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Engineering

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 the full-length Human APP 596 gene, constructed the UAS-APP-Myc with the pUAST plasmid. Through UAS/Gal4 binary system, we have successfully obtained fruit flies expressing human APP. A series of drosophila behavioral experiments were conducted and chloroquine was selected as a potential medicine to test the model. The results proved the feasibility of the Drosophila model we constructed for drug screening.


1 Plasmid construction

To make the pUAST-APP-Myc plasmid, the first step is to amplify APP (APP695) from the pCAX APP695 plasmid (Addgene#30137, USA) through Polymerase Chain Reaction (PCR), which loops Denaturation, Annealing and Extension 35 times.
To check the PCR products, we took an aliquot of DNA sample from each PCR product, mixed it with purple loading dye, and loaded it into DNA gel for electrophoresis at 120 volts for 40 min. The figure below shows the results of DNA gel electrophoresis under UV lights (The control group is on the right). 8/12 of our sample displays a DNA band at the range of 2kb, which corresponds to the size of APP.

Figure 1 PCR gel



Once we identified the PCR products with the right size, the remaining DNA of each PCR product was double digested with two restriction enzymes: SacII and XbaI. The pUAST-Myc vector was also double digested with the same two enzymes. The digested APP fragment and pUAST-Myc vector were mixed with ligation buffer and DNA ligase, incubated at 37℃ for 120 min to promote their recombination.

After ligation, recombinant plasmids were purified, mixed with E. Coli DH5α competent cells for transformation. After transformation, cells were evenly applied on the surface of LB medium with antibiotics, incubated at 37℃ overnight.

The results have been shown below, where white dots represent bacterial colonies, shows that our bacterial are successfully transformed and cultivated.

Figure 2 E.coli Flat Figure



To verify the presence of correct plasmids, DNA was extracted from each E. Coli colony, PCR and DNA gel electrophoresis were perform to check the presence of APP-containing plasmids.
(Figure below shows the result of DNA gel electrophoresis, 7 samples show DNA fragments of about 2 kb , which correspond to the size of APP)

Figure 3 Positive clone identification glue map


To make sure no mutations were introduced into the plasmids, DNA sequencing was performed to examine the obtained sample. The figures below show the DNA sequencing results (The gray part indicates that the sequencing result is the same as the APP standard sequence).

Figure 4 Graph of segmented sequencing results


After DNA sequencing verification, the pUAST-APP-Myc plasmids were microinjected into Drosophila early embryos, and transgenic flies carrying the UAS-APP-Myc transgene were obtained. These flies show orange eyes, as compared with the white eye control flies.
To check whether the APP-Myc protein is expressed in Drosophila, we extracted total proteins from Appl-Gal4 control flies and Appl-Gal4/UAS-APP-Myc flies, performed Western Blotting (WB) with anti-Myc antibody. WB can be used to test the expression of protein with combination of gel electrophoresis and immunochemical analysis techniques. The figure below shows the results of WB analysis using GE Image Quant LAS4000. The 1st lane show the Appl-Gal4 control flies, the 2nd and 3rd lanes show APP-Myc is expressed in two Appl-Gal4/UAS-APP-Myc fly lines.

Figure 5 Results of Western Blotting


2 Behavioral Experiment
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 6A). 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 6B).

Figure 6 (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 7, 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 7 (A)Results of pupation height, (B) Results of statistical analysis.



Learning Defects:

Chloroquine restores APP‐induced learning defects. Figure 8A 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 8B).

Figure 8 (A)Male learning experiment model, (B) Results of statistical analysis.


Courtship Choice:
Chloroquine salvages the courtship choice defects caused by APP over-expression. Figure 9A 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 9A).

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 9B).

Figure 9 (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 10).
These results indicate that APP expression induces autophagy in fly VNC, which could be effectively blocked 2mg/ml Chloroquine treatment.

Figure 10 (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 11).
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 11 (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 12).
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 12 (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 13).
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 13 (A) Results of AO Positive cell number (Eye disc), (B) The statistical analysis of data shown in A.


Conclusion

In summary, we successfully constructed pUAST-UAS-APP-Myc plasmid and successfully obtained the fruit fly expressing human APP. The results of chloroquine as the potential drug to test our model indicated 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 work

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, we hope our model can be used for large-scale rapid screening.


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