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Proof of Concept

Introduction

Our Drosophila model is designed to screen drugs with potential effects on neurodegenerative diseases such as Alzheimer's disease. We transferred the full-length human APP 596 gene into Drosophila, which is believed to be the main cause of AD. In order to prove the success of this model, we chose chloroquine and examined its role as a potential drug to suppress AD symptoms.

In our experiments, we constructed the UAS-APP-Myc plasmid and successfully obtained transgenic flies through the GAL4-UAS system. Drosophila carrying the human APP gene exhibited symptoms of decreased learning and memory and impaired courtship selection ability, similar to the symptoms of human Alzheimer's. We hope that in the future, this fruit fly screening model can take advantage of the short growth cycle, large number of progeny, clear phenotype and anatomical structure, and known complete genome sequence of fruit flies, and provide assistance for early large-scale drug screening in the future. Let researchers in related fields or drug research and development companies quickly screen drugs with AD treatment potential. And this point was also recognized by the experts in our HP expert interviews.

In order to verify our hypothesis, we searched the literature and selected chloroquine as a potential drug for the treatment of AD to test our model. Chloroquine, is an antimalarial drug and an inhibitor of autophagy and lysosome. In previous studies, it was found that age-dependent autophagy and degeneration of axons on the edge of APP drosophila wings increased. And it was found that feeding Chloroquine to APP Drosophila inhibits axon degeneration. We speculate that the dementia behavior of drosophila is related to autophagy [1].

Experiment Design

In order to judge whether chloroquine has an effect, we designed and conducted the following experiment.

Experiment 1: pupation height: in this experiment, we use Appl-Gal4 to drive APP expression in all larval neurons.
In this experiment, we will examine the pupation height from each group. The pupation height represents the climbing ability of Drosophila larvae. We will test whether APP-expressing larvae have impaired climbing ability and reduced pupation height.
Educational background: Junior high school or below: 8.75%, High school: 28.75%, Associate Degree: 15%, Bachelor: 30%, Master degree: 10%, PhD: 7.5%

Figure 1 Picture of pupation height of Drosophila.

Experiment 2: learning and memory test, in this experiment, we use elav-Gal4 to drive APP expression in the mushroom body (MB) of adult brain. MB is involved in fly learning and memory.
We will choose one naïve male and one non-virgin female. We will put them in an observing cell for one hour.
-Record the courtship behavior in the first 10 min
-Wait 40 min
-Record the courtship behavior in final 10 min

Male will be rejected by the non-virgin female in courtship. So, control (wild-type) males will learn from the rejection experience, and show significantly reduced courtship in the final 10 min, compared with the first 10 min. We expect to see APP-expressing flies shown no difference between the final and first 10 min, suggesting a learning and memory defect.

Experiment 3: courtship choice behavior, to test whether a male fly prefers young versus old females in the courtship. We use fru-Gal4 to drive APP expression in the courtship neurons.
In this experiment, we place one male with two young and two old virgin females in the observing cell, and measure the male’s courtship preference toward young or old virgin females. Control males will show a strong preference toward the young females, we expect that APP-expressing males will show reduced preference toward the young females.

Experiment 4: Autophagy in larval ventral nerve cord (VNC) and photoreceptor cells of eye disc.
As aberrant autophagy is frequently observed in the brain of AD patients, suggesting autophagy is likely a cause of AD, yet, this has not been investigated previously.
In this experiment, we will use Appl-Gal4 and GMR-Gal4 to drive APP expression in larval VNC and photoreceptor cells of eye disc, respectively. We will use Lyso Tracker staining to check autophagy in the control and APP-expressing VNC and eye disc, and test whether APP expression could induce autophagy in neuronal cells in VNC and eye disc.

Experiment 5: Cell death in larva ventral nerve cord (VNC) and photoreceptor cells of eye disc.
As aberrant autophagy will lead to cell death, which is also referred to as autophagy-mediated cell death.
In this experiment, we will use Appl-Gal4 and GMR-Gal4 to drive APP expression in larval VNC and photoreceptor cells of eye disc, respectively.
We will use Acridine Orange (AO) staining to check neuronal cell death in the control and APP-expressing VNC and eye disc, and test whether APP expression could induce autophagy-mediated neuronal cell death in VNC and eye disc.

Drug delivery device

A fast and efficient drug delivery device could be used for the Drosophila model. This device includes a container 1 with an opening on the upper part, a cover 2 that can be closed on the opening, a container 3 and a u-shaped capillary tube 4. The container 3 may contain a certain amount of liquid food containing candidate drugs. The plastic tube connected to the u-shaped capillary 4 contains about 200 μl of liquid food containing candidate drugs, which can feed 20 Drosophila for about 7-10 days. After 7-10 days, just add new liquid food without dismantling the device [2].

Figure 2 Drug delivery device for screening drugs using Drosophila model[2]

Experimental results

Eclosion Defects:

Chloroquine treatment does not affect the developmental progress of Drosophila, which takes about 10 days to complete the development from an egg to an adult fly (Figure 3 A). 2mg/ml Chloroquine does not affect the viability in development, while 5mg/ml Chloroquine causes 30% developmental lethality, suggesting a toxic effect of 5mg/ml Chloroquine on fly development(Figure 3 B).

Figure 3 (A) Results of eclosion defects, (B) Results of statistical analysis.

Pupation Height:

The height of pupation represents the climbing ability of Drosophila larvae.In the Figure 4, 2mg/ml (2, 5) and 5mg/ml (3, 6) Chloroquine does not affect the pupation height of Appl-Gal4 (1) and Appl>Dcr2 (4) control animals. Over-expression of APP blocks the climbing ability of larvae, which dramatically reduces the height of pupation (7). APP-induced larval climbing disability, as shown by declined pupation height, is significantly rescued by 2mg/ml Chloroquine (8), but not by 5mg/ml Chloroquine (9), further confirming the toxic effect of 5mg/ml Chloroquine on fly development. Thus, we decided to choose 2mg/ml Chloroquine for future experiments.

Figure 4 (A)Results of pupation height, (B) Results of statistical analysis.

Learning Defects:

Chloroquine restores APP‐induced learning defects. Figure 5A shows the male learning experiment model. To evaluate the learning ability, we performed the one‐hour courtship suppression training, measured the CI of the initial (CIinitial) and final (CIfinal) 10 min, and defined the learning index as

LI = (CIinitial− CIfinal)/CIinitial.

Compared with the controls (+), APP-expressing males (APP) display a learning impairment, which is improved by 2mg/ml Chloroquine(Figure 5 B).

Figure 5 (A)Male learning experiment model, (B) Results of statistical analysis.

Courtship Choice:

Chloroquine salvages the courtship choice defects caused by APP over-expression. Figure 6A shows male courtship choice experiments model, in which male flies were provided with both younger and older virgin females simultaneously. To accurately quantify the extent of males’ preference for younger or older females in courtship choice assay, we measured the preference index (PI) indicating a relative difference between males’ CI toward younger or older females(Figure 6A).

PI=CIyoung/CIold

Compared with the control males (+) that preferred to court younger females, APP-expressing males (APP) were unable to distinguish between younger and older females, and failed to show the courtship preference for young females. 2mg/ml Chloroquine could rescue the males’ choice disability induced by APP expression(Figure 6B).

Figure 6 (A)Male courtship choice experiments model, (B) Results of statistical analysis.

Lyso-tracker positive cell number (VNC):

Aberrant autophagy is frequently observed in the brains of AD patients. To confirm that APP could induce autophagy in Drosophila nervous system, and that chloroquine treatment could effectively suppress APP-induced autophagy in fly, we checked Lyso Tracker staining, which is a biomarker for autophagy, in Drosophila ventral nerve cord (VNC). Compared with the controls (A), APP expression resulted in increased autophagy (C), which was significantly suppressed by 2mg/ml Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect autophagy in the control VNC (B)(Figure 7).
These results indicate that APP expression induces autophagy in fly VNC, which could be effectively blocked 2mg/ml Chloroquine treatment.

Figure 7 (A) Results of Lyso-tracker positive cell number (VNC), (B) The statistical analysis of data shown in A.

Lyso-tracker positive cell number (Eye disc):

Same as above, we also measure autophagy in eye disc to test the effectiveness of Chloroquine. Compared with the controls (A), APP expression resulted in increased autophagy in photoreceptor cells in the eye disc (C), which was significantly suppressed by 2mg/ml Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect autophagy in the control eye disc (B)(Figure 8).
These results indicate that APP expression induces autophagy in the photoreceptor cells of eye disc, which could be effectively blocked 2mg/ml Chloroquine treatment.

Figure 8 (A) Results of Lyso-tracker positive cell number (Eye disc), (B) The statistical analysis of data shown in A.

AO Positive cell number (VNC):

To measure the effect of chloroquine on APP-induced autophagy-mediated neuronal cell death, we performed Acridine Orange (AO) staining, a biomarker for cell death, in Drosophila ventral nerve cord (VNC) to reflect the degree of neuronal damage. Compared with the controls (A), APP expression resulted in increased neuronal cell death (C), which was significantly suppressed by 2mg/ml Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect neuronal death in the control VNC (B) (Figure 9).
These results indicate that APP expression induces autophagy-mediated neuronal cell death in fly VNC, which could be effectively blocked 2mg/ml Chloroquine treatment.

Figure 9 (A) Results of AO Positive cell number (VNC), (B) The statistical analysis of data shown in A.

AO Positive cell number (Eye disc):

Same as above, we also measure photoreceptor cell death in eye disc to test the effectiveness of Chloroquine. Compared with the controls (A), APP expression resulted in increased cell death (C), which was significantly suppressed by 2mg/ml Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect photoreceptor cell death in the control eye disc (B)(Figure 10).
These results indicate that APP expression induces autophagy-mediated neuronal cell death in fly eye discs, which could be effectively blocked 2mg/ml Chloroquine treatment.

Figure 10 (A) Results of AO Positive cell number (Eye disc), (B) The statistical analysis of data shown in A.

Conclusion：

In summary, we first tested different concentrations of chloroquine on the normal development of Drosophila, and found out the suitable concentration that is not toxic to flies. Then we treated control and APP-expressing flies with the suitable concentration of chloroquine. Our conclusion is that chloroquine treatment could suppress APP-induced autophagy and neuronal cell death, and ameliorate APP-triggered behavioral defects in pupation height, learning and memory, and choice. These experiments proved the potential effect of chloroquine as an inhibitor of AD symptoms, and at the same time proved the feasibility of the Drosophila model we constructed for drug screening.

Future Prospects

Through the use of the APP transgenic Drosophila model, we proved that chloroquine has the potential to inhibit AD symptoms with experiments. Therefore, for subsequent drug screening for neurodegenerative diseases such as Alzheimer's disease, this fruit fly screening model can take advantage of the short growth cycle, large number of progenies, clear phenotype and anatomical structure, and known complete genome sequence of fruit flies, and provide assistance for early large-scale drug screening in the future.

Reference

[1]Wikipedia. (Aug.24.2021). Wikipedia. Retrieved from https://en.wikipedia.org/wiki/Chloroquine.
[2] A drug delivery device and drug delivery method and process that use fruit flies to screen drugs. 2019. Retrieved from http://www.xjishu.com/zhuanli/01/201910235932.html.