As the earth spins on its axis, most lifeforms including humans must coordinate their behaviors and internal clocks to adapt to consequent oscillations in light and temperature. Failure to adapt to these daily rhythms results in disease.

Through studies on the fruit fly, scientists at Northwestern University have discovered a new gene and molecular pathway that regulate the excitability of neural pacemaker cells in the brain. Understanding how this pathway works can offer insights on behavioral rhythms in daily human life and lead to new treatments for clock-related human diseases, such as depression and insomnia.

“Disrupted circadian rhythms contribute to a variety of human diseases including sleep disorders, neurodegenerative diseases, and metabolic disorders,” said Ravi Allada, PhD, professor and chair of the neurobiology department at the Weinberg College of Arts and Sciences at Northwestern University, and associate director of Northwestern’s Center for Sleep and Circadian Biology. “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.”

The findings are published in an article in the Proceedings of the National Academy of Sciences (PNAS) titled, “The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms.” Allada is the senior author of the paper, and Jongbin Lee, PhD, and Chunghun Lim, PhD, former postdoctoral fellows in Allada’s lab, are co-first authors of the paper.

The authors 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.

Just as the gears of a clock control its hands that mark off hours, minutes, and seconds on the dial, the core circadian molecules regulate output pathways such as neuronal activity and the consequent release of neuropeptides and neurotransmitters.

“Scientists know a lot about the clock’s ‘gears’ but not so much about the ‘hands,’ where the behavior is produced, or the connection between the two,” said Allada. “Our findings fill a molecular gap in our understanding of how the core gears of the clock control the hands.”

The molecular pathway that the study uncovers links core transcriptional oscillators to neuronal and behavioral rhythms.

“We wanted to better understand the molecular underpinnings of the daily ‘wake-up signal,’ which alerts an animal it’s time to awake,” he said. “In this study, we focused on pacemaker neurons that control the sleep-wake cycle and used genetic screening to identify genes that regulate the neurons.”

The study combines a wide range of protocols including mass spectrometry, whole-cell patch clamp electrophysiology, genetic screens, and a battery of molecular and behavioral assays. While the fly experiments were conducted in Allada’s lab, his team collaborated with Casey Diekman, PhD, associate professor of mathematics at the New Jersey Institute of Technology (NJIT), and Matthew Moye, PhD, then a PhD student at NJIT and now a postdoctoral researcher at Merck, who performed the experiments on computational modeling.

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.

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.

“Our work highlighted a role for Tango10 in controlling excitability and neuropeptide accumulation,” Allada said. “We would like to determine what the direct targets of Tango10 are, how they biochemically regulate those targets, and what the impact is on target function. One of our best candidates is a class of potassium channels.”

“The cool news here is that interactions between TANGO10 and CUL3 proteins are part of the circadian output pathway and critical for promoting circadian rhythms in the behavior of flies by regulating the excitability and signaling of pacemaker neurons,” said Andy LiWang, PhD, professor of chemistry & biochemistry at the University of California, Merced, who was not involved in the current study.

LiWang added, “In flies, normal TANGO10-CUL3 interactions decrease excitability of pacemaker neurons by increasing potassium currents through voltage-gated potassium channels. Currents through potassium channels similarly affect neuronal excitability in the suprachiasmatic nuclei, which regulates circadian behavior in mammals. Thus, this research on flies might help scientists look for homologous interactions in mammals, including humans. Once identified, defects in such homologous interactions that cause abnormal sleep-wake patterns might be addressable through drugs that compensate for such defects.”

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