Team:Worldshaper-Shanghai/Design

Home

Design

Summary

The goal of our project is to create a drug screen model for Alzheimer’s diseases and do some potential drug testing. We used full length Human APP 596 gene, which is thought to be the main cause of AD, and applied it on Drosophila. We will conduct a series of drosophila behavioral experiment to test if the creation of drug screen model on drosophila is successful. Lastly, we will examine the effect of Chloroquine as a potential medicine to inhibit the symptoms of AD.

In brief, our experiment design can be separated into three parts.
-Using molecular biology to construct the UAS-APP-Myc plasmid, and make transgenic flies bearing the UAS-APP-Myc transgene in the fly genome.
-Expressing APP-Myc in Drosophila nervous system by the Gal4/UAS binary system, and conduct behavioral experiment to test the AD model.
-With the established fly AD model, a potential drug is test for its effect on AD-like symptoms.

1 Biobrick design

Why we chose Amyloid cascade Hypothesis:

Since Kang cloned the cDNA of Amyloid Precursor Protein for the first time in 1987, APP has become a research hotspot. The following research of APP has proved that its abnormal expression and metabolism are closely related to the pathogenesis of AD [1].

Why do we transfer whole APP gene?

Since no effective drug has been developed to reverse the disease in the study of the Aβ gene, we believe that there are undiscovered disease-causing genes in the whole APP. We transfer whole APP gene into drosophila and trying to restore an entire Alzheimer’s individual.

Figure 1 Schematic diagram of APP cleavage pathways: APP is processed by two alternative pathways [2].

Biobrick design and plasmid we used.

Figure 2 shows a diagram of the plasmid we have used for experiment. The reason why we use this plasmid is because we are using the UAS/Gal4 binary system to drive APP expression in fly nervous system.

Figure 2 Diagram of the pUAST plasmid.

To obtain the UAS-APP-Myc plasmid, we started with the pUAST plasmid, and inserted APP-Myc in the poly clonal sites, with UAS next to it. This UAS-APP-Myc plasmid was microinjected into embryos of white eye flies, and the UAS-APP-Myc transgenic flies were obtained and identified by their orange eye, and confirmed by western blot.

When crossing a UAS-APP-Myc fly with the Appl-Gal4 fly, Gal4 protein will be expressed in the nervous system driven by the Appl promoter. Then Gal4, as a transcriptional activator, will bind to UAS and activate APP expression.

2 Model Organism

Why Drosophila?

Drosophila, being a common model organism, has been widely used in biomedical research. It possesses a short, simple reproduction cycle that is low cost when run. On the other hand, drosophila have a well-established researched background, with multiple Nobel winning findings. Nearly 80% of human genes have homologs in Drosophila, and Drosophila has been used as animal models for many human diseases, including cancers and neurodegenerative diseases.

Figure 3 Picture of Drosophila [3].

The first goal for us is to express human APP gene in Drosophila. For this process, we first need to construct the UAS-APP-Myc plasmid, transform it to the Bacteria E. coli for amplification, then extract the plasmid, send the plasmid to a firm for microinjection. After receiving microinjected flies, we will screen and identify the UAS-APP-Myc transgenic flies by the eye color. Embryos from white eye flies were injected, and transgenic flies carrying UAS-APP-Myc will show orange eye, because a mini-white gene was included into the plasmid.

In our design, we thought of using Drosophila to create a model for Alzheimer’s Disease medicine testing. Human Alzheimer Disease (AD) is known to have its causes related to plaques of amyloid-beta peptides that are made by cleavage of Amyloid Precursor Protein. Previous research has established fly AD models by expressing the amyloid-beta peptide (Aβ), however, recent studies suggest that Aβ is not the only cause of AD, other parts of APP, like AICD (APP intracellular domain), are also involved in AD. For this reason, we decided to express full length APP in the fly nervous system.

3 AD‐like symptoms in Drosophila

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.

Figure 4 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.

4 Drug test

Since we found that APP expression in fly nervous system causes autophagy-mediated neuronal cell death and multiple behavioral defects, including decreased pupation height, reduced learning and memory, and impaired courtship choice ability. We wonder whether these behavioral defects are the outcome of APP-induced autophagy-mediated neuronal cell death. To address this possibility, we decided to test the therapeutic effect of chloroquine on APP-induced AD-like symptoms in Drosophila, since chloroquine is a well-known inhibitor of autophagy.

We will first test different concentrations of chloroquine on the normal development of Drosophila, and to find out the suitable concentration that is not toxic to flies. Then we will treat control and APP-expressing flies with the suitable concentration of chloroquine, and test whether 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. If it can be confirmed, the symptoms of dementia may be rescued by chloroquine.

References:
[1] Peng, F. (Dec. 2015). A genetic screen for modifiers of APP in Drosophila melanogaster, Pg.7.
[2] L. Rajendran, W. Annaert. (2012) Membrane Trafficking Pathways in Alzheimer's Disease, Traffic, 13 (6): 759-770.
[3] https://en.wikipedia.org/wiki/Drosophila_melanogaster