Sleep is about as universal as it is mysterious. Animal studies as well as anecdotal human evidence indicate that sleep is necessary for survival.

But no one knows why.

From memory formation to waste clearance, sleep, Dragana Rogulja said, is thought of as “of the brain, by the brain, for the brain.”

However, sleep may be necessary for the brain, but the brain is not necessary for sleep.

“This observation that animals cannot live without sleep has been made in many model organisms,” Rogulja said, “even animals that don’t really have a brain” such as jellyfish.

The disconnect between the need for sleep and the possession of a brain is what prompted Rogulja, an assistant professor of neurobiology in the Blavatnik Institute at Harvard Medical School, and her team to take a look at multiple tissues in sleep-deprived flies and mice.

Their surprising conclusion, which they published in the June 4, 2020, issue of Cell, is that it is the accumulation of reactive oxygen species (ROS) in the gut that ultimately killed sleep-deprived animals.

Rogulja said that she did not anticipate finding the key problems with sleep deprivation in the gut. “There is no way that anybody would have suspected that,” she said.

But “we didn’t want to look under the lamppost, [and] it didn’t make sense to pin everything on the brain problems,” she added. “If animals can get sick from lack of sleep, this could be occurring anywhere.”

To pin down where sleep deprivation was turning deadly at the molecular level, the researchers used several methods to prevent flies from sleeping, including genetically engineering wakefulness-promoting neurons for permanent activity, and shaking the test tubes they lived in.

Flies that were unable to sleep at all started dying at 10 days of age, and had an average lifespan of 20 days, just about half of normal.

Looking at different tissues to find which ones changed before the flies started dying, “very quickly, the gut popped out,” Rogulja said.

Specifically, the team found that reactive oxygen species accumulated in the gut in the days before the animals started dying. To verify that this accumulation was causal, the team treated animals with antioxidants. Both oral antioxidants, including melatonin, lipoic acid and nicotine adenine dinucleotide (NAD), and genetically engineering the animals to express antioxidants specifically in the gut, allowed sleep-deprived flies to have normal or near-normal lifespans.

The team next tested whether sleep-deprivation affected mice in the same way. They showed that mice that were sleep-deprived for five days had elevated ROS levels in the small and large intestines but not in other organs, showing that ROS accumulation in response to sleep deprivation is a conserved phenomenon.

The findings are the first causal explanation for why sleep deprivation kills, and the first direct demonstration that oxidative stress due to sleep deprivation can lead to death.

The researchers plan to dig into the molecular mechanisms of both how ROS accumulate specifically in the gut in the first place, and how they kill the animals. The search for the origin of ROS accumulation may lead back to the brain, with neuronal signaling driving the gut accumulation. Likewise, the ill effects of ROS could be in the gut, or the molecules could be acting systemically.

The findings also suggest a way to ameliorate at least some of the effects of sleep deprivation.

“At least in flies, life without sleep is possible if you get rid of these molecules,” Rogulja said. Treated animals “were surviving fine,” and had normal motor activity.

However, she is not convinced that the same will turn out to be true for brain function.

In addition to working out the molecular details of ROS’ effects, the team wants to test whether animals treated with antioxidants during sleep deprivation are “cognitively any better off,” since sleep deprivation has clear effects on memory as well as other aspects of cognition.

Previous work by researchers at Columbia University has demonstrated that short-sleeping genetic mutants of drosophila were highly sensitive to oxidative stress, and that reducing ROS in the brain reduced the need for sleep. But those studies did not look at cognitive effects.

Rogulja stressed that since at this point, there is no data on whether cognition is affected as a consequence of the ROS accumulation, so predictions are purely speculative.

But “if I had to bet,” she said, “I would say they are independent processes.”

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