Aa-nat Gene Expression Research Articles

Photoperiodic adaptation of aanat and clock gene expression in seasonal populations of Daphnia pulex

ABSTRACT Changes in day-length entrain the endogenous clock of organisms leading to complex responses to photoperiod. In long-lived organisms experiencing several seasons this response of the clock to photoperiod is phenotypically plastic. However, short-lived organisms often experience a single season without pronounced changes in day-length. For those, a plastic response of the clock to different seasons would not necessarily be adaptive. In aquatic ecosystems, zooplankton species like Daphnia live only for some weeks, i.e. one week up to ca. two months. However, they often show a succession of clones that are seasonally adapted to environmental changes. Here, we found that 16 Daphnia clones per each of three seasons ( = 48 clones) from the same pond and year differed in clock gene expression with a homogenous gene expression pattern in ephippia-hatched spring clones and a bimodal expression pattern in summer and autumn populations indicating an ongoing adaptation process. We clearly demonstrate that spring clones were adapted to a short, and summer clones to a long photoperiod. Furthermore, we found that gene expression of the melatonin-synthesis enzyme AANAT was always lowest in summer clones. In the Anthropocene, Daphnia’s clock might be disturbed by light-pollution and global warming. Since Daphnia is a key-organism in trophic carbon transfer, a disruption of its clock rhythm would be devastating for the stability of freshwater ecosystems. Our results are an important step in understanding the adaptation of Daphnia’s clock to environmental changes.

CircRNA-WNK2 Acts as a ceRNA for miR-328a-3p to Promote AANAT Expression in the Male Rat Pineal Gland.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed with a daily rhythm in the rat pineal gland, are associated with the regulation of melatonin secretion and other biological functions. However, the mechanisms of these molecules in the rat pineal gland are not yet fully understood. In this study, we found that circR-WNK2 was highly expressed at night, which may be involved in the regulation of melatonin secretion through the competitive endogenous RNA (ceRNA) mechanism. By dual luciferase reporter, RNA pull-down, and fluorescence in situ hybridization (FISH) assays, we found that miR-328a-3p can target circR-WNK2 and the Aa-nat mRNA 3'UTR. Transfection experiments indicated that circR-WNK2 could competitively bind to miR-328a-3p, reduce miR-328a-3p expression, and promote Aa-nat gene expression and melatonin secretion. And by constructing a superior cervical ganglionectomy (SCGx) rat model, we found that ncRNAs expression in the pineal gland was regulated by signals from the suprachiasmatic nucleus. This finding supports the hypothesis that these noncoding RNAs may interact to shape the circadian rhythm through transcriptional processing in melatonin synthesis.

Season-dependent effects of photoperiod and temperature on circadian rhythm of arylalkylamine N-acetyltransferase2 gene expression in pineal organ of an air-breathing catfish, Clarias gariepinus

Arylalkylamine N-acetyltransferase (AANAT) activity, aanat gene expression and melatonin production have been reported to exhibit prominent circadian rhythm in the pineal organ of most species of fish. Three types of aanat genes are expressed in fish, but the fish pineal organ predominantly expresses aanat2 gene. Increase and decrease in daylength is invariably associated with increase and decrease in temperature, respectively. But so far no attempt has been made to delineate the role of photoperiod and temperature in regulation of the circadian rhythm of aanat2 gene expression in the pineal organ of any fish with special reference to seasons. Therefore, we studied effects of various lighting regimes (12L-12D, 16L-8D, 8L-16D, LL and DD) at a constant temperature (25°C) and effects of different temperatures (15°, 25° and 35°C) under a common photoperiod 12L-12D on circadian rhythm of aanat2 gene expression in the pineal organ of Clarias gariepinus during summer and winter seasons. Aanat2 gene expression in fish pineal organ was studied by measuring aanat2 mRNA levels using Real-Time PCR. Our findings indicate that the pineal organ of C. gariepinus exhibits a prominent circadian rhythm of aanat2 gene expression irrespective of photoperiods, temperatures and seasons, and the circadian rhythm of aanat2 gene expression responds differently to different photoperiods and temperatures in a season-dependent manner. Existence of circadian rhythm of aanat2 gene expression in pineal organs maintained in vitro under 12L-12D and DD conditions as well as a free running rhythm of the gene expression in pineal organ of the fish maintained under LL and DD conditions suggest that the fish pineal organ possesses an endogenous circadian oscillator, which is entrained by light-dark cycle.

Melatonin synthesis follows a daily cycle in Daphnia

In freshwater systems, Daphnia has been demonstrated to show adaptive responses following the light-dark cycle. The adjustment of these responses to the change of day and night is probably transmitted via the hormone melatonin. The rate-limiting enzyme in melatonin synthesis is the arylalkylamine N-transferase (AANAT). We identified three genes coding for insect-like AANATs in Daphnia, of which we measured the gene expression in an ecologically relevant light-dark cycle. We demonstrated that Daphnia's insect-like AANAT gene expression oscillated in a daily manner, and that the highest peak of expression after the onset of darkness was followed by a peak of melatonin production at midnight. Moreover, we could show an oscillation of endogenous melatonin synthesis in Daphnia. In most organisms, melatonin synthesis is due to rhythmic expression of genes of the circadian clock, since transcription of aanats is directly linked to a circadian transcription factor. We could demonstrate that putative clock genes and insect-like AANAT genes of Daphnia were equally expressed. Therefore, we propose that melatonin synthesis is coupled to the expression of Daphnia clock genes, and that insect-like AANATs of crustaceans have a similar function as AANATs of vertebrates: The initiation of melatonin synthesis. In future studies with Daphnia, it will be necessary to take the time of day into account since melatonin concentrations might influence stress responses.

Homeobox genes and melatonin synthesis: regulatory roles of the cone-rod homeobox transcription factor in the rodent pineal gland.

Nocturnal synthesis of melatonin in the pineal gland is controlled by a circadian rhythm in arylalkylamine N-acetyltransferase (AANAT) enzyme activity. In the rodent, Aanat gene expression displays a marked circadian rhythm; release of norepinephrine in the gland at night causes a cAMP-based induction of Aanat transcription. However, additional transcriptional control mechanisms exist. Homeobox genes, which are generally known to encode transcription factors controlling developmental processes, are also expressed in the mature rodent pineal gland. Among these, the cone-rod homeobox (CRX) transcription factor is believed to control pineal-specific Aanat expression. Based on recent advances in our understanding of Crx in the rodent pineal gland, we here suggest that homeobox genes play a role in adult pineal physiology both by ensuring pineal-specific Aanat expression and by facilitating cAMP response element-based circadian melatonin production.

Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.

Season-Related Differences in the Biosynthetic Activity of the Neonatal Chicken Pineal Gland

Influence of the season of hatch on the functional characteristics of the pineal gland was examined in neonatal Hi-Line male chickens. The pineal glands from 2-day-old birds hatched in and winter, and kept from the day of hatch in artificial lighting conditions (12L:12D), were isolated under dim red light in the middle of the day or night. The pineal glands were analyzed to characterize their melatonin biosynthetic activity: (1) expression of the Arylalkylamine- N-acetyltransferase and Hydroxyindole-O-methyltransferase genes, encoding the final two enzymes of the melatonin biosynthesis pathway; (2) the activity of AA-NAT and HIOMT; and (3) the content of the main substrates of this pathway, tryptophan (TRY) and serotonin (5-HT). Daily changes in pineal AA-NAT activity were observed in chickens hatched in both seasons, with a more pronounced nocturnal increase in summer. In contrast, the level of Aa-nat gene expression, although exhibited the same nocturnal/diurnal pattern in both seasons, was much lower in the summer. The activity of HIOMT was season- and daytime-independent. In winter chickens the pineal content of 5-HT was low and stable, while in summer birds it was correlated with levels of AA-NAT activity and Aa-nat gene expression. TRY content was very high and exhibited neither daily nor seasonal changes. The pineal gland of newly hatched chickens kept in controlled 12L:12D conditions exhibits daily variations in melatonin biosynthetic activity influenced by the season. This suggests a maternal effect on the perinatal/postnatal development of the circadian clock residing in the chicken pineal gland.

Rodent Aanat: Intronic E-box sequences control tissue specificity but not rhythmic expression in the pineal gland

Arylalkylamine N-acetyltransferase (Aanat) is the penultimate enzyme in the serotonin– N-acetylserotonin–melatonin pathway. It is nearly exclusively expressed in the pineal gland and the retina. A marked rhythm of Aanat gene expression in the rat pineal is mediated by cyclic AMP response elements located in the promoter and first intron. Intron 1 also contains E-box elements, which mediate circadian gene expression in other cells. Here we examined whether these elements contribute to rhythmic Aanat expression in the pineal gland. This was done using transgenic rats carrying Aanat transgenes with mutant E-box elements. Circadian expression of Aanat transgenes was not altered by these mutations. However, these mutations enhanced ectopic expression establishing that the intronic Aanat E-box elements contribute to the gene's pineal specific expression. A similar role of the Aanat E-box has been reported in zebrafish, indicating that Aanat E-box mediated silencing is a conserved feature of vertebrate biology.

Role of calcium in the gating of isoproterenol‐induced arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals.

Pineal circadian clocks gate arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N- acetyltransferase ( aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague–Dawley rats kept under different light–dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding ‘time of year.’

Transcription factors may frame Aa-nat gene expression and melatonin synthesis at night in the Syrian hamster pineal gland.

Pineal melatonin synthesis is stimulated at night following an increase in arylalkylamine-N-acetyltransferase (AA-NAT) activity. Depending on the species, two mechanisms of enzyme activation have been described: a cAMP/phospho-cAMP response element-binding protein-dependent stimulation of Aa-nat gene transcription in the rat, presumed to occur in all rodents, or a posttranslational regulation of AA-NAT protein in ongulates. The present data obtained in the Syrian hamster indicate another route of AA-NAT regulation. Elevated nocturnal levels of Aa-nat mRNA were strongly suppressed following light exposure or adrenergic antagonist administration, demonstrating the involvement of norepinephrine in the stimulation of melatonin synthesis. However, administration of adrenergic agonists during the day did not increase Aa-nat mRNA unless a protein synthesis inhibitor was given during the previous night. This indicates that an inhibitory protein, synthesized at night, prevents melatonin synthesis during the day. By contrast, a protein synthesis inhibitor given at the beginning of the night markedly reduced Aa-nat mRNA, suggesting that a stimulatory protein (transcription factor?) is necessary for Aa-nat gene transcription at night. Noteworthy, hamsters raised in long photoperiod were responsive to adrenergic agonist injection only in the first hour after light onset, a response that may be important in this photoperiodic species in which the melatonin peak extends into the morning hours in a short photoperiod.

Syrian hamster and rat display developmental differences in the regulation of pineal arylalkylamine N-acetyltransferase.

In the Syrian hamster, the role of noradrenaline in the regulation of melatonin synthesis is less clear than in the rat. During pineal ontogenesis in the rat, noradrenaline is the major transmitter involved in the onset of melatonin synthesis and melatonin rhythm. We analysed the involvement of noradrenaline in the ontogenesis of melatonin synthesis in the Syrian hamster and compared it with that of the rat. We followed the developmental profile of melatonin content in parallel with those of mRNA expression and activity of AA-NAT, the melatonin rhythm-generating enzyme. In addition, we tested the effect of noradrenergic drugs at early steps of pineal ontogenesis. In the Syrian hamster, the night-time Aa-nat mRNA, first detected 3 days after birth, increases progressively up to a maximum reached at 30 days of age and then decreases significantly towards adulthood. The daytime level of Aa-nat mRNA remains always low. A significant day/night rhythm appears 10 days after birth, is maximal (200-fold nocturnal increase) 30 days after birth and decreases slowly towards adulthood. Ontogenesis of the AA-NAT activity rhythm is similar, although with a much lower amplitude of day/night variations (four-fold). The developmental pattern of melatonin content is similar to that of AA-NAT and could be correlated with the appearance of sympathetic innervation in the pineal gland. However, neither alpha- nor beta-adrenergic antagonists inhibit the night-time Aa-nat mRNA transcription in the 9-day-old Syrian hamster, in contrast to what is observed in the adult. For comparison, the beta-adrenergic antagonist propranolol inhibits Aa-nat gene expression in 2-day-old rat. These results show that both species are different in the regulation of the appearance of melatonin synthesis and that Syrian hamster is peculiar from birth in term of noradrenaline involvement in the activation of melatonin synthesis.

Accumulation of rat pineal serotonin N-acetyltransferase mRNA induced by pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide in vitro.

In mammals, pineal melatonin secretion is under the control of adrenergic and peptidergic inputs regulating serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase; AA-NAT) activity. In this study, the accumulation of AA-NAT mRNA induced by norepinephrine (NE) and peptides of the secretin superfamily (pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), growth hormone releasing factor (GRF), secretin) was investigated by a new quantitative reverse transcription-PCR (RT-PCR) assay. We demonstrated that PACAP was the most potent peptide to increase the expression of AA-NAT mRNA and to induce cAMP production in rat pinealocytes. VIP was also able to elevate the AA-NAT mRNA level and cAMP efflux in a dose-dependent manner; however, it was six- and threefold, respectively, less potent than PACAP. The maximal values of AA-NAT mRNA level after PACAP and VIP exposures were similar (523.1 +/- 52.5 amol to 640.7 +/- 68.8 amol vs 461.5 +/- 54.3 amol to 579.2 +/- 72.4 amol). These saturable peak values were approximately five- to eightfold less than that after NE (3.0 +/- 0.3 fmol to 3.6 +/- 0.4fmol). GRF and secretin were less potent than VIP in inducing AA-NAT gene expression and cAMP efflux. These data suggest that the peptides act mostly on VIP(1)/PACAP (VPAC(1)) receptors of pinealocytes with different affinity. The peak cAMP efflux always preceded the elevation of AA-NAT gene expression during the 3-h infusion of VIP or NE. The cAMP efflux had declined by the time of onset of maximal AA-NAT gene expression, but remained significantly higher than its basal values. Our data indicate that even a submaximal level of cAMP is sufficient for maintaining the maximal AA-NAT mRNA accumulation. These findings show that, in addition to NE, PACAP and VIP may have an important role in the regulation of AA-NAT mRNA levels in rat pinealocytes.

The Rat Arylalkylamine N-Acetyltransferase Gene Promoter: cAMP ACTIVATION VIA A cAMP-RESPONSIVE ELEMENT-CCAAT COMPLEX

A 10-100-fold rhythm in the activity of arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) controls the rhythm in melatonin synthesis in the pineal gland. In some mammals, including the rat, the high nocturnal level of AA-NAT activity is preceded by an approximately 100-fold increase in AA-NAT mRNA. The increase in AA-NAT mRNA is generated by norepinephrine acting through a cAMP mechanism. Indirect evidence has suggested that cAMP enhances AA-NAT gene expression by stimulating phosphorylation of a DNA-binding protein (cAMP-responsive element (CRE)-binding protein) bound to a CRE. The nature of the sites involved in cAMP activation was investigated in this report by analyzing the AA-NAT promoter. An approximately 3700-base pair fragment of the 5'-flanking region of the rat AA-NAT gene was isolated, and the major transcription start points were mapped. The results of deletion analysis and site-directed mutagenesis indicate that cAMP activation requires a CRE.CCAAT complex consisting of a near-perfect CRE and an inverted CCAAT box located within two helical turns.