Altered Dynamics in the Circadian Oscillation of Clock Genes in Dermal Fibroblasts of Patients Suffering from Idiopathic Hypersomnia

<div><p>From single cell organisms to the most complex life forms, the 24-hour circadian rhythm is important for numerous aspects of physiology and behavior such as daily periodic fluctuations in body temperature and sleep-wake cycles. Influenced by environmental cues – mainly by light input -, the central pacemaker in the thalamic suprachiasmatic nuclei (SCN) controls and regulates the internal clock mechanisms which are present in peripheral tissues. In order to correlate modifications in the molecular mechanisms of circadian rhythm with the pathophysiology of idiopathic hypersomnia, this study aimed to investigate the dynamics of the expression of circadian clock genes in dermal fibroblasts of idiopathic hypersomniacs (IH) in comparison to those of healthy controls (HC). Ten clinically and polysomnographically proven IH patients were recruited from the department of sleep medicine of the University Hospital of Muenster. Clinical diagnosis was done by two consecutive polysomnographies (PSG) and Multiple Sleep Latency Test (MSLT). Fourteen clinical healthy volunteers served as control group. Dermal fibroblasts were obtained via punch biopsy and grown in cell culture. The expression of circadian clock genes was investigated by semiquantitative Reverse Transcriptase-PCR qRT-PCR analysis, confirming periodical oscillation of expression of the core circadian clock genes <i>BMAL1, PER1/2</i> and <i>CRY1/2.</i> The amplitude of the rhythmically expressed <i>BMAL1, PER1</i> and <i>PER2</i> was significantly dampened in dermal fibroblasts of IH compared to HC over two circadian periods whereas the overall expression of only the key transcriptional factor <i>BMAL1</i> was significantly reduced in IH. Our study suggests for the first time an aberrant dynamics in the circadian clock in IH. These findings may serve to better understand some clinical features of the pathophysiology in sleep – wake rhythms in IH.</p></div

Clinical characteristics of the study patients and age- gender matched controls with polysomnography findings and laboratory parameters.

<p>Clinical characteristics of the study patients and age- gender matched controls with polysomnography findings and laboratory parameters.</p

Using total RNA from primary dermal fibroblasts collected at the indicated time points were used to perform real-time PCR to measure the expression of circadian clock genes.

<p>Blue dots indicate actual measurements, the colored lines represent the gene expression profile relative to <i>18S</i> rRNA in the shape of a sine curve to prove circadian gene expression in all examined cell lines. Each colored line indicates an approximated sine wave by least squares method to 6 actual measurements (I) (3h, 6h, 12h, 24h, 27h, 30h), (II) (6h, 12h, 24h, 27h, 30h, 36h), (III) (12h, 24h, 27h, 30h, 36h, 48h). The applied method is based on the multiple components analysis which allows fitting several significant functions to the experimental data. The broken black line indicates the best fit sine curve defined as the average value of the three rhythmic functions fitted to the data. Numbers in parentheses beside the figures indicate the time points used for prediction and numbers at the right indicate root-mean-square errors (MSE). The smaller the MSE, the more accurate is the prediction of rhythmic circulation. Given examples show exemplarily the harmonic expression in two fibroblast cell lines from one healthy control (left) and one idiopathic hypersomniac (right) depicting the flattened circadian amplitude of gene expression profile in the IH group versus healthy control.</p

A direct comparison of the overall gene expression of the core circadian clock genes at the indicated time points reveal no significant difference in the absolute amount of gene expression with exception in case of <i>BMAL1</i> at time point 12h (P = 0.05) and 36h (P = 0.04).

<p>At these points the overall expressional rate in the group of healthy controls is significantly higher compared to the patient cohort. The x-axis reflects the actual interval of the sampling points. The black dots indicate the averaged overall gene expression values ± SEM of both study groups over the course of 72h.</p

Comparison of the individual overall amplitude difference of circadian gene expression rate during the 1^st 24h-period starting 6h-30h and in case of <i>BMAL1</i> during the 2^nd 24h-period starting 30h-54h between healthy controls and idiopathic hypersomniacs.

<p>On the y-axis dots indicate for each examined individual the overall amplitude defined as the half difference between maximum and minimum of the average value of the multiple sine curves. Black lines indicate the averaged values ± SEM considering P<0.05 as significant. <i>BMAL1</i> reveals the strongest damping in circadian gene oscillation in the group of idiopathic hypersomniacs in comparison to healthy controls over two consecutive 24h-periods. In case of <i>PER2</i> and <i>PER1</i> the overall amplitude in the group of idiopathic hypersomniacs is significantly dampened in the 1^st 24h-period, in the 2^nd period there is no significant damping (data not shown). Though <i>CRY1</i> also shows a clear diminished oscillation in the patient group, the difference remains not significant due to a high SEM whereas <i>CRY2</i> shows no significant amplitude difference between the two study groups.</p

Confocal scanning laser (cSLO) infrared image illustrating semi-automatic measurement tool.

<p>Three concentrial circles (blue 3.2 mm, green 3.5 mm, red 3.8 mm) are placed around the optic disc. Vessel labelling marks arteries (a) and veins (v). Measurement lines (cyan) are defined by the software user. Additional measurement lines automatically produced by the software are shown exemplary in artery two (a2; set of five lines). Yellow lines separate superior (S), inferior (I), nasal (N) and temporal (T) quadrant.</p

Data overview regarding manual retinal vessel measurements based on spectral-domain optical coherence tomography.

<p>(n) number of patients.</p><p>*measurements performed at 960 µm from the optic disc edge.</p>#<p>circular SD-OCT scan 3.5 mm in diameter.</p><p>Data overview regarding manual retinal vessel measurements based on spectral-domain optical coherence tomography.</p

Semi-automated vessel measurements based on confocal scanning laser ophthalmoscopy (cSLO): healthy controls compared to CADASIL patients.

<p>(n) number of total eyes; (v) number of total vessels; (n/a) too few vessels in the respective sector to calculate p-value.</p><p>Semi-automated vessel measurements based on confocal scanning laser ophthalmoscopy (cSLO): healthy controls compared to CADASIL patients.</p

Retinal nerve fiber layer thickness (RNFL) measured by spectral-domain optical coherence tomography: healthy controls compared to CADASIL patients.

<p>(n) number of eyes.</p><p>*superior and inferior measurements were calculated based on data from nasal superior and temporal superior quadrants and from nasal inferior and temporal inferior quadrants respectively.</p><p>Retinal nerve fiber layer thickness (RNFL) measured by spectral-domain optical coherence tomography: healthy controls compared to CADASIL patients.</p

A–D Combined simultaneous confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT).

<p><b>A–B</b> Infrared cSLO image centered on the optic disc of a healthy control subject (A) and a CADASIL patient (B). Green circle indicates the position of corresponding SD-OCT scan. Light green section inferiorly on the circle marks the localization of corresponding SD-OCT scan shown aside. <b>C–D</b> Magnified SD-OCT scans of healthy control subject (C) and CADASIL patient (D) show sections of major retinal vessels appearing as a group of heterogeneous reflectivities in a round-shaped configuration. Asterisks mark the inner and outer reflections of arterial vessel walls and diamonds indicate inner and outer reflections of venous vessel walls. Hyperreflectivities representing the vessel walls seem thicker and more accentuated in the CADASIL patient. Particularly in veins, demarcation of the inferior vessel wall (towards the retinal pigment epithelium) often remains challenging due to absorption effects also seen as acoustical shadow underneath the vessel (towards the retinal pigment epithelium). Note the typical hour-glass shaped configuration within the vessel lumen in both subjects. Lateral vessel walls cannot be visualized as OCT laser beam is not projected perpendicularly to them.</p