Naohong Kim (Project Assistant Professor, Department of Biological Sciences at the time of research / Project Lecturer, Institute for Transformative Biomolecular Research, Nagoya University)

Takahiro Iwamoto (Professor, Department of Pharmacology, Faculty of Medicine, Fukuoka University)

Yoshitaka Fukada (Professor, Department of Biological Sciences at the time of research / Project Researcher, Graduate School of Medicine)

Key points of the presentation

• He discovered the calcium ion ( ^Ca2+) regulator as the first biological clock component common to mammals, insects, plants, and bacteria, and proposed that the increase or decrease of intracellular ^Ca2+ during the daily cycle is central to the biological clock.
• Body clocks were thought to have arisen independently in animals, plants, and bacteria. However, the origin of the body clock is much older, suggesting that ^Ca2+ was ^{involved in} the body clock of the common ancestor of life.
• The discovery of drugs that manipulate the body clock through the control of calcium ions is expected to lead to the development of new drugs to overcome sleep disorders and depression. It also provided a clue to the internal clock of the final common ancestor of all living organisms ^{(Note 1)}.

Summary of presentation

A research team led by Professor Naohiro Kim at the Institute for Transformative Biomolecular Research, Nagoya University (at the time of the research: Project Assistant Professor at the Graduate School of Science, The University of Tokyo), Professor Yoshitaka Fukada at the Department of Biological Sciences, Graduate School of Science, The University of Tokyo (at the time of the research), and Professor Takahiro Iwamoto at the Department of Pharmacology, Faculty of Medicine, Fukuoka University, has identified the following factors that are common to mammals, insects, plants and bacteria The genes that make up the circadian clock ^{(Note 3)}, a 24-hour biological clock, are called clock genes ^{(Note 4).} Since the clock genes found so far are poorly conserved among animals, plants, and bacteria, it has been thought that the biological clock emerged independently in each species.

The research team focused on the temperature compensatory property of the biological clock (the property of the biological clock to maintain a constant cycle regardless of the environmental temperature), which is an important property of the biological clock, and analyzed it, and found that the regulation of intracellular ^Ca2+ by NCX is essential for the biological clock function. The research group further proposed that the increase or decrease of intracellular Ca2+ during the daily cycle is the main body clock. Furthermore, the research group found that the cycle and time of the biological clock can be manipulated by drugs that inhibit NCX activity (Fig. 1). (Figure 1)

Figure 1: Since the previously discovered clock genes are poorly conserved in mammals, insects, and other animals, molds, plants, and bacteria, it was thought that the body clock evolved independently. In this study, we found ^{Na+/Ca2+ exchange} transporter (NCX) as a clock factor common to all life. NCX is a protein that regulates intracellular ^{Ca2+ concentration}. Intracellular ^{Ca2+ concentration} increases and decreases in a daily cycle in many species, suggesting that ^{Ca2+ oscillation} served as the origin of the biological clock.

This finding will lead to the development of drugs to overcome sleep disorders and depression by manipulating the body clock. It is also a groundbreaking clue to understanding how the body clock originated in the common ancestor of our life.

Announcement

Our brain functions such as sleep, emotion, and memory are controlled by the biological clock. The 24-hour biological clock is called a circadian clock, and it exists in humans and bacteria alike. The daily rhythm produced by the circadian clock is called the circadian rhythm, in which the transcription-translation feedback loop ("transcription loop") of clock genes plays an important role. The three U.S. scientists who discovered the first clock genes in Drosophila were eligible for the 2017 Nobel Prize in Physiology or Medicine. Until now, clock genes have been found in animals, fungi, plants, and bacteria, but have been thought to have evolved independently in each species due to low homology among them.

Although it was known that the transcription loop by clock genes is essential for the generation of circadian rhythms, there was some mystery in considering it as the main body of the circadian clock. The circadian clock is temperature compensatory, meaning that its cycle is maintained at 24 hours even when the environmental temperature changes. Biochemical reactions such as transcription and translation change their reaction rates depending on temperature, for example, if the temperature is lowered by 10 degrees, the reaction rate slows down by a factor of 1/2 to 1/3. Therefore, if the biological clock is simply composed of the transcription loop alone, the clock cycle would be 48 or 72 hours when the temperature is lowered by 10 degrees Celsius. To explain temperature compensability, it was proposed nearly 70 years ago that a mechanism to compensate for the slowdown in the rate of biochemical reactions caused by a drop in temperature is necessary, but the substance of this mechanism has remained a mystery.

A research team led by Project Lecturer Naohiro Kim at the Institute for Transformative Biomolecular Cell Biology, Nagoya University (at the time of the research: Assistant Professor at the Graduate School of Science, University of Tokyo), Professor Yoshitaka Fukada at the Department of Biological Sciences, Graduate School of Science, The University of Tokyo (at the time of the research), and Professor Takahiro Iwamoto at the Department of Pharmacology, Faculty of Medicine, Fukuoka University, noticed the existence of a circadian clock in cultured cells The research team screened drugs that affect the temperature compensatory properties of the cellular clock. They found that temperature compensation was disrupted in the presence of inhibitors of two proteins involved in intracellular calcium signaling, ^{Na+/Ca2+ exchange} transporter (NCX) and ^{Ca2+/calmodulin-dependent} kinase II (CaMKII).

Further analysis revealed that NCX promotes intracellular ^{Ca2+ influx} as environmental temperature decreases, thereby preventing the activated CaMKII from decreasing the oscillation rate of the transcription loop. In other words, the transcription loop, which normally slows down with decreasing temperature, was maintained in a 24-hour cycle by a low-temperature-activated ^{Ca2+ signal}. This low-temperature ^{Ca2+ signal} was found to function in the circadian clocks of insects, plants, and bacteria as well as mammals, suggesting that NCX is a conserved mechanism in a wide range of organisms. The fact that NCX functions are conserved in the clock functions of a wide range of species suggests that NCX was involved in the clock functions of these common ancestors. In other words, while the transcriptional loop of clock genes arose after the phylogenetic divergence of animals, plants, and bacteria, the regulation of the circadian clock by calcium may have functioned from the common ancestor.

Therefore, the research team analyzed animals with impaired NCX function, assuming that NCX is involved not only in temperature compensatory properties but also in the circadian clock itself. They found that the daily behavioral rhythm was severely impaired in mice and Drosophila. In the pacemaker nerves of these animals' biological clocks, intracellular ^Ca2+ increases and decreases in a daily cycle. This has led the researchers to the view that intracellular Ca2+ oscillations may be an ancestral oscillator that functions upstream of the transcriptional loop.

Current life is thought to have diversified and evolved from the Last Universal Common Ancestor (LUCA). In 2016, German Research Students identified 355 genes that are highly conserved between bacteria and archaea and concluded that they are the LUCA gene cluster [Nature Microbiology, 1, 16116 (2016)]. Interestingly, the gene encoding NCX found in this study is included in that group of 355 genes. From this, it is conceivable that the origin of the biological clock is much older than previously thought, possibly as early as 3.8 billion years ago, the time when LUCA existed. The results of this research provide an epoch-making clue as to how biological clocks originated in the common ancestor of all life.

The common ancestor's internal clock mechanism retains a central role in present-day organisms. The research group has successfully regulated the cycle and time of the circadian clock by using NCX and CaMKII inhibitors. These discoveries have led to the development of technologies that allow us to freely control our biological clocks by regulating intracellular ^Ca2+, and have opened a new path to overcome diseases related to biological clocks, such as sleep disorders and depression.

Journal

Journal name	Science Advances
Title of paper	Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms
Author(s)	Naohiro Kon†, Hsin-tzu Wang, Yoshiaki S. Kato, Kyouhei Uemoto, Naohiro Kawamoto, Koji Kawasaki, Ryosuke Enoki, Gen Kurosawa, Tatsuto Nakane, Yasunori Sugiyama, Hideaki Tagashira, Motomomi Motomu, Yasunori Sugiyama, Hideaki Tagashira, Motomu Nakane Yasunori Sugiyama, Hideaki Tagashira, Motomu Endo, Hideo Iwasaki, Takahiro Iwamoto*, Kazuhiko Kume, Yoshitaka Fukada*
DOI No.	10.1126/sciadv.abe8132
URL	https://advances.sciencemag.org/content/7/18/eabe8132

Terminology

1 Last Universal Common Ancestor (LUCA)

The genetic phylogenetic tree of life shows that the divergence of archaea and eubacteria occurred before the divergence of prokaryotes and eukaryotes. The last common ancestor of all life (LUCA) is the common ancestor of archaea and eubacteria. Eukaryotes are thought to have arisen from the incorporation of eubacteria by a type of archaea, and many suggestions as to what kind of organism LUCA is, not only from molecular biology but also from paleontology, geology, and synthetic chemistry, suggest that it is a thermophilic bacterium with a small genome size. An analysis of a highly conserved gene cluster extracted between archaea and eubacteria has presented 355 genes that would have been retained by LUCA. Among them is the yrbG gene encoding the ancestral form of NCX. ↑

Note 2 ^{Na+/Ca2+ exchange} transporter (NCX)

An ion transporter that transports 3 ^{Na+ and} 1 ^{Ca2+ in} exchange across the cell membrane. This ion transport is bidirectional ( ^{Ca2+ efflux} and ^{Ca2+ influx} modes), and the direction of transport is determined by the concentration gradient of ^{Na+ andCa2+} inside and outside the cell and the membrane potential. ↑↑↑↑↑↑

Note 3 Circadian clock

The central mechanism that generates circadian rhythms. It oscillates autonomously with a period close to one day, and works on various physiological functions to generate circadian rhythms [ ^{see(Note 5)} below]. The three major properties of the circadian clock are autonomous oscillation, environmental entrainment, and temperature compensation. In mammals, the pacemaker of the circadian clock resides in the suprachiasmatic nuclei of the hypothalamus, which synchronizes the phases (time) of the circadian clocks in cells throughout the body. ↑up

4 Clock genes

4 Clock genes are genes that encode proteins that constitute the developmental mechanism of circadian rhythms. Most of them have been cloned from mutants or acyclic mutants of the circadian rhythm cycle. In eukaryotes, many clock genes encode transcriptional regulators, forming an autoinhibitory feedback loop; in 2017, three U.S. researchers who discovered the first clock gene, Period, were awarded the Nobel Prize in Physiology or Medicine. The transcription-translation feedback loop by clock genes is now a mechanism for generating circadian rhythms described in molecular biology textbooks. In this study, they discovered intracellular ^{Ca2+ oscillation and} its regulatory proteins as a new oscillation mechanism that exists upstream of the feedback loop. ↑up

5 Circadian rhythm

Circadian rhythm is an endogenous cyclic phenomenon in living organisms that occurs even in the absence of cyclic fluctuations in the environment. In other words, circadian rhythms continue with an approximate one-day cycle after transfer to a homeostatic environment where fluctuations are kept as constant as possible. ↑up