|
|
|
|
|
Prehistory: Suprachiasmatic
nucleus is a paired cute ovoidal nuclei displaying
robust circadian oscillation |
Okamura-lab
starts in 1995 in Kobe, when Hitoshi Okamura becomes
a professor of the Department of Anatomy II in Kobe
University School of Medicine. Thereafter, main
theme is the molecular mechanisms of mammalian circadian
clock. Before describing the achievements of the laboratory,
its prehistory is described briefly.
In the Department of Anatomy II in Kyoto Prefetural University
of Medicine (Professor Yasuhiko Ibata), Okamura
encountered the dense cluster of vasoactive intestinal
peptide (VIP) producing neurons symmetrically located
just dorsal to the optic chiasma, which strongly impressed
the dignity of the suprachiasmatic nucleus (SCN) at 1983.
At that time, we analyze SCN by histochemical and electron
microscopic techniques. During these days, we had a great
effort to established two completely original methods
which will be fruitful in later days. The first is the
highly quantitative histochemistry (Brain Research, 1987;
Mol Brain Res, 1995; J. Neuroscience, 1997), and the
second is the in vitro organotypic slice culture technique
for the study of the SCN (Neuroscience, 1994: collaboration
with Professor Shin-ichi Inouye). At this time,
we did not notice its powerfulness, but both two are
flowered after 10 years when mPer genes are discovered.
In France in Lyon and Gif-sur-Yvette supported by INSERM
and CNRS (1987-1989)(Professors Michel Jouvet, Robert
Naquet), we found that virtually all SCN neurons
are GABAergic.
|
1) |
Takahashi
Y, Okamura H, Yanaihara N, Hamada S, Fujita S,
Ibata Y: Vasoactive intestinal peptide immunoreactive
neurons in the rat suprachiasmatic nucleus demonstrate
diurnal variation. Brain Res.,
497: 374-377, 1989. |
2) |
Okamura H, Abitbol
M, Julien J.F, Dumas S, Berod A, Geffard M, Kitahama
K, Bobillier P, Mallet J, Wiklund L: Neurons containing
messenger RNA encoding glutamic acid decarboxylase
(GAD) in rat hypothalamus demonstrated by in situ
hybridization, with special enphasis on cell groups
in medial preoptic area, anterior hypothalamic
area and dorsomedial hypothalamic nucleus. Neuroscience,
39: 675-699, 1990. |
3) |
Tominaga K, Inouye
S -I T, Okamura H: Organotypic slice culture of
the rat suprachiasmatic nucleus: Sustenance of
cellular architecture and circadian rhythm. Neuroscience,
59: 1025-1042, 1994. |
4) |
Okamura H, Kawakami
F, Tamada Y, Geffard M, Nishiwaki T, Ibata Y, Inouye
S-IT: Circadian change of VIP mRNA in the rat suprachiasmatic
nucleus following p-chlorophenylalanine (PCPA)
treatment in constant darkness. Mol. Brain
Res., 29: 358-364, 1995. |
5) |
Ban Y, Shigeyoshi
Y, Okamura H: Development of circadian VIP rhythm
in the rat suprachiasmatic nucleus. J.
Neurosci., 17, 3920-3931, 1997. |
|
|
|
Although the circadian clock genes were
identified in 1984 in Drosophila, mammalian molecular
mechanism of circadian rhythms was totally unknown even
at 1995. For example, there were many groups believing
the circadian rhythms being produced by the networks of
neurons as sleep-wake cycles, since many molecular efforts
during 13 years had failed to isolate the clock genes in
mammals. In 1997, we discovered the human and mouse genes
encoding PAS-domain (PAS, a dimerization domain present
in Per, Amt and Sim)-containing polypeptides that are highly
homologous to Drosophila period by the
collaboration of Dr. Hajime Tei in Tokyo University
(Nature, 1977). We named this gene as mPer1, and
thereafter, we proceeded the molecular dissection solely
in my laboratory, and isolated another two mammalian mPer genes
(mPer2 and mPer3) and mammalian homology
of timeless (mTim) (EMBO-J, 1998). Subsequently,
we have found that mPer1 is light-inducible and
acts as a “pendulum”, committing the phase-shift of the
circadian clock by light (Cell, 1997). Collaborating with
Professor Jay Dunlap (Dartmouth University), we
found that mammalian clock is a day-type clock similar
to Neurosopra, although Drosophila’s
clock is night-type clock. Together with the discovery
of Clock gene by Professor J. Takahashi, our studies
contributed the establishment of the concept that the mammalian
circadian rhythms are generated at the gene level.
|
1) |
Tei
H, Okamura H, Shigeyoshi Y, Fukuhara C, Ozawa R,
Hirose M, Sakaki Y: Circadian oscillation of a
mammalian homologue of the Drosophila period gene. Nature 389,
512-516, 1997 |
2) |
Shigeyoshi Y, Taguchi
K, Yamamoto S, Takekida S, Yan L, Tei H, Moriya
T, Shibata S, Loros JL, Dunlap JC, Okamura H: Light-induced
resetting of a mammalian circadian clock is associated
with rapid induction of the mPerl transcript. Cell 91,
1043-1053, 1997. |
3) |
Takumi T, Taguchi
K, Miyake S, Sakakida Y, Takashima N, Matsubara
C, Maebayashi Y, Okumura K, Takekida S, Yamamoto
S, Yagita K, Yan L, Young ML, Okamura H: A light
independent oscillatory gene mPer3 in mouse SCN
and OVLT. EMBO J. 17:
4753-4759, 1998. |
|
|
Fig. 1 Clock genes and
a model of the core feedback loop in the mammalian cells. |
|
|
Although we are interested in producing
knockout mice of these mPer genes to characterize
these genes, we have neither facilities to make these animals,
nor financial support at that time. So, we were searching
low cost but scientifically important works to prove that mPer genes
really have the clock oscillating ability. It is noteworthy
that the molecular studies suggest that mammals and Drosophila
utilize similar components to generate circadian (~24 hour)
rhythms. To prove that the mPer1 and mPer2 genes
indeed have the capability to generate oscillations, we
collaborated with Professor Amita Sehgal (University
of Pennsylvania) the discoverer of Drosophila timeless,
and investigated whether the introduction of mouse mPer1 and mPer2 genes
into the arrhythmic per01 mutant of Drosophila caused
functional recovery. Behavioral assays showed that both mPer1 and mPer2 constructs
driven by Drosophila timeless promoter restored rhythms
in per01 flies that are otherwise arrhythmic due
to a lack of endogenous PERIOD protein. Thus, incorporation
of mammalian mPer1 and mPer2 genes into
Drosophila mutants demonstrates that both mPer1 and mPer2
can function as clock components. This study is unique,
and indeed appreciated from some researchers, but the recovery
rate of flies are at most 20%, thus cannot be published
in leading journals at 1999 (Later we published the results
at 2002 to Genes to Cells). Later mice homozygous
for the targeted allele of mPer1 and/or mPer2 were
reported by 3 groups (Drs. Lee, Reppert and Sassone-Corsi),
showing the severely disrupted locomotor activity rhythms
during extended exposure to constant darkness. |
1) |
Shigeyoshi
Y, Meyer-Bernstein E, Yagita K, Weili F, Chen Y,
Takumi T, Schotland P, Sehgal A, Okamura H: Restration
of circadian behavioral rhythms in a period null
Drosophila mutant (per01) by mammalian
period homologues mPer1 and mPer2. Genes
Cells, 7, 163-171, 2002. |
|
|
Fig. 2 Restoration of
circadian rhythm in per01 flies after the introduction
of mammalian mPer1 or mPer2 genes. A Actogram
(left) and its period length analysis
(right; period length is indicated in
hours at the top of periodigrams) of transgenic flies (dtim-mPer1,
dtim-mPer2) and controls (per01 and wild
type, WT). Circadian time is indicated at the
top of actograms, consecutive days at left of actograms.
The significance line denotes a 0.05 level of significance. B Frequency
distribution of activity periods in tim-mPer1- and tim-mPer2-containing
transgenic flies. C Difference of the
period length in mPer1- and mPer2- restored
rhythms. |
|
|
Just after the discovery of mPer genes,
the mammalian clock study goes Drosophila as a
model. In this line, mTim, mmammalian counter
part of Drosophila timeless, was soon be cloned.
Soon, clock and bmal1 are established
as positive transcription factors on mPer genes
following the negative feedback theory of Drosophila clock
machinery. However, the breakthrough comes from the unexpected
field.
Professor Gijsbertus van der Horst, a researcher
of DNA repair in Netherlands, made the knockout mice of mammalian
cryptochromes (Cry1, Cry2), and surprisingly found
that these mice were completely arrhythmic in behavior (Nature,
1999).
Just after they got this result, we began the collaboration
whether master clock in the SCN are working or not. Okamura
and lab members trip to Rotterdam for one week experience:
fixed smples from these mice are successfully depicted. By
the quantitative in situ hybridization methods, we found
that the mPer gene oscillation was completely abolished
in the central clock (SCN) (Science, 1999).
At the same time, clock gene oscillation was abolished in
peripheral tissues (liver) of Cry deficient mice.
Then one question arises: does the peripheral clock oscillation
influence the behavioral arrhythmicity? We performed the
transplantation of wild-type SCN to arrhythmic Cry-deficient
mice, and found that the behavioral arhhythmicity was recovered
by the transplantation of wild-type SCN (Current Biology,
2003). This rhythm emergence strongly suggests the unnessessity
of non-SCN peripheral clocks for formation of behavioral
rhythms. This result claims the SCN as the synchronizer,
but strongly supports the SCN as the rhythm generator at
behavioral rhythms, although rhythms of peripheral clocks
(endocrine, or enzyme etc) have not been addressed. abolished
it. |
1) |
Okamura
H, Miyake S, Sumi Y, Yamaguchi S, Yasui A, Muijtjens
M, Hoeijmakers JHJ, van der Horst GTJ: Photic induction
of mPer1 and mPer2 in Cry-deficient
mice lacking a biological clock. Science 286,
2531-2534, 1999. |
2) |
Sujino M, Matsumoto
K, Yamaguchi S, van der Horst G, Okamura H*, Inouye
SIT*: Suprachiasmatic nucleus grafts restore circadian
behavioral rhythms of genetically arrhythmic mice. Current
Biology 13, 664-668, 2003. (*Correspondence) |
|
|
Fig. 10 Double-plotted
locomotor activity rhythms of SCN-grafted Cry1/Cry2 double
knockout mice. Arrhythmic Cry1/Cry2 double knockout
mice first lesioned SCN, then received brain transplantation
operation of neonatal SCN. Two weeks after transplantation,
activity bouts gradually consolidated to form a free-running
rhythm with a shorter than 24 hour period. |
|
|
Since main components of core clock oscillation
had been revealed, a number of laboratories began to clarify
the molecular mechanisms of the mammalian clocks in 1998-2000.
The cellular circadian oscillation in mammals to start
with the transcription of two genes: mPer1 and mPer2.
Expression of these genes is stimulated by heterodimers
formed by the bHLH-PAS proteins CLOCK and BMAL1. We demonstrated
that mPER proteins made in the cytoplasm, translocate into
the nucleus (Genes Develop, 2000), and form a negative
complex comprised of mCRY1, mCRY2, mPER1, mPER2, mPER3
and mTIM that suppresses the transcription triggered by
CLOCK and BMAL1. Importance of posttranscriptional mechanisms
including phosphorylation and degradation are suggested.
We demonstrate that mPER1 and mPER2 proteins usually shuttle
between the cytoplasm and the nucleus and are easily degraded
by ubiquitination and the proteasome pathway (EMBO-J. 2002;
MCB, 2005). Ubiquitination of mPER proteins is inhibited
by the presence of mCRY proteins. Since mCRY protein can
also be ubiquitinated when mPER proteins are absent, the
mPER/mCRY dimer is stabilized against degradation, suppresses mPer1 and mPer2 transcription,
and shuts off mPER synthesis. Since it is speculated that
the transcription level of mPer genes is determined
essentially by the concentration of mPER/mCRY dimer in
the nucleus, re-starting mPer transcription depends
on the export of the mPER proteins out of the nucleus by
the CRM1/Exportin1 nuclear export machinery. The consequent
decrease of mPER destabilizes mCRY, and the decrease of
mCRY then allows mPer1 and mPer2 gene
transcription to restart. |
1) |
Yagita
K, Yamaguchi S, Tamanini F, van der Horst GTJ,
Hoeijmakers JHJ, Yasui A, Loros JJ, Dunlap JC,
Okamura H: Dimerization and nuclear entry of mPER
proteins in mammalian cells. Genes
Develop. 14:1353-1363, 2000. |
2) |
Yagita K, Tamanini
F, Yasuda M, Hoeijimakers JHJ, van der Horst, GTJ,
Okamura H: Nucleocytoplasmic shuttling and mCRY
dependent inhibition of ubiquitination of the mPER2
clock protein. EMBO J.
21, 1301ー1314, 2002. |
3) |
Yamamoto Y, Yagita
Y, Okamura H: Role of cyclic mPer2 expression in
mammalian cellular clock. Mol. Cell
Biol., 25, 1912-1921, 2005. |
|
|
Fig. 3 Phospholylation,
degradation and nuclear translocation of mPER proteins. |
|
|
The existence of cellular clock was indicated
by the demonstration of circadian expression of clock genes
in fibroblast cell lines after the serum-shock revealed
by Schibler’s group in Geneva. Are there any differences
of autonomously rhythmic clock genes in these cells and
those in the SCN? We addressed this question by using spontaneously
immortalized mouse embryonic fibroblasts (MEFs) from wildtype
and Cry1-/-Cry2-/- mice (Science, 2001). Both wildtype
and Cry1-/-Cry2-/- cell lines showed clock properties similar
to those found in the SCN of wildtype and Cry1-/-Cry2-/-
mice, respectively. These included: (i) temporal expression
profiles of all known clock genes, (ii) the phases of the
various mRNA rhythms (i.e. antiphase oscillation of Bmal1 and mPer
genes), (iii) the delay between maximum mRNA levels
and appearance of nuclear mPER1 and mPER2 protein, (iv)
the inability to produce oscillations in the absence of
functional mCry genes, and (v) the control of
period length by mCRY proteins. These results strongly
support the conclusion that the components and oscillatory
mechanism of central clocks (in the SCN) and of peripheral
clocks are essentially identical. |
1) |
Yagita
K, Tamanini F, van der Horst GTJ, Okamura H: Molecular
mechanisms of the biological clock in cultured
fibroblasts. Science 292,
278-292, 2001. |
|
|
Fig. 4 Endothelin-induced
time-dependent gene expressions of mRNA (black dotted line)
and proteins (red line) in mPer1 (A) and
mPer2 (B) in mouse embryonic fibroblasts
(MEF). Note 6-8 hours difference of mRNA and proteins in
both mPer1 and mPer2. (C) (Left) Circadian
expression of Per1, dbp and Bmal1 in wild type MEFs and
mCry1-/-mCry2-/- MEFs. (Right) mCry1-/-MEF (square) and
mCry2-/-MEFs (filled circle). |
|
|
An important question is how the time
information generated by the cell clock oscillator is transmitted
to the hundreds of clock controlled genes (ccg) that represent
the output of the clock. Two routes are proposed: the first
goes directly from the central loop of the mammalian clock
to the ccgs through E-box (CACGTG). The second route is
indirect, and regulates the antagonistic effects of PAR
proteins (DBP, HLF, TEF) and E4BP4 on the D-box (Mol. Cell
Biol., 2000; Genes Develop 2001). PAR proteins activate
the transcription of target genes by binding to the specific
sequence RTTAY GTAAY (R, purine; Y, pyrimidine) during
the day, and E4BP4 suppresses transcription of these target
genes in the night. E4BP4 and the PAR proteins may switch
back and forth in turning target gene transcription on
and off. Since many of genes contains D-box, it is speculated
over 100 genes are circadianly regulated by D-Box. |
1) |
Yamaguchi
S, Mitsui S, Yan L, Yagita K, Miyake S, Okamura
H: Role of DBP in the circadian oscillatory mechanism.
Mol. Cell. Biol. 20:4773-4781, 2000. |
2) |
Mitsui S, Yamaguchi
S, Matsuo T, Ishida Y, Okamura H: Antagonistic
role of E4BP4 and PAR proteins in the circadian
oscillatory mechanism. Genes Develop. 15, 995-1006,
2001. |
|
|
Fig. 5 The core, accessory
and output molecular mechanisms of the mammalian circadian
clock. BMAL1/CLOCK heterodimer binds to E-box in clock
oscillating genes and ccg, and accelerates their transcription.
The core feedback loop provided by mPer1 and mPer2 is
supplemented by an accessory PAR protein loop. DBP accelerates
the mPer1 transcription, but E4BP4 maximally expressed
in counter phase to PAR proteins suppresses mPer1 transcription. |
|
|
Next, we will show the examples how core
clock regulates cellular functions by analyzing the association
of circadian clock and cell cycle. Since the life span
of each cell is limited, cell growth and mitosis are required
to maintain organ or tissue function. There is substantial
evidence that circadian rhythms affect the timing of cell
divisions in vivo: day-night variations in both
the mitotic index and DNA synthesis were found in oral
mucosa, corneal epithelium, and bone marrow. These studies
used histochemical techniques and normal physiological
conditions, so the mitotic cells comprised only a few per
cent of the cells examined. A suitable system with a high
proportion of dividing cells is required in order to apply
biochemical techniques to study the molecular links between
regeneration and the circadian clock. We noticed that the
liver provides a suitable organ since it is known to undergo
vigorous regeneration after incomplete surgical removal.
Although the cell cycle period overall is several months
in unoperated animals, a 2/3 partial hepatectomy (PH) induces
the large majority of the remaining, pre-existing hepatocytes
to divide, and the regeneration speed is so rapid that
liver mass is restored within 7days. Moreover, expression
of mPer1 and mPer2 oscillates vigorously
in liver, and the temporal profiles and the vigorousness
of the expression of clock genes were not altered after
PH. Thus, the mouse liver is very suitable for analyzing
the molecular connection between the circadian clock and
the cell cycle.
We first compared the rate and timing of liver re-growth
after PH in mice, when PH was performed in the morning at
lights-on or in the afternoon (Science, 2003a). S-phase kinetics,
represented by the incorporation of bromodeoxyuridine (BrdU),
was similar in both morning-operated and afternoon-operated
animals. However, there was an 8 hour delay in the M-phase
(cells entering mitosis) when PH was performed at morning,
as compared to afternoon. This indicated that the timing
of the hepatectomy determines the timing of entry into M-phase
for these regenerating cells. To determine the impact of
circadian clocks on the cell cycle, clock-less arrhythmic Cry1-/-Cry2-/- mice
were subjected to PH (Science, 1999). In these mice, mitosis
was severely impaired, and liver regeneration was severely
blunted.
We performed DNA arrays and Northern blots to characterize
the molecular differences in M-phase entry and found that cyclin
B1 and cdc2 were positively correlated, and wee1,
the gene for a kinase that inhibits mitosis by inactivating
CDC2/cyclin B, was negatively correlated to M-phase. This
is interesting since all these genes are cell cycle regulators
governing G2 to M transition, and these expression profiles
correlate well with M-phase progression. In the livers of
normal mice, there were clear circadian rhythms of wee1 expression,
with very low levels in the morning and high levels in the
night. Levels of wee1 were always high in Cry-deficient
mice, whereas levels were always low in Clock mutant
mice (Clock/Clock). There are three E-boxes in the
5' UPR of wee1, which were, furthermore, activated
by CLOCK/BMAL1 and suppressed by PER2, PER3, CRY1 and CRY2.
These results suggest that wee1 transcription is
regulated directly by the core feedback loop through its
E-box elements. Changes in transcription of wee1 are
reflected at the protein level, influence CDC2 activity levels,
and are negatively correlated with the mitotic peak. WEE1
phosphorylates the cyclin-dependent kinase CDC2, a key regulator
of G2 to M transition. In order to allow entry into mitosis,
CDC2 has to be dephosphorylated by CDC25, a protein phosphatase,
and it is the competition between the activities of WEE1
and CDC25 that determines the phosphorylation status of CDC2
(and hence the propensity of cells to enter mitosis). Only
when WEE1 levels are low (normally in the morning) can CDC25
phosphatase successfully antagonize the action of WEE1. These
findings, taken together, demonstrate that the circadian
clock controls the G2 to S transition via the regulation
of WEE1. Together with the report demonstrating a high incidence
of lymphoma in mice lacking the mPer2, our present study
sheds light on the association of circadian rhythms and cell
division. |
1) |
Matsuo
T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura
H: Control mechanism of the circadian clock for
timing of cell division.
Science 302,
255-259, 2003. (published
on line in Science Express
on 21 August 2003) |
|
|
Fig. 6 Control of cell
cycle by cell clock. Schematic representation showing the
link between the circadian clock and the cell cycle (upper).
Role of CDC2, cyclin, WEE1 and CDC25 in making active MPF
(M-phase promoting factor) is depicted at the bottom. |
|
|
Above studies revealed that cell clocks
govern many of the cellular functions. Then, is the cell-time
transmitted to another cells? We want to address this issue
in the SCN, since it is the sole organ oscillate eternally.
We adopt SCN-slice culture system which we established
for monitoring peptide-release (Neuroscience, 1994). We
made a SCN slice taken from transgenic mice carrying the
luciferase gene driven by the mPer1 promoter (mPer1-luc)
(Curr Biol, 2001). In this study, we discovered that the
application of NMDA, analogous to light stimuli to the
living animals, instantly altered the phase of the core
clock oscillation of slice-SCN phase-dependently. In collaborating
with Professor Masaki Kobayashi, a specialist
enabling the visualization of very weak biophoton, we succeed
to observe the rhythmic transcription of genes at the single
cell level by a cryogenic high resolution CCD camera (Science,
2003). The SCN cells showed robust transcription rhythms
with a period length of -24 hours, with several hundreds
of cells expressing mPer1 genes synchronously.
Moreover, the individual oscillatory cells are arranged
topographically: the phase-leader with a shorter period
length is located in the dorsomedial periventricular part
of the SCN. A protein synthesis inhibitor (cycloheximide)
sets all the cell clocks to the same phase and, following
withdrawal, intrinsic interactions among cell clocks re-establish
the stable program of gene expression across the assemblage.
Tetrodotoxin, which blocks action potentials, not only
desynchronizes the cell population, but also suppresses
the level of clock gene expression, demonstrating that
neuronal network properties dependent on action potentials
play a dominant role in both establishing cellular synchrony
and maintaining spontaneous oscillations across the SCN.
Thus, the cell-rhythm oscillation generated by the core
clock oscillatory loop is coupled and amplified by the
ordered cell-cell communications in the SCN. |
1) |
Tominaga
K, Inouye SI, Okamura H: Organotypic slice culture
of the rat suprachiasmatic nucleus: sustenance
of cellular architecture and circadian rhythm. Neuroscience 59,
1025-1042, 1994. |
2) |
Asai M, Yamaguchi
S, Isejima H, Jounouchi M, Moriya T, Shibata S,
Kobayashi M, Okamura H: Visualization of mPer1
transcription in vitro by luciferase mediated bioluminescence:
NMDA induces a rapid phase-shift of mPer1 gene
in cultured SCN. Current Biology 11,
1524-1527, 2001. |
3) |
Yamaguchi S, Isejima
H, Matsuo T, Ohkura R, Yagita K, Kobayashi K, Okamura
H: Synchronization of cellular clocks in the suprachiasmatic
nucleus. Science 302,
255-259, 2003. |
|
|
Fig. 7 Temporal changes
in bioluminescence signals from 100 SCN cells randomly
chosen in wild-type (upper figure) and mPer1-luc-bearing mCry1/mCry2 double-knockout
mice (lower figure). Note variety pattern of peaks in cells
of wild type animals, but total absence of rhythmic cells
in Cry double knockout mice. |
|
|
By what route SCN regulates the peripheral
clocks? Melatonin regulates the sympathetic nervous system
by directly acting to the SCN (J. Physiol., 2003). More
importantly, sympathetic nervous system plays the key role.
In collaborating with Professor Shigenobu Shibata, we
demonstrated that hepatic gene expression of clock genes
was regulated by the sympathetic nerves (PNAS, 2003). Moreover,
we demonstrated that SCN regulates the circadian expression
of adrenal corticosterone by the activation of various
genes by the route of innervating sympathetic adrenal nerve
(Cell Metabolism, 2005). Yes, sympathetic nerve conveys
the time of central clock to peripheral organs, and the
adrenal gland is the key organ transforming circadian signals
from nerve signals to the endocrine signals. Released corticosterone
may tune the phase of peripheral clocks. |
1) |
Terazono
H, Mutoh T, Yamaguchi S, Kobayashi M, Akiyama M,
Udo R, Ohdo S, Okamura H, Shibata S: Adrenergic
regulation of clock gene expression in the mouse
liver. Proc. Natl. Acad Sci. USA 100,
6795-6800, 2003. |
2) |
Ishida A, Mutoh
T, Ueyama T, Bando H, Masubuchi S, Nakahara D,
Tsujimoto G, Okamura H: Light activates the adrenal
gland: Timing of gene expression and glucocorticoid
release. Cell Metabolism,
2, 297-307, 2005. |
|
|
Fig. 8 (Upper
figures) Light exposure induces the high level
of expression of luciferase luminescence in the adrenal
gland (arrows) in mPer1-luc mice. (Lower
figure) The schematic representation of the
neuronal routes how light resets adrenal or pineal hormones.
IML, intermediolateral cell collumn, PVN, paraventricular
nucleus of the hypothalamus, SCG, superior cervical ganglion,
SPvZ, subparaventricular zone. |
|
|
Monitoring bioactive markers in the brain
is fundamental to clarify the circadian time-keeping system
in the brain. In collaborating with Professor Daiichiro
Nakahara, we first devised a new method to monitor
mammalian melatonin in the cerebral ventricle by utilizing
the lintracerebral microdialysis probe. It is almost half
the century since the hormones such as melatonin and cortisol
have the diurnal rhythms. However, the analysis of dirunal
secretion of hormonesis hampered by that there are no system
to record continuously in a single body. We developed the
microdialysis system to analyze the melatonin secretion
for several months. By this method, we have revealed for
the first time amine-dependent and nerve-independent rhythms.
This approach will became more powerful when applied tot
the genetically altered mice. |
1) |
Nakahara
D, Nakamura M, Iigo M, Okamura H: Bimodal circadian
secretion of melatonin from the pineal gland in
a living CBA mouse. Proc. Natl. Acad.
Sci. USA 100, 9584-9589, 2003. |
|
|
Fig. 9 (Upper
figure) Transverse microdialysis probe in the
pineal glands. (Lower figures). A double
plot of melatonin levels and locomotor acivity for three
mice in LD and DD cycles. Note parallel change of melatonin
levels of locomotor activity in both LD and DD conditions. |
|
|
Finally we tried to monitor the gene
expression of clock gene st real time by inserting the
optical fiber directly to the SCN in the brain in freely
moving mice. We inserted an optical fiber just above the
SCN of mPer1-luc transgenic mice and succeeded
to record for several days oscillating luminescence that
accurately mirrored native mPer1 mRNA expression.
By this method, we first revealed mPer1 gene is activated
in the day time, and rest in the night time. These data
not only for the first time demonstrate a ticking biological
master clock in the brain of a living mammal, they also
show that real-time optical imaging of gene expression
is a new powerful tool to analyze the higher nervous function
of the brain including sleep/wake cycles(Nature, 2001). |
1) |
Yamaguchi
S, Kobayashi M, Mitsui S, Ishida Y, van der Horst
GTJ, Suzuki M, Shibata S, Okamura H: View of a
mouse clock gene ticking. Nature 409,
684, 2001. |
|
|
Fig. 11 (Upper
figure) Location of the input end of the optical
fiber in the mouse brain. (Lower figure)
Circadian fluctuation of luminescence in the SCN. A mPer1-luc transgenic
mouse, previously housed under a 12h light/12h dark cycle,
was continuously infused with luciferin (10 µM
in artificial cerebrospinal fluid) at a rate of 15 µl/h.
Luminescence was recorded under constant dark conditions
via an optical fiber positioned above the SCN. One dot
represents an average of the values of 5 minutes. Hatched and closed
bars at the bottom of the figure represent subjective
day and subjective night, respectively. |
|
|
Since Human Genome Project has finished,
the revolution of medicine is now going on: now all the
history of diseases and behavior of each person from birth
to death is referenced to its genome information. However,
even this type of correspondence studies develop, there
will be no progress how genomic information is reflected
to behavioral level. The functional significance of circadian
biology exists at this point: the unique feature of circadian
biology is that the gene transcription occurs in the SCN
reflects the behavioral and physiological rhythms almost
in a perfect state. This means that the clock gene oscillation
generated by the core loop in each SCN neuron is coupled
and amplified at the level of the SCN neuronal nuclei.
From here harmonized strongly oscillating activities are
spread into the whole brain and to all those peripheral
organs, which contain peripheral clocks. As a final result,
circadian changes are induced in behavior and hormone secretion.
Even though each neuron in the SCN generates circadian
oscillation, the system of amplification and transmission
needs to be well organized to effectively transmit the
time information to the peripheral organs. The real time
monitoring of clock gene oscillation at gene, cell, tissue,
brain, and system levels will answer the question of how
the time signal is born and integrated to the upper layers
of life. Investigations of biological clocks open the fascinating
perspective to analyze the integrational mechanism of TIME
in a vertical arrangement, providing a bridge between single
genes and the living organism as a whole.
In May 2007, we moved from Kobe University to Kyoto University.
The new laboratory is Department of Systems Biology in
the faculty of Pharmaceutical Science. Here, we want to reset
our work to clarify “How time is generated and tuned”.
We hunt this esoteric secret poco a poco by believing a fortune
to Ride the Rhythm. |
|
Fig. 12 Integration
mechanism of rhythm. “Gene” depicts rhythmic transcription
of mPer1 and mPer2. “Cell” represents
neuronal electrical activities of single SCN neurons. “SCN”
indicates the local neuronal and glial circuits. “System”
symbolizes behavior and hormonal secretion. |
|
|