A recent study, affiliated with UNIST has unveiled a new gene and molecular pathway that regulate the excitability of neural pacemaker cells in the brain. The researchers expect that understanding how this pathway works can offer insights into behavioral rhythms in daily human life and lead to new treatments for clock-related human diseases, such as depression and insomnia.
Published in the November 2021 issue of the Proceedings of the National Academy of Sciences (PNAS), this breakthrough has been jointly carried out by Jongbin Lee and Chunghun Lim from the Department of Biological Sciences at UNIST.
"Disrupted circadian rhythms contribute to a variety of human diseases including sleep disorders, neurodegenerative diseases, and metabolic disorders," noted the research team. "Surprisingly, the circadian clocks of the fruit fly are highly conserved and thus, our studies are likely to be informative for understanding human circadian rhythms."
In this study, the researchers proposed that the adaptor protein Tango10 regulates ubiquitination by the enzyme Cul3 to transduce molecular oscillations from the core molecular components of the cellular clock to the output pathway of neuropeptide release, to regulate behavioral circadian rhythms.
"Key to the body's diurnal rhythm is complex cellular machinery dependent on molecular timekeepers that are chemically modified through biochemical reactions such as phosphorylation and ubiquitination to maintain the regular fluctuations of transcriptional feedback loops," noted the research team. "The core circadian molecules regulate output pathways such as neuronal activity and the consequent release of neuropeptides and neurotransmitters, like how the core gears of the clock control its hands that mark off hours, minutes, and seconds on the dial."
In order to better understand the molecular underpinnings of the daily 'wake-up signal,' which alerts an animal it's time to awake, the research team focused on pacemaker neurons that control the sleep-wake cycle and used genetic screening to identify genes that regulate the neurons.
Using two independent genetic screens, the researchers identified flies with a poor sense of behavioral rhythm that bore a mutation in a gene called Tango10 (short for TrANsport and Golgi Organization 10). In flies bearing normal copies of the gene, the protein levels of Tango10 go up and down every day. This modulates the activity of the pacemaker neurons which in turn drives the animal's sleep-wake cycle and behavior. In flies that lack the Tango10 gene, this daily rhythm is disrupted.
The authors showed Tango10 expression in pacemaker neurons expressing a neuropeptide called pigment dispersing factor (PDF) is required for the regular rhythms in neuronal activity in these neurons. Loss of Tango10 causes PDF to pile up at the nerve terminals. This occurs even when the gears of the clock machinery are intact. The authors showed TANGO10 protein levels also fluctuate rhythmically in nerve terminals that express PDF, similar to the gear components.
To probe into the molecular partners of Tango10, the authors conducted a mass spectrometry analysis and uncovered that the protein binds to a ubiquitin ligase called CULLIN 3 (CUL3). Loss of CUL3 results in a similar loss of rhythm in mutant flies as does Tango10, noted the research team.
Through patch-clamp electrophysiology experiments in Tango10 mutant neurons, the authors demonstrated an increase of spontaneous firing that is potentially due to a decrease in voltage-gated potassium currents. These reduced potassium currents, the authors inferred, could contribute to a loss of rhythmic behavior.
Meanwhile, this research has been carried out in collaboration with Professor Ravi Allada and his research team at Northwestern University. It has been supported by the Bio·Medical Technology Development Program thorugh the National Research Foundation.
Journal Reference
Jongbin Lee, Chunghun Lim, Tae Hee Han, et al., "The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms," PNAS, (2021).
