Vol.3, No.4B, 49-52 (2013) Open Journal of Animal Sciences
Morphological abnormalities in Drosophila with
overexpression of human APP gene
Dmitry Rodin*, Olga Bolshakova, Galina Kislik, Svetlana Sarantseva
Molecular and Radiation Biophysics Department, National Research Centre “Kurchatov Institute”-B.P. Konstantinov Petersburg
Nuclear Physics Institute, Gatchina, Russia; *Corresponding Author: nomadkml@me.com
Received 16 August 2013; revised 22 September 2013; accepted 5 October 2013
Copyright © 2013 Dmitry Rodin et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Alzheimer ’s disease (AD) is the leading and one
of the most severe forms of dementia. Molecular
mechanisms underlying AD pathogenesis de-
spite much work on this subject still remain un-
clear. Cleavage of amyloid precursor protein
(APP) to amyloid beta peptide (A-beta) and fol-
lowing formation of amyloid plaques are the key
events of Alzheimer’s pathology. Thus changes
in APP expression and metabolism can lead to
patholog y development. Here we show that over-
expression of human APP in Drosophila neural
cells manifests in different morphological ab-
normalities of Drosophila imago that can be
observed immediately after fly eclosion. This
observation can help to further understand APP
molecular functions and its participation in dif-
ferent molecular pathways.
Keywords: Alzheimer’s Disease; Drosophila;
Amyloid Precursor Protein; Morphological
A-beta is a major component of one of the pathomor-
phological hallmarks of AD—senile plaques. According
to the amyloid cascade hypothesis, increased A-beta pro-
duction is the key event that triggers a cascade of reac-
tions leading to neurodegeneration and AD [1]. However,
neurodegeneration level and patient’s condition don’t
always correlate with the amount of amyloid deposits in
the patient’s brain, which possibly means that there are
alternative mechanisms leading to neurodegeneration and
disease development [2,3]. Amyloid precursor protein
(APP) is an integral membrane protein that can be in-
volved in one of such mechanisms. Overexpression of
APP or its malfunction can possibly manifest in neuron-
pathology development independently of A-beta [4].
A-beta is a product of APP cleavage and that is why in
many studies APP is considered only as a source of A-
beta. At the same time its functions remain unclear. There
are evidences proclaiming APP participation in processes
of neurodegeneration [4], cell adhesion [5], cell signaling
[6], chromatin condensation [7] etc.
Earlier we showed that APP overexpression in Dro-
sophila brain leads to different pathological changes that
manifest in vacuolization of neuronal tissues [8], degen-
eration of cholinergic and dopaminergic neurons and
disruption of cognitive functions [9].
This study is dedicated to different morphological ab-
normalities of Dros ophila imago caused by overexpres-
sion of human APP in nerve cells.
2.1. Drosophila Lines
The following lines were used: UAS-APP carrying
human APP695 (obtained from Drosophila Bloomington
Stock Center), UAS-APP-Swedish carrying human
APP695 with mutation that leads to familial form of dis-
ease (obtained from Drosophila Bloomington Stock
Center), UAS-BACE carrying human beta-secretase
(kindly provided by R. Reifegerste) APP and APP-
Swedish were expressed in Drosophila neurons using
tissue specific transcription driver elav-GAL4c155.
Flies were kept on standard yeast medium and on me-
dium containing molasses with a higher calorie content
at a temperature of 25˚C and a photoperiod of 12 h.
2.2. Preparation of Specimens for Light
Light microscopy analysis was performed in order to
study morphological changes of wings, abdomen and
heads in flies with human APP overexpression.
Morphological changes in fly heads were analyzed on
Copyright © 2013 SciRes. OPEN ACCESS
D. Rodin et al. / Open Journal of Animal Sciences 3 (2013) 49-5 2
previously prepared specimens: 1d old flies with re-
moved wings were cleared in 10% KOH at 95˚C for 30
minutes, then fly heads were separated and incubated in
9:1 mix of glycerol and 1 M Tris HCl pH 7.5 overnight
2.3. Statistics
KyPlot software (KyensLab Inc) was used for statistic-
cal analysis of obtained data. One-way ANOVA was fol-
lowed by planned multiple comparisons between rele-
vant groups with Tukey-Kramer test.
APP processing that leads to A-beta production in-
volves cleavage of the protein with β- and γ-secretases.
Drosophila has no active β-secretase (BACE) or its ac-
tivity is minimal [10,11] and Dro sophilas APP ortholog
—APPL—has no A-beta sequence [12,13]. Thus expres-
sion of human APP in Drosophila allows estimating the
effect of protein itself on Drosophilas development
while its coexpressing with BACE results in A-beta pro-
duction and deposition as shown in our previous study
To investigate the effect of APP we expressed human
APP695 gene and its mutant form APP-Swedish that
leads to familial form of disease in brain cells of Droso-
phila melanogaster.
Expression of APP and APP-Swedish separately or
together with BACE in Drosophila nerve cells resulted in
abnormal development of fly imago, which manifested in
black melanized spots on proboscis and abdomen, pro-
boscis and mouthparts deformation, crumpled wings and
abdomens swelled with hemolymph (Figur e 1).
These flies died within 1 - 3 days of eclosion. De-
formed proboscis and mouthparts (Figure 2) could be a
cause of increased mortality rate of flies with morpho-
Figure 1. Morphological abnormalities in flies expressing hu-
man APP. (a) APP expression causes dramatical deformation
and necrosis of proboscis and mouthparts, crumpled wings and
swelled abdomen; (b) Control flies (elav; +; + ) have no mor-
phological abnormalities. 1-wings, 2-abdomen, 3-proboscis and
Figure 2. Proboscis and mouthparts deforma-
tion in flies expressing APP. (a) Control flies
elav; +; + have no morphological abnormalities;
(b) APP expression causes dramatical deforma-
tion of proboscis and mouthparts. 1-proboscis;
2-labrum; 3-maxillary pulps; 4-labellum and
logical abnormalities, because of inability to consume
We analyzed morphological abnormality frequency in
flies within different genotypes cultivated on standard
yeast medium as well as on yeast medium containing
molasses with a higher calorie content to investigate whe-
ther calorie content can impact the frequency of mor-
phological abnormalities (Figure 3).
Morphological abnormalities frequency strongly de-
pended on a genotype. We observed more morphological
abnormalities in flies expressing only APP. There was a
significant drop in number of morphological abnormali-
ties both in flies expressing APP-Sw and APP together
with BACE. Flies expressing both APP-Sw and BACE
had significantly minimal number of morphological ab-
normalities. Medium containing molasses did not have
any effect on the number of morphological abnormali-
We suggest that the significant drop of morphological
abnormality number in flies expressing both APP and
BACE is due to the reduced level of APP in Drosophila
nerve cells after the APP cleavage with BACE. We sug-
gest that the smaller number of morphological abnor-
malities in flies expressing APP-Swedish is due to faster
processing of the precursor.
We also measured pupal survival rate in transgenic
flies (Figure 4) APP expression resulted in higher mor-
tality of pupae, which indicates defects at the very early
stage of Drosophila development. At the same time flies
expressing APP-Swedish or APP and APP-Swedish to-
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D. Rodin et al. / Open Journal of Animal Sciences 3 (2013) 49-5 2 51
Figure 3. Morphological abnormalities frequency (%) 1-elav;
APP; 2-elav; APP + molasses, 3-elav; APP-SW, 4-elav; APP-
SW + molasses, 5-elav; BACE/APP, 6-elav; BACE/APP + mo-
lasses, 7-elav; BACE; APPSW, 8-elav; BACE; APP-SW + mo-
lasses. 1000 flies of every genotype were inspected. Asterisks
indicate significant differences from elav; APP flies.
Figure 4. Pupal survival rate according to genotype (%) 1-elav;
APP, 2-elav; APP-SW, 3-elav; BACE/APP, 4-elav; BACE;
APPSW. 1000 pupae of every genotype were inspected. Aster-
isks indicate significant differences from elav; APP flies.
gether with BACE had significantly better survival rate
comparing to flies expressing only APP.
Our data show that APP expression itself in nerve cells
leads to severe pathological changes in Drosophila de-
velopment that manifest not only in fly brain but also
phenotypically as different morphological abnormalities.
Chakraborty et al. used the same transgenic fly lines to
study developmental defects caused by APP expression
[14]. Surprisingly he concluded that phonotypical changes
described above are directly associated with production
and deposition of A-beta in fly brains. Chakraborty et al.
suggested that these abnormalities are caused by in-
flammatory response to A-beta deposits, which is con-
troversial to our current data. In our study we showed
that morphological abnormality frequency drops signifi-
cantly in flies expressing A-beta comparing to flies ex-
pressing only full length APP or APP-Swedish. The high
level of mortality in flies expressing APP or APP-Swed-
ish (data not shown) and pupal survival rates confirm
that it’s not the A-beta factor but the expression of these
genes leads to severe defects in fly development.
Such developmental defects can indeed be due to ac-
tive immune response in transgenic flies. However, we
suggest that this immune response is not activated by
A-beta deposits but by full-length APP.
Green et al. observed similar morphological abnor-
malities—black melanized spots, abdomen swelled with
hemolymph and high mortality rate (but not crumpled
wings)—in flies with constantly activated Toll-mediated
immune response [15].
Toll-like receptors (TLR) are a part of innate immune
system. Their main function is to recognize conserved
molecules of different microorganisms. Activating of
Toll-mediated immune response results in activation of
well-known transcription factors of NF-κB family. In
case of Drosophila it activates transcription factors Dif
and Dorsal. Grilli et al. identified two identical se-
quences in the 5’-regulatory region of APP, which are
specific binding sites for transcription factors of NF-κB
family. They also showed that activity of these sites cor-
relates with APP expression [16].
Thus APP expression in flies can result in activation of
NF-κB family protein, which in turn can lead to APP
upregulation creating a loop in which expression of both
participants is co-dependent.
Stante et al. showed that full length APP is necessary
for activation of the FE65 protein and its translocation to
the nucleus [17]. FE65 is essential for the recruitment of
acetyltransferase TIP60 that plays a crucial role in DNA
strand repair. They also showed that Fe65 suppression
leads to a significant degree of chromatin de-condensa-
tion, which indicates that FE65 is involved in chromatin
compaction. As activation of FE65 is dependent on in-
teraction with APP, high levels of APP can lead to chro-
matin destabilization. However, it doesn’t explain crum-
pled wings phenotype. We suggest that this phenotype
can be due to competition between APP and its Droso-
phila ortholog APPL. Li et al. showed that APPL is nec-
essary for the development of nonneural tissues such as
wings and cuticles [18]. As APPL is expressed in the
nervous system, crumpled wings phenotype can result
from neuroendocrine dysfunction due to competition
between APP and APPL.
Thus APP upregulation can negatively affect genomic
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D. Rodin et al. / Open Journal of Animal Sciences 3 (2013) 49-5 2
Copyright © 2013 SciRes.
stability and DNA repair processes as well as tissue de-
velopment. There are many evidences showing that APP
is overexpressed in different cancer types [19,20]. Ge-
nomic destabilization and tissue development defects
resulted from APP overexpression can lead to morpho-
logical abnormalities in observed in transgenic flies.
The project is supported by the Russian Foundation for Basic
Research (grant No. 12-04-00898). The authors sincerely thank R.
Reifegerste for providing BACE Drosophila line.
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