Morphological abnormalities in <i>Drosophila</i> with overexpression of human APP gene

doi:10.4236/ojas.2013.34A2006

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Vol.3, No.4B, 49-52 (2013) Open Journal of Animal Sciences

http://dx.doi.org/10.4236/ojas.2013.34A2006

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: [email protected]

Received 16 August 2013; revised 22 September 2013; accepted 5 October 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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

Abnormalities

1. INTRODUCTION

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. MATERIALS AND METHODS

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

Microscopy

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

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.

3. RESULTS

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 sophila’s 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 Drosophila’s development

while its coexpressing with BACE results in A-beta pro-

duction and deposition as shown in our previous study

[8].

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

mouthparts.

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

pseudotracheae.

logical abnormalities, because of inability to consume

food.

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-

ties.

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-

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.

4. DISCUSSION AND CONCLUSIONS

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

D. Rodin et al. / Open Journal of Animal Sciences 3 (2013) 49-5 2

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.

OPEN A CCESS

5. ACKNOWLEDGEMENTS

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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