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

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

Abstract 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

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

Abstract 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

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/s13041-018-0394-3 Following 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

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/s13041-018-0394-3 Following 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

Interaction of OX1R and Dynlt1 in mammalian cells.

<p>HEK293 cells were transfected with expression vectors for Myc-Dynlt1 and V5-OX1R (0.5 or 2.0 µg DNA, full-length receptor) or corresponding empty vector. Whole-cell lysates were subjected to IP using an anti-V5 antibody and protein G-sepharose beads, followed by IB with anti-V5 or anti-Myc antibodies against V5-OX1R and Myc-Dynlt1, respectively. Equal transfection of Myc-Dynlt1 was verified by IB with anti-Myc antibody on cell extracts (input). The experiment was repeated 5 times, with similar results.</p

Modulation of OX1R-mediated signaling by Dynlt1.

<p>(<b>A</b>) HEK293 cells were transfected with a V5-OX1R or V5-OX1R Δ364–416 expression vector and stimulated with 500 nM OX-A for the indicated times. Protein extracts were analyzed by SDS-PAGE and Western blotting with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. ERK1/2 are quickly phosphorylated after OX-A stimulation and OX1R CTD is essential to this response. V5-OX1R Δ364–416, V5-OX1R lacking its CTD. (<b>B–D</b>) Transfected HEK293 cells were stimulated with 100 nM OX-A for the indicated times and processed as in (A). (<b>B</b>) Co-expression of Dynlt1 leads to a less sustained ERK1/2 activation in response to OX-A. Blots are from a representative experiment. The graph shows a combination of 4 independent experiments. ***: p<0.001 vs data without Dynlt1 transfected (ANOVA followed by post-hoc analysis at the different times). (<b>C</b>) Down-regulation of Dynlt1 by a siRNA (10 nM, expression reduced by 88%) leads to a sustained activation of the ERK1/2 pathway in response to OX-A. This experiment was repeated twice, each in duplicates, with similar results. The blots and the graph present the results from one of these experiments. (<b>D</b>) Dynlt1 does not lead to a less sustained ERK1/2 activation in response to OX-A when co-expressed with OX1R mutant T409A, T412A. This experiment was repeated twice, each in triplicates, with similar results. The blots and the graph are from one of these experiments.</p

Regions of orexin receptors involved in their interaction with Dynlt1 and Dynlt3.

<p>(<b>A</b>) Identification of a putative bipartite Dynlt1-binding motif in orexin receptors. The proximal portion of the structure shown here, located in the third intracellular loop of orexin receptors, is not included in the soluble orexin receptors CTD containing the distal part and used for yeast two-hybrid assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone-0026430-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone-0026430-g003" target="_blank">3</a>). Amino acid numbering refers to mouse sequences. Amino acids of the consensus delineated from other Dynlt1-binding proteins are shown in bold, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone.0026430-Mok1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone.0026430-Sugai1" target="_blank">[31]</a>. In the OX1R CTD mutant, two conserved Thr were mutated to Ala (409 and 412, numbering from full-length OX1R). (<b>B, C, D</b>) β-galactosidase (top panels) and <i>HIS3</i> (bottom panels) assays were performed on yeast transformed with plasmids expressing different combinations of orexin receptor and Dynlt1/Dynlt3. Interactions of OX1R CTD with Dynlt1 and Dynlt3 are reduced when Thr 409 and 412 of OX1R CTD are mutated into Ala. Deleting the extra amino acids of OX2R CTD favors its interaction with Dynlt1, while deleting the next 10 amino acids (comprising the distal part of the Dynlt1-binding motif) abolishes this effect. (<b>E</b>) Summary of OX1R CTD and OX2R CTD constructs tested and their relative interaction strength with Dynlt1 and Dynlt3. OX1R CTD T409, 412A, OX1R CTD with T409 and T412 mutated into alanine residues; OX1R CTD Δ407–416, OX1R CTD lacking the last 10 a.a.; OX1R CTD Δ397–416, OX1R CTD lacking the last 20 a.a.; OX1R CTD Δ387–416, OX1R CTD lacking the last 30 a.a.; OX2R CTD Δ433–460, OX2R CTD lacking the extra 28 a.a. compared to OX1R CTD; OX2R CTD Δ423–460, OX2R CTD lacking the last 38 a.a.; OX2R CTD Δ413–460, OX2R CTD lacking the last 48 a.a.; OX2R CTD Δ403–460, OX2R CTD lacking the last 58 a.a. ND, not determined. Experiments were performed 3 times and the average is presented. **: p<0.01 vs transformation with wild-type OX1R CTD or OX2R CTD.</p

Levels of OX1R in resting conditions at the plasma membrane in presence or absence of Dynlt1.

<p>Control refers to a different transfection for each experiment. Empty pCS2, 10 nM or 20 nM negative control siRNA were used respectively as control for the transfection of pCS2-Dynlt1, <i>Dynlt1</i> siRNA or a mix of <i>Dynlt1</i> and <i>Dynlt3</i> siRNAs.</p