Proof of Concept
Overview
Depression, one of the major psychological disorder in current society, has been a world-wide issue. According to WHO, “Depression is a common illness worldwide, with an estimated 3.8% of the population affected, including 5.0% among adults and 5.7% among adults older than 60 years. Approximately 280 million people in the word have depression. Over 700 000 people die due to suicide every year. Suicide is the fourth leading cause of death in 15-29-year-olds. Although there are known, effective treatments for mental disorders, more than 75% of people in low- and middle-income countries receive no treatment”
Thus, how to cope with this global disease remains to be a great challenge. As a passionate research group, willing to care for others and to contribute to the progress of global psychological health, we aim at designing solutions to fight against depression.
We tried to increase the concentration of 5-HT, the insufficiency of which is found to be connected with depression, by enhancing the production of 5-HT, so as to achieve an antidepressant effect. We have two research objectives and corresponding potential products.
1) We plan to screen effective lead compounds that may increase 5-HT in vivo. Our antidepressant target is anchored to enterochromaffin cells. Because they secrete 95% of the human body's 5-HT, it may affect the brain 5-HT content through the intestinal-brain axis, and then have a therapeutic effect on depression. In practice, we choose Rin14B cells because of their similarity to enterochromaffin cells and easy accessibility. It is planned to use patch clamp technology to establish a Rin14B cell drug screening platform and screen out candidate antidepressant compounds. The lead compounds are expected to be further developed into antidepressant drugs in the future.
2) We plan to construct a biosynthetic pathway for exogenous synthesis of serotonin in Escherichia coli and using L-tryptophan as substrate, L-tryptophan can be converted into 5-hydroxytryptophan through whole cell catalysis, therefore being able to realize the use of gene recombinant Escherichia coli to produce serotonin in vivo. The results of genetic engineering experiments will be used to develop probiotic to alleviate depression.
Supporting Experiment Results
• Screening of Lead Compounds by Rin 14b cell Screening Platform
Compound Number 21070805
After administration, the membrane potential became stable and the release frequency of AP increased, suggesting that this compound may have the ability to stably activate RIN14B cells.
Compound Number 21071106
After administration, the membrane potential decreased, the release frequency of AP increased, and the amplitude increased significantly, and the effect could not be eluted, which may indicate that this compound has a strong activation ability for RIN14B cells.
Compound Number 21061805
The membrane potential and AP release frequency increased after administration, suggesting that the compound may have the ability to activate RIN14B cells.
The patch clamp results are really encouraging, according to which three lead compounds have been obtained. These compounds will be further tested and hopefully some of them could be developed into an antidepressant drug to help depression patients.
• Construction of Recombinant E.coli Results
Figure 2: SDS-PAGE analysis for TPH and TDC proteins.
Lane 1: Culture supernatant with induction (40 μL)；
Lane 2: Culture supernatant without induction (40 μL)；
Lane 3: Without samples
Lane 4: Supernatant of cell lysate with induction (40 μL)；
Lane 5: Supernatant of cell lysate without induction (40 μL)；
Lane 6: Pellet of cell lysate with induction (40 μL)；
Lane 7: Pellet of cell lysate without induction (40 μL)；
Lane 8: Supernatant of cell lysate with induction (20 μL)；
Lane 9: Supernatant of cell lysate without induction (20 μL)；
We got two bands (43.53kDa (TPH) and 53.55kDa (TDC)) in both supernatant of cell lysate and pellet of cell lysate.This experiment result is also promising since it proves that the TPH and TDC proteins have be expressed successfully. We will work on the current experiment results to test the feasibility of constructing probiotic functional in enhancing the 5-HT concentration level in vivo. (According to iGEM Rules, our team will not do any in vivo experiments with the scope of iGEM Competition)
Reference
Depression. (2021, September, 13). Retrieved October 13, 2021, from: https://www.who.int/news-room/fact-sheets/detail/depressionDepression
GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. (2018). Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet. DOI.
Nozawa K, Kawabata-Shoda E, Doihara H, et al. TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells[J]. Proc Natl Acad Sci U S A, 2009, 106(9): 3408-13.
Pompili Maurizio,Lionetto Luana,Curto Martina,Forte Alberto,Erbuto Denise,Montebovi Franco,Seretti Maria Elena,Berardelli Isabella,Serafini Gianluca,Innamorati Marco,Amore Mario,Baldessarini Ross J,Girardi Paolo,Simmaco Maurizio. Tryptophan and Kynurenine Metabolites: Are They Related to Depression?[J]. Neuropsychobiology,2018.