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ISSN 20790597, Russian Journal of Genetics: Applied Research, 2014, Vol. 4, No. 2, pp. 113–121. © Pleiades Publishing, Ltd., 2014. Original Russian Text © O.I. Bolshakova, A.A. Zhuk, D.I. Rodin, G.A. Kislik, S.V. Sarantseva, 2013, published in Ekologicheskaya Genetika, 2013, Vol. 11, No. 1, pp. 23–31.

Effect of Human APP Gene Overexpression on Drosophila melanogaster Cholinergic and Dopaminergic Brain Neurons O. I. Bolshakova, A. A. Zhuk, D. I. Rodin, G. A. Kislik, and S. V. Sarantseva B.P. Konstantinov St. Petersburg Nuclear Physics Institute National Research Centre “Kurchatov Institute”, Gatchina, Russia email: [email protected]; [email protected] Received September 14, 2012; in final form, December 19, 2012

Abstract—We investigated the effects of overexpression of the human APP gene on the populations of cho linergic and dopaminergic brain neurons in the fruit fly, Drosophila melanogaster. The number of cholinergic neurons in the APP expressing young flies was the same as in the control and decreased significantly with age. The number of dopaminergic neurons in the APP expressing flies was significantly lower than in the control strain by the 15th day of life. Neurodegeneration was accompanied by deficiencies in memory and cognitive abilities in the flies overexpressing fulllength APP (APPSwedish), as well as in the strains with amyloidβ peptide production. Keywords: Alzeimer’s disease, Drosophila melanogaster, neurodegeneration, cholinergic neurons, dopamin ergic neurons, amyloidβ peptide DOI: 10.1134/S2079059714020026

Alzeimer’s disease (AD) is a neurodegenerative disease, which is the primary cause of dementia in the aging population and persons of advanced age. The early hypotheses for the etiology of AD proposed that the primary cause for the clinical manifestations of AD is the selective death of up to 70% of the cholin ergic neurons, predominantly, in the region of the brain defined as the nucleus basalis of Meynert (Whitehouse et al., 1982). This region in the base of the forebrain presents the main concentration of cho linergic neurons projecting towards the neocortex (Mesulam et al., 1983). However, recent studies showed that neural defects during AD involve many other neurotransmitter systems, such as the dopamin ergic, serotonin, glutamine, and adrenal synapses (Schaeffer et al., 2008; Chen et al., 2011; Sun et al., 2012). However, the genetic and molecular causes for the neurodegenerative changes during AD remain not well understood. Based on the amyloid cascade hypothesis, which to date is the preferred explanation for the etiology of AD, the accumulation of the soluble and insoluble forms of the amyloidβ (Aβ) in the brain tissues launches a cascade of pathological reactions, which lead to the death of neural cells, and, ultimately, to the clinical symptoms of dementia (Hardy and Selkoe, 2002). The Aβ is the product of proteolytical process ing of a large transmembrane protein, the Amyloid Protein Precursor (APP), by the β and γsecretases

(Thinakaran et al., 2008). Mutations in the APP gene lead to familial forms of AD. A number of studies point to alternative pathways, which can promote AD disease progression (Saura et al., 2004; Bonda et al., 2009; Mohandas et al., 2009; Pimplikar et al., 2010). It has been also reported that besides the Aβ, the fulllength APP, as well as its other processing products, can contribute to neurodegener ation (Kim et al., 2003; Stokin et al., 2008; S –l omnicki and Lesniak, 2008; Sarantseva et al., 2009). In most of the laboratory AD models, based on mammalian systems, overexpression of the APP leads to increased levels of Aβ and accumulation of the Aβ oligomers, which can promote the neurodegeneration and cognitive defects (Walsh and Selkoe, 2004). Although expression of the APP and accumulation of the Aβ oligomers in transgenic animals cannot com pletely recapitulate all clinical aspects of human dis ease, these models can provide real possibilities to investigate the genetic causes and the cellular neuro pathological defects associated with AD. In particular, these models can aid in our understanding of the con tributions of the full length APP and the Aβ towards familial forms of AD. In this regard, the Drosophila melanogaster system provides a number of advantages as it allows to differentiate between the effects of the fulllength APP and the Aβ. First, the Appl gene, the APP ortholog in Drosophila, does not contain the region that encodes the Aβ peptide. Second, Droso phila contains all the components of the enzymatic

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complexes which are necessary for the γsecretase activity, but lacks, or has negligible activity, of the β secretase (BACE). Therefore, two transgenes are neces sary to generate the Aβ in the Drosophila system: one, to produce the βsecretase, and another, to express the fulllength human APP. This allows us to distinguish the effects of the APP and Aβ by comparing the strains carrying the single APP transgene and the strains transformed with both APP and BACE. Notably, deter mination of the specific cytotoxic effects caused by the Aβ and the APP is extremely important for elucidation of the etiology of AD and development of pharmaceu tical treatments for the disease. MATERIALS AND METHODS Drosophila Strains Used in the Study In order to investigate the degeneration of the cho linergic and dopaminergic neurons, we used the fol lowing transgenic Drosophila strains: the UAS–APP (further, APP), which carries the human APP gene; the UAS–APP–Swedish (further (APP–Sw), which carries the Swedish mutation in the APP, linked to familial AD; and the UAS–BACE (further, BACE), expressing the human βsecretase gene. Transgene expression was achieved using the UAS–GAL4 system (Brand and Perrimon, 1993). Transgene expression was induced using the Gal4–Cha–UAS–GFP (further, Cha) driver, which promotes expression of genes regu lated by the UAS in the cholinergic neurons, and the ple–GAL4 (further, ple), which targets transgene expression in dopaminergic neurons. Dopaminergic neurons were visualized using the UAS–CD8–GFP (further, CD8) reporter strain. All strains were obtained from the Drosophila Bloomington Stock Center (United States). Flies were maintained on standard yeast feeding media at 29°C and a 12hour daylight schedule. Preparation of Samples for Confocal Microscopy For the preparation of the samples for confocal microscopy, the flies were placed in phosphate buffer saline (PBS), then their heads were separated, treated with octane, and fixed in 4% paraformaldehyde (PFA) for 7 min at room temperature. The samples were rinsed in PBS and their brains were dissected and placed in a well on a microscopy slide in a 1 : 1 PBS– glycerin medium. Acquisition and Analysis of Confocal Images Acquisition of confocal images was carried out using the Leica TC5 SP5 scope with a 35MBT argonne laser. All images were taken at 488 nm, and scanned under the same settings. Zstacks were scanned at 1 μM. The intensity of the fluorescence was measured using the ImageJ software (version 1.38a for

Windows). Quantitation of dopaminergic neurons was carried out with ImageJ according to the described method (Botella et al., 2008). We analyzed six Droso phila brains for each experiment, which were repeated in triplicates, for each cross and age group. Analysis of Learning and Memory Ability of Transgenic Flies The fly cognitive test for the ability to remember and distinguish specific odorants was carried out according to the method developed by Tully and Quinn (Tully and Quinn,1985). The assay is based on the fly’s ability to develop conditioned reflexes. Dur ing the learning phase of the experiment, 50–70 flies were placed in a test tube laced with a metal mesh. Then, the test odorants were supplied with air pumped through the tube. We used two strong odorants 3'octanol and 4'methylcyclohexanol. During the experiment, one odorant was pumped for 1 min, during which the flies received weak electric shocks, followed by 1 min of the flow of another odorant with no electric shocks. Then the flies were placed in a Tshaped tube at the selected point in between the flows of two odorants. Immedi ately after training, the learning ability of flies was measured as possibility of choice between 3'octanol and 4methylcyclohexanol for 120 s. The memory test was carried out the same way 1 hour after learning. The learning and memory index was calculated by sub tracting the number of flies that made the wrong choice from the number of flies that made the right choice, divided by the total number of flies and multi plied by 100. Statistical Analysis of the Data The significance of the differences between the experimental and control groups was evaluated using the oneway analysis of variance method (ANOVA) and the Tukye–Kramer multivariate comparison method, with the KyPlot software (Kyens Lab Inc., Japan). The differences were considered significant if p < 0.05. RESULTS Analysis of Neurodegeneration of the Cholinergic Neurons Confocal analysis of the cholinergic neurons was carried out in the flies’ offspring, obtained from crosses between the Cha and the APP, APP–Sw, and BACE flies and APP–Sw on the 3rd–5th, 15th–17th, and 29th–30th days of life by the confocal micro scopic method. Acetylcholine is one of the major syn aptic transmitters in the Drosophila’s central nervous system. Consistently, we detected a very high density of fluorescent signals throughout the brain and eye regions in the control Cha strain. Due to the high sig

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Fig. 1. Images of the Drosophila melanogaster’s brain, with the expression of APP in cholinergic neurons in the different age and genotypic groups. 1, Antennal lobes; 2, central complex; 3, intermediate neurons; 4, mushroom body axons; 5, internal neurons of the mushroom body (Kenyon cells); and 6, lamina neurons. a, Cha/+; b, Cha/APP; c, Cha/+; APPSw/+; d, Cha/BACE; APPSw/+.

nal levels, it was not possible, in this case, to quantitate individual cholinergic neurons. Instead, we measured the overall intensity of the fluorescent signal through out the brain (see methods for detail). Figure 1 shows the images of the fly’s brains with the expression of APP in the cholinergic neurons at different ages and depending on the genotype. Fluo rescent signals were detected in the same brain struc tures in all samples: in the antennal lobes, the central region, the intermediate neurons adjacent to the antennal lobes, and in the mushroom bodies and

Kenyon cells. In Drosophila, these brain regions are responsible for perception of external signals, locomo tion, cognition, and memory. Fluorescence levels sig nificantly decreased in all brain structures by the 30th day of the fly’s life. However, different brain structures were significantly affected in the control and transgenic flies. In the control Cha/+ strain, the largest agedependent decrease in fluorescent inten sity was localized in the central ganglia and intermedi ate neurons adjacent to the antennal lobes. In con trast, old flies’ brains expressing APP, APPSw, or APPSw and BACE, cholinergic neurons were lost pre


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Fig. 2. Effects of the expression of the APP and BACE genes on the brain’s cholinergic neurons. The graphs show the relative levels of fluorescent intensity of the fly’s brain samples of different ages (a–c) and genotypes (1–4). a, At 3–5 days of age; b, at 15– 17 days of age; c, at 27–30 days of age; 1, Cha/+; 2, Cha/APP; 3, Cha/+; APPSw/+; and 4, Cha/BACE; APPSw/+. * Indicates statistically significant results (p < 0.05).

dominantly in the antennal lobes and the mushroom bodies. Figure 2 presents graphic diagrams summarizing our data for the fluorescent signal readout in the Drosophila cholinergic neurons on days 3–5, 15–17, and 29–30, in the control Cha/+ strain, and in exper imental strains, expressing APP, APPSw, and APPSw and BACE. In the control strain, fluorescent signal remained the same in the brains at all examined ages. In young flies (3–5 days), fluorescent intensity is the same in the control and experimental samples. On days 15–17, the signal is reduced in the strains expressing APP and drops sharply on days 29–30.

There was no significant difference between the strains, expressing full length APP and the strain pro ducing the Aβ peptide. Analysis of Neurodegeneration of the Dopaminergic Neurons To visualize Drosophila dopaminergic neurons, we used the CD8 transgene that expresses a membrane tagged GFP in neural cells. Figure 3 presents images of the Drosophila’s brains expressing APP at various ages and genotypes. The dopaminergic neurons form small clusters located bilaterally throughout Drosophila brain (Fig. 3).


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Fig. 3. Images of the Drosophila melanogaster’s brain, with expression of the APP in dopaminergic neurons in the different age and genotypic groups. a, CD8/+; ple/+; b, CD8/+; APP/+; ple/+; c, CD8/+; APP/BACE; ple/+; d, CD8/+; APPSw/ple; and e, CD8/+; BACE/+; APPSw/ple.

We carried out automated quantitation of the dopaminergic neurons on days 3–5, 15–17 and 27– 30, using the ImageJ software. The results are pre sented in Fig. 4. The number of dopaminergic neurons in the control strain remains practically constant throughout the experiment. In contrast, the number of neurons is significantly reduced in the strains express ing APP, starting on days 15–17. There was no statis tically significant difference between the strains where neuronal death was caused by the full length APP and

stains that produced Aβ. Therefore, our results indi cate that APPdependent death of cholinergic and dopaminergic neurons can occur in Drosophila’s brains without the formation of the Aβ peptide. Analysis of Memory and Learning Studies of the degenerative changes in AD patients indicate that the level of cognitive dysfunction during AD is correlated to the level of the loss of cholinergic


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Fig. 4. Effects of expression of the APP and BACE genes on the brain’s dopaminergic neurons. a, Number of dopaminergic neu rons in flies on days 5–7 of age. b, Number of dopaminergic neurons in flies on days 15–17 of age. c, Number of dopaminergic neurons in flies on days 27–30 of age. 1, CD8/+; ple/+; 2, CD8/+; APP/+; ple/+; 3, CD8/+; APP/BACE; ple/+; 4, CD8/+; APPSw/ple; and 5, CD8/+; BACE/+; APPSw/ple. * denotes statistically significant results (p < 0.05).

neurons (Francis et al., 1999; Kart et al., 2004). Therefore, we examined the cognitive and memory abilities of transgenic flies on days 2–5 and 15–17 of their lives in parallel with the evaluation of the loss of the cholinergic and dopaminergic neurons. In our assay, the fly’s learning abilities were measured using the learning index and the memory index reflected animals’ memory function. The indices were calcu lated as described in the Methods section. Tables 1 and 2 present the results of the learning and memory tests. We found that fly’s capabilities for learning and mem ory in all transgenic strains were significantly lower by the 2nd day of life compared to the control strain. The

same results were obtained for strains expressing full length APP (APP or APPSw) and for flies producing the Aβ peptide. DISCUSSION Dysfunction of the cholinergic system is observed in AD patients even at the early stages of the disease (Whitehouse et al., 1982); it is manifested in a 50–70% decline in the activity of the choline acetyltransferase (ChAT), the major enzyme in the acetylcholine syn thesis pathway (Davies, 1979), and in the decrease in the density of the nicotinic acetylcholine receptors


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EFFECT OF HUMAN APP GENE OVEREXPRESSION Table 1. Effects of the expression of APP and BACE on the learning abilities of transgenic flies Age Genotype Cha/+* Cha/APP Cha/+; APPSw/+ Cha/BACE; APPSw/+ ple/+* APP/+; ple/+, APP/BACE; ple/+ APPSw/ple BACE/+; APPSw/ple

3–5 days

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15.6 ± 1.8 4.8 ± 2.6 5.6 ± 1.04 0.1 ± 1.23 12.2 ± 0.2 3.2.0 ± 0.6 1.0 ± 1.1 3.9 ± 1.3 0.25 ± 3.5

18.1 ± 0.5 2.6 ± 1.1 3.4 ± 1.4 2.5 ± 0.6 15.5 ± 2.2 1.8 ± 0.9 1.5 ± 0.6 4.3 ± 1.1 4.0 ± 1.7

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Table 2. Effects of the APP and BACE expression on the memory functions of transgenic flies Age Genotype Cha/+* Cha/APP Cha/+; APPSw/+ Cha/BACE; APPSw/+ ple/+* APP/+; ple/+, APP/BACE; ple/+ APPSw/ple BACE/+; APPSw/ple

3–5 days

15–17days

18.6 ± 2.4 4.1 ± 2.4 8.5 ± 1.3 4.4 ± 2.5 11.3 ± 2.2 6.0 ± 0.9 2.0 ± 6.6 1.25 ± 4.0 1.4 ± 1.7

16.8 ± 0.8 0.8 ± 0.2 2.6 ± 1.2 2.8 ± 1.3 12.6 ± 1.3 1.5 ± 0.6 1.3 ± 0.9 1.4 ± 0.8 1.6 ± 0.7

The table presents cognitive indices calculated as described in Materials and Methods. * Indicates the control strains. Statistical analysis was carried out using the oneway ANOVA algorithm and the Tukey–Kramer test. Experimental indices that differ signifi cantly from the control strain are shown in bold (p < 0.05).

The table presents memory indices, calculated as described in Materials and Methods. * Indicates the control strains. Statistical analysis was carried out using the oneway ANOVA algorithm and the Tukey–Kramer test. Experimental indices that differ signifi cantly from the control strain are shown in bold (p < 0.05).

(nAChR) (Guan et al., 2000). These changes are attributed to the degeneration of cholinergic neurons in specific regions of the brain. However, it has been shown that particular types of the nAChR are involved in the regulation of the dopaminergic system (Perry et al., 1995; Exley et al., 2012). Therefore, loss of these cholinergic neurons can lead to the degeneration of dopaminergic neurons during AD. Loss of dopamin ergic neurons in the substantia nigra is associated with Parkinson’s disease symptoms that are present in more than a third of AD patients (Kazee et al., 1995; Burns et al., 2005). It has been proposed that formation of the Aβ peptide plays an important role in the death and dysfunction of the cholinergic and dopaminergic neurons during AD (Perez et al., 2005).

think that the sharp drop in cognitive and memory functions at these stages point to earlier events, such as the loss of synapses. Notably, the post mortem exami nations of AD patients showed that loss of the synapses preceded the decrease in the ChaT levels (Tiraboschi et al., 2000). Although the cellular functions of the APP are still not clear, their fundamental role in the formation and maintenance of neuronal synapses is undisputed (Müller and Zheng, 2012). We suggest that the basis for the neurodegenerative changes described in our study is the disturbance in the synaptic function of the APP. Our results indicate that neurodegenera tion processes in animals’ brains and the decrease in learning abilities and memory are linked specifically to the expression of the fulllength APP (or APPSw). Many studies in Drosophila melanogaster (Iijima– Ando and Iijima, 2010) have reported neurocytotoxic effects of the Aβ. However, we did not detect a signifi cant effect of this molecule in the amplification of the investigated neuropathologies, possibly due to the moderate synthesis of Aβ in our model system.

In this study, we investigate the role of the APP gene in neurodegeneration using transgenic Drosophila melanogaster strains, which allow us to differentiate between the effects of the fulllength exogenous APP and the Aβ amyloid peptide. Expression of the APP or APPSw, or coexpression of these transgenes with BACE, leads to similar pathological phenotypes, man ifested by a significant degeneration of the cholinergic brain neurons by 30 days of age and dopaminergic neurons already by 15 days of age. The death of the neurons was particularly extensive in the strains expressing fulllength APP. Production of the Aβ amyloid also resulted in neuronal degeneration, but its levels were not statistically different from the strains expressing the fulllength APP or the mutant APPSw. The decrease in the number of cholinergic and dopaminergic neurons was accompanied by a dra matic decline in the fly’s learning abilities and mem ory. Since significant neurodegenerative effects were registered starting on the 15th and 30th days of life; we

Our results permit us to evaluate the contribution of the potentially abnormal functions of a mutant APP to the progression of AD. During expression of the human APP in the Drosophila’s neural cells, its protein product is transported into the presynaptic neuronal terminals and the neuromuscular junctions (Yagi et al., 2000) competing with the endogenous Appl product, the Drosophila homolog of the APP. Notably, the Appl is expressed predominantly in the Droso phila’s nervous system and plays a key role in the for mation and maintenance of synaptic contacts. Inacti vation of the Appl does not lead to lethality, but results in multiple behavioral changes (Luo et al., 1992). Loss of the Appl also resulted in a decrease in the number


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and size of synaptic buttons in the neuromuscular junctions in the Drosphila larvae, whereas overexpres sion leads to an increase in the number of buttons (Torroja et al., 1999). Since the APP and Appl are inte gral components of the cellular cytoskeleton (Li et al., 2004), it is quite possible that a mutation in the APP or overexpression of a wild APP type will result in cytosk eletal defects and degeneration of the synaptic com partment and, consequently, a decrease in the number of functional synaptic contacts. Furthermore, the competition between the Appl and APP can inhibit the expression of other synaptic genes through the protein kinase Gdependent mechanism (Claasen et al., 2009). In turn, the products of proteolytic cleavage of the APP also can affect transcription of the Drosophila synapse genes (Müller et al., 2007). For instance, the cytoplasmic fragment of the APP (the APP’s intracel lular domain, AICD), generated during proteolysis of the APP by the γsecretase, can translocate to the nucleus and function to regulate gene expression, syn aptic plasticity and memory (Cao and Sudhof, 2001). As a transcriptional regulator, AICD can affect chro matin remodeling by binding to the histone acetyl transferase Tip60 (Cao and Sudhof, 2001, 2004). Interestingly, transgenic mice overexpressing AICD also develop neural pathologies consistent with AD, including hyperphosphorylation of the Tau protein, neurodegeneration, and memory dysfunction (Ghosal et al., 2009). This points to the importance of consid ering the effects of multiple APP derivatives on the eti ology and pathogenesis of AD. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research, grant no. 090400647a. REFERENCES Bonda, J.D., Wang, X., GustawRothenberg, K., et al., Mitochondrial drugs for Alzheimer disease, Pharma ceuticals, 2009, vol. 2, pp. 287–298. Botella, J., Bayersdorfer, F., and Schneuwly, S., Superoxide dismutase overexpression protects dopaminergic neu rons in a Drosophila model of Parkinson’s disease, Neu robiol. Dis., 2008, vol. 30, pp. 75–73. Brand, A.H. and Perrimon, N., Targeted gene expression as a means of altering cell fates and generating dominant phenotypes, Development, 1993, vol. 118, pp. 401–415. Burns, J.M., Galvin, J.E., Roe, C.M., et al., The pathology of the substantia nigra in Alzheimer disease with extrapyramidal signs, Neurology, 2005, vol. 64, pp. 1397–1403. Cao, X. and Südhof, T.C., A transcriptionally active com plex of APP with Fe65 and histone acetyltransferase Tip60, Science, 2001, vol. 293, pp. 115–120. Cao, X. and Südhof, T.C., Dissection of amyloidbeta pre cursor proteindependent transcriptional transactiva tion, J. Biol. Chem., 2004, vol. 279, pp. 24601–24611.

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