Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway

Abstract

Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and Nacetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2 Luciferase (Per2 Luc ) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.C ircadian (from the Latin, meaning about a day) rhythms of biochemistry and physiology are innate to virtually all life forms (1, 2). Cell autonomous circadian pacemakers drive these rhythms and synchronize them each day to environmental cues. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus contains the central pacemaker that coordinates the local circadian clocks present in tissues throughout the body (3, 4). The molecular clock mechanism within individual cells is composed of transcriptional/translational feedback loops and posttranslational processes (1, 5). The bHLH-PAS transcription factors CLOCK and BMAL1 form the primary loop as they activate transcription of the Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes (1, 6). The PER and CRY proteins subsequently feed back to abrogate their own transcription by directly inhibiting the CLOCK:BMAL1 complex. A second feedback loop stabilizes the primary loop and is composed of REV-ERBα and RORa, two retinoic acid-related orphan receptors, which repress and activate, respectively, transcription of Bmal1. Whereas approximately a dozen genes involved in this timekeeping system have been identified in mammals, it is clear that other, as-yet unknown genes regulate key properties of circadian rhythms (7,8).The SCN imparts daily entraining signals to circadian clocks in cells of peripheral tissues via neural connections and humoral factors (9, 10). One well-characterized circadian rhythm synchronized by the SCN is the synthesis and release at night of the lipophilic pineal hormone melatonin (11). Light information reaches the mammalian pineal gland through a multisynaptic pathway that begins with intrinsically photosensitive melanopsin-containing retinal ganglion cells, which project to the SCN via the retinohypothalamic tract, and ultimately terminates at sympathetic afferents of the pineal gland (12, 13). Lesions of the SCN abolish the melatonin circadian rhythm (14). Two high-affi...

Psychotropic medications affecting biological rhythm

“…In these animals, locomotor activities were increased in response to novel stimuli yet overall activity in the home cage was decreased (40). It is challenging to report that the Clock mutation phenotype can be suppressed by melatonin (or the melatonin agonist, ramelteon) to stabilize circadian rhythms (41).…”

Interactions between endocrine and circadian systems

“…Similarly, timed application of melatonin can entrain blind human subjects (Arendt & Broadway 1987, Sack et al 2000. In vitro, melatonin application to cultured SCN explants affects amplitude and phase of the circadian rhythm of neuronal firing (Liu et al 1997, Shimomura et al 2010. The acute inhibitory effect of melatonin on neuronal activity seems to be mediated by MT1 receptor (Liu et al 1997), while the phase-resetting effect relies on MT2 receptor signalling (Hunt et al 2001).…”

“…Clock-D19 mutants (Vitaterna et al 1994) were back-crossed into a melatonin-proficient strain, showing that the Clock-D19 mutation leads to phase delays and dampening of the melatonin rhythm in constant darkness conditions while GC rhythms were completely abolished (Kennaway et al 2003(Kennaway et al , 2006. More recently, it has been demonstrated that the melatonin biosynthesis pathway can genetically suppress the circadian perturbations of Clock-D19 mutation (Shimomura et al 2010), suggesting a role of melatonin in contributing to the robustness of the SCN clock (see below). PER1 deficiency has been shown to enhance Aanat transcription, enzymatic activity and hence melatonin secretion (Chen & Baler 2000, Christ et al 2010.…”

Identifying potential therapeutic targets of a natural product Jujuboside B for insomnia through network pharmacology

“…The connections between molecules show molecular interactions identified in the JuB -insomnia interactome. Gene expression illustrated in coloured nodes was selected with a p < 0.05 value Insomnia is the most common sleep disturbance with difficulty initiating or maintaining sleep, along with impaired daytime function (Shimomura, 2010). There is a high prevalence of insomnia in a variety of negative consequences.…”