (Fig.4B, n = 50, 100%). Another RD gene dachshund
(dac) is expressed in two dierent domains one anterior to the MF and another posterior to the MF70,99 (Fig.4C,
n = 50, 100%). In GMR > Aβ42 + Lun background the expression of all three RD genes Ey (n = 50, 100%), Eya
(n = 50, 100%) and Dac (n = 50, 100%) was not aected (Fig.4D–F, n = 50, 100%). Our data strongly suggests
that the neuroprotective role of Lunasin is independent of RD gene function. In our transgenic model, the Aβ42
expression is triggered at the time of retinal dierentiation using a GMR-Gal4 driver27,68, which drives expression
of UAS- transgene much later than the event of eye specication, and the onset of retinal determination and
dierentiation genes expression. us, neuroprotective function of Lunasin with respect to Aβ42 mediated neu-
rodegeneration is independent of eye specication function or retinal dierentiation machinery.
Lunasin rescues Aβ42 mediated neurodegeneration independent of Wingless (Wg) or
Decapentaplegic (Dpp) signaling. Previous studies from our lab and others have shown that accumu-
lation of Aβ42 plaques (GMR > Aβ42) triggers aberrant signaling which results in neurodegeneration4,27,70,71,92.
We therefore tested (i) several signaling pathways to discern the mechanism of Lunasin mediated neuroprotec-
tive function, and (ii) known genetic modiers of Aβ42 mediated neurodegeneration27,70,71,92 (data not shown).
e evolutionarily conserved Wg signaling act antagonistically to Dpp during eye development60. Wg acts as
the negative regulator of eye fate. Gain-of-function of wg can suppress the eye fate and loss of function of wg
induces eye enlargements44,58,59,85. In the developing Drosophila eye, Wg functions antagonistically to Dpp that
promote cell survival. Dpp is also involved in retinal dierentiation60. During eye development, Wg is expressed
Figure 3. Lunasin (Lun) misexpression can restore Aβ42 mediated impairment of axonal targeting from
retina to brain. Chaoptin (MAb24B10), a marker for the axonal targeting from retina to the optic lobes of
the brain. (A) In wild-type eye imaginal discs, the retinal axons marked by MAb24B10 innervate the lamina
and medulla of the brain. (B) Misexpression of GMR > Aβ42 in the developing eye imaginal discs result in
impaired targeting of retinal axons to the brain. However, (C) misexpression of Lunasin (Lun) along with
Aβ42 (GMR > Aβ42 + Lun) resulted in signicant restoration of the axonal targeting to near wild-type.
Monoclonal antibody 22C10 marks all axonal sheath of the photoreceptors in the developing eye. 22C10
expression in (D) Wild-type, (E) GMR > Aβ42 and (F) GMR > Aβ42 + Lun background. Note that impairment
of 22C10 expression in GMR > Aβ42 background is restored signicantly in GMR > Aβ42 + Lun background.
Magnication of all eye discs is 20X.
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Figure 4. Lunasin neuroprotective function is independent of retinal dierentiation gene machinery, Dpp
and Wg signaling pathways. (AC) Wild-type expression of (A) Eyeless (Ey), (B) Eyes absent (Eya) and
(C) Dachshund (Dac), the members of RD gene machinery in developing third instar eye imaginal discs.
(A) Ey expression is anterior to MF in the third instar eye discs. (B) Eya is expressed in the dierentiating
photoreceptors and anterior to MF. (C) Dac is expressed along the MF as well as in antennal region. (D) Ey, (E)
Eya and (F) Dac expression is not aected in the GMR > Aβ42 + Lun background. (GI) In the developing eye
imaginal discs, study of dpp expression using dpp-lacZ reporter in the (G) Wild-type, (H) GMR > Aβ42, (I)
GMR > Aβ42 + Lun. Note that dpp-lacZ marks the morphogenetic furrow (MF) in the developing eye. (JL)
In the developing eye imaginal discs, study of Wg expression using a wg-lacZ reporter in the (J) Wild-type, (K)
GMR > Aβ42, (L) GMR > Aβ42 + Lun. e wg is expressed on the antero-lateral margin of the developing third
instar eye imaginal discs. Note that the Lun neuroprotection function is independent of Wg and Dpp signaling.
Magnication of all eye discs is 20X.
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on the antero-dorso-ventral eye margin (Fig.4J, n = 50, 100%) where as Dpp is expressed dynamically along with
the MF (Fig.4G, n = 50, 100%). We found that Lunasin misexpression in GMR > Aβ42 (GMR > Aβ42 + Lun)
background does not aect Wg (Fig.4L, n = 50, 100%) and/or Dpp (Fig.4I, n = 50, 100%) expression in the eye
imaginal discs. us, neuroprotective function of Lunasin with respect to Aβ42 mediated neurodegeneration is
independent of Wg and Dpp Signaling pathways.
Lunasin downregulates JNK signaling to prevent neurodegeneration. We have shown earlier that
JNK signaling is involved in Aβ42 mediated neurodegeneration27. Activation of JNK signaling triggers a cascade
of kinases, which in turn regulates the expression of puc61,63,66,100. Puc is a dual phosphatase that negatively reg-
ulates JNK signaling by a feedback loop27,63 (Fig.5A). e phospho-Jun kinase, encodes an enzyme which can
phosphorylate N-terminal its substrate Jun and can be used to study the activation status of JNK signaling. We
tested levels of JNK activation by quantifying levels of phospho-JNK in western blots27. We quantied and com-
pared the amount of phospho-Jun kinase (p-JNK) in wild-type versus GMR > Aβ42 and GMR > Aβ42 + Lu n
background. In comparison to the wild-type, p-JNK levels are upregulated in GMR > Aβ42 background as seen
earlier27. However, in comparison to GMR > Aβ42, pJNK levels were signicantly reduced in GMR > Aβ42 + Lun
background (Fig.5B,B).
We tested if JNK signaling is downregulated by Lunasin (Lun) misexpression. To activate JNK signaling,
we misexpressed Djunaspv7 and constitutively active hemipterous (hepAct) using the GMR-Gal4 driver and assay
its phenotype in the eye imaginal disc and the adult eye (Fig.5E,F,K,L). In comparison to the wild-type eye
(Fig.5C,D), misexpression of Djunaspv7 alone (GMR > Djunaspv7) results in the reduced eye phenotype (Fig.5E,F,
n = 50, 100%). The strong neurodegenerative phenotype of highly reduced eye in GMR > Aβ42 + Djunaspv7
(Fig.5G,H, n = 50, 100%) was not rescued in GMR > Aβ42 + Lun + Djunaspv7 (Fig.5I,J, n = 50, 100%) back-
ground. Similarly the strong neurodegenerative phenotype due to activation of JNK pathway by using hepAct,
as seen in GMR > hepAct (Fig.5K,L, n = 75, 100%), GMR > Aβ42 + hepAct (Fig.5M,N, n = 75, 100%) was not
rescued by misexpression of Lunasin (GMR > Aβ42 + Lun + hepAct, Fig.5O,P, n = 50, 100%). However, blocking
or downregulating JNK signaling by misexpression of puc in GMR > puc exhibits near wild-type eye (Fig.5Q,R,
n = 50, 100%). Misexpression of puc in GMR > Aβ42 + puc, exhibits signicant rescue of GMR > Aβ42 neurode-
generative phenotype (Fig.5S,T, n = 50, 45%). Furthermore, puc expression in GMR > Aβ42 + Lun background
(GMR > Aβ42 + Lun + puc) can signicantly rescue Aβ42 mediated neurodegeneration as seen in the eye imag-
inal disc and the adult eye (Fig.5U,V, n = 50, 40%). us, Lunasin, which acts upstream of JNK signaling, may
downregulate JNK signaling in rescuing Aβ42 mediated neurodegeneration in the Drosophila eye.
To test this hypothesis, we analyzed the expression of puc (Fig.5W,W’), a functional read out of JNK signaling
pathway by using a puc-lacZ reporter66,101. ere is a robust induction of puc-lacZ in GMR > Aβ42 (Fig.5X,X’,
n = 30, 100%) as compared to the wild-type puc expression in eye imaginal discs (Fig.5W,W’, n = 30, 100%).
However, in GMR > Aβ42 + Lun, puc levels are signicantly downregulated (Fig.5Y,Y’, n = 30, 70%) as compared
to GMR > Aβ42 background (Fig.5X,X’). Our data clearly validate our hypothesis that Lunasin downregulates
JNK signaling to rescue Aβ42 mediated neurodegeneration.
Lunasin increase the mortality of Aβ42 expressing ies. To rule out the possibility that these studies
are not restricted only to the retinal neurons, we employed elav-Gal4 that drives expression in the neurons of
ies77. We misexpressed Aβ42 using elav-Gal4 (elav > Aβ42), which resulted in reduced mortality rate as only
50% (n = 200) of the ies could hatch out and survive whereas remaining 50% population were arrested as lar-
vae or pupae. However, all wild-type ies hatched out and did not show any lethality (Fig.6, n = 200, 100%).
We also analyzed mortality rate when Lunasin is misexpressed in elav > Aβ42 (elav > Aβ42 + Lun) background.
Misexpression of Lun signicantly reduced the mortality rate of elav > Aβ42 background (Fig.6, n = 200, 80%).
Nearly 80% of the ies hatched out and only 20% ies failed to hatch out due to pupal and larval lethality.
Discussion
One of the hallmarks of AD is accumulation of amyloid Aβ42 plaques over a period of time, which triggers neu-
ronal death leading to neurodegeneration. is Aβ42 mediated neurodegeneration is an outcome of activation
of aberrant signaling because of stress in the neurons2,4,5,17,27. us, the Aβ42 mediated neurodegenerative phe-
notype observed in AD is not due to a single gene mutation but an outcome of impairment of several signaling
pathways4,17,19,27,70,71,92. In order to understand the complexity of this disorder, it is important to identify these
signaling pathways.
One of the approaches to discern molecular genetic basis of AD, and to nd future cures, it is important to
identify the downstream targets of signaling pathways that are triggered as an outcome of the Aβ42 accumula-
tion4. e eorts have been directed to search for chemical inhibitors that can block/downregulate these down-
stream targets of aberrant signaling pathways and thereby prevent/delay Aβ42 mediated neurodegeneration. In
this direction, the repertoire of natural product libraries comprising of plant proteins102,103 with medicinal prop-
erties provide an alternative to the chemical inhibitors which can be screened to identify the ones that can either
delay or block the onset of neurodegeneration observed in GMR > Aβ42 background.
Several plant products have been identied as therapeutic targets for cancer, inammation and various other
disease72,74,75,102,103. Chronic inammation has long been implicated in cancer and also plays major role in neu-
rodegenerative disorders like AD. e soy protein Lunasin has multiple interacting domains and may aect
dierent cell biological processes. Lunasin has been reported to have anti-metastatic and chemopreventive activ-
ity61,72,74,75,104,105. Lunasin can signicantly reduce a melanoma stem cell population106. It has been suggested that
primary anticancer mechanism of Lunasin is based on its activity as a HAT inhibitor74,107. HATs have been known
to play role in AD.
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Our studies demonstrated that Lunasin can rescue Aβ42 mediated neurodegeneration in the Drosophila eye
(Fig.1). Lunasin is known to prevent cancer but its role in neurodegenerative disorders have not been tested to
date. We tested the possibility if Lunasin is preventing accumulation of amyloid plaques and thereby preventing
Aβ42 mediated neurodegeneration. We checked Aβ42 plaque accumulation using monoclonal antibody 6E10
in wild-type, GMR > Aβ42 and GMR > Aβ42 + Lun backgrounds. We found that Aβ42 plaque accumulation
was comparable between GMR > Aβ42 and GMR > Aβ42 + Lun backgrounds (Fig.2). e fact that there is no
Figure 5. Lunasin downregulates JNK signaling to block Aβ42 mediated neurodegeneration. (A) Schematic
presentation of JNK signaling pathway. (B) Levels of phospho-JNK (pJNK) in a semi-quantitative Western
Blot can provide the status of JNK signaling. e higher levels of JNK signaling in GMR > Aβ42 as compared
to the wild-type background were signicantly downregulated in GMR > Aβ42 + Lun background. e
tubulin bands served as controls to normalize the levels of total protein loaded in all three conditions. e
p-JNK band staining intensity was calculated by ImageJ. In comparison to the wild-type (C) eye imaginal
discs and (D) adult eyes, activation of JNK signaling in GMR domain using (E,F) Djunaspv7 (GMR > jun) and
(K,L) constitutively active hepAct (GMR > hep) result in strong neurodegenerative phenotype. Furthermore,
activation of JNK signaling in GMR > Aβ42 background (G,H) GMR > Aβ42 + jun (M,N) GMR > Aβ42 + hep
exhibits stronger neurodegenerative phenotype which are not rescued by misexpression of Lunasin (I,J)
GMR > Aβ42 + Lun + jun and (O,P) GMR > Aβ42 + Lun + hep. (Q,R) Downregulation of JNK signaling
by misexpression of puc, a dual phosphatase, results in near wild-type (Q) eye imaginal discs and (R) adult
eyes. (SV) Misexpression of puc in (S,T) GMR > Aβ42 (GMR > Aβ42 + puc) and (U,V) GMR > Aβ42 + Lun
(GMR > Aβ42 + Lun + puc) results in signicant rescue as seen in eye imaginal discs and the adult eyes. e
puc-lacZ reporter is used as a functional read out of JNK signaling pathway. Expression of puc-lacZ reporter
(Green) in (W,W’) Wild-type, (X,X’) GMR > Aβ42 and (Y,Y’) GMR > Aβ42 + Lun eye imaginal discs. (W,W’)
Note that puc has weak expression in the developing photoreceptor neurons in the wild-type eye imaginal discs.
However, (X,X’) puc expression is dramatically upregulated in GMR > Aβ42 background. (Y,Y’) Misexpression
of Lunasin (Lun) along with Aβ42 (GMR > Aβ42 + Lun) can signicantly downregulate puc expression in the
developing third instar eye disc. Magnication of all eye discs is 20X.
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signicant dierence in the plaque deposition with or without Lunasin in GMR > Aβ42 background, proves that
Lunasin modulates Aβ42 toxicity indirectly, and is downstream of Aβ42 plaque accumulation.
In order to identify and characterize the mechanism behind the novel neuroprotective function ofLunasin,
we tested various genetic modiers of Aβ42 like C2H2 zinc nger transcription factor, Teashirt (Tsh), CREB
binding protein (CBP) and apical basal polarity marker, Crumbs (Crb)27,70,71,92. We found that the neuroprotective
function of Lunasin is independent of Tsh, CBP and Crb (data not shown). Interestingly, among various other
functions, CBP acts as a histone acetyl transferase (HAT)92, and it is known that Lunasin functions as inhibitor of
HAT107. However, we did not see any interaction between Lunasin and CBP in GMR > Aβ42 background (data
not shown).
In order to discern molecular genetic basis of neuroprotective function of Lunasin, we tested various signaling
pathways and found that it is independent of Wg and Dpp signaling (Fig.4). Finally, we found that neuroprotec-
tive function of Lunasin is mediated through downregulation of highly conserved JNK signaling pathway (Figs5
and 7). Earlier, we have seen that accumulation of Aβ42 plaque causes ectopic induction of JNK signaling pathway
in the neurons, which in turn triggers neuronal death27. Our data demonstrates that Lunasin misexpression can
rescue Aβ42 mediated neurodegeneration by downregulating JNK signaling in the Drosophila eye (Fig.7). us,
our studies provide evidences for the rst time that JNK signaling, an important link in onset, manifestation and
progression of AD, can be modulated by plant-based protein. Studies in various animal models of AD suggests
Figure 6. Misexpression of Lunasin can reduce the mortality rate of elav > Aβ42 ies. (A) A graph comparing
the number of ies hatched in Wild-type, elav > Aβ42 and elav > Aβ42 + Lun background validates that Lunasin
misexpression along with elav > Aβ42 (elav > Aβ42 + LunGFP) rescues the elav > Aβ42 mortality rate. We
counted 200 ies in three independent sets from each background and plotted on a graph. ese numbers are
signicant based on the calculations of P-values using the two-tailed t- test using Microso Excel 2013.
Figure 7. A model to show the mechanism by Lunasin (Lun) blocks Aβ42 mediated neurodegeneration.
Accumulation of Aβ42 plaques trigger a cascade of events, which activates JNK signaling. Activation of
JNK signaling in the neuron triggers cell death. Misexpression of Lunasin blocks JNK signaling to promote
neuroprotection of retinal neurons in the Drosophila eye.
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the involvement of JNK signaling in AD108. Our studies open up new avenues where plant proteins expressed
by transgenic approach in the neuron can prevent the onset or delay the onset of AD in the animal model of
Drosophila eye. Since JNK signaling pathway is known to be involved in developmental processes like ageing,
development, tissue homeostasis, cell proliferation, cell survival and innate immune response, the modulation of
JNK can be of signicance in other disease too like Parkinson, stroke etc109.
The results from our present study support the model of potential therapeutic benefits of Lunasin in
Alzheimer’s Disease (Fig.7). Lunasin can prevent progression of Aβ42 mediated neurodegeneration by eectively
downregulating JNK signaling. e use of plant-based product can be a promising alternative or addition to the
use of gene silencing or directly blocking signaling pathways to treat neurodegenerative disorders. us, Lunasin,
a major bioactive component of the soy-based food has potential to exert a major impact on human health.
Further studies are needed to test the ecacy of Lunasin in other vertebrate model organisms to determine if
its anti-inammatory and JNK inhibitory activities show neuroprotective eects. It will be interesting to see if
Lunasin can be developed as a potential natural product for the treatment of Aβ42 mediated neurodegeneration
observed in AD.
Data Availability
e datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request.
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Acknowledgements
We thank the Bloomington Stock Center for the Drosophila strains, K. Cho and the Developmental Studies
Hybridoma Bank (DSHB) for the antibodies. We would like to thank Michael Moran for training students and
A. Giaquinto for helping with the y crosses and for maintenance of stocks used in this paper. e authors also
thank the members of Singh lab for critical comments on the manuscript. Confocal microscopy was supported by
Biology Department central core facility. A.S. and N.G. are supported by Graduate program of Biology. is work
is supported byKBRIN-IDEA grant funded through a parent grant5P20GM103436 to A.S., Start-up support
from the University of Dayton, and asubaward on RO1 (CA183991, PI Nakano) to MKS,and National Institute of
General Medical Sciences (NIGMS) - 1 R15 GM124654-01, STEM Catalyst Grant from theUniversity of Dayton
and start-up support from the University of Dayton to A.S.
Author Contributions
A.S., N.G. and N.G. the three equal first authors performed experiments, analyzed the data and provided
comments to the manuscript. A.S. performed earlier experiments about testing the UAS-Lunasin transgene. A.S.
screened the genetic modiers and helped in manuscript writing. G.J., N.P. and A.S. were involved in generating
the UAS-Lunasin transgene. A.S. and M.K.S. were involved in developing the concept, designing the experiment,
and analyzing the data and writing the manuscript.
Additional Information
Competing Interests: e authors declare no competing interests.
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... Increased oxidative stress due to higher levels of ROS during mitochondrial and/or electron transport chain dysfunction have emerged as some of the main contributors of aging and diseases [2][3][4]. This dysfunction caused by increased ROS levels has been observed in neurodegenerative diseases including Alzheimer's disease (AD) [4][5][6][7]. Excessive ROS production in neurodegenerative diseases may be due to aberrant activation of signaling pathways that results in progressive neuronal death [6][7][8][9][10]. We have previously shown that the evolutionarily conserved Hippo pathway, known for growth regulation [11], is activated in AD and other neurodegenerative disor-Reports ders [12][13][14]. ...
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... Note: This protocol describes the use of Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay. Here we used Drosophila melanogaster's third instar larvae eye-antennal imaginal discs for TUNEL staining (Tare et al., 2011(Tare et al., , 2016Raj et al., 2020;Irwin et al., 2020;Gogia et al., 2020;Sarkar et al., 2018;Steffensmeier et al., 2013;Woodfield et al., 2013;Zhu et al., 2017). TUNEL staining can be used for other imaginal discs and tissue as well. ...
... We have also used this assay in combination with immunohistochemistry (IHC) to study protein localization in Drosophila eyeantennal imaginal discs as it allows simultaneous visualization of other markers along with assaying cell death. We have used a pan-neuronal marker, Elav to stain the nuclei of the photoreceptor neurons in the eye-antennal imaginal discs and performed TUNEL labeling to mark the dying cell nuclei (Tare et al., 2011(Tare et al., , 2016Steffensmeier et al., 2013;Singh et al., 2006;Sarkar et al., 2018;Raj et al., 2020;Moran et al., 2013;Irwin et al., 2020;Gogia et al., 2020;Yeates et al., 2020). Imaging these discs will show signals / puncta that indicate cell death i.e., exposed DNA or fragmented DNA. ...
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Cell death maintains tissue homeostasis by eliminating dispensable cells. Misregulation of cell death is seen in diseases like cancer, neurodegeneration, etc. Therefore, cell death assays like TUNEL have become reliable tools, where fragmented DNA of dying cells gets fluorescently labeled and can be detected under microscope. We used TUNEL assay in Drosophila melanogaster third-instar larval eye-antennal imaginal discs to label and quantify cell death. This assay is sensitive to detect DNA fragmentation, an important event, during apoptosis in retinal neurons. For complete details on the use and execution of this profile, please refer to Wang et al. (1999), Tare et al. (2011), and Mehta et al. (2021).
... Despite remaining hurdles, such as low bioavailability, the advantages outweigh the disadvantages, making further research worthwhile. Especially, traditional plant-based medications can be less poisonous, show fewer side effects and are less expensive compared to synthetic pharmaceuticals [26]. Bringing both together, there is initial evidence of health-promoting modulation by natural agents on stem cell formation, for example by grape-derived polyphenol resveratrol in osteoblast formation [27] or by Curcuma longa compound curcumin in chondrocyte differentiation [28]. ...
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Chapter
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Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools pose challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L² mutant and GMR-hid, GMR-GAL4 eye. L² exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly newt genes significantly restore missing photoreceptor cells both in L² and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.
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Amyloid-β (Aβ) accumulations have been identified in the retina for neurodegeneration-associated disorders like Alzheimer’s disease (AD), glaucoma, and age-related macular degeneration (AMD). Elevated retinal Aβ levels were associated with progressive retinal neurodegeneration, elevated cerebral Aβ accumulation, and increased disease severity with a decline in cognition and vision. Retinal Aβ accumulation and its pathological effects were demonstrated to occur prior to irreversible neurodegeneration, which highlights its potential in early disease detection and intervention. Using the retina as a model of the brain, recent studies have focused on characterizing retinal Aβ to determine its applicability for population-based screening of AD, which warrants a further understanding of how Aβ manifests between these disorders. While current treatments directly targeting Aβ accumulations have had limited results, continued exploration of Aβ-associated pathological pathways may yield new therapeutic targets for preserving cognition and vision. Here, we provide a review on the role of retinal Aβ manifestations in these distinct neurodegeneration-associated disorders. We also discuss the recent applications of retinal Aβ for AD screening and current clinical trial outcomes for Aβ-associated treatment approaches. Lastly, we explore potential future therapeutic targets based on overlapping mechanisms of pathophysiology in AD, glaucoma, and AMD.
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We have developed an undergraduate laboratory to allow detection and localization of proteins in the compound eye of Drosophila melanogaster, a.k.a fruit fly. This lab was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of Western Blotting technique to study protein localization in the adult eye of Drosophila. Western blotting, a two-day laboratory exercise, can be used to detect the presence of proteins of interests from total protein isolated from a tissue. The first day involves isolation of proteins from the tissue and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis to separate the denatured proteins in accordance to their molecular weight/s. The separated proteins are then transferred to the Nitrocellulose or Polyvinylidene difluoride (PVDF) membrane in an overnight transfer. The second day lab involves detection of proteins (transferred to the membrane) using Ponceau-S stain, followed by immunochemistry to detect the protein of interest along the total protein transferred to the membrane. The presence of our protein of interest is carried out by using a primary antibody against the protein, followed by binding of secondary antibody which is tagged to an enzyme. The protein band can be detected by using the kit, which provides substrate to the enzyme. The protein levels can be quantified, compared, and analyzed by calculating the respective band intensities. Here, we have used fly eyes to detect the difference in level of expression of Tubulin (Tub) and Wingless (Wg) proteins in the adult eye of Drosophila in our class. The idea of this laboratory exercise is to: (a) familiarize students with the underlying principles of protein chemistry and its application to diverse areas of research, (b) to enable students to get a hands-on-experience of this biochemical technique.
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Analysis of gene function in complex organisms relies extensively on tools to detect the cellular and subcellular localization of gene products, especially proteins. Typically, immunostaining with antibodies provides these data. However, due to cost, time, and labor limitations, generating specific antibodies against all proteins of a complex organism is not feasible. Furthermore, antibodies do not enable live imaging studies of protein dynamics. Hence, tagging genes with standardized immunoepitopes or fluorescent tags that permit live imaging has become popular. Importantly, tagging genes present in large genomic clones or at their endogenous locus often reports proper expression, subcellular localization, and dynamics of the encoded protein. Moreover, these tagging approaches allow the generation of elegant protein removal strategies, standardization of visualization protocols, and permit protein interaction studies using mass spectrometry. Here, we summarize available genomic resources and techniques to tag genes and discuss relevant applications that are rarely, if at all, possible with antibodies.
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Efforts to identify the genetic underpinnings of rare undiagnosed diseases increasingly involve the use of next-generation sequencing and comparative genomic hybridization methods. These efforts are limited by a lack of knowledge regarding gene function, and an inability to predict the impact of genetic variation on the encoded protein function. Diagnostic challenges posed by undiagnosed diseases have solutions in model organism research, which provides a wealth of detailed biological information. Model organism geneticists are by necessity experts in particular genes, gene families, specific organs, and biological functions. Here, we review the current state of research into undiagnosed diseases, highlighting large efforts in North America and internationally, including the Undiagnosed Diseases Network (UDN) (Supplemental Material, File S1) and UDN International (UDNI), the Centers for Mendelian Genomics (CMG), and the Canadian Rare Diseases Models and Mechanisms Network (RDMM). We discuss how merging human genetics with model organism research guides experimental studies to solve these medical mysteries, gain new insights into disease pathogenesis, and uncover new therapeutic strategies.
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Background: Most of the recent reports suggest that inflammatory mediators play a central role in the etiopathogenesis of Alzheimer's disease (AD) and that the conditions leading to a chronic low-grade inflammation, such as stress, depression, obesity and metabolic syndrome, increase the odds of developing Mild Cognitive Impairment (MCI) and AD. Microglia cells are the main actors in the AD process: stimuli from the microenvironment may induce microglia cells to switch to a classically activated inflammatory phenotype M1, or, on the contrary to an alternatively activated M2 phenotype characterized by the secretion of different types of cytokines. Many attempts are currently being made in order to delay the progression of AD by reducing inflammatory mechanisms underlying the disease. Several studies support a relationship among neuroinflammation and nutrients, foods or dietary patterns, taking into account the synergistic or antagonistic biochemical interactions among nutrients as well as the different food sources of the same nutrient. Natural antioxidant and anti-inflammatory compounds found in plant foods, such as fruits, particularly berries (such as strawberry, blueberry, blackcurrant, blackberry, blueberry and mulberry) have been shown to exert neuroprotective activity. It is still unclear whether the dietary bioactive compounds enter the Blood Brain Barrier (BBB) playing a direct antiinflammatory or pro-inflammatory effect on microglia and/or other Central Nervous System (CNS) cells. Another hypothesis is that they may trigger a peripheral reaction that induce indirectly a CNS' response. The subsequent synthesis of cytokines may drive microglia polarization by different ways. So, via an indirect route microglia detects and responds to immune-to-brain signaling. Conclusion: This review summarizes current evidence about the potential mechanisms of the interaction among diet, neuroinflammation and AD.
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The primordia of the thoracic imaginal discs of the Drosophila embryo originate as groups of cells spanning the parasegment boundary. We present evidence that the thoracic imaginal primordia are allocated in response to signals from the wingless (wg) and decapentaplegic (dpp) gene products. Rows of cells that express wg intersect rows of cells that express dpp to form a ladder-like pattern in the ectoderm of the germ band extended embryo. The imaginal primordia originate as groups of cells which lie near these intersection points. We have used a molecular probe derived from the Distal-less (Dll) gene to show that this population contains progenitor cells for both the dorsal (i.e. wing) and ventral (i.e. leg) discs. Although we show that Dll function is not required for allocation of imaginal cells, activation of an early Dll enhancer may serve as a molecular marker for allocation. A group of cells, which includes the imaginal progenitors, activate this enhancer in response to intercellular signals from wg and perhaps from dpp. We have used a conditional allele of wg to show that wg function is transiently required for both allocation of the imaginal primordia and for initiation of Dll expression in these cells during the brief interval when wg and dpp form the ladder-like pattern. Allocation of the imaginal primordium and activation of Dll expression appear to be parallel responses to a single set of positional cues.
Book
Many of the 14,000 genes of Drosophila are involved in the development of imaginal discs. These hollow sacs of cells make adult structures during metamorphosis, and their study is crucial to comprehending how a larva becomes a fully-functioning fly. This book examines the genetic circuitry of the well-known 'fruit fly', tackling questions of cell assemblage and pattern formation, of the hows and the whys behind the development of the fly. After an initial examination of the proximity versus pedigree imperatives, the book delves into bristle pattern formation and disc development, with entire chapters devoted to the leg, wing, and eye. Extensive appendices include a glossary of protein domains, catalogues of well-studied genes, and an outline of signaling pathways. More than 30 wiring diagrams among over 60 detailed schematics clarify the text. No student or practising scientist engaged in the study of Drosophila genetics should be without this comprehensive reference.
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The developing eye-antennal disc of Drosophila melanogaster has been studied for more than a century and it has been used as a model system to study diverse processes such as tissue specification, organ growth, programmed cell death, compartment boundaries, pattern formation, cell fate specification, and planar cell polarity. The findings that have come out of these studies have informed our understanding of basic developmental processes as well as human disease. For example, the isolation of a white-eyed fly ultimately led to a greater appreciation of the role that sex chromosomes play in development, sex determination, and sex linked genetic disorders. Similarly, the discovery of the Sevenless receptor tyrosine kinase pathway not only revealed how the fate of the R7 photoreceptor is selected but it also helped our understanding of how disruptions in similar biochemical pathways result in tumorigenesis and cancer onset. In this article I will discuss some underappreciated areas of fly eye development that are fertile for investigation and are ripe for producing exciting new breakthroughs. The topics covered here include organ shape, growth control, inductive signaling, and right-left symmetry. This article is protected by copyright. All rights reserved.
Chapter
Cells undergoing apoptosis display a number of morphological changes, including chromatin condensation, cytoplasmic shrinkage, membrane blebbing, and the formation of apoptotic bodies (1). These morphological changes are accompanied by structural changes within the cell, such as the reorganization of actin, nuclear lamin cleavage, fragmentation of DNA, and “flipping” of the phospholipid phosphatidylserine from the interior leaflet of the plasma membrane to the exterior of the cell (2–5).