Project Description
Why we choose this topic?
According to the World Health Organization(2020), depression became a major contributor to disease burden, with more than 264 million people suffering from its pain. People are constantly under stress from various aspects of life: career, socialization, study, etc. Unfortunately, in December 2019, an outbreak of CoronaVirus Disease 2019 (Covid-19) ravaged the world; nearly four billion people died during this pandemic. (Ritchie 2021). Under such an intense environment, many friends and relatives of ours fall victim to depression. Yunhe Wang, et al. (2021) used a national online survey demonstrating that people who experienced quarantine have a higher risk of being influenced by depression, especially those who have a history of mental illness and are infected by Covid-19.
These heartbreaking numbers bring students worldwide with ambition in biology and psychology to Shanghai and form this team. We, the Heartinker team, are deeply concerned about the situation of depression patients and hope to use the advanced technology of synthetic biology to discover a new antidepressive target and find a more effective and harmless antidepressive product to solve the problems of current antidepressants.
Current antidepressant drugs
SSRI (Selective serotonin reuptake inhibitor)
This kind of medicine mainly regulates the concentration of 5-HT in the synaptic cleft by inhibiting the reuptake of 5-HT by presynaptic neurons in the central nervous system. But it can also have side effects such as insomnia and anxiety.
TcA (Tricyclic Antidepressant)
TcA inhibits the reabsorption of NE(norepinephrine) and 5-HT (serotonin) in the presynaptic neurons of the nervous system. Increased the concentration of neurotransmitters in the synaptic cleft. However, it also has side effects such as insomnia, low blood pressure and an unbalanced heart rate.
MAOI (Monoamine oxidase inhibitor)
The mechanism of MAOI is to reduce the degradation of monoamine neurotransmitters in the central nervous system by inhibiting MAO(Monoamine oxidase), and to relatively increase the level of monoamine neurotransmitters in the central nervous system. MAO is involved in the catabolism of serotonin, norepinephrine, and dopamine. May cause high blood pressure. Moreover, Interactions with other drugs can also cause side effects.
Most of the current antidepressant drugs have a certain degree of addiction and withdrawal reaction. Which means there are some adverse effects after withdrawal.
Our theory
Serotonin is widely found in the natural tissues of animals, especially in the cerebral cortex and synapses in high levels, and also in plants and fungi in a small amount. In the pharmaceutical field, serotonin, as a drug, can participate in a variety of physiological functions of the organism, including emotion regulation, behavior management, sleep cycle maintenance, scavenging harmful free radicals and so on. The Monoamine hypothesis is a widely accepted hypothesis for the cause of depression at present. This theory holds that depression is caused by the decrease in the concentration or function of monoamine neurotransmitters (such as 5-hydroxytryptamine) in the synaptic space of the central nervous system. The decrease of the neurotransmitter function of 5-hydroxytryptamine(Serotonin or 5-HT) not only leads to the occurrence of depression and anxiety, but also interferes with the normal function of other neural circuits.
Even though the deficiency of 5-HT can lead to depression is still a hypothesis, after decades of research, the role of 5-HT in depression has been refined, and more scientific evidence suggests the importance of 5-HT in treating depression (Albert 2012). Therefore, we decided to choose 5-HT as a direction to develop our antidepressive product in two pathways.
Our design
Recombinant E. coli
Figure 1. Profile of our plasmid pTrc-99a-5HT
After researching, we found the humans use two enzymes, tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC), to convert Tryptophan(Trp), which is an essential amino acid for human’s body, to 5-HT. Moreover, TPH is the rate-limiting enzyme in the process, which means the amount of TPH can directly affect the production speed of 5-HT (Liu 2021). Thus, We hope to modify E. coli by inserting TPH and TDC genes, so that it can become the exogenous synthesis pathway of 5-HT, so as to increase the amount of 5-HT.
Rin 14b cell screening platform
Figure 2. EC cell releases 5-HT
We were inspired by a fascinating property of 5-HT that less than one in a million CNS neurons produce serotonin, and 95% of serotonin was released into the gut by intestinal enterochromaffin cells (Berger 2018). Hence, we hope to stimulate the EC cell to make it release more 5-HT in the blood and increase the chance to treat the depression via a gut-brain circuit. As Dr. Nozawa et al. (2009) reported in their journal, TRPA1 is an excitatory ion channel that can be stimulated by many natural compounds, like spices and herbal medicines, and these TRPA1 agonists cause Ca2+ influx and 5-HT release in EC cells. Luckily, this ion channel is also highly expressed in gastrointestinal tissues in humans, mice, and rats. In other words, if we can stimulate the TRPA1 agonists on the EC cells, we can make it release more 5-HT.
However, it’s impossible to achieve a completely pure EC cell culture to test. Then we turn our attention to another cell with a similar function as the EC cell, the Rin 14b cell. Dr. Nozawa and his colleagues found the gene expression markers of EC cells such as TPH1, chromogranin A, VMAT1, and synaptophysin are also highly expressed in Rin 14b cells. Moreover, the agonists, like Ca2+ and ionomycin, which can trigger the release of 5-HT from EC cells, can also activate Rin 14b cells, indicating that RIN14B cells share functional similarities with EC cells and can be used as a model to test the potential compounds.
In this part of our project, we will use the patch-clamp techniques to find compounds that can stimulate the Rin 14b cells, which can increase the attitude and frequency of its action potential and mark it as a possible direction that pharmacologists can keep researching on.
Reference
Gillman, P. K. (2009, January 29). BPS Publications. British Pharmacological Society | Journals. https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1038/sj.bjp.0707253#b88.
J, N. D., S, A., J, N., J, S., A, B., & S, F. (1999, July 9). Mechanisms of action of selective serotonin reuptake inhibitors in the treatment of psychiatric disorders. European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology. https://pubmed.ncbi.nlm.nih.gov/10523062/.
V, R. M., & Z, P. W. (1999, June 19). Metabolism of tricyclic antidepressants. Cellular and molecular neurobiology. https://pubmed.ncbi.nlm.nih.gov/10319193/.
Gillman, P. K. (2009, January 29). BPS Publications. British Pharmacological Society | Journals. https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1038/sj.bjp.0707253.
PJ;, H. C. J. D. R. S. C. (2017, May 4). How do antidepressants work? New perspectives for refining future treatment approaches. The lancet. Psychiatry. https://pubmed.ncbi.nlm.nih.gov/28153641/.
Shulman, K. I., Herrmann, N., & Walker, S. E. (2013, August 10). Current Place of Monoamine Oxidase Inhibitors in the Treatment of Depression. CNS Drugs. https://link.springer.com/article/10.1007/s40263-013-0097-3.
Du, Y., Gao, X.-R., Peng, L., & Ge, J.-F. (2020). Crosstalk between the microbiota-gut-brain axis and depression. Heliyon, 6(6). https://doi.org/10.1016/j.heliyon.2020.e04097
Nozawa, K., Kawabata-Shoda, E., Doihara, H., Kojima, R., Okada, H., Mochizuki, S., Sano, Y., Inamura, K., Matsushime, H., Koizumi, T., Yokoyama, T., & Ito, H. (2009). TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proceedings of the National Academy of Sciences, 106(9), 3408–3413. https://doi.org/10.1073/pnas.0805323106