Laboratory of Biological Rhythms

Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock with unknown function. We used genetic mouse models (WT, mPer2Luc, ChP-specific Bmal1 knockout) combined with surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. We found that the ChP clock regulates cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases. In ChP of SCNx mice, the rhythms were severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout; they were restored by daily injections of dexamethasone. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

Fig: A. Workflow. B. Period of locomotor activity in Control and SCNx mice. C. SCN after sham surgery and after lesion. D. Rhythms in SCNx ChP are dampened to similar extent as rhythms in ChP from tissues-specific Bmal1-KO. E. Rhythmic genes in Control (left) or SCNx (right) ChP samples. F. Phase histogram of genes Control (left) or SCNx (right) ChP samples. G. PER2::LUCIFERASE rhythms in single-cell sized ROIs across an explanted ChP from either Control or SCNx mice.

Choroid plexus (ChP) in brain ventricles plays significant role in brain homeostasis. ChP cells contain robust circadian oscillators that control its function. Chronodisruption in the form of exposure to constant light (cLL) and chronic advance of the light-dark cycle (cLD-shifts) impaired the ChP clock due to disrupted signals originating from the SCN involving glucocorticoid signaling. Sleep patterns and immune markers in the brain were also affected. Interestingly, LPS systemic inflammation did not influence the ChP clock, but disrupted the liver clock. The data highlight the role of ChP in protecting the brain from neuroinflammation. Our results provide a novel link between human lifestyle-induced chronodisruption and the impairment of ChP-dependent brain homeostasis.

Fig. Choroid plexus (ChP) chronodisruption. (A) Total locomotor activity, (B) relative amplitude, and (C) sleep duration of mice reared on

normal light-dark regime (LD12:12), on a chronic phase-shifted light-dark (cLD-shifts) cycle regime, or on a constant light (cLL) regime.

(D-E) 6-day luminescence microscopy of the circadian expression of PER2 protein imaged in ChP explants of analyzed mice,

quantitative analysis of amplitude (F) and period (G) of individual ChP cells. (I) Multiplex analysis of cytokines in plasma of analyzed mice.

It remains unclear, however, whether social jetlag has any negative health effects. By investigating a unique population-representative dataset, including 1957 blood samples analyzed for nine biomarkers, we report significant associations between sleep phase preference, social jetlag and cardio-metabolic biomarkers. We identify novel factors affecting social jetlag and demonstrate that flexible working schedule efficiently alleviates social jetlag.

Karolína Liška , Tereza Dočkal , Pavel Houdek , Martin Sládek, Vendula Lužná , Kateryna Semenovykh , Milica Drapšin and Alena Sumová.

Lithium affects the circadian clock in the choroid plexus – A new role for an old mechanism. Biomedicine & Pharmacotherapy 159 (2023), 114292

Lithium is an effective mood stabilizer used in the treatment of Bipolar Disorder (BD), but the mechanism of its therapeutic action is not well understood. We investigated the effect of lithium on the circadian clock located in the ventricle barrier complex containing the choroid plexus (CP), a part of the glymphatic system that influences gross brain function via the production of cerebrospinal fluid. Using in vivo and ex vivo methods, analyzing clock gene expression and probing cell-signaling pathways of the CP, we demonstrated that lithium strongly affected the circadian clock in the CP. The effect of lithium was not mediated by GSK3b-exclusive mechanism, but rather by modulation of PKC activity. The effects on the CP clock may be involved in the therapeutic effects of lithium and improve brain function in BD patients by aligning the function of the CP clock-related glymphatic system with the sleep-wake cycle. Importantly, our data argue for personalized timing of lithium treatment in BD patients.

Fig: Lithium affects the circadian clock in the choroid plexus independently of GSK3, resulting in time-dependent phase shifts that may affect CSF production, brain function and its therapeutic outcome.

doi:10.1016/j.neuropharm.2021.108455. The paper provides new insights into the modulatory role of endocannabinoid signaling during the light entrainment of the SCN.

Suprachiasmatic nucleus (SCN) of the hypothalamus is the master clock that drives circadian rhythms in physiology and behavior and adjusts their timing to external cues. Neurotransmitter glutamate and glutamatergic receptors sensitive to N-methyl-D-aspartate (NMDA) play a dual role in the SCN by coupling astrocytic and neuronal single cell oscillators and by resetting their phase in response to light. Recent reports suggested that signaling by endogenous cannabinoids (ECs) participates in both of these functions. ECs, such as 2-arachidonoylglycerol (2-AG), act via presynaptic CB1 receptors to modulate synaptic activity and affect the SCN response to light-mimicking NMDA stimulus in a time-dependent manner. We demonstrate that circadian clock in the rat SCN regulates expression of 2-AG transport, synthesis and degradation enzymes as well as its receptors. Inhibition of the major 2-AG synthesis enzyme, diacylglycerol lipase, enhances the phase delay and lowers the amplitude of explanted SCN rhythm in response to NMDAR activation. Using PER2 bioluminescence imaging, we show how single cell oscillators reflect the phase response of the whole SCN. Additionally, we present strong evidence that the zero amplitude behavior of the SCN in response to single NMDA stimulus in the middle of subjective night is the result of a loss of rhythm in individual SCN cells. The paper provides new insights into the modulatory role of endocannabinoid signaling during the light entrainment of the SCN.

Fig 1. Explanted SCN from mPer2Luc mouse oscillating in vitro was treated by diacylglycerol lipase inhibitor and NMDA during daytime (left) or in the middle of subjective nighttime (right).

Fig 2. PER2 levels in a single representative neuron from the explants shown in Fig. 2. Red line shows the signal before treatment.

Daily rhythm of BMAL2 protein in the liver is phase advanced in SHR compared to Wistar (A, B). Locomotor activity, just before the food is accessible (C), is significantly higher in SHR.

Nováková M, Sládek M, Sumová A. (2013): Human chronotype is determined in bodily cells under real-life conditions. Chronobiol Int. 30(4):607-17.

http://informahealthcare.com/doi/abs/10.3109/07420528.2012.754455

The chronotype is influenced both genetically and by the environment, mainly by the light. It is dependent on the central clock located in the hypothalamus. The chronotype affects e.g. the individual tolerance to shift work. Therefore, it is useful to know the individual chronotype to assign the best working schedule. The chronotype can be determined from the timing of sleep and the secretion of melatonin which are controlled by the central clock in the brain. In our study, we found that the individual chronotype also determines the timing of clock gene expression in peripheral clocks in cells acquired by scrubbing the mucosa of the inner cheek. Our study showed that the analysis of clock gene expression in peripheral cells can be used as a novel marker of the phase of the inner clock. This method can be used also in real-life field conditions.

Phase advanced melatonin secretion in early chronotypes (“larks“) as compared to late chronotypes (“owls“) (Nováková et al., Chronobiology International, 2013)

Although individuals are exposed to the same lighting conditions, they differ in preferred sleep/wake schedule.

Sládek, M. & Sumová, A. Entrainment of Spontaneously Hypertensive Rat Fibroblasts by Temperature Cycles. PLoS One 8, e77010 (2013).

Regular daily changes in body temperature are one of the signals that entrain the circadian clock in peripheral organs. In this paper, we used isolated cell cultures from Wistar rats and SHR transfected with circadian luminescent reporter to investigate the sensitivity of the peripheral clock to the phase setting through an external 2.5 ° C temperature cycle. We have shown that a mere two-day temperature cycle is sufficient to restore dampened circadian rhythms and we constructed a curve of phase response to the temperature cycle.

The phase change after the temperature cycle depends on the phase of the clock at the beginning of the temperature cycle.