The term circadian rhythm was originally coined by Halberg to indicate the near-24-hour (h) endogenous oscillations of biological processes in organisms associated with the earth’s daily rotation cycle. Such endogenous rhythms, observed in organisms ranging from photosynthetic prokaryotes to higher eukaryotes, reflect the existence of an intrinsic circadian clock that temporally orchestrates physiological and behavioral processes with the specific function of coordinating and adapting the internal environment with the external cues. The entrainment of circadian rhythms, which consists of the alignment of the endogenous circadian oscillator to external stimuli, relies on external cues, such as the light pattern and food intake. In particular, the daily light–dark cycle represents the primary external synchronizer of circadian rhythms. In mammals, light is processed through the eye and transmitted through the retinohypothalamic tract to hypothalamic suprachiasmatic nucleus (SCN), the master internal pacemaker. In the retina, the intrinsically photoreceptive retinal ganglion cell (ipRGC) expressing the photopigment melanopsin, which renders them photosensitive to short-wavelength irradiation, together with the retinal rod and cone photoreceptors, conveys photic information to entrain SCN clocks. Notably, SCN is composed of bilateral nuclei containing approximately 10,000 neurons, each of them displaying a cell-autonomous circadian oscillator. A number of papers have demonstrated that the SCN is both necessary and sufficient for the generation of circadian rhythms in rodents. SNC neurons have distinct and topographically organized coupling mechanisms that allow them to remain synchronized to one another. They generate a pronounced circadian rhythm of neuronal firing frequency, which allows them to synchronize other cells throughout the body.
Notably, in the brain, beside the master clock in SCN operating as self-sustaining clocks, other functional nuclei have been found to act as semiautonomous clocks (i.e. olfactory bulb, dorsomedial hypothalamus, arcuate nucleus, habenula) or as slave oscillators (i.e. bed nucleus of the stria terminalis, amygdala, preoptic area, paraventricular nucleus, nucleus accumbens), both coordinated by the SCN.
The central pacemaker clock synchronizes multiple peripheral clocks expressed in nearly every mammalian organ (i.e. such as lungs, liver, heart, and skeletal muscle). Noteworthy, while in the SCN circadian rhythms are similar between diurnal and nocturnal species, a phase shift, matched to their active period, in the rhythms has been observed in their peripheral tissues. The synchronization of peripheral clocks is not only hierarchically and vertically controlled by the hypothalamic master clock through the peripheral nervous system, but it also horizontally achieved through both humoral and non-humoral pathways. In particular, SCN controls peripheral oscillators through the autonomic innervation of peripheral tissues, endocrine signaling (glucocorticoids), body temperature, and feeding-related cues. The neural control of peripheral oscillators requires both sympathetic and parasympathetic pathways. SCN projections, through the paraventricular nucleus–superior cervical ganglia (PVN-SCG) pathway, deliver the entraining signal for the submandibular salivary glands. Autonomic pathways derived from the SCN provide photic information to oscillators in the adrenal gland and liver. Moreover, sympathetic pathways modulate the sensitivity of the adrenal to adrenocorticotropic hormone (ACTH) and the release of glucocorticoids. In the adrenal cortex and medulla, cellular oscillators respond to neural inputs deriving from the SCN. Furthermore, glucocorticoids are humoral entraining signal for peripheral clocks entraining signals for peripheral oscillators. In particular, since glucocorticoids-response elements (GREs) are present in promoter regions of the core clock components glucocorticoids regulate the transcriptional activation of clock genes and clock-related genes.
At the molecular level, the circadian clock consists of multiple sets of transcription factors resulting in autoregulatory transcription-translation feedback loops (TTFLs) that represent the core mechanism of the circadian clock in mammals. A central role in the regulation of this loop is played by the heterodimeric partnership between two transcription factors, i.e. the brain and muscle Arnt-like protein 1 (ARNTL, also known as BMAL1) and the circadian locomotor output cycles kaput (CLOCK). In particular, transcription of BMAL1 and CLOCK genes, or its related gene NPAS2 (neuronal PAS domain containing protein-2), which is mainly expressed in the forebrain, leads to the heterodimerization in the cytoplasm of the BMAL1:CLOCK complex, which translocates into the nucleus where it binds to canonical Enhancer Box (E-Box)-sequences containing the consensus sequence CACGTG or noncanonical E-Boxes of clock-regulated genes. Among the different target genes, BMAL1 and CLOCK also promote the expression of the components of the negative arm of the molecular clock, such as period (PER1, PER2, PER3) and cryptochrome (CRY1, CRY2). PER and CRY form a complex in the cytoplasm that translocates into the nucleus. Following their translation and nuclear accumulation, PER and CRY inhibit the transcriptional activity of BMAL1:CLOCK complex. At post-transcriptional level, the stability of PER and CRY proteins is regulated by Skp1-Cullin-F-box protein (SCF) E3 ubiquitin ligase complexes, involving β-TrCP (β-Transducin Repeat Containing E3 Ubiquitin Protein Ligase) and F-Box and Leucine Rich Repeat Protein 3 (FBXL3), respectively. The casein kinase 1ε/δ (CK1ε/δ) and adenosine 3’,5’-monophosphate (AMP) kinase (AMPK) phosphorylate PER and CRY proteins, respectively, thus promoting polyubiquitination by their respective E3 ubiquitin ligase complexes, which tag PER and CRY proteins for degradation by the 26 S proteasome complex. Decrease in PER and CRY protein levels relieves the suppression of BMAL1:CLOCK activity, thereby permitting to establish a new oscillatory cycle. In addition to such core loop, further key regulators of the circadian clock, such as the nuclear receptors REV-ERBα (also known as nuclear receptor subfamily 1, group D, member 1, NR1D1) and REV-ERBβ (also known as nuclear receptor subfamily 1, group D, member 2, NR1D2) (REV-ERBs), as well as the retinoic acid orphan receptor (ROR) (RORα, RORβ, and RORγ) establish another feedback loop. In particular, while REV-ERBs act as transcriptional repressors of BMAL1 expression, RORs positively regulate the expression of BMAL1 by binding to sites Retinoic acid receptor-related Orphan Receptor Element (RORE) elements in the BMAL1 gene promoter.
On the other hand, the biological clock is not only based on transcriptional mechanisms, but also, membrane depolarization, intracellular calcium flux, and activation of cyclic AMP (cAMP) signaling appear to be important regulators of the mammalian transcriptional clock. Accordingly, intracellular calcium is fundamental for neuronal firing rhythms in SCN slices and sufficient membrane depolarization, periodic calcium influx, and daily activation of cAMP signaling are required for the rhythmic expression of the core clock components in SCN neurons. Notably, such effects are mediated by the phosphorylation-dependent activation of the calcium/cAMP response element binding protein (CREB) that binds to calcium/cAMP regulatory elements (CREs) on DNA. In particular, CRE sequences have been found in the promoters of several clock genes, such as PER1 and PER2. Since membrane potential, calcium flux, and activation of cAMP signaling have been reported to be also rhythmic themselves in SCN, they represent both outputs of and inputs to the transcriptional clock, by possibly establishing positive feedback loops participating in rhythm generation.
However, the mechanisms underlying the molecular clock cannot be simplified in such a reductionist fashion. Indeed, clock genes integrate a plethora of different signals to produce an integrated output over the 24-h cycle, in preparation for the diverse tasks related to periods of light or darkness, wherein gene expression ch
