180712 電気通信大学BLSCセミナー

生物時計のサイエンス：
時計遺伝子の働きから睡眠障害の理解まで
1) 非24時間睡眠覚醒症候群マウス
2) 概日時計の分子発振メカニズム

金 尚宏 (Naohiro Kon)
東京大学大学院理学系研究科
生物科学専攻 深田吉孝研究室

Terminology

Human FRP (24.18 h)

Zeitgeber: German for “Time giver”.
Environmental stimulus that can entrain clock
Free running period, FRP: Period length of clock under nozeitgeber signals
T: Period length of zeitgeber cycle
Phase relationship: Relationship between phase (time) of clock
and that of zeitgeber cycle
Entrain: From French entrainer, “to carry along”.
T = FRP with appropriate phase relationship.
Science, 284, 2177 (1999); Chronobiol Int, 20, 741 (2003)

Entrainment is explained by phase shift of clock
Phase response curve (PRC)

Under entrained condition→ Phase‐dependent response
to single zeitgeber pulse

Daily phase shift = FRP - TSleep disorder is representative problem caused by abnormal clock
International Classification of Sleep Disorders (ICSD‐3, 2014)

Delayed sleep phase syndrome, DSPS

Activity watch rhythm

Features・ 3‐6 hours delay relative to desired or socially acceptable schedules
・ Sleep onset insomnia
Prevalence・ 0.13‐0.17% in general population
・ 7‐16 % in adolescent (13‐17 years old)・ 7% of patients with chronic insomnia in sleep clinics
Pathophysiology (Unclear)
・ Normal FRP in general DSPS patients (Kitamura et al., 2013)
・ Long FRP (0.5 hr) in familial DSPS with Cry1 mutation (Patke et al, 2017)
・ More sensitive melatonin suppression to evening light (Aoki et al., 2001)
Sleep Med 8, 566 (2007)

Advanced sleep phase syndrome, ASPS

Activity watch rhythmFeatures
・ Several hours early relative to conventional and desired time
・ Early morning awakenings and sleepiness in late afternoon
Prevalence・ Unclear・ Increase with age (1 % in middle‐aged and older adults)
Pathophysiology (Unclear)・ Short FRP in familial ASPS
Sleep Med 8, 566 (2007)

Non‐24‐h sleep‐wake syndrome (Non‐24)

Activity watch rhythmFeatures
・ Daily drift of sleep and wake times
Prevalence
・ 50% of total blind people
・ Although rare, observed in sighted people (> 10 years old)
Pathophysiology (Unclear)
・ Long free running period (0.31hr)
・ In sighted non‐24, 26% patients shows DSPD before the onset
Sleep Med 8, 566 (2007)

Non‐24 with normal visual function

Sleep 28, 945 (2005)

Circadian clock generates behavioral rhythm

Tight‐coupling of morning and evening oscillators
establishes temporal organization of daily activities

Central clock in the SCN controls
behavioral rhythms and peripheral clock

Interneuronal couplingTranscriptional feedback loop for oscillation

Nature Reviews Genetics 9, 764 (2008)

Exploring of bioactive molecule affecting cellular clock
Monitoring of cellular rhythm

Effect of KN93 on rhythm

KN93(CaMKII inhibitor)

Kon et al., Communicative & Integrative Biology, 2015.

CaMKII activity is essential for cellular clock
Small-molecule inhibitor of CaMKII

Knock-down of CaMKII

KN‐92(Inactive analog)

KN‐93(CaMKII inhibitor)
Rat‐1 Bmal1‐luc cells

NIH3T3 Bmal1‐luc reporter

CaMKII is Ca2+‐dependent molecular switch

J Neurosci, 24, 8394 (2004).

CaMKII is essential for brain function

・ Intellectual disability
・ Epileptic seizure
・ Ataxia

Transcriptional feedback loops for cellular clock

What component(s) is affected by CaMKII?

CaMKII activity is essential for E‐box‐dependent gene expression
Chronic treatment of CaMKII inhibitor

Overexpression of CaMKIINIH3T3 cells
Rat‐1 cells

E‐box‐dependent transcription may be a target of CaMKIICaMKII Inhibition decreases phosphorylation of CLOCK
and heterodimerization of CLOCK and BMAL1
Immunoprecipitation by anti‐CLOCK antibody

Synchronized Rat‐1 cells
Phosphorylation level of CLOCK

KN93

CLOCKantibody

CaMKI
PI
CLOCK
BMAL1

CLOCK‐BMAL1 interaction

CaMKII phosphorylates CLOCK
Co‐immunoprecipitation assay

In cell phosphorylation assay

Nuclear lysate of mouse liver
In vitro kinase assay
CLOCK and BMAL1
expressed in HEK293T cell
Partial fragment of CLOCKIs CaMKII a linker between two oscillation mechanism?

Liver from Per1-luc. TG mice

SCN from Per1-luc. TG miceCa2+ is essential for the transcriptional feedback loops.

Roles for upstream regulators of CaMKII

Ca2+/CaM is important for cellular rhythm and E‐box gene expression

Morning activation of Ca2+‐CaMKII‐E‐box gene expression in SCN
Neuronal
firingCa2+ level

Neuron, 38, 253, (2003).

CaMKII activity

E-box gene expression (Per1)

circadian time

Neurochemistry Int., 44, 617, (2004).

Cell, 91, 1043, (1997).

Do CaMKII activity levels determine phase of clock?

State variables define the state of of the oscillation
The fundamental modeling concept is that components
underlying rhythm generation change rhythmically in time.
Chronobiology: Biological Timekeeping Jay C. Dunlap, Jennifer J. Loros, and Patricia J. DeCoursey

Chronobiol Int, 20, 741 (2003)

If CaMKII activity is a state variable,
modulation of the level might reset phase of clock.

Setting CaMKII activity to trough level leads the oscillatorto trough level of E‐box gene expression

Rat‐1 Bmal1‐luc cells

Kon et al., Genes and Development, 2014.

Behavioral analysis of CaMKII kinase‐dead knock‐in mice
K42R

CaMKIIK42R
K42R prevents ATP binding for phosphorylation

Yamagata et al., J. Neurosci. 29, 7607 (2009)

CaMKII mutants show long period andtemporal decrease in periodogram amplitude

3/13 mice show
Arrhythmicity

CaMKII mutants show decoupling of onset and offset
activity rhythms, resulting in prolongation of activity time.

Temporally delaying morning activity prolongsactivity time in CaMKIIK42R mice
Activity timeEMENME; evening peak N; night peak M; morning peak

CaMKII mediates coupling of morning and evening rhythms

Attenuated amplitude of individual cellularoscillation in the SCN of CaMKIIK42R mice
Single neuronal rhythms of SCN

Amplitude of individual rhythms

Desynchronization of individual cellularoscillation in the SCN of CaMKIIK42R mice
Single neuronal rhythms of SCN
Phase plotting of individual rhythms

Decoupling of oscillations between left and right
SCN in CaMKIIK42R mice
Bioluminescence from left or right SCN nuclei

Kon et al., Genes and Development, 2014.

CaMKII regulates circadian clock at multiple levels
Cellular Oscillation

Left-Right SCN coupling

SCN Network

Evening-Morning behavioral CouplingAcknowledgement

Yoshitaka Fukada
Wang Hsintzu
Hikari Yoshitane
Kimiko Shimizu
Chihiro Hara
Jun Nakano
Arisa Hirano
Masamitsu Iino
Kazunori Kanemaru

Yoko Yamagata

Tomoko Yoshikawa
Sato Honma
Kenichi Honma

Benjamin Rusak

Joseph Takahashi
Marleen de groot
Yasunori SugiyamaIsamu Kameshita