Biology subjects

Fujishima, H.

Publications and source records attributed to Fujishima, H..

4 recordsLinked to original sources

bioRxiv2023

Post-synaptic competition between calcineurin and PKA regulates mammalian sleep-wake cycles

Phosphorylation of synaptic proteins is a pivotal biochemical reaction that controls the sleep-wake cycle in mammals. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigated a pair of kinases and phosphatases that reciprocally regulate sleep duration. Through comprehensive screening of Protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes via the generation of 40 gene knockout mouse lines including post-natal CRISPR targeting, we identified a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase (PP) 1 (Pppr1r9b), and catalytic and regulatory subunits of PP2B (calcineurin) (Ppp3ca and Ppp3r1) as sleep control genes. AAV-mediated stimulation of PKA and PP1/calcineurin activities confirmed PKA as a wake-promoting kinase, while PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the dramatic changes in sleep duration associated with their increased and decreased activity, ranging from approximately 17.3 hours/day (PP1 expression) to 6.7 hours/day (post-natal CRISPR targeting of calcineurin). For these phosphatases to exert their sleep-promoting effects, localization signals to the excitatory post-synapse were necessary. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of calcineurin, suggesting that PKA and calcineurin construct a hierarchical phosphorylation control network for sleep regulation at excitatory post-synapses.

neuroscience↗

bioRxiv2023

Cortical parvalbumin neurons are responsible for homeostatic sleep rebound through CaMKII activation

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. We show here that CaMKII-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in sleep homeostasis regulation. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. We propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

neuroscience↗

bioRxiv2022

Circadian ribosome profiling reveals a role for the Period2 upstream opening reading frame in sleep

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered post-transcriptionally. We used ribosome profiling to examine post-transcriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA post-transcriptionally, which can impact physiological rhythms and sleep. Significance StatementPeriod (Per) is an iconic gene in the field of circadian rhythms since its discovery in 1971 by Seymour Benzer and Ronald Konopka in fruit flies. The inhibitory feedback loop of PER protein drives circadian rhythms. We show that Per2 is regulated by an upstream open reading frame (uORF) in the 5 untranslated region of Period2 mRNA. Mutation of the Per2 uORF altered the amplitude of luciferase reporter expression in well-characterized cell culture models. Per2 uORF mutant mice had significantly elevated Per2 mRNA levels and exhibited sleep loss, particularly during light-to-dark and dark-to-light transitions, which suggests a role for uORFs in modulating molecular and physiological circadian rhythms.

cell biology↗

bioRxiv2021

Realization of phosphorylation hypothesis of sleep by mammalian CaMKIIβ

The reduced sleep duration observed in Camk2a and Camk2b knockout mice revealed the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII)/CAMKII{beta} as sleep-promoting kinases and lead to the phosphorylation hypothesis of sleep. However, the underlying mechanism of sleep regulation by kinases and protein phosphorylation is largely unknown. Here, we demonstrate that the phosphorylation states of CaMKII{beta} regulates sleep duration and sleep needs. Importantly, the activation or inhibition of CaMKII{beta} can increase or decrease sleep duration by almost two-fold, supporting the role of CaMKII{beta} as a core sleep regulator in mammals. This sleep regulation depends on the kinase activity of CaMKII{beta} in excitatory neurons. Furthermore, CaMKII{beta} mutants mimicking different phosphorylation states can regulate various sleep steps including sleep induction, sleep maintenance, and sleep cancelation. Key CaMKII{beta} residues responsible for the mode switch undergo ordered (auto-)phosphorylation. We thus propose that ordered multi-site phosphorylation of CaMKII{beta} underlies multi-step sleep regulation in mammals.

neuroscience↗