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Pineal Gland and Chronobiology

• David C. Klein, PhD, Head, Section on Neuroendocrinology
• Steven L. Coon, PhD, Staff Scientist
• Joan L. Weller, BA, Senior Research Assistant
• Diego Bustos, PhD, Postdoctoral Fellow
• Sam Clokie, PhD, Postdoctoral Fellow
• Margaret Ochocinska, PhD, Postdoctoral Fellow
• Jiri Pavlicek, PhD, Postdoctoral Fellow
• Surajit Ganguly, PhD, Guest Researcher
• M.A.A. Namboodiri, PhD, Guest Researcher
• Harvey Pollard, MD, PhD, Guest Researcher
• Qiong Shi, PhD, Guest Researcher
• Martin Rath, Special Volunteer¹
• Louise Rovsing, Special Volunteer¹

We focus primarily on the pineal gland and regulation of the production of melatonin, which is the pineal hormone. Our work has broad implications for vertebrate biology and is of special interest to clinical scientists studying human diseases relating to circadian rhythms, including endocrine pathologies, sleep and mood disorders, and deficiencies in alertness. We also address the factors controlling remarkable global changes in gene expression that occur in the pineal gland on a 24-hour basis in addition to factors associated with establishment of the pineal phenotype.

The timezyme: arylalkylamine N-acetyltransferase

Pavlicek, Coon, Namboodiri, Pollard, Weller, Clokie, Shi, Ganguly, Klein; in collaboration with Aitken, Chemineau, Cole, Dawid, Dyda, Falcón, Gothilf, Hickman, Hwang, Iuvone, Jaffe, Koonin, Malpaux, Obsil, Sackett, Schuck, Szewczuk, Toyama, Zhen

A pivotal advance in our investigations into arylalkylamine N-acetyltransferase (AANAT), the second enzyme in the pathway for melatonin synthesis from serotonin, was our finding that the enzyme is critical to the control of the rhythm in melatonin synthesis. In all species examined to date, the large increase in melatonin synthesis at night causes an increase in the production of melatonin, which is a highly conserved feature of vertebrate physiology.

Transcriptional mechanisms. The transcriptional mechanisms that control expression of the AANAT gene include interactions of cyclic AMP–response elements in the promoter region; other response elements in the promoter, including the E-box and photoreceptor conserved elements, appear to determine the marked tissue-specific pattern of expression of the gene, which is limited to the pineal gland and retina. We determined that, in rodents, expression of AANAT rises about 100-fold at night; however, this feature of regulation is not highly conserved. We discovered little or no nocturnal increase in AANAT expression in ungulates and primates. Thus, transcriptional control mechanisms are not essential in regulating melatonin synthesis in all vertebrates.

Post-translational control. Sequence analysis revealed that all vertebrate AANATs contain C- and N-terminal sites for phosphorylation by cyclic AMP–dependent protein kinase. We determined that the sites control the stability and biological half-life of the enzyme. Phosphorylation of the sites controls binding of AANAT to 14-3-3 protein, which protects the enzyme from destruction; when the enzyme is released, it can be destroyed rapidly. The phosphorylation state of AANAT and binding to 14-3-3 appear to govern the balance between destruction and protection. In collaboration with Fred Dyda and Alison Hickman, we determined the molecular basis of this interaction by analyzing the crystal structure formed by AANAT and 14-3-3- proteins. Surajit Ganguly and Joan Weller, in collaboration with Benoit Malpaux and Philippe Chemineau, demonstrated that the interaction is critical for regulation of the enzyme in vivo (Ganguly et al., Proc Natl Acad Sci USA 2005;102:1222).In collaboration with Alastair Aitken and based on both mRNA and protein analysis, Qiong Shi used a comparative approach to analyze and establish the developmental appearance of each 14-3-3 isoform in the pineal gland. He found marked differences in the relative abundance of each isoform and in the binding and activation capacities of the 14-3-3 isoforms.

Recent research has focused on one of the phosphorylation sites of AANAT located in the C-terminal region. Joan Weller developed highly specific antisera to determine the phosphorylation state of the enzyme and revealed that the C-terminal site is phosphorylated at night and that the phosphorylation state of the enzyme decreases immediately after animals are exposed to light, most likely leading to destruction of the enzyme. Surajit Ganguly established that both the C- and N-terminal PKA sites are important for binding to 14-3-3 proteins. A remarkable on/off mechanism centered around the C-terminal PKA site swiches the enzyme from highly active to highly inactive. In collaboration with P. Michael Iuvone, we found that the same mechanism operates in the retina to control both the activity of AANAT and melatonin production. In the retina, light acts directly on photoreceptor cells to control cyclic AMP, thereby controlling AANAT. We suspect that light acts through a similar mechanism in photosensitive pinealocytes. In collaboration with Philip Cole, Larry Szewcuk, Yousang Hwang, and Weiping Zhen, we used novel synthetic approaches to examine the role of phosphorylation in controlling AANAT. We made AANATs that contain non-hydrolyzable phosphate at the N-terminal PKA site, injected them into cells, and measured their biological half-life by using antisera. The results indicate that the non-hydrolyzable phosphate at the N-terminal PKA site prevents destruction of AANAT. We have also pursued new approaches to the development of AANAT inihibitors in order to identify lead compounds for drug development.

Steven Coon established that, in the monkey pineal, large changes occur in the abundance of AANAT protein and activity, even though AANAT mRNA, i.e., gene expression, does not change, underscoring the importance of post-translational regulatory mechanisms in primate biology.

• Humphries A, Wells T, Baler R, Klein DC, Carter DA. Rodent Aanat: intronic E-box sequences control tissue specificity but not rhythmic expression in the pineal gland. Mol Cell Endocrinol 2007;270:43-49.
• Hwang Y, Ganguly S, Ho AK, Klein DC, Cole PA. Enzymatic and cellular study of a serotonin N-acetyltransferase phosphopantetheine-based prodrug. Bioorg Med Chem 2007;15:2147-2155.
• Klein DC. Arylalkylamine N-acetyltransferase: “the Timezyme.” J Biol Chem 2007;282:4233-4237.
• Pozdeyev N, Taylor C, Haque R, Chaurasia SS, Visser A, Thazyeen A, Du Y, Fu H, Weller J, Klein DC, Iuvone PM. Photic regulation of arylalkylamine N-acetyltransferase binding to 14-3-3 proteins in retinal photoreceptor cells. J Neurosci 2006;26:9153-9161.
• Zilberman-Peled B, Appelbaum L, Vallone D, Foulkes NS, Anava S, Anzulovich A, Coon SL, Klein DC, Falcón J, Ron B, Gothilf Y. Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream. J Neuroendocrinol 2007;19:46-53.

Molecular evolution of AANAT

Pavlicek, Coon, Ganguly, Weller, Klein; in collaboration with Sackett, Hassan, Gaildrat, Falcón

In a line of research spearheaded by Steven Coon and subsequently led by Jiri Pavlicek, Pavlicek focused on an important element of the AANAT molecule, a floppy loop described as Loop 1, which forms a portion of the arylamine-binding pocket. Loop 1 has undergone notable changes during the course of the enzyme’s evolution. With Daniel Sackett and Sergio Hassan, Pavlicek evaluated the importance of specific residues in the floppy loop to determine their contribution to the greater than 1,000-fold increase in specific activity of AANAT that occurred during chordate evolution. With Pascaline Gaildrat and Jack Falcón, he analyzed a primitive form of AANAT in the cephalochorate Amphioxus. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL65, which is responsible for increased enzyme activity. Removal of the CPL tripeptide markedly reduced activity. Moreover, increasing the local flexibility of the tripeptide region by P64G and P64A mutations had a counterintuitive effect, reducing both activity and the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that the mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis without becoming immobilized by intramolecular interactions.

• Pavlicek J, Coon SL, Ganguly S, Weller JL, Hassan SA, Sackett DL, Klein DC. Evidence that proline focuses movement of the floppy loop of arylalkylamine N-acetyltransferase (EC 2.3.1.87). J Biol Chem 2008;283:14552-14558.

Pineal/retinal evolution, AANAT, and the formation of conjugates of arylalkylamine and retinaldehyde

Klein, Coon; in collaboration with Kirk, Falcón, Iyer, Koonin

According to our theory of the evolution of the pineal gland, both the pineal gland and retina evolved from the same primitive photoreceptor cell after that cell acquired AANAT and HIOMT, the enzymes required to make melatonin. We believe that, originally, the two enzymes were important only in the detoxification of arylalkylamines, which can be dangerous in all tissues because of the amine’s reactivity and the aldehyde that arises from the amine’s oxidation. Detoxification would have led to the production of melatonin and eventually to the development of the rhythm in melatonin as a day/night signal. However, our theory proposes that high levels of melatonin would have been destructive to the primitive photorecetor by requiring high levels of the melatonin precursor serotonin; serotonin would have been especially toxic to photoreceptor function because it can react with and thus remove retinaldehyde, the key photodetection molecule. A2S, the theoretical product formed by this reaction, would contain two molecules of retinaldehyde and one molelcule of serotonin; homologous compounds would be formed from other arylalkylamines. Such products belong to a larger family of N-bis-retinyl compounds, including bis-retinal-ethanolamine (A2E), which is thought to be toxic to the retina through effects of the retinal side chains. The formation of A2S in the primitive photoreceptor would reduce photosensitivity by removing retinal; in addition, the product would be toxic. Segregation of these processes to the pinealocyte and retinal photoreceptor would have resolved the conflict, permitting melatonin production and photodetection to evolve and improve.

In collaboratin with Ken Kirk, we have synthesized A2S and related compounds and are monitoring their formation in order to test the hypothesis that their formation in the retina is a function of AANAT activity. In addition, we are examining whether the compounds might play a role in human retinal disease, specifically macular degeneration.

• Coon SL, Klein DC. Evolution of arylalkylamine N-acetyltransferase: emergence and divergence. Mol Cell Endocrinol 2006;252:2-10.
• Klein DC. Evolution of the vertebrate pineal gland: the AANAT hypothesis. Chronobiol Int 2006;23:5-20.

Global analysis of gene expression

Coon, Ganguly, Weller, Rath, Klein; in collaboration with Dawid, Gaildrat, Gothilf, Morin, Kim, Toyama, Baler, Blackshaw, Carter, Hogenesch, Humphries, Møller, Munson, Sugden

We have initiated several projects aimed at (1) obtaining a global picture of differences in gene expression that occur on a night/day basis and (2) identifying genes that are highly enriched in the pineal gland. We began our work by analyzing a relatively small number of genes. Now, with microarrys, we can examine tens of thousands of genes and ESTs (expressed sequence tags). Together with Igor Dawid, we faciliated the NICHD-sponsered commercialization of an Affymetrix chip for analysis of gene expression in zebrafish and Xenopus . In collaboration with Dawid, Yoav Gothilf, and Reiko Toyama, we are characterizing pineal gene expression in the zebrafish and other species as a function of time of day and of development. We have already identified a set of genes highly expressed in the pineal gland. Steven Coon and Michael Bailey are coordinating the work, which involves a collaboration with Peter Munson, David Carter, and Ruben Baler and is providing a basis for analysis of the control of gene expression in second-messenger cascades. The work has triggered several investigations that analyze the expression of genes that previously had not appeared in the pineal literature, including those encoding methionine adenosyl transferase, MAP kinase phosphatase-1, PepT1, phoshodiesterase 4D2, dopamine D4R receptor, and a subunit of the IgE receptor.

The group of pineal-specific genes expressed in the pineal gland of several vertebartes includes genes known to be associated with melatonin production and visual signal transduction. In addition, we have identified many genes (over 600), new to the pineal literature, that exhibit more than 2-fold night/day differences in expression and another set of genes that are exceptionally highly expressed in the pineal gland as compared with other tissues. This work is leading to a rapid increase in knowledge of the biochemical profile, conserved across species, of the pineal gland. It is also pointing to new transcriptional pathways controlled by previously unrecognized transcription factors; by analyzing these transcription factors and the promoters of genes that are either upregulated at night or highly expressed in the pineal gland, we will be able to construct a regulatory network that describes the cascade of transcription factors underlying the control of pineal gene expression. We have also identified potentially new and important pathways involved in cell-cell communication and signal transduction in the pineal gland; expression of some genes points to new functions for the pineal gland.

Using quantitative RT-PCR, our collaborator David Sugden has confirmed the results of our microarray studies and has provided a more detailed and quantitative description of the 24-hour patterns of gene expression, revealing that the patterns are not all similar and in phase but rather exhibit novel features. Work by Morten Møller and Martin Rath, using radiochemical in situ hybridization histology, has also confirmed the 24-hour patterns of rhythmic gene expression as well as the marked tissue specificity of genes of interest.

Our results permit a full-scale biochemical and physiological analysis of the control of genes in the pineal gland. Results from organ culture work revealed that the vast majority of genes that exhibit night/day changes in expression are also regulated by norepinephrine acting through a cyclic AMP mechanism that may involve cyclic AMP–response element binding sites in regulated genes and less specific epigenetic mechanisms.

Regulation of S-adenosyl methionine synthesis

Kim,² Klein; in collaboration with Charlton, Møller, Cepko, Blackshaw, Zhao

S-adenosylmethionine is the co-factor of the last enzyme in the melatonin biosynthesic pathway. Jong-So Kim determined that the expression of the enzyme that catalyzes the synthesis of this co-factor in the pineal gland—methionine adenosyl transferase 2a (MAT)—increases at night and is accompanied by increased enzyme activity and enzyme protein. The molecular basis of the increase involves neural stimulation of the pineal gland by norepinephrine, which results in the elevation of cyclic AMP (Kim et al., J Biol Chem 2005;280:677).

The nocturnal rise in S-adenosylmethine synthesis is obviously linked to the increased requirement for this methyl donor. While the literature has yet to describe regulation of the synthesis of S-adenosylmethionine by neural mechanisms, the co-factor does play a central role in the synthesis and metabolism of many transmitters (catecholamines, indoles, histamine, and so forth). Accordingly, evidence from the pineal gland indicating that MAT 2a activity can be regulated by a neural circuit via a cyclic AMP mechanism suggests that activityof MAT 2a might be regulated by transmitters in other brain regions and that the levels of S-adenosyl methionine might be controlled through pharmacological manipulation of MAT 2a expression. Our work provides evidence of strong and rapid control of melatonin production downstream of the enzyme commonly thought to be the only regulated element of the serotonin-to-melatonin pathway. Accordingly, it appears that adrenergic cyclic AMP control of melatonin production from serotonin involves at least two targets: AANAT and S-adenosymethionine production.

Regulation of phosphodiesterase 4B2

Kim,² Klein; in collaboration with Baler, Carter, Munson, Møller

The microarray analysis of the pineal suggested that expression of the gene encoding a cyclic AMP–selective phosphodiesterase, termed PDE4B2 , is elevated at night. Jong-So Kim found that the level of mRNA encoding this isoform is 5 times higher at night than during the day and that expression in the pineal gland is higher than in other tissues, as confirmed by Morten Møller with in situ hybridization. Kim found that the increase in PDE4B2 mRNA is associated with increased protein and activity, which influence the accumulation of cyclic AMP. Expression of the PDE4B2 gene is thus controlled by the same neural pathway that regulates AANAT and MAT; in addition, cyclic AMP controls expression of PDE4B2 , representing a negative feedback mechanism. Our work provides the first demonstration of a negative feedback mechanism involving cyclic AMP destruction in the pineal gland. It also represents a means of telling time—an internal interval timer.

• Kim JS, Bailey MJ, Ho AK, Møller M, Gaildrat P, Klein DC. Daily rhythm in pineal phosphodiesterase activity reflects adrenergic/cAMP induction of the PDE4B2 variant. Endocrinology 2007;148:1475-1485.

Induction of the oligopeptide transporter PEPT1

Gaildrat,³ Klein; in collaboration with Ganapathy, Inui

One outcome of our microarray studies was the finding that expression of the oligopeptide transporter (PEPT1) gene increases markedly at night (about 100-fold). In collaboration with Vadivel Ganapathy, Pascaline Gaildrat discovered that expression of the gene at night in the pineal gland truncates the gene. Regulation reflects neural cyclic AMP activation of the pineal gland and large changes in both the mRNA encoding the protein and the protein itself. PEPT1 is relatively unstable and disappears rapidly. Gaildrat also found that the expression of PEPT1 is highly restricted to the pineal gland and identified a section of the gene that appears to be responsible for the restricted pattern of tissue distribution and for the night/day pattern of expression, namely, cyclic AMP–response elements and putative sites for the binding of CRX/OTX transcription factors, features shared with the AANAT gene and located in an internal promoter (Gaildrat et al., J Biol Chem 2005;280:16851). It appears that we have uncovered a mechanism that selectively provides the pinealocyte with a membrane-linked function and that may involve a regulatory role of the PEPT1 product in melatonin production.

NeuroD-1, CRX, OTX, Pax4, and Pax6 expression in the pineal gland

Bustos, Coon, Ochocinska, Rath, Klein; in collaboration with Carter, Munson, Baler, Møller, Muñoz

Through our microarray analyses and as confirmed with independent techniques, we revealed that the pineal gland expresses high levels of several transcription factors, including NeuroD1, CRX OTX, Pax4, and Pax6, that play a role in the development of the eye and other tissues. With collaborators under the direction of Morten Møller, we undertook a detailed description of the developmental appearance of these transcripts (Figure 4.9); the long-term plan is to determine the transcripts’ role in developmental and adult expression of genes in the pineal gland.

Otx2 is a vertebrate homeobox gene essential for the development of rostral brain regions. It also appears to play a role in the development of retinal photoreceptor cells and pinealocytes. Work led by Martin Rath revealed the temporal expression pattern of Otx2 in the rat brain, with exceptionally strong expression in the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, despite a distinct lag in time of onset. We identified the Otx2 protein in pineal extracts and found it to be localized in pinealocytes. Total pineal Otx2 mRNA neither showed night/day variation nor was influenced by the removal of sympathetic input, suggesting that the level of Otx2 mRNA is independent of the photoneural input to the gland. Our observations are consistent with the view that pineal expression of Otx2 is required for development and plays a role in the adult by controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis.

NeuroD1/BETA2, a member of the bHLH transcription factor family, influences the fate of specific neuronal, endocrine, and retinal cells. In collaboration with Estela Muñoz, we found that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo, at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during night and day and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identified 127 transcripts that are more than 2-fold downregulated and 16 that are more than 2-fold upregulated. According to quantitative RT-PCR, the most dramatically downregulated gene is KIF5C (kinesin-family member 5C) (about 100-fold); the most dramatically upregulated gene is that encoding glutamic acid decarboxylase 1 (about 4-fold). Other affected transcripts encode proteins involved in differentiation, development, signal transduction, and trafficking. To elucidate the role of NeuroD1 in the rodent pinealocyte, Margaret Ochocinska is using a Cre-Lox conditional knockout stategy to block expression of NeuroD1 in both the pineal gland and retina.

Studies by Martin Rath and other laboratory members revealed that Pax4 is strongly expressed in the rat pineal gland and retina. The levels of pineal Pax4 transcripts are low in the fetus and increase postnatally; Pax6, on the other hand, exhibits an inverse pattern of expression and is more strongly expressed in the fetus. In the adult, the abundance of Pax4 mRNA exhibits a diurnal rhythm in the pineal gland, with maximal levels occurring late during the light period. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA. At night, the release of norepinephrine by sympathetic innervation adrenergically stimulates the pineal gland. We found that treatment with adrenergic agonists suppresses pineal Pax4 expression both in vivo and in vitro. As indicated by the observation that treatment with a cyclic AMP reduces pineal Pax4 mRNA levels, suppression of pineal Pax4 appears to be mediated by cyclic AMP, a second messenger of norepinephrine in the pineal gland. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by the sympathetic neural pathway that, in turn, controls pineal function via an adrenergic-cyclic AMP mechanism. The daily changes in Pax4 expression may influence gene expression in the pineal gland.

• Muñoz E, Bailey MJ, Rath MF, Shi Q, Morin F, Coon SL, Møller M, Klein DC. NeuroD1: developmental expression and regulated genes in the rodent pineal gland. J Neurochem 2007;102:887-899.
• Rath M, Bailey MJ, Kim J-S, Ho AK, Gaildrat P, Coon SL, Møller M, Klein DC. Developmental and diurnal dynamics of Pax4 expression in the mammalian pineal gland: nocturnal down-regulation is mediated by adrenergic-cyclic AMP signaling. Endocrinology 2008; [E-pub ahead of print].
• Rath MF, Morin F, Shi Q, Klein DC, Møller M. Ontogenetic expression of the Otx2 and Crx homeobox genes in the retina of the rat. Exp Eye Res 2007;85:65-73.
• Rath MF, Muñoz E, Ganguly S, Morin F, Shi Q, Klein DC, Møller M. Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland. J Neurochem 2006;97:556-566.

Studies on the pineal-immune link

Rath, Ganguly, Klein; in collaboration with Møller

Analysis of gene expression in the pineal gland also revealed a large daily rhythm in the expression of the gene encoding a subunit of the IgE receptor. We confirmed expression of the gene with several techniques and established that such expression reflects adrenergic/cyclic AMP signaling in pinealocytes. Our current work aims to determine the functional role played by such expression in physiology and to examine the hypothesis that the pinealocyte is a sentinel that modulates the immune response on a circadian schedule. In addition, we found that perivascular phagocytes in the vertebrate pineal gland serve as antigen-presenting cells. These findings, together with evidence of expression of the peptide transporter PEPT1, provide the initial molecular building blocks of a system mediating the relationship between antigens and the pineal gland. Microarray technology studies have revealed that the pineal gland expresses a set of genes known to be associated with immune/inflammation responses in other tissues. Accordingly, our goal is to determine how the pineal gland may be involved in these responses.

• Ganguly S, Grodzki C, Sugden D, Møller M, Odom S, Gaildrat P, Gery I, Siraganian RP, Rivera J, Klein DC. Neural adrenergic-cyclic AMP regulation of the IgE receptor alpha-subunit expression in the mammalian pinealocyte: a neuroendocrine-immune response link? J Biol Chem 2007;282:32758-32764.
• Møller M, Rath MF, Klein DC. The perivascular phagocyte of the mouse pineal gland: an antigen-presenting cell. Chronobiol Int 2006;23:393-401.

Regulation of dopamine D4R receptor: cAMP + T3 “AND” gate regulation

Kim,² Bailey, Klitten,⁴ Klein; in collaboration with Møller, Celi

Preliminary cDNA microarray studies suggested that D4R expression increases at night. Morten Møller confirmed such expression by in situ analysis and Jong-So Kim and Michael Bailey by Northern blot and quantitative PCR. Other than trace levels, we could not find expression of other dopamine receptors in the pineal gland. D4R expression in the pineal gland is many-fold higher than in most other tissues, suggesting that the D4R receptor plays an important role in pineal signal transduction, perhaps as an inhibitor of adrenergic stimulation of adenylate cyclase. Regulation of the expression of this gene is unusual; in the case of other genes studied to date, including AANAT, MAT2, MKP-1, Type II deiodinase, ICER, and FRA-2, the nocturnal increase in mRNA can be reproduced in organ culture by treatment with either norepinephrine or a cyclic AMP agonist. However, in the case of the D4R receptor, D4R mRNA does not increase following treatment with either norepinephrine or a cyclic AMP agonist. Rather, co-treatment with the thyroid hormone triiodothyronine (T3) and either norepinphrine or cyclic AMP is required to increase D4R mRNA, thereby establishing that expression of the D4R receptor is regulated by an “AND” gate operated by cAMP and T3 and providing the first evidence of a role for the thyroid gland in regulating a specific gene in the pineal gland. This finding is consistent with the knowledge of an increase in the capacity to convert thyroxine (T4) to T3 through induction of type II deiodinase at night in the pineal gland. In indicating that melatonin synthesis might be a function of thyroid status, our study has broad implications for dopamine signal transduction in other tissues.

As part of a broader effort to understand the similarities between the retina and pineal gland, Laura Klitten led a semiquantitative in situ hybridization study that revealed prominent night/day variation in Drd4 expression in the retina of the rat, with a peak during the night. Drd4 expression is seen in all retinal layers, but the nocturnal increase is confined to the photoreceptors. Removal of sympathetic input to the eye does not affect retinal Drd4 expression, but triiodothyronine treatment induces Drd4 expression in the photoreceptors. In a developmental series, we found that expression of Drd4 is restricted to postnatal stages with a peak at postnatal day 12. The high Drd4 expression in the rat retinal photoreceptors during the night supports physiological and pharmacologic evidence that the Drd4 receptor is involved in the dopaminergic inhibition of melatonin synthesis upon light stimulation. The sharp increase of Drd4 expression at a specific postnatal time suggests that dopamine is also involved in retinal development.

• Klitten LL, Rath MF, Coon SL, Kim JS, Klein DC, Møller M. Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina. Exp Eye Res 2008; [E-pub ahead of print].

Role of islet antigen 2 and islet antigen 2beta in circadian biology

Klein; in collaboration with Notkins, Schnerman, Constance, Weaver, Piggins

We discovered that the elimination of two proteins, islet antigen 2 and islet antigen 2beta, removes daily rhythms in activity, cardiovascular function, and temperature. The proteins are found in secretory vesicles and are best recognized by their association with type 1 diabetes; autoantibodies against the proteins are a predictor of the disease. Our studies suggest that the absence of daily rhythms may be attributable to a combination of effects on neurotransmission and downstream effects.

¹ Student from the University of Copenhagen
² Jong-So Kim, PhD, former Postdoctoral Fellow
³ Pascaline Gaildrat, PhD, former Guest Researcher
⁴ Laura Klitten, former Student Volunteer from the University of Copenhagen

Collaborators

• Alastair Aitken, PhD, University of Edinburgh, Edinburgh, UK
• Michael J. Bailey, PhD, Texas A&M University, College Station, TX
• Ruben Baler, PhD, Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD
• Stefano Bertuzzi, MD, Mammalian Development Section, NINDS, Bethesda, MD
• Seth Blackshaw, PhD, The Johns Hopkins University, Baltimore, MD
• David Carter, PhD, University of Wales, Cardiff, UK
• Francesco S. Celi, MD, Clinical Endocrinology Branch, NIDDK, Bethesda, MD
• Connie Cepko, PhD, Harvard University, Cambridge, MA
• Clivel Charlton, PhD, Florida A&M University, Tallahassee, FL
• Philippe Chemineau, PhD, Institut National de la Recherche Agronomique, Nouzilly, France
• Constance L. Chik, MD, University of Alberta, Edmonton, Canada
• Philip Cole, MD, PhD, The Johns Hopkins University, Baltimore, MD
• Cara M. Constance, PhD, College of the Holy Cross, Worcester, MA
• Igor Dawid, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
• Fred Dyda, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
• Jack Falcón, PhD, Observeratoire Oceanologique/CNRS, Banyuls, France
• Takaishi Furukawa, PhD, Osaka Bioscience Institute, Osaka, Japan
• Pascaline Gaildrat, PhD, Faculté de Médecin, Centre Hospitalier Universitaire, Rouen, France
• Vadivel Ganapathy, PhD, Medical College of Georgia, Augusta, GA
• Sandra Goebbels, PhD, Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany
• Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
• Sergio A. Hassan, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
• Alison Hickman, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
• Anthony Ho, PhD, University of Alberta, Edmonton, Canada
• John Hogenesch, PhD, Genome Institute of Novartis Foundation, San Diego, CA
• Ann Humphries, PhD, University of Wales, Cardiff, UK
• Yousang Hwang, PhD, The Johns Hopkins University, Baltimore, MD
• Ken-Ichi Inui, PhD, Kyoto University Hospital, Kyoto, Japan
• P. Michael Iuvone, PhD, Emory University School of Medicine, Atlanta, GA
• Lakshminarayan M. Iyer, PhD, Computational Biology Branch, NCBL, Bethesda, MD
• Howard Jaffe, PhD, Laboratory of Neurochemistry, NINDS, Bethesda, MD
• Jong-So Kim, PhD, Pohang University of Science and Technology, Pohang, South Korea
• Ken Kirk, PhD, Laboratory of Chemistry, NIDDK, Bethesda, MD
• Eugene V. Koonin, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
• Benoit Malpaux, PhD, Institut National de la Recherche Agronomique, Nouzilly, France
• Sanford Markey, PhD, Laboratory of Neurotoxicology, NIMH, Bethesda, MD
• Morten Møller, PhD, Panum Institutet, Københavns Universitet, Copenhagen, Denmark
• Fabrice Morin, PhD, Université de Rouen, Mont-Saint-Aignan, France
• Estela Muñoz, PhD, Instituto de Histología y Embriología, Universidad Nacional de Cuyo, Mendoza, Argentina
• Peter Munson, PhD, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
• Abner Notkins, MD, Oral Infection and Immunity Branch, NIDCR, Bethesda, MD
• Tomas Obsil, PhD, Charles University, Prague, Czech Republic
• Hugh Piggins, PhD, Manchester University, Manchester, UK
• Zoila Rangel, MS, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
• Daniel Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
• Jürgen Schnermann, MD, Kidney Disease Branch, NIDDK, Bethesda, MD
• Peter W. Schuck, PhD, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
• Peter J. Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
• David Sugden, PhD, Kings College, University of London, London, UK
• Larry Szewczuk, PhD, The Johns Hopkins University, Baltimore, Maryland
• Reiko Toyama, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
• David Weaver, PhD, University of Massachusetts, Worcester, MA
• Pin-Xian Xu, PhD, Mount Sinai School of Medicine, New York, NY
• Al Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD
• Weiping Zhen, PhD, The Johns Hopkins University, Baltimore, MD

For further information, contact klein@helix.nih.gov or visit http://sne.nichd.nih.gov.

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