Tiredness, more than hunger, thirst, or pain, may just be the most difficult condition for a human to bear. In his classic short story “Sleepy,” Anton Chekhov describes the loss of humanity caused by deep fatigue: “And Varka is sleepy. Her eyes are glued together, her head droops, her neck aches. She cannot move her eyelids or her lips, and she feels as though her face is dried and wooden, as though her head has become as small as the head of a pin” (translation by Constance Garnett).
Sleep is a vital need that is common to all organisms with a nervous system, including flies, worms, and even jellyfish. But what is tiredness? A newly published study from Bar-Ilan University is the first to describe the underlying biological mechanism. “The question is, why do we sleep?” says sleep researcher Prof. Lior Appelbaum. “It’s a strange thing; you can die [from lack of it]. It seems to defy evolution. If you’re a fish in the ocean, sleeping can get you eaten by a shark. But for some reason, it’s very important, since all animals sleep.” He says that this is an evolutionary riddle that has not yet been completely solved – science still does not know exactly how sleep helps the brain or individual cells.
The new study, by Appelbaum and his postdoctoral fellow David Zada, both of them from the Goodman Faculty of Life Sciences and the Gonda Multidisciplinary Brain Research Center at Bar-Ilan University, brings science closer to solving the mystery. Their team of researchers has discovered a mechanism that leads to tiredness in the nervous system of the zebrafish, with supportive evidence for the existence of such a mechanism in mice. The study was published this week in the journal Molecular Cell.
During wakefulness, the body builds up “sleep pressure,” which dissipates during sleep. But what causes this pressure, which leads to a sensation of tiredness? The researchers showed that during waking hours, DNA damage accumulates in nerve cells. This damage is caused by the activity of these cells, as well as by other causes, such as radiation exposure.
The importance of DNA repairs during sleep
The researchers explain that DNA is a long chain of nucleic acids and that damage manifests as tears in that chain. Repairs are then necessary to reattach the torn sections without changing the sequence of these acids. Throughout the day, DNA damage response proteins – which act as repair systems – are active in each cell, reconnecting the ruptures in the DNA chain. But during wakefulness, these tears occur at a greater rate than the repairs, and damage continues to accumulate.
Too much of this damage in the brain can be dangerous to an organism – be it a jellyfish or a human – which is why tiredness builds up, pushing the organism to stop its activities and fall asleep. DNA repairs during sleep are more efficient, enabling the organism to begin a new day.
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The researchers conducted a series of experiments in which they tested the hypothesis that the accumulation of damaged DNA is what leads to a build-up in sleep pressure. They caused deliberate damage to the DNA of zebrafish, for example by activating their nerve cells, to see how this affects their sleep. They did this by using specific drugs or optogenetic tools, which can be used to load target cells with light-sensitive molecules using genetic manipulation and then activate isolated nerve cells using pulses of light.
They found that the greater the DNA damage, the more the fish needed to sleep. When the accumulated damage exceeded a certain threshold, sleep pressure was so intense that the fish fell asleep instantly to enable efficient DNA repair.
In another experiment, the researchers tried to establish the minimum period of sleep required to reduce sleep pressure and DNA damage in the fish. “We gradually shortened the time we allowed them to sleep at night and found that the minimum, surprisingly, was six hours. That’s exactly the time we were allotted for sleep in the army,” says Appelbaum. “If you let them sleep less than that, the DNA damage is not reduced enough, and they fall asleep even under the light, despite being very sensitive to light.”
Appelbaum believes that in the future, these discoveries may lead to the development of a system that will measure the damage to human DNA. The extent of the damage will alert us to the amount of sleep we need. “Perhaps it will be possible to create a ‘tiredness clock’ so that we don’t get too little sleep – or too much,” he says.
The researchers also managed to discover the brain mechanism that induces tiredness and tells the organism that it has to sleep. They found that a protein called poly(ADP-ribose) polymerase 1 (Parp1) attaches to broken DNA in cells and gathers the systems required to remove the damage. “This protein is like an antenna that signals ‘this is where the tears are in the DNA,” says Appelbaum. More Parp1 accumulates where DNA is damaged during wakefulness, while the amount of it declines during sleep.
The researchers even showed that repairs are carried out while the organism is asleep. “Zebrafish have transparent bodies, skulls, and brains, so their isolated cells and even clusters of proteins inside nerve cells can be photographed,” says Appelbaum. “That way, we were able to record the actual repair proteins that arrive during sleep, and show that, at night, those repairs are more efficient.”
Dr. Alex Gileles-Hillel from the Faculty of Medicine at the Hebrew University of Jerusalem and Hadassah Hospital, who was not involved in this study, says that “this is phenomenal work. The researchers did more than was necessary to prove that the Parp1 mechanism is responsible for the sensation of tiredness. They used the customary model, which uses experiments with fish, and added experiments on mice. This is very impressive work, due to the variety of approaches that were used in order to prove a causal relationship between DNA damage, the Parp1 protein, and sleep pressure,” he says.
The researchers indicated this causal relationship through pharmacological and genetic manipulation. They first overexpressed Parp1 and established that it induces sleep and enhances sleep-dependent DNA repair. They also under expressed this protein (a maneuver called knockdown) or blocked its action with a drug, which blocks the signal for repairing damaged DNA. As a result, the fish didn’t know they were tired, they didn’t fall asleep and their bodies did not undergo a DNA repair process. “Through this mechanism, the brain knows it’s tired,” concludes Appelbaum. “The protein builds up during waking hours, and beyond a certain threshold, it promotes the need to sleep.”
In order to bolster their findings, the researchers tested the role of Parp1 in the regulation of sleep in mice. This part of the work was done in collaboration with Prof. Yuval Nir of Tel Aviv University. Just as in the bodies of zebrafish, the inhibition of the protein shortened the duration of sleep in mice. According to Nir, they also found that this protein also affects the quality of sleep. Its inhibition reduced the amount of slow brain waves during sleep, waves which indicate deep and restorative sleep in mammals (NREM sleep).
The brain’s sewage system
The findings provide a detailed description of the chain of events that explain sleep at the cellular level. Gileles-Hillel says that this is a microscopic peek into sleep, but adds that he is sure that this is not the only mechanism for inducing sleep, “since the brain operates as an organ, not only on the level of individual cells.”
He notes that only a decade ago, a human glymphatic system was discovered. These brain lymphatics carry cerebrospinal fluid, which drains waste materials from the central nervous system and takes them back into the bloodstream. “It’s like the brain’s sewage system,” says Gileles-Hillel, “and the system is more active during sleep.” He adds that the new study describes only the intracellular system and that it’s likely that these two systems complement each other.
Gileles-Hillel notes that Parp1 is also relevant to oncology. With cancer, cells divide at a rapid pace, causing more DNA damage than in healthy cells. Cancerous cells, therefore, require more repair system activation than healthy cells. Parp1 inhibition is used in breast cancer treatment, for example. “It’s known that Parp1 inhibitors cause fatigue among women with breast cancer,” says Gileles-Hillel. And, he adds, it is known that sleep disorders increase the risk of getting cancer and of having more aggressive tumors.
“It could be that just like with nerve cells in the brain, sleep disruption hinders the repair of DNA mutations in other systems of the body. Cells divide all the time, and mutations constantly occur. If DNA damage is repaired during sleep, this is diminished when sleep is disturbed, which could lead to the development of cancerous cells. If this is so, the mechanism described in this study may be relevant not just to neurons, but to cells in general,” he says.
Appelbaum and Zada mention in their article that disturbed sleep has an effect not only on cancer but also degenerative neurological diseases such as Parkinson’s or Alzheimer’s disease as well. “Sleep disorders are a major symptom of aging,” Appelbaum says. “It may be that if sleep is disturbed for a span of many years, it leads to an increase in DNA damage and even to the death of nerve cells, ultimately leading to diseases associated with old age.”
Gileles-Hillel says that the link between sleep disorders and the development of Alzheimer’s disease is well-known. “People who don’t sleep well in their sixties have a higher chance of developing this disease in their next decade,’ he explains. “This doesn’t necessarily point to a causal connection, but animal studies have shown that if you deprive mice of sleep, their brains accumulate compounds like those found in Alzheimer’s patients.”
According to Gileles-Hillel, “if this new study has any significance for degenerative diseases, that’s where the money will be. They’re really saying that damage is caused to DNA, which is repaired during sleep. If the damage is not repaired, a degenerative disease may follow. As soon as you understand the mechanism, you can find a cure.’