Molecular Components of mammalian Circardian Rhythm
Mammals synchronize their circadian activity primarily to the cycles of light and darkness in the environment. This is achieved by ocular photoreception relaying signals to the suprachiasmatic nucleus (SCN) in the hypothalamus. Signals from the SCN cause the synchronization of independent circadian clocks throughout the body to appropriate phases. Signals that can entrain these peripheral clocks include humoral signals, metabolic factors, and body temperature. At the level of individual tissues, thousands of genes are brought to unique phases through the actions of a local transcription/translation-based feedback oscillator and systemic cues. In this molecular clock, the proteins CLOCK and BMAL1 cause the transcription of genes which ultimately feedback and inhibit CLOCK and BMAL1 transcriptional activity. Finally, there are also other molecular circadian oscillators which can act independently of the transcription-based clock in all species which have been tested.
Introduction
As the sun sets, nocturnal rodents begin to forage, nocturnal birds of prey begin their hunt while diurnal birds of prey sleep, filamentous fungi begin their daily production of spores, and cyanobacteria begin nitrogen fixation in an environment of low O2 after the day’s photosynthesis. As the sun rises the next morning many plants have positioned their leaves to catch the first rays of light and many humans sit motionless in cars on a nearby gridlocked highway. It is now understood that the obedience to temporal niches in these and all organisms is governed by a molecular circadian clock. These clocks are not driven by sunlight, but are rather synchronized by the 24 hour patterns of light and temperature produced by the earth’s rotation. The term circadian is derived from “circa” which means “approximately” and “dies” which means “day”. A fundamental feature of all circadian rhythms is their persistence in the absence of any environmental cues. This ability of clocks to “free-run” in constant conditions at periods slightly different than 24 hours, but yet synchronize, or “entrain”, to certain cyclic environmental factors allows organisms to anticipate cyclic changes in the environment. Another fundamental feature of circadian clocks is the ability to be buffered against inappropriate signals and to be persistent under stable ambient conditions. This robust nature of biological clocks is well illustrated in the temperature compensation observed in all molecular and behavioral circadian rhythms. Here temperature compensation refers to the rate of the clock being nearly constant at any stable temperature which is physiologically permissive. The significance of temperature compensation is especially evident in poikilotherimic animals that contain clocks that need to maintain 24 hour rhythmicity in a wide range of temperatures. Combined, the robust oscillations of the molecular clocks (running at slightly different rates in different organisms) and their unique susceptibility to specific environmental oscillations contribute to and fine-tune the wide diversity of temporal niches observed in nature.
However, the circadian clock governs rhythmicity within an organism far beyond the sleep: activity cycle. In humans and most mammals there are ~24 hour rhythms in body temperature, blood pressure, circulating hormones, metabolism, retinal electroretinogram (ERG) responses, as well as a host of other physiological parameters . Importantly, these rhythms persist in the absence of light:dark cycles and in many cases in the absence of sleep:wake cycles. On the other side of the coin, a number of human diseases display a circadian component, and in some cases, human disorders and diseases have been shown to occur as a consequence faulty circadian clocks. This is evident in sleep disorders such as Delayed Sleep Phase Syndrome (DSPS) and Advanced Sleep Phase Syndrome (ASPS) in which insomnia or hypersomnia result from a misalignment of one’s internal time and desired sleep schedule . In familial ASPS (FASPS), the disorder cosegregates both with a mutation in the core circadian clock gene PER2 and independently with a mutation in the PER2-phosphorylating kinase, CK1 δ. Intriguingly, transgenic mice engineered to carry the same single amino acid change in PER2 observed in FASPS patients recapitulate the human symptoms of a shortened period. Although, these mutations are likely not the end of the story for these disorders, they give insight into the way molecular clocks affect human well-being. Jet lag and shift work sleep disorder are other examples of health issues where the internal circadian clock is desynchronized from the environmental rhythms. In addition to sleep-related disorders, circadian clocks are also directly linked with feeding and cellular metabolism, and a number of metabolic complications may result from miscommunication with the circadian clock and metabolic pathways. For example, loss-of-function of the clock gene, Bmal1, in pancreatic beta cells can lead to hypoinsulinaemia and diabetes. Finally, some health conditions show evidence of influence of the circadian clock or a circadian clock-controlled process. For example, myocardial infarction and asthma episodes show strong nocturnal or early morning incidence. Also, susceptibility to UV light-induced skin cancer and chemotherapy treatments varies greatly across the circadian cycle in mice.
In mammals the suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock for the entire body. However, the SCN is more accurately described as a “master synchronizer” than a strict pacemaker. Most tissues and cell types have been found to display circadian patterns of gene expression when isolated from the SCN. Therefore, the SCN serves to synchronize the individual cells of the body to a uniform internal time more like the conductor of an orchestra rather than the generator of the tempo themselves. The mammalian SCN is entrained to light cycles in the environment by photoreceptors found exclusively in the eyes . The SCN then relays phase information to the rest of the brain and body via a combination of neural, humoral, and systemic signals which will be discussed in more detail later. Light information influencing the SCN’s phase, the molecular clock within the SCN, and the SCN’s ability to set the phase of behavior and physiology throughout the body constitute the three necessary components for a circadian system to be beneficial to an organism: 1) environmental input, 2) a self-sustained oscillator, and 3) an output mechanism.
Temperature and circadian clocks
The influence of temperature on circadian clocks is important to discuss here both because of the ubiquity of temperature regulatory mechanisms in circadian clocks but also as potential targets for chronotherapeutics. First, as mentioned in the introduction to this chapter, all circadian rhythms are temperature compensated. This fundamental property allows the clock to maintain a stable period of oscillation regardless of the ambient temperature. A circadian clock would not be reliable if its period changed every time the sun went down or ran at a different period in the winter than in the summer. Temperature compensation is expressed as the coefficient Q10which represents the ratio of the rate of a reaction at temperatures 10°C apart. The Q10 of periods of various circadian rhythms of many species of broad phyla are between 0.8 and 1.2. Most chemical reactions within cells are affected by temperature; for example, most enzymatic reactions increase in rate as temperature is increased. In fact, the kinases CK1ε and δ increase their rate of phosphorylation of some protein targets at higher temperatures as would be expected; however, their rates of phosphorylation of clock proteins are stable at those same temperatures. This temperature compensation is yet another example of the robustness of the molecular clock to retain precision in varying conditions. Even with broad reduction in global transcription, the clocks in mammalian cells remain rhythmic with only slightly shorter periods.
The mechanisms of temperature compensation are still not understood, but great strides have been taken using the Neurospora crassa fungus. These organisms are routinely exposed to wide variations in temperature in their natural environment. The levels of the clock protein FRQ (which plays the negative limb role in fungus as PER and CRY do in mammals) are elevated at warmer temperatures and a long-form splice variant is observed at warm temperatures. Mutants of the kinase CK-2, which phosphorylates FRQ, display either better temperature compensation than wild-type or opposite “overcompensation”. In our own work we observed an impairment in temperature compensation of PER2 rhythms in the SCN and pituitary of mice when the Heat Shock Factors (HSF) were pharmacologically blocked. These results fit with a model in which positive and negative effects of temperature on rates of cellular activity balance out to a net null effect. However, other findings suggest that this balancing model may be more complicated than necessary. Other extremely simple circadian rhythms, such as the in vitro phosphorylation of KaiC in Synechococcus, demonstrate beautiful temperature compensation with the presence of just the three proteins and ATP. Also, the transcription/translation-free rhythms of oxidation in peroxiredoxins in human red blood cells are temperature compensated. These results suggest that very simple oscillators may be temperature compensated purely by the robustness inherent in the individual processes rather than requiring balancing agents.
Although, circadian clocks run at the same period at various temperatures, this does not mean that circadian clocks ignore temperature. Most species, particularly poikilotherimic organisms, are exposed to wide daily temperature oscillations, and they use the change in temperature as an entraining cue. In fact, in Neurospora if a temperature cycle and light:dark cycle are out of phase, the fungus will entrain to the temperature cycle more strongly than to the light. In the fruit fly Drosophila melanogaster, the entrainment of global transcription rhythms appears to use a coordinated combination of light:dark cycles and temperature cycles so that the phase of light entrainment slightly leads the phase set by temperature of the same genes. The importance of temperature changes is most strikingly observed at the behavioral level. In standard laboratory conditions with a light:dark cycle at a stable temperature, the flies show strong crepuscular activity with a large inactive period during the middle of the day. When more natural lighting is paired with a temperature cycle, the flies show a strong afternoon bout of activity and behaviorally act like a different species.
Environmental temperature cycles act as extremely weak behavioral entrainment cues in warm-blooded animals, or “homeothermic” animals, which maintain their body temperature regardless of ambient temperature. However, the internal body temperature of homeothermic animals undergoes circadian fluctuations with amplitudes of approximately 1°C and 5°C depending on the species. As mentioned earlier, the surgical ablation of the SCN abolishes the circadian component to body temperature fluctuation along with behavioral and sleep rhythms in mice, rats, and ground squirrels. Although it is hard to isolate effects that activity, sleep, and the SCN have on body temperature oscillations, both human and rodent examples exist. In humans, the circadian oscillation of rectal temperature persists if a person is restricted to 24 hour bed rest and is deprived of sleep. In hibernatory animals, such as the ground squirrel, a low amplitude SCN-driven body temperature rhythm is observed during bouts of hibernation in which there is an absence of activity for days at a time.
As discussed in the Peripheral Clocks section, these rhythms of body temperature fluctuation are sufficient to entrain the peripheral oscillators of homeothermic animals in all cases that have been reported. The most recent evidence suggests that this effect on the molecular clock mammals by temperature cycles is regulated by the heat shock pathway. Briefly, after heat exposure the Heat Shock Factors (HSF1, HSF2, and HSF4) initiate the transcription of genes with Heat Shock Elements (HSE) in their promoters. The genes of Heat Shock Proteins (HSP) contain HSEs and once translated these proteins chaperone, or sequester the HSFs from further transcription. This feedback loop maintains a transient response to temperature changes. Although commonly associated with heat tolerance to extreme temperatures, the dynamic range heat shock pathway can include temperature changes within the physiologic range. Blocking HSF transcription transiently with the pharmacological agent KNK437 mimicked the phase shifts caused by a cool temperature pulse and blocked the phase shifting effects of warm pulses . Also, a brief exposure to warm temperatures caused an acute reduction of Per2levels followed by an induction when returned to a cooler temperature in the liver . Along with being a temperature sensor for phase setting, it is also evident that the HSF family and the circadian clock are more intimately related. Although the levels of HSF proteins have not been found to have a circadian oscillation, their binding to target motifs certainly does even in the absence of temperature cycles. Additionally, the promoter of the Per2 gene contains HSEs that are conserved among multiple species, and a number of hsp genes oscillate with a phase similar to Per2 . Finally, deletion of the Hsf1 gene lengthens the free-running behavioral period of mice by about 30 min, and pharmacologic blockade of HSF-mediated transcription ex vivo causes the molecular clock to run >30 hr in SCN and peripheral tissues . Clearly the heat shock response pathway exerts both phase and period influence on the circadian clock. It will be exciting to see how this relationship is further elucidated in the future.
