Calcineurin goes to sleep

The need for sleep is evolutionarily conserved across animals (1). During sleep, animals give up opportunities for food, water, and sex, and become more vulnerable to predation. Although we know why we eat, drink, or reproduce, we do not yet know why we sleep. The ubiquity of sleep across species suggests 1) an ancient origin of sleep, perhaps coincident with the evolution of the first nervous system(s), and 2) that sleep must provide an essential function, or functions, maintained through hundreds of millions of years of animal evolution. Increasingly, sleep is understood to be a fundamental pillar of health, as insufficient sleep amount or quality increases the risk of numerous neuropsychiatric, metabolic, and immune conditions. Therefore, understanding the molecular basis for the need to sleep remains an essential research question to advance basic biology and human health. In this issue of PNAS, Yin et al. (2) use mice to identify the evolutionarily conserved phosphatase calcineurin as a critical regulator of baseline sleep and homeostatic increase in sleep depth following sleep deprivation. Indeed, Yin et al. (2) showed that increased or decreased calcineurin activity in neurons drives a striking increase (~100%) or decrease (~70%) in baseline sleep respectively, and near abrogation of homeostatic sleep response upon calcineurin ablation.

Over 40 years ago, Alexander Borbély proposed the longstanding two-process model of sleep regulation whereby sleep is controlled by a homeostatic sleep drive (process S), that promotes sleep in proportion to time spent awake, and the circadian rhythm (process C), that promotes sleep at the ecologically appropriate time of day (3). Since that time, considerable advances have been made in understanding the molecular basis of process C through discovery and elucidation of “clock genes” (4). Major advances have also been made in identifying discrete neural circuits of sleep- and wake-promoting neurons (5, 6) which controls the sleep–wake transition. In contrast, the conserved molecular basis for process S, the homeostatic need for sleep, has largely remained a mysterious black box. Process C, the circadian rhythm, is now understood as a cellular phenomenon, utilizing a molecular clock present in most cells of the body. Recent advances are starting to reveal that sleep need also has a molecular and cellular basis, located within neurons broadly across the nervous system. A cellular-level of sleep need is inspired by the conserved need for sleep across animals with divergent sleep circuitry, and the brain-wide deleterious effect of sleep loss across species. Cellular-level sleep need is evidenced by genetic identification of conserved sleep-regulating kinases in excitatory neurons (7–9) and sleep need–driven brain-wide phosphorylation (10–12). Thus, kinase activity in excitatory neurons is a significant determinant of cellular-level sleep need. It is, therefore, reasonable to ask whether phosphatases, which antagonizes kinases to drive protein dephosphorylation, are similarly important in mammalian sleep need.

Yin et al. (2) addressed these issues with viral-mediated brain-wide induction or ablation of the phosphatase calcineurin, using a virus optimized for broad brain expression through retro-orbital delivery (13). The resulting adult brain chimeric (ABC) mice enable high-throughput one-step sleep analysis for all calcineurin subunits, many of which are encoded by potentially redundant or lethal genes (7, 13). Using well-established electroencephalogram (EEG) signatures of sleep, Yin et al. (2). found ABC-induction of a constitutively active mutant of calcineurin catalytic subunit (CNAα) in brain neurons strongly promoted non-rapid eye movement (NREM) sleep (~100%), while its ablation strongly suppressed NREM (~50%). Furthermore, ABC-ablation of the calcineurin regulatory subunit CNB1, which destabilized and decreased all catalytic subunits (α/β/γ), massively suppressed NREM (~75%). These results are striking given that sleep-regulating kinases typically alter NREM by ~30% (9), but consistent with severe insomnia of calcineurin knockout in fruitfly (14). Furthermore, ablation of CNAα or CNB1 nearly abrogated NREM slow wave activity (SWA) rebound after sleep deprivation. EEG-SWA increases proportionally with sleep loss and dissipates only during NREM, and thus has been widely regarded as a physiological marker of process S, the homeostatic need for sleep in rodents and humans. Therefore, neuronal calcineurin governs both baseline sleep and the homeostatic sleep drive, providing a major advance toward opening the black box of process S.

In PNAS, Yin et al. use mice to identify the evolutionarily conserved phosphatase calcineurin as a critical regulator of baseline sleep and homeostatic increase in sleep depth following sleep deprivation.

Yin et al. (2) analyzed calcineurin signaling in sleep regulation through epistatic analysis against known wake-regulating cAMP-dependent protein kinase A (PKA) and sleep-promoting salt-inducible kinase 3 (SIK3). This builds on recent advances in forward genetics that identified a SIK3 mutant lacking an inhibitory phosphorylation site (S551), regulated by PKA, resulting in NREM hypersomnia (sleepy, SIK3^Slp) (7–9). A phosphodeficient mutant of this site conferring resilience to PKA phosphorylation (SIK3^S551A) also promotes sleep in mammals (15). Yin et al. (2) clearly show that SIK3 is also a substrate of calcineurin phosphatase activity, providing a putative mechanism whereby PKA promotes wake, and calcineurin promotes sleep in part through antagonistic regulation of shared substrates (Fig. 1). Consistent with these findings and with data that PKA suppresses sleep in flies (16), Yin et al. (2) document strong sleep suppression (~70%) or promotion (~30%) in ABC-induced gain- or loss-of PKA function, respectively. Further, the loss of baseline sleep in CNB1^KO is rescued by a sleep-promoting dominant-negative PKA mutant (dnPKA) expressed under the same promoter or partially rescued by PKA-resistant SIK3^Slp. Importantly, suppression of the homeostatic response to sleep deprivation (EEG-SWA) upon calcineurin ablation is not rescued by SIK3^Slp. Therefore, calcineurin likely regulates baseline NREM sleep and the homeostatic response to sleep deprivation through partly separate mechanisms.

Fig. 1.

Wake-promoting PKA and sleep-promoting Calcineurin form an antagonistic relationship through regulation of shared substrates.

How do Yin et al.’s conclusions fit into the larger picture? Yin et al.’s (2) results add to the growing body of evidence for a cellular-level of sleep need in mammals controlled by brain-wide reversible phosphorylation (7, 8, 17, 18). The primary findings of Yin et al. (2), regulation of mammalian sleep need by calcineurin, through brain-wide or forebrain-specific intracellular signaling, is in strong agreement with recent independent work on calcineurin/PKA (17–19), and similar studies on SIK3 (7, 8) and other sleep regulating kinases (20, 21). Furthermore, a privileged role for cortical excitatory neurons in mammalian sleep need is observed here (2) and by others for calcineurin (17, 18), PKA (17), SIK3 (7, 8) and other sleep regulating kinases (20), indicating cortical excitatory neurons may be a primary depository of sleep regulating molecules and major site for the accumulation of sleep need at the cellular level. Recent advances also show that the responses to sleep deprivation are developmentally regulated (22, 23), raising the question of how the mechanisms of cellular sleep need are shaped during brain maturation. Considering that sleep loss induces global cognitive impairments that can only be reversed by sleep, Yin et al. (2) inspires one to imagine a future where the molecular basis of cellular level sleep need, often called “process S,” is understood at a comparable level to the circadian rhythm, process C. How does cellular-level sleep need integrate upstream biological signals in vivo, such as synaptic activity to promote processes such as memory consolidation? How does cellular-level sleep need mediate the restorative benefits of sleep? How do we target cellular-level sleep need to develop procognitive sleep medicine? Yin et al. (2), and contemporary scientists in this field are now providing critical steps in opening the mysterious black box of the molecular basis for why we sleep, ushering in an era of advances in sleep medicine and improved lifelong human health.