Nobody likes staying up late on Sundays because feeling lethargic on a Monday morning is rough. But on a more serious note – sleep deprivation is associated with severe health problems, and can even be lethal. Given that sleep helps maintain proper brain function, one might guess that sleep deprivation is lethal because of brain dysfunction/damage. However, this is just a hypothesis, and the mechanism underlying this lethality is unknown. The laboratory of Professor Dragana Rogulja (Assistant Professor, Harvard Medical School) decided to study this question, and found an unexpected answer.

From the very beginning, Prof. Rogulja was skeptical of the hypothesis that sleep was required to prevent brain damage. This is because simple organisms like jellyfish, with simple nervous systems and no clearly-defined brain, also engage in sleep-like behaviors. While they might not dream like humans do, they stop paying attention to their surroundings and don’t respond to external stimuli. Prof. Rogulja argues that our approach to understanding the effects of sleep deprivation is inherently flawed because it’s too anthropocentric. And this myopic lens might hamper our quest to understand why sleep is required. Given that many of nature’s simplest organisms also sleep, we must look outside of the brain to understand its importance.

“The simple animals like jellyfish and Hydra that have very primitive nervous systems…it is very clear that they engage in these forms of behavior that are like sleep.”

Prof. Rogulja for Quanta Magazine

The assignment Prof. Rogulja gave her lab was simple – deprive fruit flies (a common model organism used in research) of sleep and then perform a complete pathology to look for clues. The instructions were clear: dissect the fly, take out all the organs, and look for evidence of damage – markers of cell death, DNA damage, etc.

And so her students did just that – flies were deprived of sleep by stimulation of sleep-suppressing neurons (by a process called thermogenetic stimulation). The first thing they noticed is that, compared to control groups that slept normally, sleep deprived flies died much earlier (Figure 1, below). Not just that, the longer they were sleep deprived, the sooner they died. And this effect was very pronounced: whereas normal flies live up to 40 days, flies with 90% sleep loss started dying around Day 10, and by Day 20, they were all dead. We’re talking about a brutal 50+% reduction in life-span.

Figure 1. Survival curve of sleep-deprived flies (green, blue) and non-deprived parental controls (greys). Mean and standard error of the mean reported. 29 C is temperature used for thermogenetic stimulation. Figure adapted from Vaccaro, et al. Cell. 2020.

These results led to her next question: what exactly was causing sleep-deprived flies to die sooner? By performing pathologies on all the flies looking at as many organs as possible, the lab looked at what “bad things” were happening in all parts the body. Crucially, they didn’t restrict themselves to studies of the brain and/or nervous system. What they found was completely unexpected.

One of the damage markers they looked at are called Reactive Oxygen Species (ROS) – a type of highly reactive oxygen-containing molecule that easily reacts with other molecules even when it’s not supposed to. They looked for ROS production all over the body and found that ROS were significantly upregulated only in the gut (Figure 2, below). When the flies were allowed to sleep afterwards, ROS levels slowly decreased. These findings were also confirmed in mouse models where markers of DNA damage and cell death were observed alongside ROS elevation.

Figure 2. Reactive oxygen species production (measured as oxidized dihydroethidium build up) in the gut of sleep-deprived flies on Day 10. Figure adapted from Vaccaro, et al. Cell. 2020.

So – sleep deprivation in flies and mice causes a build up of ROS in the gut and can be fatal. The next question on Prof. Rogulja’s was – can we do something about the ROS build up? One known treatment for ROS is antioxidants. Using food supplemented with antioxidants, they found that 11 out of 53 antioxidants could clear ROS build up. Curiously, sleep-deprived flies could survive comparably long with non-deprived flies. Lastly, compounds like melatonin and NAD showed no effect on the survival of non-deprived flies (suggesting that nothing about these compounds inherently increases lifespan in flies).

Summary: Sleep deprivation causes ROS build up in the gut, which can be lethal. Antioxidant treatment can prevent this ROS build up, and improve survival. Figure: graphical abstract adapted from Vaccaro, et al. Cell. 2020.

So what does this mean for you and me?

– You might’ve heard the saying “I’ll sleep when I die.” Well, if you don’t sleep, you might die sooner than you expected. Ensure you’re getting enough sleep.
– You’ve also probably heard that antioxidants are good for you. This is true. One antioxidant that receives a lot of media attention is resveratrol found in red wine. Enjoy that glass of merlot but know that the resveratrol concentrations are too low to be beneficial. I’m sorry.
– In general, antioxidants are good for you and eating food rich in antioxidants (leafy greens, carrots, potatoes, artichokes, avocados, pumpkin, etc.) is a good idea. But your body has also evolved over thousands of years to regulate its own antioxidant levels – you don’t need to overcompensate with supplements.

As a scientist, what am I still curious about?

– Do these sleep-deprivation results apply to humans? Experiments in mice (also mammals) suggest that they might, but we don’t know yet.
– What specifically is causing the ROS build up? Is it ROS production by the organism or is the gut microbiome involved?
– If the microbiome is involved, how are gut bacteria sensing that the host is sleep-deprived?
– If the microbiome is not involved, my hypothesis is that gut cells need to be frequently replaced because they’re constantly being used, eroded, and dying. Perhaps these cells are not able to replicate effectively and hence show markers of cell death and DNA damage?
– I eat more when I’m sleep-deprived. Perhaps oxidative damage reduces nutrient assimilation in the gut and that’s my attempt at compensating for the lost nutrition?
– This study addressed acute sleep-deprivation. What about chronic sleep-deprivation?

Here is a link to Prof. Rogulja’s Laboratory if you would like to learn more. I hope you found this article insightful, intriguing and/or interesting! As always, feel free to reach out with questions, queries, comments or concerns.

A scientist

Vaccaro, et al. Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. 2020, Cell 181, 1307–1328.

Link to Prof. Rogulja’s interview with Quanta Magazine

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