About basics of Circadian clock moleculer mechanisms in Drosophila and humans.

1. CIRCADIAN
RHYTHMS
BY- HARSH VARDHAN CHARAN
M.S.(PHARM.)
PHARMACOLOGY AND
TOXICOLOGY
18PCM2783

2. CONTENT
S Introduction
 The brain’s master clock: SCN
 Regulation of Melatonin Secretion
 Synthesis and Functions of Melatonin
 Nobel Prize for the discovery of molecular mechanism of the
circadian clockwork in Drosophila
 Molecular mechanisms of the circadian clockwork in mammal
 Critical role of PER protein in organismal homeostasis
 Circadian rhythm sleep disorders

3. What Is the Circadian Rhythm?
 Circadian rhythms are physical, mental and behavioural changes that
follow a roughly 24-hour cycle, responding primarily to light and
darkness in an organism’s environment.
 This internal body clock is affected by environmental cues, like
sunlight and temperature.
 Circadian rhythms can influence sleep-wake cycles, hormone release,
body temperature and other important bodily functions. They have
been linked to various sleep disorders, such as insomnia. Abnormal
circadian rhythms have also been associated with obesity, diabetes,
depression, bipolar disorder and seasonal affective disorder.
 The circadian rhythm is often referred to as the "body clock.

4. The body clock regulates
every aspect of human physiology

5. The brain’s ‘master’ clock:
The suprachiasmatic nucleus (SCN)
 A master clock in the brain coordinates
all the biological clocks in a living thing,
keeping the clocks in sync.
 In vertebrate animals, including
humans, the master clock is a group of
about 20,000 nerve cells (neurons) that
form a structure called the
suprachiasmatic nucleus, or SCN.
 The SCN is located in a part of the
brain called the hypothalamus and
receives direct input from the eyes.

6. Evidences to support that SCN is the controlling
centre of the Circadian Rhythms
Stephen and Zucker removed SCN from rats which resulted in loss of
normal circadian cycle.
Friedman proved a pathway called Retinohypothalamic tract between retina
and SCN.
Fulton and Bailey observed that people with brain tumours damaging the
SCN caused sleep/waking disorders and that proves SCN is the controlling
centre of circadian rhythms.

7. REGULATION OF MELATONIN SECRETION
Melatonin Secretion by the Pineal Gland, Bioninja.

8. Tryptophan
5-Hydroxy
tryptophan
Serotonin(5-
Hydroxy
tryptamine)
N-Acetyl
Serotonin
Melatonin
 Synthesis of Melatonin
 Functions of Melatonin
*Regulation of : * Photoprotection
 Skin immune system * Protection against oxidative stress
 Vasculature * Induction of DNA Repair
 Metabolism *Regulation in release of sex hormones in
females and maintainence of menstrual cycle
 Adrenal function
 Melanin pigmentation
 Barrier function

10.  Using fruit flies as a model organism, the Nobel laureates isolated a gene that controls the normal daily
biological rhythm.
 Subsequently, they identified additional protein components of this machinery, exposing the mechanism
governing the self-sustaining clockwork inside the cell.

11. Molecular mechanism of the clock in
Drosophila
 Period gene was the first gene identified in connection with circadian rhythms by
Seymour Benzer by using 3 mutant Drosophila flies.
 Mutation was in the gene encoding for the period protein. These mutant flies showed
abnormal circadian rhythms i.e. changes in the normal 24 hour cycle and these are
measured by eclosion (emergence of adult flies from pupae) or locomotor activity.
 This period gene was later cloned and sequenced by Jeffrey Hall, Michael Rosbash and
Michael Young.
 For a long time per was the only circadian gene known, but rapid progress in the
circadian field in the 1990s led to the identification of timeless (tim), Clock (Clk) and
cycle (cyc), which, together with per, make up the core transcriptional feedback loop
that drives circadian rhythms.

12. Transcription-Translation feedback loops
Circadian Rhythms and Sleep in Drosophila melanogaster by Christine Dubowy and Amita Sehgal
Genetics.

13. COMPARISON OF TTFL IN MAMMALS AND DROSOPHILA
Transcriptional networks of circadian clock genes in Drosophila and mammals by Hoyeon Lee, ResearchGate.

14. Transcription-Translation feedback loop in Mammals
 In mammals, the master transcription factors BMAL1/2 (brain and muscle Arnt-like protein)
dimerize with CLOCK (circadian locomotor output cycles kaput) in the brain which drives the
transcription of Period (Per1-3) and Cryptochrome (Cry1/2) genes via interactions with E-box
promoter elements.
 PER and CRY proteins in turn form complexes in the cytoplasm, and at a certain threshold, the
PER/CRY complex migrates to the nucleus where it inhibits the action of BMAL and CLOCK, and
thus PER and CRY transcription.
 The inhibitory PER/CRY complexes are subsequently degraded in the proteasome following
phosphorylation by casein kinase Iε (CKIε) and then ubiquitination which removes the inhibition
on CLOCK and BMAL, allowing the feedback loop to restart again in a 24 h loop.
 CLOCK/BMAL1 also activate genes encoding nuclear receptors of the REV-ERB and ROR families,
which regulate the expression of Bmal1 (and also Cry1 and Clock).
 These retinoic acid-related orphan nuclear receptors, REV-ERBα and RORα subsequently
compete to bind retinoic acid-related orphan receptor response elements (RREs) present in
Bmal1 promoter. It has been shown that members of ROR (α, β and γ) and REV-ERB (α and β)
are able to regulate Bmal1 through RREs. RORs activate transcription of Bmal1, whereas REV-
ERBs repress the transcription process.

15. Molecular mechanisms of the circadian clockwork in mammal I.Robinson, A.B.Reddy

16. Critical role of PER protein in organismal homeostasis
 PER proteins are negative elements of the transcription-translation feedback loop and are
candidate tumour suppressors. In flies, Period deficiency leads to a reduced health span, while
Period overexpression may increase health span.
 PER-deficient mice showed an increased rate of tumorigenesis and reduced life span. Per
deficiency also cooperates with p53 deficiency in tumour formation.
 Genetic ablation mPER1 and mPER2 function results in a complete loss of circadian rhythm
control based on wheel-running activity in mice. In addition, these animals also display apparent
premature aging and a significant increase in neoplastic and hyperplastic phenotypes.
 Studies have demonstrated that the circadian clock function is very important for cell cycle,
DNA damage response, and tumour suppression in vivo. The temporal expression of genes
involved in cell cycle regulation and tumour suppression, such as c-Myc, Cyclin D1, Cyclin A,
Mdm-2, and Gadd45α, is deregulated in mPer2 mutant mice.
 Familial advanced sleep-phase syndrome is also known to be associated with mutations in the
mammalian Per2 gene.
 The period gene is essential for maintaining a robust anti-oxidative defence. It was proved
experimentally by deleting the per gene in mutant flies and it was observed that lack of per
increased susceptibility to H2O2 compared to wild-type flies, coinciding with enhanced
generation of mitochondrial H2O2 and decreased catalase activity due to oxidative damage.

17. Circadian rhythm sleep disorders
 Circadian rhythm sleep disorders are caused by desynchronization between internal
sleep-wake rhythms and the light-darkness cycle. Patients typically have insomnia,
excessive daytime sleepiness, or both, which typically resolve as the body clock
realigns itself.
 The cause of this misalignment may be internal (delayed or advanced sleep phase
syndrome) or external (jet lag, shift work).
 Patients with circadian rhythm disorders often misuse alcohol, hypnotics, and
stimulants.
 Circadian rhythm disorders include the following:
• Circadian rhythm sleep disorder, jet lag type (jet lag disorder)
• Circadian rhythm sleep disorder, shift work type (shift work disorder)
• Circadian rhythm sleep disorder, altered sleep phase types

18. Medications for Circadian Rhythm Disorders
 Melatonin- Melatonin supplements have been reported to be useful in treating jet-
lag and sleep-onset insomnia in elderly persons with melatonin deficiency. However,
melatonin supplements have not been approved by the FDA; therefore, it is not clear
as to how much melatonin is safe and effective.
 Melatonin Receptor Stimulant- Rozerem is a FDA approved drug used to promote
the onset of sleep and help normalize circadian rhythm disorders.
 Benzodiazepines- Short-acting benzodiazepines are often prescribed in the early
treatment of a circadian rhythm disorder and are used in conjunction with
behavioural therapy.
 Orexin receptor antagonists- Orexins are chemicals that are involved in regulating
the sleep-wake cycle and play a role in keeping people awake. This type of drug
alters the action of orexin in the brain. The only approved drug in this class is
Belsomra.
 CNS Stimulant- Provigil is a stimulant indicated to treat workers with sleep disorders
caused by their shift work.

19. REFERENCES
 Robinson.I and Reddy.A.B,2014 Molecular mechanisms of the circadian clockwork in
mammals.Elsevier. 588: 2477-2483.
 Dubowy.C and Sehgal.A,2017 Circadian Rhythms and Sleep in Drosophila
melanogaster.Genetics. 205: 1373-1397.
 Press release,2017 for the discovery of molecular mechanisms controlling the
circadian rhythm.The Nobel Prize.
 Ko.C.H and Takahashi.J.S,2006 Molecular components of the mammalian circadian
clock.Human Molecular Genetics. 15: 271-277.
 Chan.M-C, Spieth.P.M, Quinn.K, Parotto.M, Zhang.H and Slutsky.A.S,2012 Circadian
rhythms: From basic mechanisms to the intensive care unit.PMC. 40: 246-253.
 Krishnan.N, Davis.A.J and Giebultowicz.J.M,2008 Circadian regulation of response to
oxidative stress in Drosophila melanogaster.PMC. 374: 299-303.

20. THANK YOU