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THE PINEAL GLAND AND
CHRONOBIOLOGY
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David C. Klein, PhD, Head, Section on Neuroendocrinology Steven
L. Coon, PhD, Staff Scientist Surajit Ganguly, PhD, Research Fellow Pascaline
Gaildrat, PhD, Postdoctoral Fellow Jong-So Kim, PhD, Postdoctoral Fellow Fabrice
Morin, PhD, Postdoctoral Fellow Estella
Munoz, PhD, Postdoctoral Fellow Qiong
Shi, PhD, Postdoctoral Fellow Joan L.
Weller, BA, Senior Research Assistant Jack
Falcón, PhD, Guest Researchera Anthony
Ho, PhD, Guest Researcherb M.A.A.
Namboodiri, PhD, Guest Researcherc Harvey Pollard, MD, PhD, Guest Researcherc |
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Arylalkylamine
N-acetyltransferase, the melatonin rhythm enzyme Ganguly, Coon, Namboodiri, Falcón, Ho,
Morin, Pollard, Weller, Klein; in collaboration with Baler, Bernard,
Chemineau, Chong, Cole, Dawid, Dyda, Gothilf, Hickman, Iuvone, Jaffe, Koonin,
Malpaux, Schomerus, Toyama, Zhen We pioneered investigations into
arylalkylamine N-acetyltransferase (AANAT), the first enzyme in melatonin
synthesis from serotonin (Figure 12.1). A major advance in our work was the
early identification of that enzyme as 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. Another major advance was the cloning of the gene encoding AANAT,
allowing significant progress in understanding the transcriptional and
translational control of the enzyme.
The regulation of the enzyme has two features:
the mechanisms that generate large daily changes in the enzyme and those that
control the selective expression of the AANAT-encoding gene both in the
pineal gland and, to a lesser and more variable degree, in the retina; the
gene is essentially silent in other tissues. Regulation of the daily rhythm
in expression of the enzyme occurs in some but not all vertebrates. In
rodents, birds, and some fish (e.g., pike), a 10- to 100-fold change occurs
in the abundance of mRNA encoding the enzyme. However, in primates and
ungulates, mRNA levels remain relatively similar during the day and night.
The reason appears to be that, in the first group, the requirement for new
mRNA synthesis provides a “security” system that prevents rapid
changes in melatonin production during the day. In addition, in the second
group, the availability of mRNA at the beginning of the night provides an
opportunity for melatonin production to increase immediately at night,
without a delay reflecting the time necessary to synthesize sufficient
amounts of mRNA to support protein synthesis. A group led by Ruben Baler discovered that the
daily increase in mRNA that occurs in the rat pineal gland reflects the
effect of cyclic AMP, which acts via cyclic AMP–response elements in
the AANAT gene. Marianne Bernard, Nelson Chong, and Mike Iuvone have found
that, in birds, cyclic AMP plays a lesser role; rather, expression of the
AANAT gene is turned on by the biological clock, which is also the case in
fish as indicated by the work of Yoav Gothilf and Jack Falcón. In both cases,
E-box elements in the AANAT gene mediate the effects of the biological clock.
Tissue-selective expression of the pineal
gland clock components appears to reflect a contribution from photoreceptor conserved
elements (PCE), which also control retinal-specific expression of genes. This
expression is a reflection of the common origin of the pinealocyte and
retinal photoreceptor from a primitive photoreceptor cell. Current studies by
Reiko Toyama and Igor Dawid in conjunction with Yoav Gothilf, together with
studies by Ruben Baler, have provided evidence that the PCE elements function
by mediating the effects of members of the CRX/OTX family of homeobox genes. In situ analysis,
performed by Fabrice Morin in collaboration with Morten Møller, of OTX2, a
member of this family, has revealed a high level of expression in the rat
pineal gland. The international group involving workers in Tel Aviv and NICHD
has identified a specific region in the zebrafish AANAT gene that confers
rhythmicity and tissue specificity. The region, as depicted in Figure 12.2,
is termed the Pineal-Restrictive Downstream Module (PRDM). It contains a
functional E-box (E) and three PCEs (P) and is of interest because it is
atypically located somewhat distantly downstream of the coding region of the
gene. As indicated above, transcriptional factors
controlling the expression of AANAT are important in some but not all
species. In contrast, regulation of the stability and activity of the enzyme
is controlled in all species by cyclic AMP, which causes phosphorylation of
the enzyme and leads to the immediate binding of the enzyme to the protein
14-3-3. Joan Weller has developed a set of highly specific antisera to
monitor phosphorylation of two cyclic AMP–dependent protein kinase
sites in the AANAT, T31, and S205. Using the antisera, she has established
that both sites are phosphorylated at night; further work by Suragit Ganguly
has made it clear that phosphorylation leads to binding of 14-3-3 proteins
and that phosphorylation at both sites is required to position AANAT
precisely in the 14-3-3 binding pocket so as to prevent destruction of the
enzyme and to activate it by enhancing the affinity for arylalkylamine
substrates. As outlined in Figure 12.3, a balance exists between the
destruction and protection of AANAT. Figure 12.3 does not show, however, that
the kinetics of the enzyme also change when AANAT is bound to 14-3-3. The
changes are thought to reflect structural changes in the conformation of a
floppy loop of protein that is part of the arylalkylamine binding site, as
revealed by structural studies performed in collaboration with Fred Dyda and
Allison Hickman. Recent studies with Philip Cole that used semisynthetic
AANATs have underscored the importance of phosphorylation in controlling the
degradation of AANAT and demonstrated that the substitution confers
stability. There are seven isoforms of 14-3-3 proteins; Qiong Shi has
initiated efforts to identify which isoform has the highest affinity of AANAT
and to determine the molecular basis of their specificity. More specifically,
Shi, working with Alastair Aitken and Suragjit Gangluly, is using selective
antisera and expression vectors to probe the interaction. The importance of
such post-translational regulatory mechanisms is clear from studies on the
monkey pineal AANAT performed by Steven Coon. Those studies have clearly
established that large changes occur in the abundance of AANAT protein and
activity and that AANAT mRNA, i.e., gene expression, does not change.
Accordingly, it appears that in primates the primary mechanism for regulation
of AANAT protein is post-translational.
Appelbaum L, Toyama R, Dawid IB, Chong NW, Chaurasia SS, Haque R, Gothilf Y, Zheng W, Zhang Z, Ganguly S, Weller JL, Klein
DC, Cole PA. Cellular stabilization of the melatonin rhythm enzyme induced by
nonhydrolyzable phosphonate incorporation. Nat Struct Biol 2003;10:1054-1057. Global
analysis of pineal, retinal, and brain gene expression Coon, Gaildrat, Ganguly, Morin, Kim,
Weller, Munoz, Klein; in collaboration with Baler, Blackshaw, Carter,
Hogenesch, Humphries, Møller, Munson We have initiated several projects aimed at
both obtaining a global picture of differences in gene expression that occur
on a night/day basis and identifying genes that are highly enriched in the
pineal gland. Use of microarrays has provided an opportunity to analyze tens
of thousands of genes. In addition to using commercially available
microarrays, we played a key role in establishing an NICHD/Affymetix
partnership, which resulted in the development of a zebrafish microarray that
is now in use in laboratories throughout the world and that we and our
collaborators are using to study developmental and rhythmic expression of
genes in the pineal gland, retina, and brain. Our efforts toward understanding pineal gene
expression have involved several species and have led to the identification
of a set of genes highly expressed in the pineal gland. Steven Coon is
coordinating the work, which involves most members of the section and a
collaborative effort with Peter Munson. The group of pineal-specific genes
expressed in the pineal gland of several vertebrates includes the well-known
genes that are associated with both melatonin production and visual signal
transduction. In addition, we have identified a number of genes that are new
to the pineal literature, leading to a rapid increase in knowledge of the
biochemical profile of the pineal gland conserved across species and of
features not found in all species. A striking observation is that large
rhythmic changes in gene expression in several hundred genes occur in the rat
pineal gland, but not in the primate pineal gland, suggesting a global
difference in the role of transcriptional control between primates and
rodents in the pineal gland. The microarray work is pointing to new
transcriptional pathways controlled by transcription factors that have not
been previously studied in the pineal gland. An example is the transcription
factor NeuroD, which is strongly enriched in the pineal gland and has been
known to exist in the cerebellum (see Figure 12.4). The existence of NeuroD
in the pineal gland as revealed by pineal microarray analysis has led Estella
Munoz to knock down the expression of the gene and study downstream effects.
Humphries A, Klein DC, Baler R, Carter DA.
cDNA array analysis of pineal gene expression reveals circadian rhythmicity
of the dominant negative helix-loop-helix protein-encoding gene, Id-1. J Neuroendocrinol 2002;14:101-108. Humphries A, Weller J, Klein DC, Baler R,
Carter DA. NGFI-B (Nurr77/Nr4a1) orphan nuclear receptor in rat pinealocytes:
circadian expression involves an adrenergic-cyclic AMP mechanism. J Neurochem 2004;91:946-955. Regulation
of S-adenosyl methionine synthesis Kim, Klein; in collaboration with
Charlton Our gene profiling efforts have focused on the
enzyme that produces the S-adenosylmethionine (SMA), methionine
adenosyltransferase (MAT). As the co-factor of the last enzyme in the
melatonin pathway, SAM is essential for melatonin synthesis. Work spearheaded
by Jong-So Kim and involving Clivel G. Charlton has revealed that the level
of MAT2A, one form of MAT, rises at night as a result of increased gene
expression. The increase is a response to neural stimulation of the pineal
gland by norepinephrine, which causes elevation of cyclic AMP. The increase
in SMA synthesis at night is obviously linked to the increased requirement
for this methyl donor. Regulation of the synthesis of SMA by neural
mechanisms has not been previously described, although SMA plays a central
role in the synthesis and metabolism of many transmitters (catecholamines,
indoles, histamine, and so forth). Accordingly, evidence from the pineal
gland that the activity of MAT 2a can be regulated by a neural circuit via a
cyclic AMP mechanism points to the possibility that activity of MAT 2a is
regulated by transmitters in other brain regions and that SMA levels are
controlled through pharmacological manipulation of MAT 2a expression. Kim JS, Coon SL, Blackshaw S, Charlton CG,
Klein DC. Neural regulation of a 24-hour rhythm in methionine adenosyl
transferase (MAT): beta-adrenergic receptor-mediated regulation of MAT
expression in the pineal gland. J Biol
Chem 2005, in press. Regulation
of acetyl coenzyme A synthesis Morin, Klein; in collaboration with
Benjamin In collaboration with William Benjamin and
Morten Møller, Fabrice Morin has studied ATP-dependent citrate lyase (ACL),
the enzyme that regulates the formation of acetyl coenzyme A. The work
clearly established that the pineal gland expresses much higher levels of the
enzyme relative to other tissues (see Figure 12.5) and that it represents a
major protein in the pineal gland (about 2 percent of total protein). Morin
obtained evidence of a physical interaction between ACL and AANAT, which
would bring the source of acetyl coenzyme A directly into contact with AANAT and
promote efficient ace-tylation of serotonin as well as melatonin production.
It appears possible that this association might be regulated by
phosphorylation and that it could provide an important element in the general
activation of the pineal gland in support of the nocturnal increase in
melatonin production. Morin recently developed methods to analyze acetyl
coenzyme A and found very rapid light-induced changes at night, pointing to a
new mechanism to control melatonin production.
Induction
of membrane protein Gaildrat, Klein; in collaboration with
Ganapathy, Inui One outcome of our microarray studies was the
finding that the expression of the gene encoding the oligopeptide transporter
(PEPT1) is markedly increased at night (about 100-fold). In collaboration
with Vadivel Ganapathy, Pascaline Gaildrat has found that the gene is
expressed at night in the pineal gland and that this expression produces a
truncated version of the intestinal gene product (see Figure 12.6).
Regulation reflects neural activation of the pineal gland by cyclic AMP,
which produces changes in both the mRNA encoding the protein and the protein
itself. These actions represent a unique mechanism of regulation of a
membrane protein; it is also unusual that the protein product, which is
associated with the membrane, is relatively unstable and disappears rapidly.
Moreover, Gaildrat discovered that the expression of the truncated gene
product is highly restricted to the pineal gland and identified the section
of the gene (an internal promoter) that is responsible for this pattern of
tissue distribution, for the night/day pattern of expression, and for the
truncated nature of the gene product. The internal promoter shares features
with the rat AANAT promoter, such as the presence of cyclic
AMP–response elements and putative sites for the binding of CRX/OTX
transcription factors. These results have uncovered a unique mechanism for
selectively providing the pinealocyte with a membrane-linked function that
may involve a regulatory role of the PEPT1 product that is linked to
melatonin production.
Metal
biology Klein, Shi, Gaildrat, Ganguly Enzymes involved in indole synthesis and
metabolism require metals. The concentration of divalent ions including
copper and zinc are governed by metallothionines, small proteins dedicated to
binding and buffering these ions. Qiong Shi discovered that expression of one
form of these proteins is governed by an adrenergic cyclic AMP mechanism in
the pineal gland. He determined that both the protein and mRNA encoding the
enzyme are under the control of this second messenger. These developments
point to broad integration of the biochemical function of the pineal gland
and demonstrate that, although large changes in the production of melatonin
are controlled by changes in AANAT activity, there is little doubt that other
factors contribute to the rhythm in melatonin. Formation
of conjugates of arylalkylamine and retinaldehyde Klein, Coon; in collaboration with Kirk According to our revolutionary 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. Originally, the enzymes were
thought to be important only in detoxification of arylalkylamines, which can
be dangerous in all tissues because of amine’s reactivity and that of
the aldehyde that arises from oxidation of the amine. Detoxification led to
the production of melatonin and eventually to the development of the rhythm
in melatonin as a day/night signal. However, the theory proposes that the
requirement for high levels of melatonin was destructive to the primitive
photoreceptor because it required high levels of serotonin, the melatonin
precursor, which was especially toxic to photoreceptor function; serotonin
could react with and remove retinaldehyde, the key photodetection molecule.
The theoretical product formed by the reaction would contain two molecules of
retinaldehyde and one molecule of serotonin (see Figure 12.7); homologous
compounds would be formed from other arylalkylamines. These products belong
to a larger family of N-bis-retinyl
compounds, including bis-retinal-ethanolamine, 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 retinaldehyde; in addition, the product would be toxic. Segregation
of the processes to the pinealocyte and retinal photoreceptor made it
possible for melatonin production and photodetection to evolve and improve.
Steve Coon and David Klein, working with Ken Kirk, have synthesized A2S and
related compounds by using LC/MS/MS to monitor their formation. They are
testing the hypothesis that the compounds’ formation in the retina is a
function of AANAT activity. They are also examining whether the compounds
might play a role in human retinal disease, specifically macular
degeneration. Iyer LM, Aravind L, Coon SL, Klein DC, Koonin
EV. Evolution of cell–cell signaling in animals: did late horizontal
gene transfer from bacteria have a role? Trends
Genet 2004;20:292-299. aCNRS,
Université Curie, bUniversity
of Alberta, Edmonton, cUniformed
Services University of the Health Sciences, Bethesda, MD COLLABORATORS Alastair Aitken, PhD, Ruben Baler, PhD, Laboratory of Cellular and Molecular
Regulation, NIMH, William B. Benjamin, PhD, SUNY, Stony Marianne
Bernard, PhD, Laboratoire de
Neurobiologie et Neuroendocrinologie Cellulaires, Université Poitiers, France Seth Blackshaw, PhD, David Carter, PhD, Clivel G. Charlton, PhD, Philippe
Chemineau, PhD, INRS, Nouzilly, France Constance L. Chik, MD, Nelson Chong, PhD, Philip Cole, MD, PhD, The Igor Dawid, PhD, Laboratory of Molecular Genetics, NICHD, Fred Dyda, PhD, Laboratory of Molecular Biology, NIDDK, Vadivel Ganapathy, PhD, Medical Yoav Gothilf, PhD, Allison Hickman, PhD, Laboratory of Molecular Biology, NIDDK, Anthony Ho, PhD, John Hogenesch, PhD, Genome Institute of the Novartis
Foundation, Ann Humphries, PhD, Ken-Ichi Inui, PhD, P. Michael Iuvone, PhD, Howard Jaffe, PhD, Laboratory
of Neurochemistry, NINDS, Ken Kirk, PhD, Laboratory
of Chemistry, NIDDK, Eugene V. Koonin, PhD, Benoit Malpaux, PhD, INRS, Nouzilly, France Sandford Markey, PhD, Laboratory
of Neurotoxicology, NIMH, Morten Møller, PhD, Panum
Institute, Randall T. Moon, PhD, Howard
Hughes Medical Institute, Peter Munson, PhD, Tomas, Obsil, PhD, Laboratory
of Molecular Biology, NIDDK, Benjamin Ron, PhD, Christof Schomerus, PhD, J.W.
Goethe Universität, Reiko Toyama, PhD, Laboratory
of Molecular Genetics, NICHD, Weiping Zhen, PhD, The For further information, contact klein@helix.nih.gov |
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