Team:Jiangsu High School/Results

Home

Results

Method:
Activation on autophagy was investigated by using mTagRFP-mWassbi-LC3 translocation assay. Briefly, SH-SY5Y cells were transfected with mRFP-GFP-LC3 plasmid via Lipofectamine 2000  according to the manufacturer's protocol. The cells were treated with different testing compounds and then fixed with 4% paraformaldehyde. The images were acquired using a Leica DMI8 confocal microscope and analyzed by the Image J software. Autophagy activator Nitazoxanide (NTZ) and inhibitor Chloroquine (CQ) were applied to an assay of SH-SY5Y as positive and negative control, respectively.

Result:
At early stage of autophagosomes biogenesis, puncta of mTagRFP-mWasabi-LC3 accumulated and displayed both green and red fluorescent signals (mRFP+GFP+ yellow). Notably, when autophagosomes were fused with lysosomes, they formed acidic autolysosomes (mRFP-GFP+ red), and the co-located mTagRFP-mWassbi-LC3 emitted only a red signal because the green signal quenched immediately under acidic conditions. Thus an increase in numbers of red and yellow puncta would be an indicator of promoted autophagy. Compound C4, C5, C7, C8, C9, C10 show an increase in numbers of both yellow and red puncta compared with the control, indicated they are the activators of autophagy.

Scale bar: 5 µm

 

Future Plans
Of course, this would not be the end of AD research we could accomplish; rather, this would be the beginning. Future research might entail experimenters testing the effectiveness of the compound C4-Paroxetine, C13-Amitriptyline with animal models. Testing might include investigating potential side effects of the compound such as level of toxicity within the animal model. Another future possibility may be utilizing proteins that indicate autophagy flux to prove that the compound really incites autophagy. For example, p62 proteins are degraded into autolysosomes (thus, the protein could be seen as a marker for degeneration in autophagy); in other words, decreased levels of p62 is equivalent to autophagy activation (Klionsky et al. 2012). This knowledge obtained would aid further explorations regarding the compound's action mechanism against AD and the plausibility of it becoming a full-fledged medicine to repress the disease's symptoms.

Reference
•      Klionsky, Daniel, Abdalla, Fabio, Abeliovich, Hagai et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 8. 445-544. 10.4161/auto.19496.