Neuroligin-2 shapes individual slow waves during slow-wave sleep and the response to sleep deprivation in mice.

BACKGROUND: Sleep disturbances are a common comorbidity to most neurodevelopmental disorders and tend to worsen disease symptomatology. It is thus crucial to understand mechanisms underlying sleep disturbances to improve patients' quality of life. Neuroligin-2 (NLGN2) is a synaptic adhesion protein regulating GABAergic transmission. It has been linked to autism spectrum disorders and schizophrenia in humans, and deregulations of its expression were shown to cause epileptic-like hypersynchronized cerebral activity in rodents. Importantly, the absence of Nlgn2 (knockout: KO) was previously shown to alter sleep-wake duration and quality in mice, notably increasing slow-wave sleep (SWS) delta activity (1-4 Hz) and altering its 24-h dynamics. This type of brain oscillation is involved in memory consolidation, and is also a marker of homeostatic sleep pressure. Sleep deprivation (SD) is notably known to impair cognition and the physiological response to sleep loss involves GABAergic transmission. METHODS: Using electrocorticographic (ECoG) recordings, we here first aimed to verify how individual slow wave (SW; 0.5-4 Hz) density and properties (e.g., amplitude, slope, frequency) contribute to the higher SWS delta activity and altered 24-h dynamics observed in Nlgn2 KO mice. We further investigated the response of these animals to SD. Finally, we tested whether sleep loss affects the gene expression of Nlgn2 and related GABAergic transcripts in the cerebral cortex of wild-type mice using RNA sequencing. RESULTS: Our results show that Nlgn2 KO mice have both greater SW amplitude and density, and that SW density is the main property contributing to the altered 24-h dynamics. We also found the absence of Nlgn2 to accelerate paradoxical sleep recovery following SD, together with profound alterations in ECoG activity across vigilance states. Sleep loss, however, did not modify the 24-h distribution of the hypersynchronized ECoG events observed in these mice. Finally, RNA sequencing confirmed an overall decrease in cortical expression of Nlgn2 and related GABAergic transcripts following SD in wild-type mice. CONCLUSIONS: This work brings further insight into potential mechanisms of sleep duration and quality deregulation in neurodevelopmental disorders, notably involving NLGN2 and GABAergic neurotransmission.

Transcriptional regulation of the mouse EphA4, Ephrin-B2 and Ephrin-A3 genes by the circadian clock machinery.

Circadian rhythms originate from molecular feedback loops. In mammals, the transcription factors CLOCK and BMAL1 act on regulatory elements (i.e. E-boxes) to shape biological functions in a rhythmic manner. The EPHA4 receptor and its ligands Ephrins (EFN) are cell adhesion molecules regulating neurotransmission and neuronal morphology. Previous studies showed the presence of E-boxes in the genes of EphA4 and specific Ephrins, and that EphA4 knockout mice have an altered circadian rhythm of locomotor activity. We thus hypothesized that the core clock machinery regulates the gene expression of EphA4, EfnB2 and EfnA3. CLOCK and BMAL1 (or NPAS2 and BMAL2) were found to have transcriptional activity on distal and proximal regions of EphA4, EfnB2 and EfnA3 putative promoters. A constitutively active form of glycogen synthase kinase 3ß (GSK3ß; a negative regulator of CLOCK and BMAL1) blocked the transcriptional induction. Mutating the E-boxes of EphA4 distal promoter sequence reduced transcriptional induction. EPHA4 and EFNB2 protein levels did not show circadian variations in the mouse suprachiasmatic nucleus or prefrontal cortex. The findings uncover that core circadian transcription factors can regulate the gene expression of elements of the Eph/Ephrin system, which might contribute to circadian rhythmicity in biological processes in the brain or peripheral tissues.

Correction to: The effect of Neuroligin-2 absence on sleep architecture and electroencephalographic activity in mice.

Correction to: Molecular Brain (2018) 11:52 https://doi.org/10.1186/s13041-018-0394-3Following publication of the original article [1], the authors reported that the article was mistakenly submitted with the omission of two authors: Feng Cao and Zhengping Jia. The authors declare that this was an error made in good faith. The corrected author list and list of affiliations are used in this Correction. The changes made to the author list and list of affiliations are also listed below, as well as the revised 'Acknowledgements' section and 'Authors' contributions' section.

The effect of Neuroligin-2 absence on sleep architecture and electroencephalographic activity in mice.

Sleep disorders are comorbid with most psychiatric disorders, but the link between these is not well understood. Neuroligin-2 (NLGN2) is a cell adhesion molecule that plays roles in synapse formation and neurotransmission. Moreover, NLGN2 has been associated with psychiatric disorders, but its implication in sleep remains underexplored. In the present study, the effect of Nlgn2 knockout (Nlgn2-/-) on sleep architecture and electroencephalographic (EEG) activity in mice has been investigated. The EEG and electromyogram (EMG) were recorded in Nlgn2-/- mice and littermates for 24 h from which three vigilance states (i.e., wakefulness, rapid eye movement [REM] sleep, non-REM [NREM] sleep) were visually identified. Spectral analysis of the EEG was performed for the three states. Nlgn2-/- mice showed more wakefulness and less NREM and REM sleep compared to wild-type (Nlgn2+/+) mice, especially during the dark period. This was accompanied by changes in the number and duration of individual episodes of wakefulness and sleep, indexing changes in state consolidation, as well as widespread changes in EEG spectral activity in all states. Abnormal 'hypersynchronized' EEG events have also been observed predominantly in Nlgn2-/- mice. These events were mainly observed during wakefulness and REM sleep. In addition, Nlgn2-/- mice showed alterations in the daily time course of NREM sleep delta (1-4 Hz) activity, pointing to modifications in the dynamics of sleep homeostasis. These data suggest that NLGN2 participates in the regulation of sleep duration as well as EEG activity during wakefulness and sleep.

Regulation of the Neuroligin-1 Gene by Clock Transcription Factors.

NEUROLIGIN-1 (NLGN1) is a postsynaptic adhesion molecule involved in the regulation of glutamatergic transmission. It has been associated with several features of sleep and psychiatric disorders. Our previous work suggested that transcription of the Nlgn1 gene could be regulated by the transcription factors CLOCK and BMAL1 because they bind to the Nlgn1 gene promoter in vivo. However, whether CLOCK/BMAL1 can directly activate Nlgn1 transcription is not yet known. We thus aimed to verify whether CLOCK/BMAL1, as well as their homologs NPAS2 and BMAL2, can activate transcription via the Nlgn1 promoter by using luciferase assays in COS-7 cells. We also investigated how Nlgn1 expression was affected in Clock mutant mice. Our results show transcriptional activation in vitro mediated by CLOCK/BMAL1 and by combinations with their homologs NPAS2 and BMAL2. Moreover, CLOCK/BMAL1 activation via the Nlgn1 gene fragment was repressed by GSK3ß. In vivo, Nlgn1 mRNA expression was significantly modified in the forebrain of Clock mutant mice in a transcript variant-dependent manner. However, no significant change in NLGN1 protein level was observed in Clock mutant mice. These findings will increase knowledge about the transcriptional regulation of Nlgn1 and the relationship between circadian rhythms, mental health, and sleep.

EphA4 is Involved in Sleep Regulation but Not in the Electrophysiological Response to Sleep Deprivation.

STUDY OBJECTIVES: Optimal sleep is ensured by the interaction of circadian and homeostatic processes. Although synaptic plasticity seems to contribute to both processes, the specific players involved are not well understood. The EphA4 tyrosine kinase receptor is a cell adhesion protein regulating synaptic plasticity. We investigated the role of EphA4 in sleep regulation using electrocorticography in mice lacking EphA4 and gene expression measurements. METHODS: EphA4 knockout (KO) mice, Clock(Δ19/Δ19) mutant mice and littermates, C57BL/6J and CD-1 mice, and Sprague-Dawley rats were studied under a 12 h light: 12 h dark cycle, under undisturbed conditions or 6 h sleep deprivation (SLD), and submitted to a 48 h electrophysiological recording and/or brain sampling at different time of day. RESULTS: EphA4 KO mice showed less rapid eye movement sleep (REMS), enhanced duration of individual bouts of wakefulness and nonrapid eye movement sleep (NREMS) during the light period, and a blunted daily rhythm of NREMS sigma activity. The NREMS delta activity response to SLD was unchanged in EphA4 KO mice. However, SLD increased EphA4 expression in the thalamic/hypothalamic region in C57BL/6J mice. We further show the presence of E-boxes in the promoter region of EphA4, a lower expression of EphA4 in Clock mutant mice, a rhythmic expression of EphA4 ligands in several brain areas, expression of EphA4 in the suprachiasmatic nuclei of the hypothalamus (SCN), and finally an unchanged number of cells expressing Vip, Grp and Avp in the SCN of EphA4 KO mice. CONCLUSIONS: Our results suggest that EphA4 is involved in circadian sleep regulation.

Neuroligin-1 links neuronal activity to sleep-wake regulation.

Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.

Dynein light chain Tctex-type 1 modulates orexin signaling through its interaction with orexin 1 receptor.

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization.

Morning and evening-type differences in slow waves during NREM sleep reveal both trait and state-dependent phenotypes.

Brain recovery after prolonged wakefulness is characterized by increased density, amplitude and slope of slow waves (SW, <4 Hz) during non-rapid eye movement (NREM) sleep. These SW comprise a negative phase, during which cortical neurons are mostly silent, and a positive phase, in which most neurons fire intensively. Previous work showed, using EEG spectral analysis as an index of cortical synchrony, that Morning-types (M-types) present faster dynamics of sleep pressure than Evening-types (E-types). We thus hypothesized that single SW properties will also show larger changes in M-types than in E-types in response to increased sleep pressure. SW density (number per minute) and characteristics (amplitude, slope between negative and positive peaks, frequency and duration of negative and positive phases) were compared between chronotypes for a baseline sleep episode (BL) and for recovery sleep (REC) after two nights of sleep fragmentation. While SW density did not differ between chronotypes, M-types showed higher SW amplitude and steeper slope than E-types, especially during REC. SW properties were also averaged for 3 NREM sleep periods selected for their decreasing level of sleep pressure (first cycle of REC [REC1], first cycle of BL [BL1] and fourth cycle of BL [BL4]). Slope was significantly steeper in M-types than in E-types in REC1 and BL1. SW frequency was consistently higher and duration of positive and negative phases constantly shorter in M-types than in E-types. Our data reveal that specific properties of cortical synchrony during sleep differ between M-types and E-types, although chronotypes show a similar capacity to generate SW. These differences may involve 1) stable trait characteristics independent of sleep pressure (i.e., frequency and durations) likely linked to the length of silent and burst-firing phases of individual neurons, and 2) specific responses to increased sleep pressure (i.e., slope and amplitude) expected to depend on the synchrony between neurons.