Researchers have found that in sleep-deprived flies, specific neurons associated with sleep exhibit changes in their mitochondria (responsible for cellular energy production) and energy use. These molecular changes, which reverse with rest, indicate that the need for sleep is linked to the accumulation of metabolic stress. Manipulating these mitochondrial processes can influence the pressure to sleep, suggesting that sleep is, in part, a response to the metabolic stress that builds up during wakefulness.
“Sleep pressure” is a central variable in the mechanism of sleep regulation, but it is not yet fully understood in terms of how it physically works in the brain.
Sleep pressure is what makes people increasingly sleepy after many hours awake, but scientists still do not know exactly how this sensation of “needing to sleep” is produced in brain cells.
What is known is that when we are awake for long periods, several changes occur in the brain. These include changes in the firing patterns of neurons (the way they send signals), changes in the strength of the connections between them (synapses), changes in the concentrations of certain chemicals, such as metabolites, and even changes in which genes are turned on or off in cells.
However, it is not yet clear whether these changes cause the feeling of needing sleep or whether they are simply the result of not sleeping for a long time.
To investigate this, a good approach is to look at specific types of neurons that play a direct role in getting us to sleep and staying asleep. These “specialized” neurons are likely to be the most direct sources of insight into why we need sleep since they regulate the process of sleep.
To better understand the molecular (i.e., cell- and molecule-level) changes that occur in the brain that may be linked to sleep, researchers at the University of Oxford analyzed what’s called the transcriptome—the complete set of active genetic messages in cells.
They used a technique called “single-cell sequencing” to study the genetic messages in specific neurons in flies, both in well-rested flies and in flies that had been sleep-deprived.
The researchers also used a fluorescent marker that allows them to precisely identify neurons that induce sleep, called dFBNs.
These neurons, which project to a specific area of the fly brain known as the dorsal fan body, were enriched in the study to compare how they respond to sleep deprivation compared to other types of brain cells.
In parallel, a second study confirmed that these neurons (dFBNs) play an important role in promoting sleep, using advanced genetic tools based on detailed information about which genes were active in these neurons.
For the brain to properly control sleep, it needs a “signal” that indicates the need for sleep while we are awake and a “relief” of that need during sleep.
To understand this, the researchers examined the transcriptomes of brain cells isolated from well-rested flies and from sleep-deprived flies.
They observed that, in dBNF neurons, sleep deprivation leads to an increase in the activity of genes that produce proteins essential for the functioning of mitochondria — cellular structures responsible for generating energy in the form of ATP (adenosine triphosphate).
In addition, these genetic changes were accompanied by physical changes in the mitochondria: they fragmented, underwent a process called mitophagy (where damaged mitochondria are eliminated), and increased the number of points of contact with another cellular structure, the endoplasmic reticulum.
These points of contact create “bridges” for the mitochondria to repair damage caused by lipid oxidation, a consequence of the stress generated by sleep deprivation. Interestingly, these changes in the mitochondria were temporary and reversed after a period of recovery from sleep.
However, if the researchers interfered with the mitochondria’s “breathing” by installing mechanisms to flush out excess electrons, the flies did not experience as much pressure to sleep.
To test the impact of mitochondrial fragmentation on these dBNF neurons, the researchers artificially induced or inhibited the mitochondrial fission (division) process. When mitochondria fused excessively, neuron excitability and sleep time increased.
In contrast, when mitochondria were fragmented, both neuron excitability and sleep duration decreased. This suggests that mitochondrial fusion and fission directly influence sleep pressure.
Sleep alters mitochondrial dynamics. a, b) Maximum intensity projections of automatically detected mitochondria (a, black) and their morphometric parameters (b) in dBNF dendrites from rested flies, sleep-deprived flies, flies allowed to recover for 24 h after sleep deprivation, and sleep-deprived flies coexpressing AOX or TrpA1. The mitochondrial number is not altered by sleep deprivation but is elevated after recovery sleep and in sleep-deprived flies coexpressing activated AOX or TrpA1. c) Visualization of events in mitochondrial fission. d) Drp1 recruitment. Sleep deprivation increases anti-FLAG fluorescence (intensity-coded according to the key on the right) within automatically detected mitochondrial contours. e) Mitochondria–ER contacts. f) Mitophagy. Summed intensity projections of dBNF dendrites expressing mito-QC.
Another important finding was that, after sleep deprivation, ATP levels in dBNF neurons increased. This increase was caused by reduced ATP consumption, which results from the suppression of activity in these neurons due to the stress of being awake for long periods.
However, this process also increases susceptibility to oxidative stress, which damages mitochondria.
Finally, the study showed that the pressure to sleep can be altered by manipulating the energy use of cells. When the scientists prevented the cell’s energy from being used to create ATP, the pressure to sleep was reduced. Conversely, when they stimulated excess ATP production using a light-activated “proton pump,” the need for sleep increased.
These findings suggest that sleep may be an unavoidable physiological need associated with mitochondrial metabolism, similar to what occurs during the aging process.
In other words, metabolic stress accumulated during waking hours appears to be relieved by sleep, and the need for sleep itself may be a consequence of this accumulation of metabolic stress.
READ MORE:
Mitochondrial origins of the pressure to sleep
Raffaele Sarnataro, Cecilia D. Velasco, Nicholas Monaco, Anissa Kempf, Gero Miesenböck
Science, 4 NOV 2024. preprint doi: https://doi.org/10.1101/2024.02.23.581770
Abstract:
The neural control of sleep requires that sleep need is sensed during waking and discharged during sleep. To obtain a comprehensive, unbiased view of molecular changes in the brain that may underpin these processes, we have characterized the transcriptomes of single cells isolated from rested and sleep-deprived flies. Transcripts upregulated after sleep deprivation, in sleep-control neurons projecting to the dorsal fan-shaped body (dFBNs) but not ubiquitously in the brain, encode almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis. These gene expression changes are accompanied by mitochondrial fragmentation, enhanced mitophagy, and an increase in the number of contacts between mitochondria and the endoplasmic reticulum, creating conduits for the replenishment of peroxidized lipids. The morphological changes are reversible after recovery sleep and blunted by the installation of an electron overflow in the respiratory chain. Inducing or preventing mitochondrial fission or fusion in dFBNs alters sleep and the electrical properties of sleep-control cells in opposite directions: hyperfused mitochondria increase, whereas fragmented mitochondria decrease, neuronal excitability and sleep. ATP levels in dFBNs rise after enforced waking because of diminished ATP consumption during the arousal-mediated inhibition of these neurons, which predisposes them to heightened oxidative stress. Consistent with this view, uncoupling electron flux from ATP synthesis relieves the pressure to sleep, while exacerbating mismatches between electron supply and ATP demand (by powering ATP synthesis with a light-driven proton pump) promotes sleep. Sleep, like ageing, may be an inescapable consequence of aerobic metabolism.
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