New methods to assess circadian clocks in humans

Transcription

1 Indian Journal of Experimental Biology Vol. 52, May 2014, pp Mini Review New methods to assess circadian clocks in humans Marta Nováková 1,2 & Alena Sumová 2,* 1 2 nd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic 2 Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic Proper function of the circadian system seems crucial for human health. New advances in methods for assessment of the functional state of the human circadian system facilitate our understanding of the relationship between the disruption of the circadian system and various diseases. Based on the results of such studies, new directions for the diagnosis and treatment of diseases emerge. This communication aims to summarize current methods for evaluating the human circadian system in the laboratory as well as in field studies. The advantages and limitations of the current methods and various approaches used for both in vivo and in vitro assessment of the human circadian system are discussed. Keywords: Circadian, Clock, Human, Methods The experimental approaches that are currently used for the assessment of the circadian system in humans are summarized in this communication. It aims to provide basic information about progress in approaches to study the human circadian system, especially for those who are not experienced in the field and desire to introduce these techniques into their research. The circadian system ensures the adaptation of bodily functions to the rhythmically changing environment, namely, to daily alternations in light and darkness. As a result, many processes in the body are driven rhythmically and repeat over a period of approximatelly 24 h. Moreover, the circadian system adjusts the actual phase of these rhythmic processes relative to the external light/dark cycle. Proper function of the circadian system seems crucial for maintaining homeostatic balance among various physiological, metabolic and behavioural processes, which is an important prerequisite for health. Therefore, assessment of the functional state of the circadian system in humans is needed not only for experimental but also for therapeutic purposes. The human circadian system Circadian oscillations are driven at the cellular level via rhythmic expression of so called clock genes and their protein products. The mechanism of these molecular rhythms is based on the principle that the * Correspondent author Telephone: sumova@biomed.cas.cz clock proteins control expression of their own genes as well as other genes encoding various transcription factors 1. As an outcome of this mechanism, up to 10% of the transcriptome is under circadian control 2. Some of these rhythmically driven genes are crucial for metabolism, the cell cycle and the immune response. Therefore, disruptions to circadian regulation may be potentially associated with various diseases 3. Almost every cell in the body contains its own oscillatory mechanism which is self-sustaining and these cells are referred to as peripheral clocks. The cellular clocks require neuronal and humoral cues from a central, hierarchically superior oscillator to maintain the same phase at the organ or tissue level 4,5. In humans, as in all mammals, the central clock is located in the hypothalamic suprachiasmatic nuclei (SCN) 6. The endogenous circadian period differs greatly among individuals; the reported mean period length in humans varies around h, depending on the protocol used for its assessment The circadian rhythms are entrained to the 24 h period of the solar day, namely by the light/dark cycle 13. The light signal is transduced directly from the retina to the central clock in the SCN 14, which entrains the period of its rhythmicity with the solar day and sends these signals to the peripheral oscillators. The SCN are also entrainable by cues of non-photic origin, though these cues are much less efficient than the light/dark cycle. For example, the human SCN clock may be entrained by administration of melatonin in a manner dependent on the time of delivery 15. Melatonin is a hormone released from the pineal gland in a rhythmic manner

2 NOVÁKOVÁ & SUMOVÁ: METHODS TO ASSESS CIRCADIAN CLOCK IN HUMANS 405 with high levels during the night and low levels during the day. The rhythm is a direct output of the central circadian clock and provides the organism with information not only about the time of day but also about the season 16. In contrast to the central clock, peripheral clocks are sensitive to changes in the food intake regime 17. Under natural conditions, the timing of food intake is controlled by the SCN. However, people in modern society often need to adapt their behaviour to various socioeconomic demands, which forces them to be active during the night hours. Typically, this situation occurs with shift workers. When they eat at night, their peripheral oscillators might receive conflicting information arising from the SCN, which are dominantly entrained by the light/dark cycle and, at the same time, from food-processing signals set by food intake at an improper time of a day. Such a situation may cause internal desynchrony within the circadian system of the shift worker and result in aberrant temporal control of various physiological processes. The same problem may also occur when humans with temporal disruption of the sleep/wake cycle, such as those suffering from so called circadian sleep disorders like advanced sleep phase syndrome, delayed sleep phase syndrome, irregular sleep-wake rhythm, or a free-running sleep/wake cycle 18, are forced to adapt their behaviour according to the rest of the society. Changes in lifestyle, including misalignment of activity with the proper time of day, have been associated with disorders like metabolic syndrome, obesity, type 2 diabetes, cardiovascular disease 19 and various types of cancer, namely breast cancer 20,21. The relationship between the circadian system and metabolism or the cell cycle has been extensively reviewed elsewhere 22,23. In addition, circadian abnormalities have been associated with psychiatric disorders like seasonal affective disorder, major depressive disorder, bipolar disorder and others. Given the strong connection between these disorders and circadian misalignment, chronotherapies based on readjustment of the circadian system with melatonin receptor agonists, sleep deprivation or bright light are being successfully used in addition to traditional medication Recent advances in our knowledge of the associations between the disruption of the circadian system in humans and many diseases, as mentioned above, make assessment of the functional state of the human circadian system in both healthy subjects and patients very important. Choice of the proper method to assess the human circadian system Human samples suitable for detection of the functional state of the circadian system The central oscillator is located deep in the brain and therefore, the only way to evaluate its function in humans is by observing the rhythmic processes that are driven by the central clock, i.e., its output rhythms like body temperature or hormone levels. Nevertheless, in some studies the function of the human central SCN clock has been determined in samples collected postmortem 27,28. The main disadvantage of this approach is the potential lack of detailed information about the subject s behaviour and lighting conditions shortly before death, especially in the case of samples collected after accidents. The fact that most cells in the body possess the circadian clock enables investigations of human peripheral clocks that are accessible for sampling. These clocks may be collected by either invasive (e.g., blood or biopsy samples) or non-invasive (e.g., saliva, hair follicle, buccal mucosa samples) means. Development of new molecular techniques allowing for highly sensitive detection of clock gene expression or protein levels will likely expand the range of samples available for evaluation of the human circadian system. Real-life conditions or standard laboratory protocol? The experimental design of the assessment of the human circadian system depends on which question is to be answered. For assessment of the basic properties of circadian clocks, such as their endogenous period, it is crucial to separate the human subjects from any environmental factors that may potentially mask or entrain them, namely, the exposure to external light, cycles in food intake, activity, etc. For these experiments, standard laboratory protocols like a constant routine or a forced desynchrony protocol are the best choice These approaches require specialized clinics or sleep laboratories equipped with facilities for long-term indoor stay and staff to care for the subjects around the clock. Therefore, these studies are labor-intensive, costly and may cause discomfort to subjects because of the many restrictions they are subjected to for several days (for more information see 30 ). While these approaches currently represent the best way to assess the endogenous function of the human circadian system, they cannot reveal the actual state of the circadian system in a subject who is exposed to

3 406 INDIAN J EXP BIOL, MAY 2014 various environmental and social factors that vary from day to day. Additional research may reveal how the circadian system is able to adapt to these factors and cope with situations where the subjects must adjust to these changes. In this case, a field study approach for the assessment of the circadian system in real-life conditions is necessary. In these studies, subjects are asked to behave normally (i.e., keep to their habitual sleep/wake schedule, maintain their activity and go to work or attend the school) before the collection of samples. To avoid masking factors that might acutely affect the marker used for assessment of the circadian system, sampling is performed under semi-controlled conditions that can be achieved even in their homes. The main constraint subjects face is to avoid light exposure during sampling because most markers are directly sensitive to light. The subjects are instructed to protect themselves from direct light in the morning and evening on the day of sampling, and collect samples at night under dim-light conditions. Although these studies are much easier to perform than the constant routine protocol, interpretation of the data requires the consideration of various factors that potentially affect or mask the results. In vivo or in vitro approach? The marker used for assessment of the human circadian system (see below) may be followed under in vivo or in vitro conditions. For the in vivo approach samples are collected at regular intervals throughout the 24 h cycle (or longer). Thus, the protocol is time-consuming for subjects and staff. Moreover, during sampling the subjects must meet certain restrictions depending on the protocol used, as described above. Additionally, repeated sampling often disturbs sleep patterns, especially in the selfsampling protocol. Nevertheless, this approach is the best way to determine the actual levels of circadian markers (e.g. melatonin, cortisol, body temperature or clock gene expression) under complex in vivo conditions. In vitro assessment of circadian parameters in humans has been introduced 7 as an effective alternative to the repeated sampling (see below). Once a sample is collected from a subject, its rhythmicity may be studied in a long-term experiment. Moreover, this approach allows for easy manipulation of experimental conditions, which opens new avenues for exploring the properties of human circadian clocks. However, the cells are taken from the complex environment and cultured in vitro for a long period, which means they are maintained under conditions that cannot fully imitate the in vivo situation. Moreover, the circadian clock is highly sensitive to the cultivation conditions, including serum exchange, medium composition, temperature, ph, etc. 32. Therefore, the question of to what extent does the results of in vitro experiments reflect the real in vivo state must still be clarified. In vivo methods for assessment of the human circadian system Sleep timing, locomotor activity and core body temperature Preferred sleep patterns vary greatly among humans. The preference of sleep/wake timing is defined as an individual's chronotype 33. In fact, the individual s chronotype reflects the phase angle of entrainment of the sleep/wake cycle relative to the daytime and is influenced by genetic, environmental and social factors 34. Some studies suggested that individuals with shorter endogenous periods tend to be morning chronotypes, whereas those with longer periods are often evening chronotypes 8,35. To assess the individual chronotype, various questionnaires, namely the Horne-Ostberg Morningness- Eveningness Questionnaire (MEQ) 36 and the Munich Chronotype Questionnaire (MCTQ) 37, are commonly used. Whereas the MEQ asks for the preferred time of rest and activity, the MCTQ follows individual sleep times while considering work and free days separately. Both questionnaires show a strong correlation in the evaluation of the individual's chronotype 38. Self-reported sleep diaries are another method to assess individual sleep timing over a short period, usually several weeks 39,40. Because both chronotype questionnaires and sleep diaries are limited by self-report subjectivity, they are preferably supplemented by locomotor activity recordings. Wrist actigraph devices are commonly used to detect the activity. Modern devices combine a sensor that records the wrist movement with a sensor that records the light exposure of probands 41. Core body temperature fluctuation is a well-defined circadian output rhythm, and its assessment became a traditional way to establish the phase and amplitude of the circadian clock over the last decades 42. Thermometers are a relatively easy and inexpensive way to measure the circadian rhythm; however, the rhythm is significantly masked by many factors, such as activity, food intake and sleep. Therefore, valid estimates of the phase and amplitude of the circadian

4 NOVÁKOVÁ & SUMOVÁ: METHODS TO ASSESS CIRCADIAN CLOCK IN HUMANS 407 rhythm need to be measured using a constant routine protocol when continuous recording with a rectal thermometer can be performed. Currently, new devices enable other ways of recording body temperature, such as via ingestible, temperature-sensitive, comercially available capsules These capsules are costly, but their use is more tolerable for subjects. New methods recording human circadian rhythms in vivo are still under development. Recently, new devices capable of simultaneously and non-invasive measurement of many physiological, behavioural, and environmental variables were introduced 46,47. These approaches are especially promising for future research directed to precise estimation of the circadian phase in humans under real-life conditions. Hormones, melatonin Many hormones, e.g., melatonin, corticosteroids, leptin, growth hormone, testosterone and prolactin, are secreted under the circadian control 48,49. Melatonin and cortisol hormone levels in bodily fluids are often used as markers of the circadian system. Melatonin is the marker of choice because its secretion from the pineal gland is governed directly by the SCN and its levels exhibit a robust rhythm with low levels during the day and high levels at night 15. Importantly, secretion of melatonin is resistant to external and internal cues, except for light exposure. Thus, melatonin levels are often used as a reliable and precise marker of the circadian system. Therefore, when humans are shielded from exposure to external light, the daily profile of melatonin levels provides a precise marker of SCN clock function. The rise and decline in melatonin levels may signal the beginning of subjective night and subjective day, respectively. Light suppresses melatonin levels in a dose-dependent manner 50,51, and even low-intensity light may be effective 52. Moreover, the spectral characteristics of light are also important and light of nm was found to be the most effective 53,54. The ability of light of different intensities and wavelengths to acutely suppress melatonin levels has often been used as a tool to study human circadian photoreception 15. Hence, light intensity needs to be carefully checked in all experiments where endogenous melatonin levels are used as a marker of the functional state of the SCN circadian clock. The methods used for the detection of melatonin levels are specific antibody-based techniques, namely, radioimmunoassay and enzyme-linked immuno sorbent assay 55,56. Melatonin is preferably detected in saliva and blood, and its metabolite 6-sulfatoxymelatonin is detected in urine. Although plasma levels of melatonin are generally 3 to 10 times higher than those found in saliva 55,57, determination of salivary melatonin is advantageous in cases when a non-invasive procedure is needed, like in field studies (see above). There are two experimental approaches using melatonin as a marker, each of which yields different information. One approach is to detect melatonin levels at regular intervals over a 24 h period or longer. The frequency of sampling greatly varies among studies, and generally, more frequent sampling allows for a more precise estimate of the phase, amplitude and duration of the melatonin rhythm. The other approach, which allows for assessment of circadian phase, is to detect dim light melatonin onset (DLMO), i.e., the time when melatonin levels rise above basal levels 58. Samples are frequently collected around the time of expected endogenous melatonin rise under the dim-light conditions. Basically, there are two methods of how to read DLMO. The first involves reading the absolute threshold, i.e, the time when melatonin reaches a reference level (e.g., 3 pg/ml in saliva; 10 pg/ml in blood, or 2 standard deviations above the average baseline). The second method uses a relative threshold, i.e., the time when melatonin levels reach a certain percentage of maximum levels in the rising phase (e.g., 20, 25 or 50% of maximum) The latter method requires sample collection throughout the 24 h period, but it is more appropriate when the DLMO of subjects with significantly different melatonin profile amplitudes are compared. Expression of clock genes Advances in molecular biology techniques facilitated direct exploration of the molecular mechanism of human peripheral clocks by assessing expression profiles of clock (or clockcontrolled) genes, namely Per1, Per2, Per3, Reverbα, Bmal1, Cry1 and Clock. Therefore, not only the output rhythms (see above), but also transcript levels of genes responsible for rhythmicity are studied. In the in vivo approach, tissue samples are typically collected in intervals of one to several hours throughout the day, and the gene expression is determined by quantitative real-time polymerase chain reaction (qpcr). The interval for sample collected depends on the conditions of the study and the level of invasiveness of the sampling. For example, samples could be collected at 1 h intervals when blood is collected in subjects maintained under constant routine

5 408 INDIAN J EXP BIOL, MAY 2014 protocol 62, but only at 4 h intervals when buccal scrubs are sampled in subjects assessed in a field study 63,64. Because the search for an optimal compromise between the sample quality and low invasiveness has proven to be challenging, an effort has been made to find the most suitable human tissue for detection of clock gene expression. Until now, various methods to collect several tissue samples have been developed. The most invasive approach, biopsy, has the advantage of providing a sufficient amount of high-quality sample. However, repeated collection of such samples to obtain 24 h gene expression profiles is often unacceptable for the subjects. In several studies, biopsies of adipose tissue 65,66 and oral mucosa 67 were collected to assess the clock gene expression profiles. The most commonly used samples for detection of clock gene expression profiles are various blood cells 62, When the subject is cannulated, blood can be collected in very short intervals. However, this approach is not suitable for field studies because it requires the subject to stay in the hospital for the duration of at least the sampling period. Generally, subjects are more willing to participate in studies when non-ivasive methods are used. Moreover, the non-invasive collection of samples is suitable for field studies because it can also be performed under real-life conditions at home (see above). For these reasons, non-invasive methods of obtaining samples for clock gene expression profiles from oral mucosa scratches 63,64,80 or hair follicles 78,81 were introduced. Although oral mucosa samples are sensitive to RNA degradation, this method has been proven reproducible and suitable for detection of the phase of the circadian clock 64. In contrast to melatonin levels, the clock gene expression profiles are likely not acutely sensitive to light exposure. The phases of the expression profiles of some clock genes correlate well with the phases of melatonin levels and sleep timing 62,64, which apparently reflects their synchronization with the central SCN clock. In fact, disturbances in phasing can provide valuable information about the strength of the entraining signal from the SCN to the peripheral clock. For example, findings of disturbances in mutual phasing among clock genes in the peripheral clocks in buccal mucosa of patients with Smith Magenis syndrome suggests aberrant pathways derived from the SCN, which is consistent with the weak temporal control of melatonin and the sleep/wake cycle previously observed in these patients 63. The circadian phases of the individual peripheral clocks are not same and they also differ to that of the central clock in the SCN. This has to be taken in account when making general conclusions about the actual timing of the human circadian system based on the clock gene expression determined in the peripheral tissues. In vitro methods for assessment of the human circadian system Clock gene expression The endogenous period, phase and amplitude of clock gene expression profiles in vitro were commonly determined using an approach that consisted of culturing human cells (adipose cells 82, islet cells 83, blood cells 84, mesenchymal stem cells 85 ), synchronizing them and collecting them at regular intervals around the clock for subsequent detection of gene expression by qpcr. Lately, a method for detection of the period of the human peripheral clock has been introduced 7 in parallel with animals studies 86 in which the circadian period and phase can be reliably estimated by detection of clock gene expression rhythms in real time. Detection occurs in transfected cell lines or tissues explanted from transgenic animals where the promoter of a clock gene drives expresion of a reporter gene, like luciferase. This novel approach allows continuous observation of clock gene expression in real time for several days. For assessment of human peripheral clocks, mainly skin fibroblasts cultivated from skin biopsies are used 7,8,32,87. After transduction of fibroblasts with a lentiviral vector that contains the promoter of a clock gene fused with the luciferase gene, real-time bioluminescence that is directly proportional to the expression of the clock gene is recorded. Although this method is technically more demanding than around-the-clock sample collection, the main advantage is continuous recording from a single dish for several days without the need for repeated collection of samples. Luciferase bioluminescence reporters also can be used for other human cell types, namely, islet cells 88 or retinal pigment epithelial cells 89. Although the approach appears to be the best choice for exploring the human circadian system, there might be some limitations. The cells in the dish are taken out of the internal environment and become hypersensitive to culturing conditions 32. Therefore, the main issue remains whether the parameters of the human circadian clock we observe in vitro exactly reflect the situation in vivo. Some

6 NOVÁKOVÁ & SUMOVÁ: METHODS TO ASSESS CIRCADIAN CLOCK IN HUMANS 409 studies confirmed a significant correlation between the period length measured by plasma melatonin profiles, (i.e., in vivo) and fibroblasts bioluminenscence profiles (i.e., in vitro) 90 but others reported an absence of correlation 87. Therefore, more studies are needed to verify whether the in vitro rhythms precisely reflect the behavior of the clock in its natural environment. Nevertheless, this method represents a useful experimental tool for the exploration of the human circadian rhythms. Conclusion The huge impact of the circadian system on regulation of numerous functions in our body and its significance for our health demonstrate the need to expand the range of methods for reliable assessment of the human circadian system. Approaches to study the output rhythms driven by the central clock in the SCN and also how to evaluate properties of the peripheral clocks in the body s cells have been developed. Each method has advantages and limitations; therefore, the choice of the best approach always depends on the question to be answered. These techniques were employed to study the functional state of the circadian system in both healthy volunteers and patients and resulted in new discoveries about the relationship between the disturbances to the circadian system and various diseases. Further research is needed to reduce the technical limitations and cost of these methods to make them more accessible for routine use in clinics. Subsequently, the methods could be applied to the diagnosis and the prevention of various diseases. It would also allow for better identification of factors in our external environment that are potentially harmful to the circadian system and reveal the mechanisms by which they influence human health. This would enable better translation of the enormous experimental knowledge of the circadian system accumulated in animal studies to effective therapy for various diseases in humans. Acknowledgement The study was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic, grant no. NT /2010; the Grant Agency of Charles University in Prague, no ; and research project nos. AV0Z and RVO: The authors declare that they have no conflicts of interests. 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