Cell Networking Keeps Brain’s Master Clock Ticking

May 4, 2007 • Science Update

Each day, a master clock in the brain synchronizes the timing of lesser clocks in cells throughout the body to the rising and setting of the sun, regulating such daily rhythms as sleep, body temperature, eating, and activity. Scientists funded in part by the National Institute of Mental Health have now discovered that the secret to this master clock’s robust time-keeping ability lies in the unique way its cells work together.

“Unlike lesser clocks, the brain’s master clock is more than just the sum of its parts,” explained NIMH grantee David Welsh, M.D., Ph.D., a practicing psychiatrist as well as a neurobiologist at Scripps Research Institute. “By working in concert, the master clock’s neurons can compensate for genetic glitches that would otherwise cripple them if they had to function by themselves.”

The master clock, it turns out, can take a lickin’ and keep on tickin’.

Welsh, Andrew Liu, Steve Kay, Scripps Research Institute, and colleagues report on their study online in Cell, May 3, 2007.

Daily rhythm disturbances seen in mood disorders, sleep disorders, jet lag, and shift work have been traced to mismatches between the day/night cycle and the master clock, located in the brain’s hypothalamus .

Scientists attempting to understand how such mismatches occur have been unraveling the intricate workings of several genes involved. They genetically engineer mice to lack a gene coding for a clock part and then infer the missing gene’s function from the animal’s daily activity rhythms. Yet the behavioral effects of such gene “knockouts” may be very subtle and difficult to interpret.

So Welsh and colleagues devised a way to peer into the activity of single isolated mouse clock cells in which a key clock gene had been knocked out. Then, to another clock gene they attached the gene that makes fireflies glow. Each cell became a rhythmically glowing beacon of the ticking of its daily clock. This revealed whether the knocked out gene played an essential role in the cell’s rhythmicity.

Even though the whole master clock and the animals’ daily behavioral rhythms seemed relatively normal, the isolated clock cells sometimes lacked rhythms, depending on which gene was knocked out. For example, with the Cry2 gene knocked out, cells glowed rhythmically for a week, indicating that Cry2 is not required for cell rhythmicity. But with the Cry1 gene knocked out, the cells could only generate a few feeble cycles now and then, signaling Cry1’s critical importance for generating rhythmicity in single cells (see videos above).

Cells with any particular gene knockout behaved the same whether they came from the brain’s master clock or from lesser clocks in other body tissues. This indicated that all the cells had the same clock mechanism, but that networking of cells in the master clock represents a higher level of regulation, resulting in more robust rhythmicity than in lesser clocks. The neuronal network compensates for the glitches that gene deletions or other mutations cause in single cells.

One potential implication of the new findings for treating disorders of daily rhythms is that drugs affecting connections among the master clock cells might re-set errant clocks, Kay suggested.

In another NIMH-funded clock gene study, published online in Cell on April 26, 2007, a newly discovered mutation in mice was dubbed “overtime” because it stretched the normal 24-hour daily rhythm cycle to 26 hours. Dr. Joseph Takahashi, Northwestern University, and colleagues traced the mutation to a gene, Fbx13, that fine-tunes the clock system’s machinery by helping to orchestrate the appearance and disappearance of certain proteins — key regulators of cell function — at the proper times in the daily cycle. By disrupting this intricate process, the mutation delayed the switching-on of a key clock gene, Period, which would normally trigger the start of a new cycle after 24 hours.

I've Got Rhythm

Neurons molecularly engineered to lack the Cry2 clock gene turn on-and-off rhythmically despite being disconnected from each other. The time-lapse bioluminescence movie compresses a week of activity in disconnected cells from a mouse brain’s master clock into two seconds. By taking the clock apart, the researchers found that its unique coupling maintains daily rhythms and protects its time-keeping from genetic glitches. Uncoupling the cells revealed what different parts of the clock do. In this case, it showed that the Cry2 clock gene is not critical for rhythmicity.

I Don't

Unlike the Cry2 gene knockout above, activity of master clock neurons molecularly engineered to lack the Cry1 clock gene failed to show any daily rhythms in this time-lapse movie of a week of activity. Instead, some of the cells died after two days (the abrupt drop-off in luminescence), but most just remained static. Cry1 clock genes are thus critical for autonomous time-keeping, as they maintain rhythm without help from their neighbors.

Source: Scripps Research Institute.

References

Siepka SM, Yoo SH, Park J, Song W, Kumar V, Hu Y, Lee C, Takahashi JS. Circadian Mutant Overtime Reveals F-box Protein FBXL3 Regulation of Cryptochrome and Period Gene Expression . Cell. 2007 Apr 25; [Epub ahead of print]

Liu AC, Welsh DK, Ko CH Tran HG, Zhang EE, Priest AA, Buhr ED, Singer O, Meeker K, Verma IM, Doyle III FJ, Takahashi JS, Kay SA. Intercellular Coupling Confers Robustness Against Mutations in the SCN Circadian Clock Network. Cell. 2007 May 4; [Epub ahead of print]]