Evaluation of circadian phenotypes utilizing fibroblasts from patients with circadian rhythm sleep disorders

Circadian rhythm sleep disorders (CRSDs) are defined by persistent or recurrent disturbed sleep–wake patterns and consist of several subtypes including advanced sleep–wake phase disorder (ASWPD), delayed sleep–wake phase disorder (DSWPD), and non-24-hour sleep–wake rhythm disorder (N24SWD).ASWPD is characterized by extremely early involuntary sleep timing, whereas DSWPD is characterized by significantly delayed sleep timing, and N24SWD has sleep timing that occurs with a 30 min to 1 h delay each day. CRSDs have a high rate of comorbidity with various psychiatric disorders, especially mood disorders.A previous study showed that approximately half of their patients with N24SWD developed psychiatric problems before or after the onset of N24SWD.CRSDs are thought to result from impairment of the circadian clock system and/or a misalignment between the endogenous circadian rhythm and exogenous entrainment factors that affect sleep timing. Patients with CRSDs are mostly treated with chronotherapy, in which intense light exposure or melatonin administration is performed during the phase-advance or phase-delay portion, respectively, of the sleep–wake cycle. In mammals, the central oscillator in the suprachiasmatic nucleus of the hypothalamus incorporates environmental cues, such as light exposure, and coordinates the phase of oscillators in peripheral tissues.The molecular mechanisms underlying the circadian clock system involve the transcription–translation negative feedback loops of multiple clock genes including,,,,and. ,,, ^{1 2 3 4} ,,, ^{5 6 7 8 7} , ^{9 10} BMAL1 CLOCK CRY PER ROR REV-ERB

Evaluating the circadian phenotype is crucial for establishing a precise clinical diagnosis and for understanding the pathophysiology of diseases that are associated with disturbed biological rhythms such as CRSDs. The intrinsic circadian period,(the free-running period of circadian rhythms in the absence of external cues), is considered to be a critical factor in the pathophysiology of CRSDs.In fact, we found that thedetermined under a forced desynchrony protocol was longer in patients with N24SWD than it was in healthy subjects with an intermediate chronotype.However, the forced desynchrony protocol is costly and laborious,and thus different approaches that can provide more convenient and feasible methods of evaluating circadian phenotypes in a clinical setting are needed. τ τ ,,, ^{1 2 3 4 11} , ^{12 13}

Surrogate measurement techniques, such as using cultured cells derived from biopsy samples of an individual, have been developed and tested for assessing circadian phenotypes.We recently measured clock gene expression rhythms (rhythms) in primary fibroblasts obtained from skin biopsy samples of healthy subjects and compared the period length ofrhythms (theperiod) with the subjects' circadian/sleep parameters, as evaluated by questionnaires, sleep logs and actigraphy.The results showed that theperiod was significantly correlated with subjects' chronotypes and habitual sleep time. Our data suggest that evaluating theperiod may be useful for predicting circadian phenotypes. However, the approach of using isolated cultured cells has not yet been applied to assess patients with CRSDs. Therefore, in the present study, we examined the circadian phenotypes of patients with DSWPD and N24SWD by evaluating-(-) rhythms in skin fibroblast cells from individual patients. ,,,,, ^{14 15 16 17 18 19 14} in vitro in vitro in vitro in vitro in vitro Bmal1 luciferase Bmal1 luc

Therhythms were measured in the fibroblast cells derived from patients with DSWPD, patients with N24SWD and control subjects. The period length of the-rhythm (period) varied among individuals. Theperiod ranged from 21.35 to 24.04 h (mean±s.d.: 22.67±0.67 h) in DSWPD fibroblast samples, from 22.05 to 24.83 h (mean±s.d.: 23.18±0.70 h) in N24SWD fibroblast samples and from 21.96 to 24.08 h (mean±s.d.: 22.80±0.47 h) in control fibroblast samples (). Theperiod differed significantly among the three groups (F(2,114)=4.37,=0.003). A prolongedperiod was observed in the N24SWD group compared with the control (Bonferroni-corrected=0.029) or DSWPD group (Bonferroni-corrected=0.002). In contrast, no difference in theperiod was observed between the DSWPD and control groups. Bmal1-luc Bmal1 luc in vitro in vitro in vitro P in vitro P P in vitro Figure 2

Patients with CRSDs underwent chronotherapy. In some patients, the CRSD was intractable and they were considered to be non-responders. The relationship between treatment response andperiod were assessed in the DSWPD and N24SWD groups (and). Theperiod did not differ between the responders and the non-responders in the DSWPD group (22.73±0.77 h vs 22.58±0.51 h;=0.69, df=39, one-tailed=0.246) but did differ in the N24SWD group (22.97±0.47 h vs 23.59±0.89 h;=−1.95, df=10.42, one-tailed=0.039 (Welch correction)). Theperiod of the non-responders was longer than that of the responders in the N24SWD group. Detailed information of the control subjects and patients is shown in. in vitro in vitro t P t P in vitro Table 1 Figure 3 Supplementary Tables S1–S3

To our knowledge, this is the first study to assess the circadian phenotypes of patients with CRSDs by performing surrogate measurements using fibroblast cells in culture. Our data showed that theperiod was significantly longer in the N24SWD group than it was in the control group. These findings are consistent with our previous study, which demonstrated that theof the melatonin rhythms in patients with N24SWD was longer than thein control subjects.Ourandfindings demonstrating a longer circadian period in the N24SWD group strongly support the notion that prolongation of the circadian period contributes to the N24SWD phenotype. in vitro τ τ in vitro in vivo ¹¹

Theoretically, a longdelays the phase of circadian rhythms.A significantly longerwould result in continuous phase delays in the sleep–wake cycle, which is the typical phenotype of N24SWD. However, a prolongedperiod was not necessarily observed in all of the N24SWD fibroblast samples. This indicates that other factors have a role in the N24SWD phenotype. For instance, impaired photic entrainment of the circadian clock is considered to be one factor that leads to the onset of N24SWD, as nearly 50% of completely blind individuals show free-running sleep–wake patterns.In contrast, theperiods of the DSWPD and control groups did not differ. This finding suggests that circadian period length is not a primary factor in patients with the DSWPD phenotype. It has been proposed that alterations in circadian entrainment mechanisms could lead to the onset of DSWPD.Possible altered mechanisms include reduced phase advance and/or enhanced phase delay of the sleep–wake cycle and decreased phase-advance portion and/or increased phase-delay portion of the sleep–wake cycle. τ τ in vitro in vitro ^{13 1 17}

In accordance with the results of previous reports,our patients with CRSDs varied in their responses to chronotherapy. Notably, the chronotherapy non-responders had a longerperiod compared with the responders. This finding suggests that longerperiod might predict poorer treatment response in patients with N24SWD. McCarthypreviously measuredrhythms in the fibroblast cells from bipolar disorder patients and examined the effect of the mood stabilizer lithium onrhythms. They found prolonged period length ofrhythm and reduced responsiveness to lithium in bipolar disorder fibroblast samples. The relationship betweenperiod length and clinical response to lithium treatment was not assessed as the clinical data were not available in their study. Pharmacological assays using isolated fibroblasts might be useful for evaluating responsiveness to therapeutic agents and impairment of circadian entrainment mechanism in patients with CRSDs. ,,, ^{11 28 29 30 31} in vitro in vitro et al. in vitro in vitro in vitro in vitro

Previous studies have generally shown thatrhythms are not strongly correlated withrhythms such as the melatonin-secretion rhythm.Nevertheless, correlations between fibroblast period and human daily behavior have been observed.The cultured cells are dissociated from other tissues and are isolated from any external and internal circadian signals. By contrast,tissues are co-dependent and interact closely with one another. It has been suggested thatrhythms reflect the molecular mechanisms of the circadian oscillators in peripheral cells, whereasrhythms reflect the physiological mechanisms of the circadian clock system of an individual. In addition, a missense mutation in thegene has been identified in a large pedigree with familial ASWPD.Vanselowdemonstrated that alteredrhythms were observed in fibroblast cells that express thegene variant that has the same mutation found in the familial ASWPD pedigree. These findings indicate thatrhythms represent intrinsic clock properties in cells and suggest thatrhythm assays may be utilized for assessing individual genetic (but not physiological) differences in the circadian clock system. Further analyses usingrhythm assays could provide new insights into the molecular pathology of CRSDs and may serve as a system that could be used to screen for potential therapeutic agents on a personalized basis. in vitro in vivo in vivo in vitro in vivo PER2 et al. in vitro PER2 in vitro in vitro in vitro ,, ^{14 15 17} ,, ^{14 18 19} , ^{32 33 34}

There are some limitations to this study. Sex and age were not matched between patients with CRSDs and control subjects. It has been previously reported, though, that theperiod did not differ between young healthy subjects (11 men and 7 women; mean±s.d. age: 25.44±3.58 years) and old healthy subjects (11 men and 7 women; mean±s.d. age: 67.89±7.32 years).Thus, we believe that neither sex nor age would have influencedperiod. Responders and non-responders to chronotherapy were classified on the basis ofof the sleep–wake cycle. Previous studies have, however, shown that sleep–wake rhythms do not always synchronize with the melatonin-secretion rhythm known as a circadian phase marker.Evaluating physiological rhythms as well as sleep–wake rhythms would be required to demonstrate thatperiod could serve as a reliable molecular marker for the treatment outcome of CRSDs. in vitro in vitro τ in vitro ^{16 35}