Before You Read, take note: God Is Brilliant, in all of his creations. Disregard not that, and heed it. The genes sense time? A clock ticks inside us!
-S. Valeri
Until I peeled apart my eye lids, didn't realize that it is nothing but the cosmic rays emanating from sun only has interrupted my cozy early morning dreams. Every morning is the same, there is a literal "soft-war" inside the brain, arguing for and against to get up and start the daily routines. Though there always remains a residual desire to sleep more in the warmth of the plumage, the irresistible pull from within never lets us to lay down beyond a limit. Assumably most of us might have been experiencing it in every morning. Have you ever wondered about what really exerts from within, and demands to wake up out of the bed every morning? If it is said that nothing but the clock ticks inside hauls us from within, it won't be an exaggeration at all. Yes, there really exists a clock inside our skull! Now, a counter question may pop up - could the springs and cogs of the whole clock machinery identifiable inside the network of myriad of neurons in the brain? It should be a perplexing task to convince for a moment, but daring to answer 'yes' would be appropriate in case it can be agreeable to imagine that the networking of proteins is analogous to the 'springs' of a clock and the genes, which are responsible for the expression of these proteins, function as the main cogs of the clock mechanism. In this era of proteomics, the chronomics (the science of time) has gained much impetus than ever since. In this brief account, a cross-section of the important developments in the chronobiology and its implications on human health and welfare are being presented.
The only known life hosting planet, earth, revolves on its own axis. This planetary movement has endowed a rhythmically oscillating level of light on earth. Consequently all the organisms on earth experience the rhythmic changes in day and night, which is an easily perceivable signal for most organisms on earth and not to miss at all. This rhythmic change in day and night has constrained the organisms on earth to organize their daily activities into a defined temporal pattern, which repeats in cycles roughly close to a period of 24 h. Therefore, this rhythmic activities of an organism is termed circadian rhythm (from Latin 'circa' = about; dies = day). For e.g. the daily rest-activity rhythm in Drosophila have a period of ~ 24.3 h (Fig.1).
figure 1 depicts the daily rest-activity pattern of a fruit fly - Drosophila. Top black and white bars represent 12 h night and 12 h day respectively. The multiple small horizontal black bars filled in the figure represent the event records. The fly was kept in a small tube that contain food in one end and was capped at the other end with a cotton plug. When the fly moves along the tube, it crosses a laser beam, that recorded as an event. The contiunous horizontal black bar represents the constant darkness. The yellow shaded area indicates further the time which is in dark period. The fly exhibits a typical bimodal activity pattern, when light comes 'on' and 'off'. It continues through also in constant darknessIn classical terms, circadian rhythm is defined as oscillations in the biochemical, physiological, and behavioral functions of an organism with a periodicity of ~ 24 h. Circadian rhythms have three basic features. First, the rhythm is an innate property of the organism and is maintained under constant conditions. Second, the period length is temperature compensated, thus it is ensured to maintain the rhythm at a constant rate throughout the physiological range of external temperature. Third, it is synchronizable with the outside world by external cues. Light plays a major role as a circadian zeit geber (German:zeit =time, geber = giving), may be because light is an essential factor to maintain the life on earth. The circadian rhythms are exhibited by organisms from every level of the 'life-tree', the blue green algae, cyanobacteria, to the advanced level of life, the mammals, and indeed humans too. The conservation of biological clocks throughout evolution has suggested that it confers a selective advantage to the organism. It is important that the organism must be well fit to the environmental settings to survive through the pressures of life challenges. Therefore, the organism should have a highly functional biological clock, which can sense and respond quickly to the environmental fluctuations. The activities of different species of organisms on earth have been temporally discrete, in such a way that it should benefit the organism maximum. For e.g. the bees go in search for nectar only after the dawn, because the blossom is in correlation to the sun light. In a different perspective, some lower animals are avoided being preyed by higher predatory animals by simply scheduling their foraging out of phase with the predator, thus compromising for the potential risk of predation and feeding. It might be noticed that humans are normally active during the day time (diurnal), with a period of 25.1 h, whereas some lower animals like mice are active during the night (nocturnal), with a period of 23.7 h.
History: The concept of time has always perplexed and fascinated people. The research on biological rhythms has drawn the attention of scientists since 1729, when Jean Jacques d'Ortous de Mairan (a French astronomer) has started to look at the changes in plants in relation to day and night (1) . Thereon the quest was continuing relentlessly through the centuries. Though it was widely accepted only ~ 60 years ago that the life forms have an internal timekeeping device, a genetic underpinning for circadian clock in plants was first proposed by Buenning in 1935 (2). It took another 30 years for Pittendrigh (1967) to infer that the animals (insects) also have an internal clock that too geared by a genetic gadgetry (3).
The 1971 have witnessed a discovery that ever since connected a behavioral trait to a gene, thereby gave birth to a novel branch of biology- the behavioral neurogenetics. Konopka R J (Caltech, USA) was the first to report a 'clock gene' from Drosophila melanogaster (the common fruit-fly)(4). His discovery of period mutant (a defective gene) has marked the real beginning of molecular chronobiology. Flies carrying mutation in this gene exhibited an altered rhythm in their daily rest-activity pattern. However, as recently as 1994 only the clock gene period in Drosophila was molecularly characterized (5). In last decade there was an explosion in this field which was aptly dubbed by some clock researchers as "Clockwork Explosion". As a culmination of the efforts of many researchers, presently there is a wealth of knowledge available to the scientific community. A family of genes called clock controlled genes (ccg) exists today. The ccg exceed more than seven by now and every day breaks into a new surprise as well. Most of the discoveries about circadian clock have been achieved in the model organism Drosophila, because of the vast repertoire of genetic and molecular tools available for this simple fruit fly. Its genome was completely sequenced and many genes have already been identified and much more are annotated. For better correlation of the findings in fruit fly to higher animals researchers have embarked onto mammals, like rodents, in the recent past, nevertheless the progress made is amazing than predicted. The uncanable achievements in the field of bioinformatics and molecular biology have fuelled the pace of rhythm research exponentially.
This realm of research has come to light when its paramount importance has been emphasized by recognizing 'biological clock' as the first runner-up in the list of break-through of the year in 1998 by the American Association for the Advancement of Science.
Modeling the circadian clock: The simple clock model accommodates three components, an input, an oscillator and an output. The most potent input in the environment is the light signals. However, temperature and even the food could work as an input to set the clock. The input feeds the internal oscillator, and informs about the external environment, where the organism lives. The oscillator 'reads' and 'translates' the information about the external environment and generates an output in compliance with the external settings. The ability to read correctly the external world is very important for an organism to reorganize its physiology and behavior to have a better fitness. Fitness is the key to survival of an organism according to the classic theory of Darwin. The well organized will have better survival in an ever-changing world, where it can swiftly adapt to the challenges.
Photoreceptors like rhodopsin and cryptochrome function as the input into the central oscillator. Mutants for various photoreceptor molecules or genes down stream to them (in a functional cascade) have proven that light is an important signal for setting the internal clock. The eyes play a major role in circadian photoreception, besides its primary function, vision.
The central oscillator is composed of multiple genes and their proteins. In Drosophila the ccg genes express in clock neurons in the brain and other peripheral tissues. These genes show a rhythmic expression pattern, which is the basis of circadian functions. Drosophila has two clusters of important clock neurons called lateral and dorsal neurons. The clock proteins peak in the clock neurons in midnight (Fig. 2).
Figure 2 shows a full brain of Drosophila. The brain was stained with anti-PER antibody, which is having a fluorescent label. The brain was dissected out at midnight to check for the expression level of PERIOD protein. The arrows indicate the various clock neurons in the brain, which express the clock protein PERIOD. sLNvs means small lateral ventral neurons; lLNvs, large lateral ventral neurons; LNds, lateral neurons dorsal; DNs, dorsal neurons. The sLNvs are believed to be the pacemaker neurons in Drosophilaand trough in the daytime. This could be visualized by staining these proteins using fluorescent-labeled antibodies against the proteins.
The output is the general behavioural or physiological function of an organism. Drosophila researchers routinely study two main outputs. First, the daily rest-activity rhythm, which have a period of ~ 24.3 h; it continues in constant darkness as well. Second, the eclosion rhythm (emergence of a fly from a puparium).
A clock-mutant (having defect in a clock gene) fails to show an overt rhythm in rest-activity pattern or even the eclosion rhythm. That's how really in the beginning researchers have 'spotted' the clock mutants, and later on identified and characterized the 'clock genes' involved in the clock mechanisms.
Molecular mechanisms: The basic mechanisms behind the orchestration of circadian timekeeping are achieved by the unique wiring of proteins. Presently it has been envisaged that the clock machinery works based on a transcriptional-translational feed back loop (6 - recent review; references therein). That literally means, the transcription of a gene - the copying of DNA sequences, is negatively influenced by the translation of the same gene- the process of production of a protein from a gene. There is a temporal separation of approximately 6 h between these two processes. Besides, at least one of the clock protein is sensitive to light signals. The interplay of light and the clock proteins eventually accomplishes a circadian pattern of clock gene expression.
Presently, there are at least seven clock controlled genes known in Drosophila. The concerted action of these genes achieve the 'jerk and torque' of the clock machinery. They are namely, period (per), timeless (tim), Clock (Clk), cycle (cyc), vrille (vri), double-time (dbt) and shaggy (sgg). Most of them have counterparts in higher animals including man. For e.g. there are three period genes in humans, hper1, hper2 and hper3.
The principles of molecular timekeeping in a nutshell: A dimer formed by the mutual binding of CYCLE (CYC) and CLOCK (CLK) proteins reach and attach to the promoter (a specific DNA sequence that drives a gene for expression) of per and tim genes. In turn, the transcription of the per and tim switched on. This event happens in the nucleus. Then the mRNAs of per and tim are transported for protein synthesis into the cytoplasm, where PERIOD (PER) and TIMELESS (TIM) proteins are generated. They get accumulated from early evening through the night in the cytoplasm. As their levels shoot up in the cytoplasm, some 'modifier' proteins like DOUBLE-TIME (DBT) and SHAGGY (SGG) change the physical properties of the PER and TIM by adding a phosphate group (phosphorylation) onto them, later on the PER and TIM form a heterodimer complex. The phosphorylation of PER-TIM initiated the translocation of the PER-TIM heterodimer into the nucleus. As long as the PER-TIM dimer is shuttled to the nucleus, their production switch will be kept on. However, once the concentration of PER-TIM exceeds a limit in the nucleus (by mid night), it interferes with CLK-CYC dimer molecules present in the nucleus, which have been driving the per and tim. The binding of PER-TIM to CLK-CYC distorts the latter's functioning, and results in the switching off the per and tim transcription. Consequently, the further production of PER and TIM is suspended. This state remains as long as the levels of PER and TIM are high in nucleus. That is one side of the story, on the other side, when the light irradiates in the dawn, the TIM in the nucleus (already phosphorylated) gets degraded through a light mediated action. The cryptochrome (a photoreceptor molecule) transduces the light signals to the TIM. Once the TIM is degraded, the PER loses its existence as such. In conclusion, the light rays eliminate the protein complex, PER-TIM, from the nucleus and in turn releases their 'disturbing' action on CLK-CYC. Once the CLK-CYC relieved from the interference of PER-TIM, the former resumes its functioning once again, thus paves the way for another bout of the same expression of per and tim in the day light. (Fig. 3).
Figure 3 is the schematic representation of the clock mechanism in Drosophila. At night, the CLK-CYC dimer binds to the per and tim genes' promoter and start their transcription - produces the respective mRNAs. The mRNAs are translated into proteins. The proteins get phosphorylated and form a dimer:PER-TIM. This dimer translocated into the nucleus, where it binds with the CLK-CYC and disturbs the latters action and stops the production of mRNAs. At day, the dimer PER-TIM is degraded by the light, through a CRY mediated mechanism. This relieves the negative impact on CLK-CYC dimer, thus resumes the transcription of per and tim again.
Medical implications of circadian clock: The circadian clock has far reaching implications on human health and welfare. There are many emerging issues, which have greater concern in the present day scenario due to the advanced life styles adopted by many populations around the world. In this competitive world, to increase the production rate people have been put through in constant shift work routines; and to promote more business the personals have been travelling across the meridians quite frequently. These activities have bestowed few serious health risks like sleep disturbances, jetlag etc. to the subjects involved in. It is debatable that whether our internal clock machinery has evolved that quickly as we have technically advanced. Few major issues of the human health connected to the circadian clock have been discussed briefly.
The melantonin, which displays a clear circadian pattern, peaks during the night, has been routinely tested for most circadian rhythm related investigations (7). The recent report that circadian clock genes hPer1, hPer2, and hPer3 are expressed in a circadian manner in human peripheral blood mononuclear cells (PBMCs), with the peak level occurring during the habitual time of activity (
, offers few more candidates for the studies on human circadian rhythms and their associated disorders.
Reproductive fecundity: The incidence of infertility cases are rapidly increasing among the human population. But, the exact reason for this phenomenon has not yet been identified. It was always believed that there is a relation between the fitness of partners and the success of a pregnancy. In this context, it is well recognized today that circadian clock influences the fitness of an organism (9). The reproductive fecundity is the best output to assess the fitness of an organism. The elegantly designed experiments in Drosophila have proved that defect in a clock gene(s) afflicted the sperm release from the testes to seminal vesicles (these tissues displayed rhythmic and autonomous expression of clock genes), which in effect reduced the successful fertilization frequencies. However, the influence of circadian clock on human reproductive function has not yet been investigated.
FASPS: The human sleep-wake cycle remains as a mystery black box to the entire medical world. Though most of us have a regular pattern of sleep-onset at the fall of night and awakening by about 1-2 h after sunrise, there do exist peculiar marginal cases. In a particular case, called familial advanced sleep phase syndrome (FASPS), the subject goes into bed an hour after sunset and is wide-awake by 4.00 in the morning. Recently, FASPS has been linked to a mutation in human clock gene, hper2, the homolog to Drosophila per (10). The mutation in hper2 blocks a phosphorylation site, which is required for the nuclear translocation of the protein.
DSPS: The opposite case of FASPS is the delayed sleep phase syndrome (DSPS), where the patients go to bed around 4:00 in the morning and wake up around the noon. Though the genetic mechanism underlying DSPS is yet to be elucidated, it is conceivable that there is a 'misinterpretation' about the day by the internal clock mechanism. Either the input or the oscillator may have been corrupted, because the bright light therapy to this patients has found to be quite efficacious.
Fragile X syndrome: The absence of a protein, called fragile X mental retardation protein (FMRP), predisposed the new born baby to become mentally retarded. As the name indicates it is a X chromosome linked inheritable disease. It has been hypothesized that FMRP is involved in the processing and/or translation of mRNAs. To better understand the molecular mechanisms behind fragile X syndrome, a Drosophila homolog of hFMR1, the dfmr1 has been mutated and studied in depth. It is well documented that the fragile X syndrome patients show a variety of sleep disorders. The studies on Drosophila has revealed that the mutation in dfmr1 affects an output function of the circadian clock (11), and the Drosophila become arrhythmic in constant darkness (normally rhythmic). Moreover, the Drosophila model for fragile X syndrome provides new insights into the sleep-wake cycles of animals, because it revealed additional molecular mechanisms connected to this particular genes.
SAD: The seasonal affective disorder (SAD)- winter depression- is a syndrome characterized by recurrent depressions, mainly in countries where there is a severe winter, and relatively less sun light. This syndrome harbors hypersomnia, hyperphagia and carbohydrate craving. SAD prevalence in the United States is estimated to be ~6%, about 11 million people. The females are more affected than males (12). Bright light therapy has been suggested to be beneficial for these patients to recover from their depressive mood, at least to certain extent. Assumably, some circadian clock functioning is impaired in these patients. The sleep and melatonin release is under the control of a central clock in the brain; the supra chiasmatic nuclei (SCN) is believed to receive some light signals from the retina, and that triggers the internal circadian clock function.
Jetlag: Approximately 78% of the subjects have reportedly been suffered sleep disturbances during the first night after a transmeridian flight (13). An accompanying gastrointestinal disturbance has also been reported by ~60% of these subjects. It has been demonstrated that the transmeridian flying is an acute stress for female aircrews, they have been suffering a delayed ovulation and associated menstrual disturbances, at in 30-35% of them. Traveling across meridians imposes a new light-dark phase on the traveler. In this newer day-night phase, the traveler's internal clock is set to the earlier time zone, and he/she needs more time to re-synchronize to local environment. As a consequence, the individual suffers from insomnia, fatigue and irritability. At this moment, the social demands in a new time zone and the person's ability to coping with it are in conflict, because the internal clock is out of phase. The individual takes at least a day or more to adjust to the local environment. In mammals including humans, the pineal hormone melatonin level peak during the night, and its release could be suppressed by exposure to light pulses. The SCN expresses high-affinity melatonin receptors; and to circumvent the effects of jet-lag melatonin has been widely prescribed in clinics, though its usefulness in treatment of jetlag and other ailments is controversial. Studies on pilots and cabin crews have exhibited cognitive deficits, possibly in working memory that became apparent after several years of chronic disruption of circadian rhythms due to the constant flights across the transmeridian (14).
For airline pilots, sleep deprivation and circadian desynchronization due to rapidly rotating shift schedules, rapid time zone changes, and unorganized sleep/wake cycles can result in severe performance decrements. Twelve airline accident cases in the past suggest that pilot scheduling might have been a factor for these accidents; and the investigation committees have came up with suggestions for the chronohygiene of the pilot scheduling (15). In conclusion, the rapid time-zone transitions impaired performances and consequently the safety of both aircrews and the frequent flyer. Therefore, more prudent practices should be implemented in the air traffic systems to avoid further incidence of airline crashes.
Rotating Shiftwork: Impaired mental and physical performance is always a spin-off of the sleep deprival, which is inevitable in a shiftwork routine. One of the compounded effect of sleep disturbance is fatigue. The individual's ability to perform a task optimally as dictated by the circadian clock is often skewed from the demand of a shiftwork regime. In realistic terms, the person fails to pay attention to the minute and often important details of his task. Many dreadful disasters occurred in the past were regarded as errors committed by workers who had not been synchronized to work in the night shift. For example the disasters at Three Mile Island, Chernobyl, and Bhopal were occurred between midnight and dawn (16). The ailments arising out of a shiftwork routine could be ameliorated to a greater extent by simply exposing the subject to high-intensity light during the night, to shift the phase of the circadian clock (17).
Breast Cancer: The incidence of breast cancer has steadily increased during this century in industrialized countries. The projected reason is that the individuals are exposed to light for longer periods than that afforded by the natural daily light-dark cycle. The melatonin hypothesis proposes that the suppression of melatonin secretion at night by artificial light increase the chances for breast cancer by increasing the exposure to estrogen (18). The lifetime exposure to estrogen is a major risk factor for breast cancer. To further support this hypothesis, it has been reported that severely blind women had about half the incidence of breast cancer than women with normal vision (19). Further research is warranted to arrive at a precise answer to the many open questions.
Learning and memory: per mutation in Drosophila has been linked to robust changes in certain drug sensitivities and to defects in the patterns of expression of some genes that are tightly connected to the control of learning and memory (20). It is well known that the mental acuity is not the same throughout the day. Therefore, it is advisable to find out that at what time a person can learn the best and according to that he/she should pursue his/her learning. Understanding more about the role of different genes on learning and memory would open novel avenues for pharmacological interventions to manipulate the learning process in the future.
Issues related to diagnosis and treatment: The success of any treatment depends on how accurately the physician can diagnose the cause of the problem. The art of diagnosis relies basically on observing the physiological and behavioural variables. These variables change in a rhythmic manner over the course of a day. The relevance of considering the circadian rhythmic variance, while performing diagnosis is not yet clear. But, physicians keeping in mind this fact would certainly give an advantage. The efficacy of cancer treatment drugs has been found to be largely depend on the time of delivery (21). Thus, it is possible to improve the therapeutic potential and minimize toxic side effects by optimizing schedules for administering drugs. As a matter of fact, it is reminded that in Ayurveda practices the time of administration of a drug is strigently followed.
Conclusion: Since the discovery of period gene, the circadian clock research has undergone a quantum leap. Presently our understanding about the circadian clock both in lower animals and mammals is quite appreciable. The components of the circadian clock, once seemed to be quite elusive and mysterious, were brought to light by ingenious experiments. The clock picture is more clear now with a transcriptional-translational feedback loop functioning in the center. A family of clock genes and their functions are widely known today. However, the clock puzzle is not yet finished absolutely. The bridging of knowledge on molecular mechanisms of the circadian clock in Drosophila and human illnesses like fragile X syndrome is a commendable achievement. It points toward the feasibility to extract valuable information from lower model systems, that can find applications in medical science. Rational drug design, aimed at many disorders connected to the circadian clock, like insomnia and FASPS could be further rectified by unraveling the molecular mechanisms under laying these disorders. Eventually, it is presumable that further development in this filed may be improving the general well being of the modern man, who abodes in a sophisticated environment or rather in a controlled environment where programmed machines set all the boundaries. The future generations may be in a position to program, as per wish, their internal clock. We can anxiously await to watch what time holds in its store!
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