Charlotte von Gall

Mammalian melatonin receptors: molecular biology and signal transduction

Abstract. The pineal hormone, melatonin, is an important regulator of seasonal reproduction and circadian rhythms. Its effects are mediated via high-affinity melatonin receptors, located on cells of the pituitary pars tuberalis (PT) and suprachiasmatic nucleus (SCN), respectively. Two subtypes of mammalian melatonin receptors have been cloned and characterized, the MT1 (Mel1a) and the MT2 (Mel1b) melatonin receptor subtypes. Both subtypes are members of the seven-transmembrane G protein-coupled receptor family. By using recombinant melatonin receptors it has been shown that the MT1 melatonin receptor is coupled to different G proteins that mediate adenylyl cyclase inhibition and phospholipase Cβ activation. The MT2 receptor is also coupled to inhibition of adenylyl cyclase and additionally it inhibits the soluble guanylyl cyclase pathway. In mice with a targeted deletion of the MT1 receptor, the acute inhibitory effects of melatonin on SCN multiunit activity are completely abolished, while the phase-shifting responses to melatonin (given in physiological concentrations) appear normal. Furthermore, melatonin inhibits the phosphorylation of the transcription factor cyclic AMP response element binding protein, induced by the pituitary adenylate cyclase-activating polypeptide in SCN cells predominantly via the MT1 receptor. However, a functional MT2 receptor in the rodent SCN is partially able to compensate for the absence of the MT1 receptor in MT1 receptor-deficient mice. These findings indicate redundant and non-redundant roles of the receptor subtypes in regulating SCN function. In the PT, a functional MT1 receptor is essential for the rhythmic synthesis of the clock gene product mPER1. Melatonin produces a long-lasting sensitization of adenylyl cyclase and thus amplifies cyclic AMP signaling when melatonin levels decline at dawn. This action of melatonin amplifies gene expression rhythms in the PT and provides a mechanism for reinforcing rhythmicity in peripheral tissues which themselves lack the capacity for self-sustained oscillation. Mice with targeted deletion of melatonin receptor subtypes provide an excellent model to understand cellular mechanisms through which melatonin modulates circadian and photoperiodic rhythmicity.

Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin

In mammals, many daily cycles are driven by a central circadian clock, which is based on the cell-autonomous rhythmic expression of clock genes. It is not clear, however, how peripheral cells are able to interpret the rhythmic signals disseminated from this central oscillator. Here we show that cycling expression of the clock gene Period1 in rodent pituitary cells depends on the heterologous sensitization of the adenosine A2b receptor, which occurs through the nocturnal activation of melatonin mt1 receptors. Eliminating the impact of the neurohormone melatonin simultaneously suppresses the expression of Period1 and evokes an increase in the release of pituitary prolactin. Our findings expose a mechanism by which two convergent signals interact within a temporal dimension to establish high-amplitude, precise and robust cycles of gene expression.

Targeted disruption of the mouse Mel(1b) melatonin receptor.

ABSTRACT Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel1a melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel1b melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel1b receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel1b receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel1a receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel1a receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel1a-Mel1b double-mutant mice, indicating that the Mel1b receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

Phosphorylation of CREB Ser142 Regulates Light-Induced Phase Shifts of the Circadian Clock

Biological rhythms are driven in mammals by a central circadian clock located in the suprachiasmatic nucleus (SCN). Light-induced phase shifting of this clock is correlated with phosphorylation of CREB at Ser133 in the SCN. Here, we characterize phosphorylation of CREB at Ser142 and describe its contribution to the entrainment of the clock. In the SCN, light and glutamate strongly induce CREB Ser142 phosphorylation. To determine the physiological relevance of phosphorylation at Ser142, we generated a mouse mutant, CREB(S142A), lacking this phosphorylation site. Light-induced phase shifts of locomotion and expression of c-Fos and mPer1 in the SCN are significantly attenuated in CREB(S142A) mutants. Our findings provide genetic evidence that CREB Ser142 phosphorylation is involved in the entrainment of the mammalian clock and reveal a novel phosphorylation-dependent regulation of CREB activity.

Melatonin Plays a Crucial Role in the Regulation of Rhythmic Clock Gene Expression in the Mouse Pars Tuberalis

Abstract: Circadian rhythms in physiology and behavior are driven by a central clock residing within the hypothalamic suprachiasmatic nucleus (SCN). Molecularly, the biological clock is based on the transcriptional/translational feedback loop of clock genes (mPer, mCry, Clock, and Bmal1). Circadian expression of clock genes is not limited to the SCN, but is found in many peripheral tissues. Peripheral rhythms depend on neuroendocrine/neuronal output from the SCN. Melatonin, the hormone of darkness, represents an important neuroendocrine output of the circadian clock. The hypophyseal pars tuberalis (PT) is one of the main target regions for melatonin. The aim of the study was to test whether mPer, mCry, Clock, and Bmal1 are rhythmically expressed in the mouse PT and how the absence of melatonin receptors affects clock gene expression. We analyzed clock gene expression by in situ hybridization and compared wild‐type (WT), melatonin 1 receptor knockout (MT1 ko), and melatonin 2 receptor knockout (MT2 ko) mice. mPer1, mCry1, Clock, and Bmal1, but not mPer2 and mCry2, were rhythmically expressed in the PT of WT and MT2 ko mice. In the PT of MT1 ko mice, expression of mPer1, mCry1, Clock, and Bmal1 was dramatically reduced. We conclude that melatonin, acting through the MT1 receptor, is an important regulator of rhythmic clock gene expression in the mouse PT.

Of Rodents and Ungulates and Melatonin: Creating a Uniform Code for Darkness by Different Signaling Mechanisms

Melatonin synthesis in the mammalian pineal gland is one of the best investigated output pathways of the circadian clock because it can be readily measured and is tightly regulated by a clearly defined input, the neurotransmitter norepinephrine. In this system, a regulatory scenario was deciphered that is centered around the cyclic AMP pathway but shows peculiar species-specific differences. In rodents, the cyclic AMP–mediated, temporally sequential up-regulation of two transcription factors, the activator CREB (cyclic AMP–responsive elementbinding protein) and the inhibitor ICER (inducible cyclic AMP–dependent early repressor), is the core mechanism to determine rhythmic accumulation of the mRNA encoding for the rate-limiting enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase (AA-NAT). Thus, in rodents, the regulation of melatonin synthesis bears an essential transcriptional component, which, however, is flanked by posttranscriptional mechanisms. In contrast, in ungulates, and possibly also in primates, AA-NAT appears to be regulated exclusively on the posttranscriptional level. Here, increasing cyclic AMP levels inhibit the breakdown of constitutively synthesized AA-NAT protein by proteasomal proteolysis, leading to an elevated enzyme activity. Thus, self-restriction of cellular responses, as a reaction to external cues, is accomplished by different mechanisms in pinealocytes of different mammalian species. In such a temporally gated cellular adaptation, transcriptionally active products of clock genes may play a supplementary role. Their recent detection in the endogenously oscillating nonmammalian pineal organ and, notably, also in the slave oscillator of the mammalian pineal gland underlines that the mammalian pineal gland will continue to serve as an excellent model system to understand mechanisms of biological timing.

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice

In rodents, the nocturnal rise and fall of arylalkylamine N‐acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)‐inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP‐response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin‐deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin‐deficient C57BL mouse and the melatonin‐proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin‐deficient and that this melatonin‐deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.

Rhythms in clock proteins in the mouse pars tuberalis depend on MT1 melatonin receptor signalling

Melatonin provides a rhythmic neuroendocrine output, driven by a central circadian clock that encodes information about phase and length of the night. In the hypophyseal pars tuberalis (PT), melatonin is crucial for rhythmic expression of the clock genes mPer1 and mCry1, and melatonin acting in the PT influences prolactin secretion from the pars distalis. To examine further the possibility of a circadian clockwork functioning in the PT, and the impact of melatonin on this tissue, we assessed circadian clock proteins by immunohistochemistry and compared the diurnal expression in the PT of wild type (WT), and MT1 melatonin receptor‐deficient (MT1–/–) mice. While in the PT of WT mice mPER1, mPER2, and mCRY1 showed a pronounced rhythm, mCRY2, CLOCK, and BMAL1 were constitutively present. Despite reported differences in maximal levels and timing of mCry1, mPer1, and mPer2 RNAs, the corresponding protein levels peaked simultaneously during late day, suggesting a codependency for their stabilization and/or nuclear entry. MT1–/– mice had reduced levels of mPER1, mCRY1, CLOCK and BMAL1, consistent with the earlier reported reduction in mRNA expression of these clock genes. Surprisingly, mPER2‐immunoreaction was constitutively low, although mPer2 was rhythmically expressed in the PT of MT1–/– mice. This suggests that mPER2 is degraded due to the reduced levels of its stabilizing interaction partners mPER1 and mCRY1. The results show that melatonin, acting through the MT1, determines availability of the circadian proteins mPER1, mPER2 and mCRY1 and thus plays a crucial role in regulating rhythmicity in PT cells.

Analysis of cell signalling in the rodent pineal gland deciphers regulators of dynamic transcription in neural/endocrine cells*

In neurons, a temporally restricted expression of cAMP‐inducible genes is part of many developmental and adaptive processes. To understand such dynamics, the neuroendocrine rodent pineal gland provides an excellent model system as it has a clearly defined input, the neurotransmitter norepinephrine, and a measurable output, the hormone melatonin. In this system, a regulatory scenario has been deciphered, wherein cAMP‐inducible genes are rapidly activated via the transcription factor phosphoCREB to induce transcriptional events necessary for an increase in hormone synthesis. However, among the activated genes is also the inhibitory transcription factor ICER. The increasing amount in ICER protein leads ultimately to the termination of mRNA accumulation of cAMP‐inducible genes, including the gene for the Aa‐nat that controls melatonin production. This shift in ratio of phosphoCREB and ICER levels that depends on the duration of stimulation can be interpreted as a self‐restriction of cellular responses in neurons and has also been demonstrated to interfere with cellular plasticity in many non‐neuronal systems.

Degeneration of the Cerebellum in Huntington's Disease (HD): Possible Relevance for the Clinical Picture and Potential Gateway to Pathological Mechanisms of the Disease Process

Huntingtons disease (HD) is a polyglutamine disease and characterized neuropathologically by degeneration of the striatum and select layers of the neo‐ and allocortex. In the present study, we performed a systematic investigation of the cerebellum in eight clinically diagnosed and genetically confirmed HD patients. The cerebellum of all HD patients showed a considerable atrophy, as well as a consistent loss of Purkinje cells and nerve cells of the fastigial, globose, emboliform and dentate nuclei. This pathology was obvious already in HD brains assigned Vonsattel grade 2 striatal atrophy and did not correlate with the extent and distribution of striatal atrophy. Therefore, our findings suggest (i) that the cerebellum degenerates early during HD and independently from the striatal atrophy and (ii) that the onset of the pathological process of HD is multifocal. Degeneration of the cerebellum might contribute significantly to poorly understood symptoms occurring in HD such as impaired rapid alternating movements and fine motor skills, dysarthria, ataxia and postural instability, gait and stance imbalance, broad‐based gait and stance, while the morphological alterations (ie ballooned neurons, torpedo‐like axonal inclusions) observed in the majority of surviving nerve cells may represent a gateway to the unknown mechanisms of the pathological process of HD.