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A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration

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Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration.
| Activation of Hippo signaling upon amyloid-beta 42 (Aβ42) accumulation also activates c-Jun-amino-terminal kinase (JNK) signaling. (A-E) shows the expression of embryonic lethal abnormal vision (ELAV; red), JNK signaling pathway reporter puc-lacZ (green, gray), and discs large (Dlg; blue) in eye discs from (A,A') glass multiple repeat (GMR)> Aβ42, (B,B') GMR> Aβ42 + hpo, (C,C') GMR> Aβ42 + hpo RNAi , (D,D') GMR> Aβ42 + yki RNAi , and (E,E') GMR> Aβ42 + yki. For comparison between genotypes, (A'-E') shows puc-lacZ (Gray) levels. (F) A semiquantitative Western blot is presented to show phospho-JNK (p-JNK) levels in the wild-type, GMR> Aβ42, GMR> Aβ42 + hpo, and GMR> Aβ42 + hpo RNAi background. The samples were loaded in the following sequence: Lane 1-Molecular weight marker, Lane 2-Wild-type (Canton-S), Lane 3-GMR> Aβ42, Lane 4-GMR> Aβ42+hpo (gain-of-function), Lane 5-GMR> Aβ42+hpo RNAi (loss-of-function). Alpha-tubulin is used as a loading control, and (F') graph shows the quantification of p-JNK levels, which were calculated from a set of three (n = 3) in wild-type and other indicated genotypes from the Western blot (F). The p-values for estimation of p-JNK levels in all combination in a semiquantitative Western blot was calculated in a set of three (n = 3) using Student's t-test in Microsoft Excel software. The p-value between wild-type and GMR> Aβ42 was significant (p < 0.001; ***), wild-type and GMR> Aβ42+hpo was significant (p < 0.001; ***), and between wild-type (Canton-S) and GMR> Aβ42+hpo RNAi (loss-of-function) was significant (p < 0.001; ***). The p-value between GMR> Aβ42 and GMR> Aβ42+hpo was significant (p < 0.001; ***) and between GMR> Aβ42 and GMR> Aβ42+hpo RNAi was significant (p < 0.01; **).
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ORIGINAL RESEARCH
published: 13 March 2020
doi: 10.3389/fcell.2020.00117
Frontiers in Cell and Developmental Biology | www.frontiersin.org 1March 2020 | Volume 8 | Article 117
Edited by:
Cesare Indiveri,
University of Calabria, Italy
Reviewed by:
Johanna O. Ojala,
University of Eastern Finland, Finland
Wen Fu,
University of Alberta, Canada
*Correspondence:
Madhuri Kango-Singh
mkangosingh1@udayton.edu
Amit Singh
asingh1@udayton.edu
Present address:
Madison Irwin,
Department of Pharmacy, Michigan
Medicine, Ann Arbor, MI,
United States
Meghana Tare,
Department of Biological Sciences,
BITS Pilani Campus, Pilani, India
Aditi Singh,
University of Toledo School of
Medicine, Toledo, OH, United States
Specialty section:
This article was submitted to
Cellular Biochemistry,
a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 15 November 2019
Accepted: 11 February 2020
Published: 13 March 2020
Citation:
Irwin M, Tare M, Singh A, Puli OR,
Gogia N, Riccetti M, Deshpande P,
Kango-Singh M and Singh A (2020) A
Positive Feedback Loop of Hippo- and
c-Jun-Amino-Terminal Kinase
Signaling Pathways Regulates
Amyloid-Beta-Mediated
Neurodegeneration.
Front. Cell Dev. Biol. 8:117.
doi: 10.3389/fcell.2020.00117
A Positive Feedback Loop of Hippo-
and c-Jun-Amino-Terminal Kinase
Signaling Pathways Regulates
Amyloid-Beta-Mediated
Neurodegeneration
Madison Irwin 1†, Meghana Tare 1†, Aditi Singh 1†, Oor vashi Roy Puli 1, Neha Gogia 1,
Matthew Riccetti 1, Prajakta Deshpande 1, Madhuri Kango-Singh 1,2,3, 4
*and
Amit Singh 1,2,3, 4,5
*
1Department of Biology, University of Dayton, Dayton, OH, United States, 2Premedical Program, University of Dayton,
Dayton, OH, United States, 3Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton,
Dayton, OH, United States, 4The Integrative Science and Engineering Center, University of Dayton, Dayton, OH,
United States, 5Center for Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN, United States
Alzheimer’s disease (AD, OMIM: 104300) is an age-related disorder that affects
millions of people. One of the underlying causes of AD is generation of hydrophobic
amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These
plaques induce oxidative stress and aberrant signaling, which result in the death
of neurons and other pathologies linked to neurodegeneration. We have developed
aDrosophila eye model of AD by targeted misexpression of human Aβ42 in
the differentiating retinal neurons, where an accumulation of Aβ42 triggers a
characteristic neurodegenerative phenotype. In a forward deficiency screen to look
for genetic modifiers, we identified a molecularly defined deficiency, which suppresses
Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a
member of evolutionarily conserved Hippo signaling pathway that regulates growth.
Activation of Hippo signaling causes cell death, whereas downregulation of Hippo
signaling triggers cell proliferation. We found that Hippo signaling is activated
in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues
the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling
enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that
c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that
activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas
downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration.
We tested the nature of interactions between Hippo signaling and JNK signaling
in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data
suggest that Hippo signaling and JNK signaling, two independent signaling
pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell
death. Our studies demonstrate a novel role of Hippo signaling pathway in
Aβ42-mediated neurodegeneration.
Keywords: neurodegeneration, Alzheimer’s disease, cell death, amyloid-beta 42, Hippo signaling, growth
regulation, c-Jun-amino-terminal kinase (JNK) signaling, Drosophila eye
Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
INTRODUCTION
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder that affects the aging population and is predicted
to continually increase in prevalence and incidence in the
United States (Barnes and Yaffe, 2011). The hallmark of AD and
other neurodegenerative diseases is loss of cognitive function
due to neuronal death (Hardy, 2009; Hirth, 2010; O’Brien and
Wong, 2010; Selkoe and Hardy, 2016). AD is characterized by
accumulation of two types of protein aggregates in AD brains,
viz., extracellular plaques of amyloid-beta (Aβ) peptides and
intracellular tangles of hyper-phosphorylated and cleaved forms
of tau, the microtubule-associated protein (MAP). Abnormal
cleavage of the amyloid precursor protein (APP) results in 42
amino acid long polypeptides hereafter referred to as Aβ42
peptides (Crews and Masliah, 2010; O’Brien and Wong, 2010;
Selkoe and Hardy, 2016). As per the amyloid hypothesis, the
accumulation of the Aβ42 peptides into plaques initiates a
pathological cascade eventually leading to neurodegeneration
(Tare et al., 2011; Selkoe and Hardy, 2016; Yeates et al., 2019).
Since this hypothesis was postulated, several signaling pathways
and genetic modifiers have been implicated in Aβ42-mediated
neurodegeneration (Moran et al., 2013; Steffensmeier et al., 2013;
Cutler et al., 2015; Yeates et al., 2019).
Since the genetic machinery is highly conserved, many
AD animal models including mouse, rodents, flies, fish, dogs,
and non-human primates have been developed to discern
mechanisms of neurodegeneration (Iijima-Ando and Iijima,
2010; Pandey and Nichols, 2011; Tare et al., 2011; Sabbagh
et al., 2013). These animal models also allow testing for
therapeutic targets (Pandey and Nichols, 2011; Sabbagh et al.,
2013; Sarkar et al., 2016, 2018b; Deshpande et al., 2019).
Drosophila melanogaster, the fruit fly, has served as a versatile
model to study neurodegenerative diseases (Hirth, 2010; Prussing
et al., 2013). The adult Drosophila eye arises from a monolayer
epithelium, which is housed inside the larva and is referred to
as the eye-antennal imaginal disc (Kumar, 2011; Singh et al.,
2012; Tare et al., 2013). The adult eye is comprised of nearly 800
unit eyes or ommatidia (Ready et al., 1976; Kumar, 2011; Singh
et al., 2012). After retinal differentiation, few undifferentiated
cells undergo programmed cell death (PCD) during pupal
development (Brachmann and Cagan, 2003). It is notable that
PCD does not normally occur during early eye development;
however, cell death may occur due to abnormal signaling
(Mehlen et al., 2005; Singh et al., 2006; Tare et al., 2016). We have
developed a Drosophila AD model by misexpressing high levels
of human Aβ42 polypeptide in the differentiating photoreceptor
neurons of the developing Drosophila eye. Misexpression of Aβ42
in the developing Drosophila eye results in progressive loss of
Abbreviations: AD, Alzheimer’s disease; Aβ42, amyloid-beta 42; Hpo/MST1/2,
Hippo; Yki/YAP, Yorkie; Wts/LATS1, Warts; Sav/SAV1/WW45, Salvador;
Mobs/hMob1, Mob-as-tumor-suppressor; Sd/TEAD1, Scalloped; DIAP1/BIRC5,
Drosophila inhibitor of apoptosis; rpr, reaper;hid, head involution defective; JNK, c-
Jun-amino-terminal kinase; MAPK, mitogen-activated protein kinase; Egr/TNFα,
Eiger; Hep/JNKK, hemipterous; Bsk/JNK, basket; dJun/c-Jun, Jun-related antigen;
puc, puckered; GMR, glass multiple repeat; PBS, phosphate buffered saline; ELAV,
embryonic lethal abnormal vision; Dlg, discs large.
photoreceptor neurons and aberrant morphology that mimics
the neuropathology of atrophy and loss of neurons linked to AD
(Tare et al., 2011; Sarkar et al., 2016).
Activation of the c-Jun-amino-terminal kinase (JNK)
signaling pathway is implicated in Aβ42-mediated
neurodegeneration (Tare et al., 2011; Sarkar et al., 2016).
JNK signaling, which belongs to the mitogen-activated protein
kinase (MAPK) superfamily, is a stress-activated protein kinase
that triggers apoptosis upon activation (Adachi-Yamada and
O’connor, 2004; Stronach, 2005; Dhanasekaran and Reddy,
2008). The JNK cascade is initiated by the binding of the ligand
Eiger (Egr), the Drosophila homolog of the human tumor
necrosis factor (TNF) to TNF receptors, named Wengen and
Grindelwald in flies (Igaki et al., 2002; Kanda et al., 2002; Moreno
et al., 2002). Upon receptor activation, the signal is transmitted
by hemipterous (hep), the Drosophila JNKK that phosphorylates
basket (bsk), the Drosophila JNK (Glise et al., 1995; Sluss et al.,
1996; Holland et al., 1997). Bsk phosphorylates and activates
Drosophila Jun-related antigen (Jra or dJun). The transcription
factor Jun translocates to the nucleus to induce target genes of
the JNK pathway (Sluss et al., 1996; Kockel et al., 2001). A key
transcriptional target of JNK signaling is puckered (puc), which
is a dual-specificity phosphatase that negatively regulates bsk
and thereby forms a negative feedback loop (Martin-Blanco
et al., 1998; Adachi-Yamada, 2002; Stronach, 2005). When
activated, JNK signaling triggers cell death by phosphorylation
of reaper (rpr) and head involution defective (hid), as well as
caspase-independent mechanisms (Martin-Blanco et al., 1998;
Stronach, 2005; Singh et al., 2006; Igaki, 2009).
Interestingly, a key interactor of the JNK is the Hippo
pathway, which is a conserved signaling pathway primarily
involved in the regulation of organ size (Kango-Singh and
Singh, 2009; Pan, 2010; Halder and Johnson, 2011; Staley
and Irvine, 2012). The Hippo and JNK pathways interact in
several contexts, for example, in the regulation of growth, cell
survival, and regeneration (Grusche et al., 2011; Sun and Irvine,
2011). The Hippo pathway is comprised of several upstream
regulators that relay the signal through a core kinase cascade
that ultimately controls the activation of Yorkie (Yki), the
effector of the Hippo pathway (Huang et al., 2005). Yki acts
as a transcriptional coactivator and requires Scalloped (Sd), a
TEAD/TEF family transcription factor to induce the expression
of Hippo pathway target genes (Wu et al., 2008; Zhang et al.,
2008; Ren et al., 2010). The core kinase cassette is comprised
of Hippo (Hpo) and Warts (Wts), two serine-threonine protein
kinases of the mammalian Ste-20 and nuclear Dbf-2-related
(NDR) kinase family, respectively. Hpo phosphorylates and
complexes with the WW-domain containing adaptor protein
Salvador (Sav). The Hpo–Sav complex interacts with the
downstream kinase Wts and its binding partner Mob-as-tumor-
suppressor (Mats). Following Hpo-mediated phosphorylation,
Wts undergoes autophosphorylation and in turn phosphorylates
Yki (Justice et al., 1995; Kango-Singh et al., 2002; Tapon et al.,
2002; Harvey et al., 2003; Jia et al., 2003; Pantalacci et al., 2003;
Udan et al., 2003; Wu et al., 2003; Huang et al., 2005; Lai et al.,
2005; Wei et al., 2007; Kango-Singh and Singh, 2009). Overall,
activation of the Hippo pathway sequesters Yki in the cytoplasm
Frontiers in Cell and Developmental Biology | www.frontiersin.org 2March 2020 | Volume 8 | Article 117
Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
and results in induction of cell death and decreased organ size
(Harvey et al., 2003; Pantalacci et al., 2003; Udan et al., 2003; Wu
et al., 2003; Wei et al., 2007; Verghese et al., 2012). In contrast,
inactivation or downregulation of Hippo pathway allows Yki to
translocate to the nucleus, bind Sd, and regulate expression of
target genes. These target genes include Myc and bantam, the
two promoters of growth, and Diap1, a Drosophila inhibitor of
apoptosis protein 1 (Nolo et al., 2006; Thompson and Cohen,
2006; Wu et al., 2008; Zhang et al., 2008; Peng et al., 2009; Neto-
Silva et al., 2010; Oh and Irvine, 2011). Other phosphorylation-
independent mechanisms of Yki regulation are also known that
mainly involve physical association of Yki with Hippo signaling
components, which prevents its nuclear localization (Oh and
Irvine, 2008, 2009, 2010; Zhang et al., 2008). While Hippo
signaling plays a role in several diseases like cancer, polycystic
kidney disease, and heart disease, its role in neurodegenerative
diseases such as AD remains poorly understood.
In a genetic modifier screen, we identified a deficiency,
Df(2R)BSC782/+, which rescued the Aβ42-mediated
neurodegeneration phenotype. This deficiency uncovers 10
genes including the hpo gene. Further testing with the candidate
genes uncovered by Df(2R)BSC782 revealed hpo as the causal
genetic modifier of the neurodegeneration phenotype of Aβ42
overexpression. This suggested that hpo and other components
of the Hippo signaling pathway may impact the Aβ42-mediated
neurodegeneration phenotype. Here we report that the Hippo
pathway affects Aβ42-mediated neurodegeneration phenotypes
as hyperactivation of Hippo signaling leads to enhancement of
Aβ42 toxicity, whereas downregulation of Hippo signaling
rescues Aβ42-mediated neurodegeneration phenotype.
Previously, we had reported that Aβ42 induced neuronal
apoptosis via activation of a JNK–caspase-dependent pathway.
Recently, JNK and Hippo pathway were shown to interact in
several contexts, which prompted us to study if JNK–Hippo
interactions affected the Aβ42-mediated neurodegeneration
phenotype. Here we report that misexpression of Aβ42 induces
JNK signaling, which in turn, induces Hippo signaling by
blocking Yki activation. Activation of Hippo signaling in Aβ42-
mediated neurodegeneration activates puc-lacZ, a reporter of
JNK signaling. Here we present evidences to support a role
for a positive feedback loop between JNK and Hippo signaling
pathways that promotes Aβ42-mediated neurodegeneration in
the Drosophila eye.
MATERIALS AND METHODS
Fly Stocks
All fly stocks used in this study are listed at FlyBase (http://
flybase.bio.indiana.edu). Fly stocks used in this study were:
GMR-Gal4 (Moses and Rubin, 1991), UAS-Aβ42 (Tare et al.,
2011; Sarkar et al., 2018b), UAS-hpo (Udan et al., 2003), UAS-
hpoRNAi (Pantalacci et al., 2003), UAS-wts13F (Kwon et al.,
2015), UAS-wtsRNAi (Trip Line), UAS-yki (Oh and Irvine,
2009), UAS-ykiRNAi(N+C) (Zhang et al., 2008), diap1-4.3-green
fluorescent protein (GFP) (Ren et al., 2010), hid 5-GFP (Tanaka-
Matakatsu et al., 2009), ex697-lacZ (Boedigheimer et al., 1997),
UAS-puc, pucE69 (Martin-Blanco et al., 1998), UAS-junaspv7
(Treier et al., 1995), UAS-hepAct (Glise et al., 1995), UAS-bskDN
(Adachi-Yamada et al., 1999). For the genetic screen, we used
molecularly defined deficiencies. We identified Df(2R)BSC782/+,
a deficiency, which is located on the right arm of the second
chromosome, and uncovers βTub56D,par-1,CG16926,CG7744,
CG15120,mei-W68,oseg6,TBCB,rep, and hpo genes (listed in
Flybase). For wild-type control, we used the Canton-S stock of
D. melanogaster in this study. Fly stocks were maintained at 25C
on the regular cornmeal, yeast, molasses food medium.
Genetic Crosses
We employed a Gal4/UAS system for targeted misexpression
studies (Brand and Perrimon, 1993). All Gal4/UAS crosses
were maintained at 18, 25, and 29C, unless specified, to
sample different induction levels (Singh and Choi, 2003;
Singh et al., 2005). The GMR-Gal4 driver used in this
study targets misexpression of transgenes in the differentiating
retinal precursor cells of the developing eye imaginal disc
and pupal retina (Moses and Rubin, 1991). Misexpression of
Aβ42 in the differentiating retina (GMR-Gal4 >UAS-Aβ42,
referred to as GMR>Aβ42 throughout the text) exhibits a
stronger neurodegenerative phenotype at 29C (Tare et al.,
2011; Sarkar et al., 2018b). For all other genetic interaction
studies involving the JNK and Hippo pathway, UAS lines that
upregulate or downregulate pathway components were tested
using appropriate transgenes by crossing to the GMR>Aβ42 flies
though appropriate genetic crosses.
Immunohistochemistry
Eye-antennal imaginal discs were dissected from the wandering
third-instar larvae in 1X phosphate buffered saline (PBS), fixed
in 4% paraformaldehyde in PBS for 20 min, and washed in
PBS. We stained the tissue with a combination of antibodies
following a previously published protocol (Singh et al., 2002;
Sarkar et al., 2018a). The primary antibodies used were
rat anti-Embryonic Lethal Abnormal Vision (ELAV) (1:50;
Developmental Studies Hybridoma Bank, DSHB), mouse anti-
discs large (Dlg) (1:100; DSHB), rabbit anti-Dlg (1:200; a gift
from Dr. K. Cho), mouse anti-6E10 (1:100), rabbit anti-β-
galactosidase (1:200; Cappel), mouse anti-22C10 (1:100; DSHB),
and mouse anti-Chaoptin (MAb24B10) (1:100; DSHB) (Zipursky
et al., 1984). Secondary antibodies (Jackson Laboratory) used
were goat anti-rat IgG conjugated with Cy5 (1:250), donkey anti-
rabbit IgG conjugated with fluorescein isothiocyanate (FITC)
(1:200), donkey anti-mouse IgG conjugated with FITC (1:200),
and donkey anti-mouse IgG conjugated with Cy3 (1:250). We
mounted the tissues in Vectashield (Vector Laboratories). The
immunofluorescent images were captured by laser scanning
confocal microscopy (Singh and Gopinathan, 1998). We took
the images at 20×magnification unless stated otherwise. We
analyzed and prepared the final figures with images using Adobe
Photoshop CS6 software.
Detection of Cell Death
We performed terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) assays using a cell death detection kit from
Roche Diagnostics. TUNEL assays allow identification of the cells
Frontiers in Cell and Developmental Biology | www.frontiersin.org 3March 2020 | Volume 8 | Article 117
Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
undergoing cell death where the cleavage of double- and single-
stranded DNA is labeled by a fluorescent tag (TMR Red) (White
et al., 1994; Mccall and Peterson, 2004). The fluorescently labeled
nucleotides are added to 3OH ends in a template-independent
manner by terminal deoxynucleotidyl transferase (TdT). The
fluorescent-tagged fragmented DNA within a dying cell can be
detected by fluorescence microscopy. After secondary antibody
staining, eye antennal discs were blocked in 10% normal donkey
serum in PBS with 0.2% Triton X-100 (PBT) and processed
for TUNEL labeling (Singh et al., 2006). For each genotype, we
counted TUNEL-positive nuclei from five sets of eye imaginal
discs to determine dying cell population. We used these cell
counts for statistical analysis using Microsoft Excel 2013. We
calculated the p-values using Student two-tailed t-test, and the
error bars represent standard deviation from mean.
Adult Eye Imaging
We prepared the adult flies for imaging by freezing at 20C
for 2 h followed by mounting the fly on a dissection needle.
The needle was secured with mounting putty to suspend the fly
horizontally over a glass slide. We took adult eye images on the
Axiomager.Z1 Zeiss Apotome and obtained the Z-stacks (Oros
et al., 2010; Wittkorn et al., 2015; Singh et al., 2019). Final images
are projections of Z-stacks using the extended depth of focus
function of the Axiovision software 4.6.3.
Western Blot
Protein samples were prepared from (n= ∼50) adult eyes
from Canton-S (wild-type), GMR>Aβ42, GMR>Aβ42+hpo
following standardized protocols (Gogia et al., 2017). The
samples were loaded in the following sequence: Lane 1-Molecular
weight marker (BIORAD Precision Plus Protein Kaleidoscope
Prestained Catalog Number #1610375), Lane 2-Wild-type
(Canton-S), Lane 3-GMR>Aβ42, Lane 4-GMR>Aβ42+hpo
(gain-of-function), Lane 5-GMR>Aβ42+hpoRNAi (loss-of-
function). We incubated the blots with the Phospho SAPK/JNK
(81E11) (1:3,000, Cell Signaling) and after appropriate washes
incubated with horseradish peroxidase conjugated goat anti–
rabbit IgG (1:5,000) secondary antibody. Signal was detected
using super signal chemiluminescence substrate (Pierce). We
captured the images using the BioSpectrum R
500 Imaging
System and analyzed the blot images and band intensity. We
used Microsoft Excel 2017 software for statistical analysis. We
calculated the p-values using the Student two-tailed t-test. The
error bars represent standard deviation from means.
RESULTS
Genetic Modifier of Amyloid-Beta
42-Mediated Neurodegeneration
The wild-type eye imaginal disc (Figure 1A) develops into the
adult compound eye (Figure 1B). The eye-antennal imaginal
discs were stained with a membrane-specific marker Dlg (green)
and pan neural marker ELAV (red), which marks the nuclei of
the photoreceptor neurons (Figure 1A). Targeted misexpression
of human Aβ42 in the differentiating photoreceptor neuron
of the developing eye imaginal disc using GMR-Gal4 driver
(GMR>Aβ42) results in the loss of photoreceptor neurons on
the posterior margin of the eye imaginal disc (Figure 1C). The
neurodegeneration phenotype worsens with time, which results
in a highly reduced adult eye with glazed appearance [n=112,
all of them (112/112, 100%) showed highly reduced adult eye
phenotype, Figure 1D] (Tare et al., 2011).
We performed a forward genetic screen using molecularly
defined deficiencies to find genetic modifiers of Aβ42
neurodegeneration phenotype. In this screen, we identified
a deficiency, Df(2R)BSC782, which in transheterozygous
combination (Df(2R)BSC782/+) rescues the GMR>Aβ42-
mediated neurodegeneration phenotype both in the eye imaginal
disc (Figure 1F) and the adult eye (n=117, 89/117, 76% of the
adult eye showed rescue phenotype; Figure 1G). Interestingly,
Df(2R)BSC782 deficiency is located on the right arm of the
second chromosome and uncovers 10 genes including hippo
(hpo), a member of highly conserved Hippo growth regulatory
pathway (Figure 1E). We individually tested genes uncovered
by Df(2R)BSC782 (Figure 1K) using gain-of-function and
loss-of-function approaches to identify which gene(s) functions
as the genetic modifier(s) of Aβ42 (GMR>Aβ42)-mediated
neurodegeneration in the Drosophila eye. Misexpression of
hpo (GMR>hpo) results in the loss of photoreceptor neurons
on the posterior margin of the eye imaginal disc (Figure 1H),
resulting in a highly reduced or “No-eye” phenotype in
the adult fly (n =119, 108/119, 91% showed reduced or
“No-eye” phenotype; Figure 1I). As compared to GMR>
Aβ42 (Figures 1C,D), misexpression of hpo along with Aβ42
(GMR>Aβ42+hpo) enhances neuronal loss and results in
a stronger neurodegeneration phenotype in the eye imaginal
disc (Figure 1J) and the adult eye (n=136, 136/136, 100%
showed strong neurodegenerative phenotype; Figure 1K).
The GMR>Aβ42+hpo adults, which showed strong pupal
lethality, were dissected out from their pupal cases as they failed
to close. To further validate the role of Hippo signaling, we
downregulated hpo gene function by misexpressing hpoRNAi
(GMR>hpoRNAi) that results in mild overgrowth both in the
eye disc (Figure 1L) and in the adult flies (n=98, 61/98,
62%; Figure 1M). Coexpression of hpoRNAi with Aβ42 (GMR>
Aβ42+hpoRNAi) shows a strong rescue of Aβ42-mediated
neurodegeneration both in the eye imaginal disc (Figure 1N)
and the adult eye (n=107, 71/107, 66%; Figure 1O). The adults
of GMR>Aβ42+hpoRNAi show a dramatic rescue to near wild-
type adult eye and significantly reduced the pupal lethality as
compared to GMR>Aβ42 or GMR>Aβ42+hpo. These results
validate our findings from deficiency screen that hpo is a genetic
modifier of Aβ42-mediated neurodegeneration in the Drosophila
eye. In order to understand the mechanism of Aβ42-mediated
neurodegeneration, it is important to understand the impact of
Hippo signaling on the Aβ42 and its downstream effects.
Modulation of Hippo Activity Does Not
Affect Amyloid-Beta 42 Plaque
Accumulation
Since loss-of-function of hpo can rescue Aβ42-mediated
neurodegeneration, we therefore tested the effects of
Frontiers in Cell and Developmental Biology | www.frontiersin.org 4March 2020 | Volume 8 | Article 117
Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 1 | hippo is a genetic modifier of amyloid-beta 42 (Aβ42)-mediated neurodegeneration in the Drosophila eye. Panels show images of eye imaginal discs
stained for the proneural marker embryonic lethal abnormal vision (ELAV; red) and a membrane-specific marker discs large (Dlg; green), and the resulting eye
phenotype in the adult from (A,B) wild-type and (C,D) glass multiple repeat (GMR)>Aβ42. (D) Note that the GMR>Aβ42 adult eyes are highly reduced and have
glazed morphology with black necrotic spots. (E) A map showing the deficiency BSC782 identified in the forward genetic screen and position of hpo and other genes
within this deficiency is depicted. (F–O) Panels show the eye disc stained with Dlg (green) and ELAV (red) and accompanying adult eye phenotypes from (F,G) GMR>
Aβ42 +Df(2R)BSC782/+,(H,I) GMR>hpo, (J,K) GMR>Aβ42 +hpo,(L,M) GMR>hpoRNAi, and (N,O) GMR>Aβ42 +hpoRNAi . Note that downregulation of Hippo
signaling (N,O) GMR>Aβ42 +hpoRNAi significantly rescues the GMR>Aβ42 neurodegenerative phenotype, whereas activation of Hippo signaling (J,K) GMR>Aβ42
+hpo enhances the GMR>Aβ42 neurodegenerative phenotype. The orientation of all imaginal discs is identical with posterior to the left and dorsal up. Magnification
of the eye disc or adult eye images is the same across all panels.
modulation of Hippo signaling on Aβ42 accumulation. We
used the 6E10 antibody that specifically detects the Aβ42
polypeptide. We observed robust Aβ42 accumulation in
the eye imaginal discs from GMR>Aβ42 (Figures 2A,A),
GMR>Aβ42+hpo (Figures 2B,B), or GMR>Aβ42+
hpoRNAi (Figures 2C,C), suggesting that upregulation or
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 2 | hpo does not affect the amyloid-beta 42 (Aβ42) accumulation. Panels show eye imaginal discs and pupal retinae stained with the proneural marker
embryonic lethal abnormal vision (ELAV; shown in red); 6E10, an anti-Aβ42 antibody (green or gray), and the membrane-specific marker discs large (Dlg; blue). (A–C)
shows confocal images of eye discs for all markers (Dlg, 6E10, ELAV), whereas the 6E10 expression alone (gray) is shown in panels (A’–C’). Eye discs (A–C) and
pupal retinae (D–F) of the following genotypes were compared: (A,A’,D,D’) glass multiple repeat (GMR)>Aβ42, (B,B’,E,E’) GMR>Aβ42 +hpo, and (C,C’,F,F’)
GMR>Aβ42 +hpoRNAi. Note that activation (GMR>Aβ42 +hpo) or downregulation (GMR>Aβ42 +hpoRNAi ) of Hippo signaling in GMR>Aβ42 background does
not affect the accumulation of Aβ42 plaques.
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
downregulation of Hpo does not directly affect the levels of
Aβ42 in the eye disc. It is noteworthy that changes in Hpo levels
have a significant effect on GMR>Aβ42 (Figures 2A,A)
phenotypes where GMR>Aβ42+hpo (Figures 2B,B)
enhances neurodegeneration, and GMR>Aβ42+hpoRNAi
(Figures 2C, C) show a significant rescue of the Aβ42-mediated
neurodegenerative phenotype.
GMR enhancer drives expression in the differentiating retinal
neurons, which initiates in early third instar of eye development,
and continues in pupal retina. Since accumulation of Aβ42
exhibits a progressive neurodegenerative phenotype, we analyzed
the effect of modulation of hpo levels at a later time window
of pupal development. Interestingly, Aβ42 accumulation is not
affected in the pupal retina of GMR>Aβ42 (Figures 2D,D),
GMR>Aβ42+hpo (Figures 2E,E), or GMR>Aβ42+hpoRNAi
(Figures 2F,F). This suggests that changes in the hpo activity
do not affect Aβ42 expression or levels during larval or
pupal stages. Thus, hpo may affect the Aβ42 phenotypes by
acting downstream of Aβ42 accumulation likely by modifying
downstream signals.
Hippo Signaling Is Activated in
Amyloid-Beta 42-Mediated
Neurodegeneration
One possibility is that Aβ42 accumulation may cause
neurodegeneration by affecting Hippo pathway activity.
Therefore, we investigated if expression of Hippo pathway
reporters is affected in GMR>Aβ42 background (Figure 3).
diap1- 4.3-GFP and ex-lacZ serve as functional readouts
of the Hippo signaling pathway (Hamaratoglu et al., 2006;
Kango-Singh and Singh, 2009; Ren et al., 2010). diap1-4.3-
GFP is a Hippo response element mapped to the regulatory
regions of diap1 gene, which reports diap 1 endogenous
gene activity in response to Hippo signaling by changes in
the expression of a GFP reporter (Ren et al., 2010). In the
wild type, diap1 is expressed uniformly in the eye region
of the imaginal disc (Figures 3A,A) and ubiquitously in
the pupal retina (Figures 3C,C). Loss-of-function of hpo
results in cell proliferation along with upregulation of diap1
reporter in the eye disc (Ren et al., 2010). Misexpression
of Aβ42 (GMR>Aβ42+diap1-4.3-GFP) results in a strong
suppression of the diap1-4.3-GFP reporter expression in the
GMR>Aβ42 eye imaginal disc (Figures 3B,B) and pupal retina
(Figures 3D,D). Downregulation of diap1-4.3-GFP reporter
suggests that Hippo signaling is activated in the GMR>Aβ42
background. Similarly, another reporter of Hippo signaling
activity, ex-lacZ, is expressed ubiquitously in the wild-type
eye imaginal disc (Figures 3E,E) and in the pupal retina
(Figures 3G,G). However, ex-lacZ expression is downregulated
in GMR>Aβ42 background both in the eye imaginal disc
(Figures 3F,F) and in the pupal retina (Figures 3H,H).
Activation of Hippo pathway is known to upregulate hid
expression (Udan et al., 2003). Therefore, we tested the hid5-
GFP reporter. In comparison to the wild-type eye imaginal
disc (Figures 3I,I), hid5-GFP is robustly induced in GMR>
Aβ42 eye imaginal disc (Figures 3J,J). Similarly, hid5-GFP
was strongly upregulated in the pupal retina of GMR>
Aβ42 (Figures 3L,L) as compared to the wild-type pupal
retina (Figures 3K,K). Thus, misexpression of Aβ42 (GMR>
Aβ42) activates Hippo signaling that results in induction of
cell death.
Hippo Signaling Levels Affect
Amyloid-Beta 42-Mediated Neuronal Cell
Death
We tested if activation of Hippo signaling is responsible for
triggering cell death of neurons in GMR>Aβ42 background.
So we tested if other components of the pathway that act
downstream of Hpo affect Aβ42-mediated neurodegeneration.
We employed TUNEL staining that labels the fragmented DNA
and thereby mark the nuclei of the dying neurons (White et al.,
1994; Cutler et al., 2015). Misexpression of Aβ42 results in
induction of cell death as evident from TUNEL-positive nuclei in
the eye imaginal disc and the adult eye (Figure 4A). Activation of
Hippo signaling by misexpressing hpo along with Aβ42 (GMR>
Aβ42+hpo) results in 2-fold increase in cell death (Figure 4A)
as evident from the number of TUNEL-positive nuclei in
eye imaginal disc (Figure 4B) and pupal retina (Figure 4C).
The adult fly of GMR>Aβ42+hpo genotype failed to hatch
out and exhibits a “no-eye” phenotype (Figure 4D). Similar
effects were observed when the Hippo pathway was activated by
overexpression of wts (GMR>Aβ42+wts13F;Figures 4A,H–J) or
downregulation of yki (GMR>Aβ42+ykiRNAi;Figures 4A,N–P).
In contrast, downregulation of Hippo signaling by hpoRNAi
(GMR>Aβ42+hpoRNAi;Figures 4A,E–G) and wtsRNAi (GMR>
Aβ42+wtsRNAi;Figures 4A,K–M) or overexpression of yki
(GMR>Aβ42+yki;Figures 4A,Q–S) results in the converse
phenotype of a significant rescue as evident from the highly
reduced cell death (Figure 4A) in the eye imaginal disc
(Figures 4A,E,K,Q), pupal retina (Figures 4F,L,R), and in the
adult eye (Figures 4G,M,S), respectively. These data suggest
that other pathway components, specially the effector Yki,
also modify the effects of Aβ42-mediated neurodegeneration.
Genetic interactions also suggest that the Hippo pathway
acts downstream of Aβ42 accumulation. Aβ42-mediated
neurodegeneration is dependent on the JNK signaling
pathway activity (Tare et al., 2011). Therefore, we explored
the JNK pathway as we have previously shown a similar
downstream role of JNK signaling in Aβ42-mediated cell death
(Tare et al., 2011).
JNK Activity Is Modulated by Hippo
Pathway Levels in Amyloid-Beta
42-Mediated Neurodegeneration
Hippo signaling can activate JNK signaling (Ma et al., 2015,
2017). We first tested if changes in Hippo signaling activity
affect JNK signaling activity in the GMR>Aβ42 background.
puc-lacZ serves as a reporter for the JNK signaling activity
(Martin-Blanco et al., 1998). Earlier we reported that puc-lacZ is
robustly induced in Aβ42 (GMR>Aβ42) background, suggesting
increased JNK activity (Figures 5A,A;Tare et al., 2011). As
compared to GMR>Aβ42, gain-of-function of hpo (GMR>
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 3 | Accumulation of amyloid-beta 42 (Aβ42) activates Hippo signaling. Expression levels of Hippo pathway reporters were tested in eye discs and pupal
retinae. Expression of diap1 4.3-green fluorescent protein (GFP) (green, gray) is shown for eye discs and pupal retinae from (A,A’,C,C’) wild-type, (B,B’,D,D’) glass
multiple repeat (GMR)>Aβ42, respectively. diap1-4.3-GFP expression is shown in a split channel in gray in (C’,D’) panels. Changes in ex-lacZ (green, gray) levels is
shown in eye discs and pupal retinae from (E,E’,F,F’) wild type, (G,G’,H,H’) GMR>Aβ42, respectively. ex-lacZ expression is shown in a split channel in gray in
(E’–H’).(I–L) shows the expression of hid-5’ GFP (green), a reporter for cell death in wild-type (I,I’) eye imaginal disc and (K,K’) pupal retina and GMR>Aβ42 (J,J’)
eye imaginal disc and the (L,L’) pupal retina. Gray panels (I’–L’) show hid-5’ GFP expression in indicated genotypes. In (I–L), nuclei are marked by the nuclear dye
TOPRO (red).
Aβ42+hpo,puc-lacZ) results in a strong upregulation of puc-lacZ
reporter expression (Figures 5B,B), whereas downregulation of
hpo by misexpression of hpoRNAi (GMR>Aβ42+hpoRNAi,puc-
lacZ) results in downregulation of puc-lacZ reporter expression
(Figures 5C,C). puc-lacZ expression coincides with the area
of highest neuronal loss. To study the effects of downstream
pathway components, we checked effects of upregulation or
downregulation of Yki. Blocking Hippo signaling by yki
misexpression (GMR>Aβ42+yki,puc-lacZ) shows robust
induction of puc-lacZ reporter in GMR expression domain
(Figures 5 E,E). Consistent with this, downregulation of Yki
(GMR>Aβ42+ykiRNAi,puc-lacZ) did not induce high levels
of puc-lacZ (Figures 5 D,D). Our data suggest that activation
of Hippo signaling in GMR>Aβ42 background results in
enhancement of neurodegeneration, which is accompanied by
induction of JNK signaling pathway.
We further verified our immunohistochemistry results with a
semiquantitative Western blot to assess levels of phospho-JNK,
the activated form of JNK (Mehan et al., 2011). pJNK levels were
compared in protein extracts made from eye discs from wild type,
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 4 | Hippo activation triggers cell death in glass multiple repeat
(GMR)>amyloid-beta 42 (Aβ42) background. For each genotype, we counted
terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive
nuclei from five (n=5) eye imaginal discs to determine dying cell
(Continued)
FIGURE 4 | population. (A) Quantification of TUNEL-positive nuclei in
indicated genotypes is shown (n=5, p0.05). The p-values obtained from
Student’s t-test between wild-type and GMR>Aβ42 was significant (p<
0.001; ***), GMR>Aβ42 and GMR>Aβ42+hpo (gain-of-function) was
significant (p<0.001; ***), and between GMR>Aβ42 and
GMR>Aβ42+hpoRNAi (loss-of-function) was significant (p<0.001; ***). The
p-value obtained from Student’s t-test between GMR>Aβ42 and GMR>
Aβ42+wts was significant (p<0.05; *), between GMR>Aβ42 and GMR>
Aβ42+wtsRNAi was significant (p<0.001; ***), between GMR>Aβ42 and
GMR>Aβ42+yki was significant (p<0.001; ***), and between GMR>Aβ42
and GMR>Aβ42+ykiRNAi was significant (p<0.001; ***). (B–S) shows the
extent of cell death based on TUNEL assays (red, gray) in indicated genotypes
in imaginal discs, pupal retinae, and adult eyes. The eye discs (B,E,H,K,N,Q)
were assessed for dying cells using TUNEL assay (red) and stained with Dlg
(green) and embryonic lethal abnormal vision (ELAV; blue). The pupal retinae
(C,F,I,L,O,R) assessed for dying cells using TUNEL assays (gray). Adult eye
phenotypes are shown in panels (D,G,J,M,P,S). Panels show the extent of cell
death in (B–D) GMR>Aβ42+hpo,(E–G) GMR>Aβ42+hpo RNAi,(H–J)
GMR>Aβ42 +wts,(K–M) GMR>Aβ42 +wtsRNAi,(N–P) GMR>Aβ42 +
ykiRNAi, and (Q–S) GMR>Aβ42 +yki. The orientation of all imaginal discs is
identical with posterior to the left and dorsal up. Magnification of all eye
imaginal discs is 20×.
GMR>Aβ42, GMR>Aβ42+hpo, and GMR>Aβ42+hpoRNAi.
In comparison to the wild type, p-JNK levels were upregulated
in GMR>Aβ42, GMR>Aβ42+hpo background, whereas it
was reduced in GMR>Aβ42+hpoRNAi background. The alpha-
tubulin served as the loading control. The quantification of
pJNK levels shows that compared to GMR>Aβ42, pJNK levels
are higher when Hippo pathway is activated (GMR>Aβ42+
hpo;Figures 5F,F). Taken together, these data present evidence
for activation of both Hippo and JNK pathways during Aβ42-
mediated neurodegeneration. Genetic interaction and Western
blot analysis shows that Hippo pathway can activate JNK
signaling in the GMR>Aβ42. However, given the complex
context-dependent nature of interactions between the Hippo and
JNK pathways, it is important to test whether JNK signaling
pathway can also affect Hippo signaling pathway.
Activation of Hippo Signaling in
Amyloid-Beta 42 Background Is Dependent
on c-Jun-Amino-Terminal Kinase Signaling
Pathway
To further explore the relationship between Hippo and JNK
signaling pathways in Aβ42-mediated neurodegeneration, we
tested Hippo signaling activity when levels of JNK signaling
pathway are modulated in GMR>Aβ42 background. diap1-
4.3-GFP serves as a functional readout of the Hippo signaling
pathway (Ren et al., 2010). In comparison to diap1-4.3-
GFP expression in the control eye disc (Figures 6A,A), the
diap-1-4.3-GFP reporter is downregulated in GMR>Aβ42
background (Figures 6C,C). The GMR>Aβ42 adults exhibit
strong neurodegeneration phenotype in the eye (Figure 6D) as
compared to the wild-type adult eye (Figure 6B). Upregulation
of JNK signaling activity by misexpressing activated Jun
in the GMR>Aβ42 background (GMR>Aβ42+junaspv7;
Figures 6E,E) or by expression of activated Hep (GMR>
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 5 | Activation of Hippo signaling upon amyloid-beta 42 (Aβ42) accumulation also activates c-Jun-amino-terminal kinase (JNK) signaling. (A–E) shows the
expression of embryonic lethal abnormal vision (ELAV; red), JNK signaling pathway reporter puc-lacZ (green, gray), and discs large (Dlg; blue) in eye discs from (A,A’)
glass multiple repeat (GMR)>Aβ42, (B,B’) GMR>Aβ42 +hpo,(C,C’) GMR>Aβ42 +hpoRNAi,(D,D’) GMR>Aβ42 +ykiRNAi , and (E,E’) GMR>Aβ42 +yki. For
comparison between genotypes, (A’–E’) shows puc-lacZ (Gray) levels. (F) A semiquantitative Western blot is presented to show phospho-JNK (p-JNK) levels in the
wild-type, GMR>Aβ42, GMR>Aβ42 +hpo, and GMR>Aβ42 +hpoRNAi background. The samples were loaded in the following sequence: Lane 1-Molecular weight
marker, Lane 2-Wild-type (Canton-S), Lane 3-GMR>Aβ42, Lane 4-GMR>Aβ42+hpo (gain-of-function), Lane 5-GMR>Aβ42+hpoRNAi (loss-of-function).
Alpha-tubulin is used as a loading control, and (F’) graph shows the quantification of p-JNK levels, which were calculated from a set of three (n=3) in wild-type and
other indicated genotypes from the Western blot (F). The p-values for estimation of p-JNK levels in all combination in a semiquantitative Western blot was calculated
in a set of three (n=3) using Student’s t-test in Microsoft Excel software. The p-value between wild-type and GMR>Aβ42 was significant (p<0.001; ***), wild-type
and GMR>Aβ42+hpo was significant (p<0.001; ***), and between wild-type (Canton-S) and GMR>Aβ42+hpoRNAi (loss-of-function) was significant
(p<0.001; ***). The p-value between GMR>Aβ42 and GMR>Aβ42+hpo was significant (p<0.001; ***) and between GMR>Aβ42 and GMR>Aβ42+hpoRNAi was
significant (p<0.01; **).
Aβ42+hepAct;Figures 6G,G) exhibits downregulation of diap1-
4.3-GFP reporter activity. However, when JNK signaling is
downregulated by misexpression of dominant-negative Bsk in
GMR>Aβ42 background (GMR>Aβ42+bskDN;Figures 6I,I)
or Puc (GMR>Aβ42+puc;Figures 5K,K), it results in
a strong upregulation of diap1-4.3-GFP reporter expression
in the GMR domain. This correlates with the adult eye
phenotypes where activating JNK signaling by misexpressing
activated Jun (GMR>Aβ42+junaspv7;Figure 5F) and Hep
(GMR>Aβ42+hepAct;Figure 6H) enhances the Aβ42-mediated
neurodegeneration, whereas downregulating JNK signaling by
misexpressing dominant negative Bsk (GMR>Aβ42+bskDN;
Figure 6J) and puc (GMR>Aβ42+puc;Figure 6L) rescues
the Aβ42-mediated neurodegeneration in adult eye. Thus,
modulating JNK signaling can (i) modulate neurodegeneration
phenotype of GMR>Aβ42 and (ii) also regulate Hippo signaling
as evident from the changes in diap1-4.3-GFP reporter expression
in the eye. Our data suggest that both Hippo and JNK can affect
each other in Aβ42-mediated neurodegeneration.
Hippo and c-Jun-Amino-Terminal Kinase
Signaling May Interact in Amyloid-Beta
42-Mediated Neurodegeneration
To explore the relationship between Hippo and JNK pathways
further, we used genetic epistasis. We sampled the effects at
two developmental stages of third instar eye-antennal imaginal
disc and the adult eye. In an Aβ42 background (GMR>
Aβ42), we activated JNK signaling and blocked Hpo signaling
at the same time by misexpression of activated hep and
yki (GMR>Aβ42+hepAct +yki). This resulted in stronger
neurodegeneration in the eye imaginal disc (Figure 7A) and
the adult eye (Figure 7B) as compared to GMR>Aβ42 alone.
These flies failed to hatch out of the pupal case and exhibited a
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 6 | Activation of c-Jun-amino-terminal kinase (JNK) signaling pathway in amyloid-beta 42 (Aβ42) background activates Hippo pathway. Eye imaginal discs
from larvae of indicated genotypes assessed for changes in expression of diap1-4.3-green fluorescent protein (GFP; green), a reporter for Hippo pathway, are shown.
All discs show expression of discs large (Dlg; red), diap1-4.3-GFP (green), and embryonic lethal abnormal vision (ELAV; blue). (A,A’) show wild-type control glass
multiple repeat (GMR)>diap1-4.3-GFP eyes discs and (B) wild-type adult eye phenotypes; and (C,C’) GMR>Aβ42 eye discs and (D) adult eye phenotype. (E–K)
shows effects of modulating JNK activity on Hippo pathway reporter diap1-4.3-GFP in eye-antennal imaginal disc of (E,E’) GMR>Aβ42 +junaspv7,(G,G’) GMR>
Aβ42 +hepAct,(I,I’) GMR>Aβ42 +bskDN ,(K,K’) GMR>Aβ42 +puc backgrounds. Note that (E,E’,G,G’) diap1-4.3-GFP exhibits robust induction when JNK
signaling is activated, whereas (I,I’,K,K’) diap1-4.3-GFP levels are significantly reduced when JNK signaling is downregulated in GMR>Aβ42 background. The adult
eye phenotypes associated with (D) GMR>Aβ42, (F) GMR>Aβ42+junaspv7,(H) GMR>Aβ42+hepAct ,(J) GMR>Aβ42+bskDN , and (L) GMR>Aβ42+puc. Note
that (J,L) downregulation of JNK signaling showed significant rescues in the adult eye phenotypes, whereas (F,H) activation of JNK signaling enhanced the
neurodegenerative phenotype of (D) GMR>Aβ42.
strong neurodegenerative phenotype. We also tested the effects
of activation of Hippo signaling and downregulation of JNK
signaling in the GMR>Aβ42 background by misexpressing
hpo and dominant negative bsk (GMR>Aβ42+hpo+bskDN).
This also resulted in strong neurodegeneration both in the eye
imaginal disc (Figure 7C) as well as the adult eye (Figure 7D).
These flies also failed to hatch out of the pupal case and were
dissected out from their pupal case. These data suggest that
increasing levels of either Hippo or JNK signaling pathways do
not compensate for the downregulation of the other. Therefore,
both pathways may act in a feed forward/feedback loop. We
further tested this hypothesis by blocking both Hippo and
JNK signaling pathways by misexpressing yki and bskDN , which
resulted in a rescue in the eye imaginal disc (Figure 7E) as well
as the adult eye (Figure 7F). These adults hatch out from the
pupal case, although they showed some black necrotic spots.
Our data suggest that the Hippo and JNK pathways interact
synergistically in a positive feedback loop during Aβ42-mediated
neurodegeneration (Figure 7G).
DISCUSSION
AD, an age-related progressive neurodegenerative disorder,
manifests by progressive neuronal loss, brain atrophy, and
cognitive impairments (Barnes and Yaffe, 2011). However, the
mechanism of neurodegeneration observed in AD and related
dementia (ADRD) has not been fully understood (Sarkar et al.,
2016; Deshpande et al., 2019). Accumulation of Aβ42 plaques
over a period of time triggers neuronal death due to the
induction of aberrant signaling in neurons (Hardy, 2009; Tare
et al., 2011). In addition, the causal genetic defects in AD are
not due to a single gene mutation but are thought to involve
genetic alterations that cause impairment of several signaling
pathways (Sarkar et al., 2016). Currently, identification and
characterization of downstream target(s) of aberrant signaling
induced by abnormally high levels of Aβ42 are a growing area
of research.
Several animal AD models have shown promise; however, our
Drosophila model allows us to test other signaling pathways using
genetic epistasis approaches (Pandey and Nichols, 2011; Lenz
et al., 2013; Sabbagh et al., 2013; Sarkar et al., 2016; Deshpande
et al., 2019). We previously reported the neuroprotective effects
of the apical basal polarity gene crumbs (crb) (Moran et al.,
2013; Steffensmeier et al., 2013) and the homeotic gene teashirt
(tsh) (Moran et al., 2013) in Aβ42-mediated neurodegeneration.
Interestingly, these genetic modifiers are members of the Hippo
signaling pathway. In the present study, we employed our
Drosophila eye model of AD to identify genetic modifiers
in a forward genetic screen using the molecularly defined
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
FIGURE 7 | Hippo and c-Jun-amino-terminal kinase (JNK) signaling act synergistically to induce amyloid-beta 42 (Aβ42)-mediated neurodegeneration. Panels show
eye discs stained with discs large (Dlg; green) and embryonic lethal abnormal vision (ELAV; red) and the resulting adult eye phenotypes when JNK or Hippo pathway is
blocked. (A,B) Activation of JNK signaling along with inactivation of Hippo signaling in the glass multiple repeat (GMR)>Aβ42 background (GMR>Aβ42+hepAct+yki)
does not rescue the neurodegenerative phenotype. (C,D) Activation of Hippo signaling along with inactivation of JNK signaling in the GMR>Aβ42 background
(GMR>Aβ42+hpo+BskDN) does not rescue the neurodegenerative phenotype. (E,F) Inactivation of both JNK signaling and Hippo signaling (GMR>Aβ42+yki +
BskDN ) together in the GMR>Aβ42 background exhibits significant rescue of the neurodegenerative phenotype. (G) A model that reconciles the data from this study,
which shows that Hippo signaling and JNK signaling act synergistically via a positive feedback loop to induce Aβ42-mediated neurodegeneration.
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Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
deficiencies set. We identified a deficiency Df(2R)BSC782/+,
which can rescue Aβ42-mediated neurodegeneration. This
deficiency uncovers hpo and other genes (Figure 1), and we
confirmed that hpo was the specific genetic modifier of Aβ42
phenotypes. These data strongly support a role of Hippo signaling
in Aβ42-mediated neurodegeneration.
Hippo Acts Downstream of Amyloid-Beta
42 Plaque
We analyzed accumulation of Aβ42 protein in differentiating
retinal neurons of eye imaginal disc from various genetic
backgrounds of GMR>Aβ42 alone as well as where Hippo levels
have been modulated. The rescue of the neurodegeneration
phenotype due to downregulation of Hippo signaling occurs by
a mechanism downstream of Aβ42 accumulation (Figure 2). We
tested the levels of diap1-4.3-GFP (Ren et al., 2010) and ex-lacZ
(Boedigheimer et al., 1993; Hamaratoglu et al., 2006), which serve
as the reporter for Hippo signaling, in GMR>Aβ42 background
(Figure 3). When Hippo signaling is activated, it triggers cell
death (Verghese et al., 2012), and diap1-4.3-GFP and ex-lacZ
levels are downregulated. We found that diap1-4.3-GFP and
ex-lacZ levels were downregulated in GMR>Aβ42 background,
which suggests that Hippo signaling is activated (Figure 3).
Furthermore, we found that cell death levels were increased in
GMR>Aβ42 background when Hippo levels were upregulated
and vice versa (Figure 4). Thus, our study suggests that Aβ42
plaques induce Hippo signaling to trigger neurodegeneration
(Figure 7G).
Activation of c-Jun-Amino-Terminal Kinase
Signaling
Previously, we and others have shown that JNK signaling
is activated in GMR>Aβ42 background. (Tare et al., 2011;
Sarkar et al., 2016). It is known that Hippo can regulate
JNK signaling (Ma et al., 2015, 2017). To understand if the
two pathways work independently or together in triggering
Aβ42-mediated neurodegeneration, we tested the reporters like
puc-lacZ, a reporter for JNK activity in our study (Martin-
Blanco et al., 1998). We found that JNK signaling is activated
during Aβ42-mediated neurodegeneration. Furthermore, when
we activate Hippo signaling in GMR>Aβ42 background, we
found that the reporters for JNK signaling were robustly activated
(Figure 5). We also tested the levels of p-JNK and found
that when Hippo pathway is downregulated, JNK signaling
is also reduced. Thus, it is possible that activation of Hippo
signaling can trigger activation of JNK signaling in GMR>
Aβ42 background.
Furthermore, we explored the converse relation. We tested
if JNK signaling can activate Hippo signaling in GMR>Aβ42
background (Figure 6). Interestingly, we found that activation
of JNK signaling in GMR>Aβ42 background further enhances
the neurodegenerative phenotype of GMR>Aβ42. Interestingly,
the reporter of Hippo signaling pathway, diap1-GFP, showed
robust activation where we activated JNK signaling in GMR>
Aβ42 background (Figure 6). This suggests that activation of
JNK signaling can further enhance the effects of Hippo activation
and vice versa. Also, it suggests that these two pathways can
in turn activate each other (Figures 5,6). We then explored
the roles of Hippo and JNK signaling in Aβ42-mediated
neurodegeneration to understand if these key pathways interact
or act independently.
Positive Feedback Loop of Hippo and
c-Jun-Amino-Terminal Kinase Signaling
Regulate Neuroprotective Function
We previously reported the neuroprotective effects of the
apical basal polarity gene crumbs (crb) (Moran et al., 2013;
Steffensmeier et al., 2013) and the homeotic gene teashirt
(tsh) (Moran et al., 2013), both members of the Hippo
signaling pathway. However, the neuroprotective function of
Hippo or JNK signaling interactions together has not been
fully understood. We tested if neuronal death observed in
Aβ42-mediated neurodegeneration uses both JNK and Hippo
signaling independently or in epistatic interactions to trigger
neurodegeneration. We employed classical genetic approaches
to determine this relation between two signaling pathways. We
found that if we activate JNK signaling and downregulate Hippo
signaling at the same time in GMR>Aβ42 background or vice
versa, it does not rescue neurodegenerative phenotypes both
in the third instar eye-antennal imaginal disc and the adult
eye (Figures 7A–D). This observation fits with our prior results
(Figures 5,6) that activation of one pathway can activate the
other. Finally, we found that blocking both Hippo signaling
and the JNK signaling at the same time exhibits a significant
rescue of the GMR>Aβ42-mediated neurodegeneration during
both developmental stages (Figures 7E,F). Based on our results,
we propose a model that accumulation of Aβ42 triggers
Hippo and JNK signaling (Figure 7G). There is ample evidence
for the involvement of JNK signaling in AD (Yarza et al.,
2015). We report a role for Hippo signaling in Aβ42-
mediated neurodegeneration. Furthermore, based on our genetic
interaction studies, we found that JNK and Hippo signaling
are involved in a positive feedback loop in Aβ42-mediated
neurodegeneration, and inactivation of both cascades rescues
the phenotype (Figure 7G). Both of these pathways are crucial
for normal development and in disease pathology. It will
be interesting to explore the therapeutic value of pathway
components in the future.
Novel Role of Hippo in Neuroprotection
A growing body of epidemiological evidence and molecular
investigations has shown some interesting links between cancer
and AD (Battaglia et al., 2019; Nudelman et al., 2019). For
example, several studies have shown that autophagy, ubiquitin
proteasome system, and cell death are common biological
hallmarks shared by AD and cancer (Nudelman et al., 2019).
Hippo signaling has been shown to regulate organ size growth,
cell proliferation, and cell death (Justice et al., 1995; Xu et al.,
1995; Tapon et al., 2002; Harvey et al., 2003; Jia et al., 2003;
Pantalacci et al., 2003; Wu et al., 2003; Lai et al., 2005; Kango-
Singh and Singh, 2009; Halder and Camargo, 2013; Snigdha
et al., 2019) and neural development (Wittkorn et al., 2015).
Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 March 2020 | Volume 8 | Article 117
Irwin et al. Role of Hippo Signaling in Alzheimer’s Disease
Recently, Hippo signaling was implicated in many disease models
where it plays a role in apoptosis, autophagy, regeneration, and
cell survival (Pfleger, 2017; Ma et al., 2019; Sahu and Mondal,
2019). Thus, it is interesting to find a role for Hippo pathway
in Aβ42-mediated neurodegeneration. Since several components
of the Hippo pathway are ubiquitously expressed in flies, it is
possible that Hippo signaling downregulation not only promotes
cell proliferation but also may be providing neuroprotection.
In case of neurons, which are postmitotic cells, the cell
survival or neuroprotective function is utilized. Activation of
MST1/2 has been associated with the progression of AD, where
amyloid precursor protein (APP) promotes the interaction of
transcription factor FOXO3a with MST1, triggering Bim (a
proapoptotic member of te Bcl-2 family)-mediated neuronal
death (Sanphui and Biswas, 2013). Recently, it has been shown
that the amyloid βprecursor proteins (AβPPs) like APLP1
and APLP2 can use YAP/TAZ, the mammalian orthologs of
Yki, as signal transducers (Orcholski et al., 2011). Improper
cleavage of amyloid precursor proteins (APP) by beta- and
gamma secretase produces amyloid-beta polypeptide(s), which
are prone to aggregation and are involved in AD. Dysfunction
of PP2ACα, a key member of the protein phosphatase family
that negatively regulates Hippo pathway, also results in AD-like
conditions (Liu et al., 2018). Hippo pathway is also implicated
in other neurodegenerative disorders, for example, MST1/2, the
mammalian orthologs of Hpo, play a key role in amyotrophic
lateral sclerosis (ALS). Loss of MST1 (e.g., in MST1 knockout
mice) shows increased motor neuron viability, delayed symptom
onset, and extended survival (Lee et al., 2013). Other examples
include Huntington disease (Mueller et al., 2018) and retinal
degeneration (Murakami et al., 2014). Thus, our findings on the
Hippo and JNK pathways open new avenues of research in the
AD field and may help find better targets for devising therapeutic
interventions in the future.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/supplementary material.
AUTHOR CONTRIBUTIONS
MI and MT performed the experiments and data analysis.
AdS, OP, NG, MR, and PD performed the experiments. MK-S
performed data analysis and manuscript writing. AS developed
the concept, performed data analysis and manuscript writing.
FUNDING
Confocal microscopy was supported by the core facility at
the University of Dayton. MI and MR are supported by
the Honors program of the University of Dayton. MT, OP,
NG, and PD are supported by the University of Dayton
graduate program. This work was supported by start-
up research funds from the University of Dayton, and a
subaward from NIH grant R01CA183991 (PI Nakano) to
MK-S. This work is supported by NIH1R15GM124654-
01 from NIH, Schuellein Chair Endowment Fund, and
STEM Catalyst Grant from the University of Dayton
to AmS.
ACKNOWLEDGMENTS
We thank Bloomington Drosophila Stock Center (BDSC) for
Drosophila strains and the Developmental Studies Hybridoma
Bank (DSHB) for antibodies. We also thank Justin Kumar, Y.
Henry Sun, and Kyung Ok Cho for gifts of fly strains and
antibodies and members of Singh and Kango-Singh lab for
critical comments on the manuscript.
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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.
Copyright © 2020 Irwin, Tare, Singh, Puli, Gogia, Riccetti, Deshpande, Kango-Singh
and Singh. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original author(s) and the copyright owner(s)
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 17 March 2020 | Volume 8 | Article 117
... 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. ...
... image was opened in the Fiji/ImageJ software and a region of interest was drawn. The split channel function was used and the number of TUNEL positive nuclei were counted manually in at least 5 discs of each genotype (Singh et al., 2005;Gogia et al., 2020;Irwin et al., 2020;Raj et al., 2020;Yeates et al., 2020). Average was calculated and used to plot graphs. ...
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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).
... The activated Hippo pathway could contribute to Aβ42induced neural cell death. 325 Additionally, in Fused in Sarcoma (FUS)-mediated ALS, activated Hippo participated in neuronal cell death by further activating c-JUN amino-terminal kinase (JNK). Cell death could be rescued by downregulating the Hippo pathway. ...
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As an evolutionarily conserved signalling network, the Hippo pathway plays a crucial role in the regulation of numerous biological processes. Thus, substantial efforts have been made to understand the upstream signals that influence the activity of the Hippo pathway, as well as its physiological functions, such as cell proliferation and differentiation, organ growth, embryogenesis, and tissue regeneration/wound healing. However, dysregulation of the Hippo pathway can cause a variety of diseases, including cancer, eye diseases, cardiac diseases, pulmonary diseases, renal diseases, hepatic diseases, and immune dysfunction. Therefore, therapeutic strategies that target dysregulated Hippo components might be promising approaches for the treatment of a wide spectrum of diseases. Here, we review the key components and upstream signals of the Hippo pathway, as well as the critical physiological functions controlled by the Hippo pathway. Additionally, diseases associated with alterations in the Hippo pathway and potential therapies targeting Hippo components will be discussed.
... The cholesterol metabolism pathway (hsa04979), Hippo signaling pathway (hsa04722), neurotrophin signaling pathway (hsa04392) TGF-beta signaling pathway (hsa04350), signaling pathways regulating pluripotency of stem cells (hsa04550), and Rap1 signaling pathway (hsa04015) were enriched robustly in subgroup III, which indicated that these pathways contributed to the deterioration of AD. In the past, several investigators have illustrated that cholesterol regulation is related to the severity of AD (Akram et al. 2010); the Hippo signaling pathway functions in Aβ42-mediated neurodegeneration in AD (Irwin et al. 2020); neurotrophic signaling deficiency aggravates environmental risks for AD pathogenesis (Wu et al. 2021); upregulation of the TGF-beta signaling pathway contributes to cell degeneration in AD; and Rap1 signaling modification is neuroprotective in AD (Dumbacher et al. 2018). However, the function of signaling pathways regulating the pluripotency of stem cells (hsa04550) has not been explored in AD. ...
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Alzheimer’s disease (AD) is a progressive cognitive disorder that occurs worldwide, and the lack of disease-modifying targets and pathways is a pressing issue. This study aimed to provide new targets and pathways by performing molecular subgroup classification. After normalizing the collected data, the subgroup number was confirmed with consensus clustering. Comparisons of clinical features among subgroups were conducted to clarify the clinical traits of each subgroup. Subgroup-specific genes were identified to perform weighted gene coexpression analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out. Next, gene set enrichment analysis (GSEA) was performed. Protein–protein interaction networks were built to screen core genes and in each subgroup to perform Spearman correlation analysis with clinical traits. Sequencing profiles of 1068 AD samples collected from 2 datasets were classified into 3 subgroups. Clinical comparisons revealed that patients in subgroup III tended to be younger, while their pathological grades were the most severe. WGCNA detected four gene modules, and the turquoise module, where the dopaminergic synapse pathway was enriched, was related to subgroup I. The neurotrophin signaling pathway and TGF-beta signaling pathway were robustly enriched in the blue and brown modules, respectively, in subgroup III. Moreover, 3 hub genes in subgroup I were negatively correlated with the sum of neurofibrillary tangle (Nft) density. Conversely, hub genes in subgroups II and III exhibited positive correlations with the sum of Nft density. These results provide new pathways and targets for AD treatment. Graphic Abstract
... As a member of the mitogen-activated protein kinase family, C-Jun amino terminal kinase (JNK) plays an important role in the regulation of stress, cell differentiation, and apoptosis [13]. In addition, it is shown to be activated in models of neuronal apoptosis under excitotoxicity, nutrient withdrawal, and ischemia inducements [14]. ...
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Chapter
RNA is an important connecting link between DNA and proteins. Levels of RNA within a cell or a tissue serve as the unique genetic signatures, which can help in correlating gene expression to the resultant phenotype(s) during development and disease. Transcriptomics is the study of all RNAs expressed/available in cells or tissues that allow study of (1) differences in gene expression patterns among various cell types or organs, (2) identify novel messenger RNAs and transcripts, and (3) study epigenetic changes within the transcriptome. This knowledge can be applied to human disease(s) by developing disease markers or to study developmental landmarks using biomarkers. In this chapter, we have highlighted the history of transcriptomic and genetic engineering, the available transcriptomic techniques to study various types of RNA, their analysis using gene ontology tools, and finally the utility of genetic engineering tools and model organisms in transcriptomics research.
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Background: Aging-related cognitive decline is an early symptom of Alzheimer's disease and other dementias, and on its own can have substantial consequences on an individual's ability to perform important everyday functions. Despite increasing interest in the potential roles of extracellular microRNAs (miRNAs) in central nervous system (CNS) pathologies, there has been little research on extracellular miRNAs in early stages of cognitive decline. We leverage the longitudinal Normative Aging Study (NAS) cohort to investigate associations between plasma miRNAs and cognitive function among cognitively normal men. Methods: This study includes data from up to 530 NAS participants (median age: 71.0 years) collected from 1996 to 2013, with a total of 1,331 person-visits (equal to 2,471 years of follow up). Global cognitive function was assessed using the Mini-Mental State Examination (MMSE). Plasma miRNAs were profiled using small RNA sequencing. Associations of expression of 381 miRNAs with current cognitive function and rate of change in cognitive function were assessed using linear regression (N = 457) and linear mixed models (N = 530), respectively. Results: In adjusted models, levels of 2 plasma miRNAs were associated with higher MMSE scores (p < 0.05). Expression of 33 plasma miRNAs was associated with rate of change in MMSE scores over time (p < 0.05). Enriched KEGG pathways for miRNAs associated with concurrent MMSE and MMSE trajectory included Hippo signaling and extracellular matrix-receptor interactions. Gene targets of miRNAs associated with MMSE trajectory were additionally associated with prion diseases and fatty acid biosynthesis. Conclusions: Circulating miRNAs were associated with both cross-sectional cognitive function and rate of change in cognitive function among cognitively normal men. Further research is needed to elucidate the potential functions of these miRNAs in the CNS and investigate relationships with other neurological outcomes.
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Chapter
A hallmark of development, aging, represents the changes in the body over a period of time. At cellular level, the basic unit of organization in multicellular organism, aging is associated with deterioration of cellular function(s) and replicative ability. These changes include accumulation of genetic damage, shortening of telomere length, metabolic waste and oxidative stress, altered protein synthesis/processing, impaired immune system, repair mechanisms, and epigenetic control. Such changes result in the loss of molecular, morphological, and physiological integrity of cells that ultimately affect the tissue(s) and organ(s). Aging results in progressive loss of organ function leading to diseases like diabetes, neurodegeneration, and cancer. In this chapter, we summarize the changes occurring at the cellular, tissue, and organ levels and discuss some of the models available to study aging and associated diseases. Additionally, we also briefly emphasize on some of the antiaging therapeutic strategies that help in reducing the morbidity from age-related diseases.
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Background: Neuronal cell cycle re-entry (CCR) is a mechanism, along with amyloid-β (Aβ) oligomers and hyperphosphorylated tau proteins, contributing to toxicity in Alzheimer's disease (AD). Objective: This study aimed to examine the putative factors in CCR based on evidence corroboration by combining meta-analysis and co-expression analysis of omic data. Methods: The differentially expressed genes (DEGs) and CCR-related modules were obtained through the differential analysis and co-expression of transcriptomic data, respectively. Differentially expressed microRNAs (DEmiRNAs) were extracted from the differential miRNA expression studies. The dysregulations of DEGs and DEmiRNAs as binary outcomes were independently analyzed by meta-analysis based on a random-effects model. The CCR-related modules were mapped to human protein-protein interaction databases to construct a network. The importance score of each node within the network was determined by the PageRank algorithm, and nodes that fit the pre-defined criteria were treated as putative CCR-related factors. Results: The meta-analysis identified 18,261 DEGs and 36 DEmiRNAs, including genes in the ubiquitination proteasome system, mitochondrial homeostasis, and CCR, and miRNAs associated with AD pathologies. The co-expression analysis identified 156 CCR-related modules to construct a protein-protein interaction network. Five genes, UBC, ESR1, EGFR, CUL3, and KRAS, were selected as putative CCR-related factors. Their functions suggested that the combined effects of cellular dyshomeostasis and receptors mediating Aβ toxicity from impaired ubiquitination proteasome system are involved in CCR. Conclusion: This study identified five genes as putative factors and revealed the significance of cellular dyshomeostasis in the CCR of AD.