Lecture 12: Genetics of sleep disorders

Question 1

where are the main outputs of the SCN?

Answer 1

PVN: paraventiruclar nucleus
cortisol, melatonin

MPO: Medial preoptic nucleus
thermoregulation, GnRH

DMH: Dorsomedial hypothalamus
feeding

all via SPZ or direct to SCN.

Rhythmic SCN output creates an environment permissive to sleep or wake:
Low body temp. during inactive
High cortisol at start of active phase
High melatonin during night

Question 2

role of MPO

Answer 2

sex hormone secretion and thermoregulation, reduces temp during sleeping.

Question 3

role of PVN

Answer 3

neuroendocrine control, increased melatonin at night, increased cortisol just before waking.

Question 4

role of DMH

Answer 4

most important, feeding.
has outputs to lateral hypothalamus (orexin).
and VLPO (sleep promoting neurons).

Question 5

DMH lesions in rodents?

Answer 5

arhythmic sleep/wake.

Question 6

what controls sleep?

Answer 6

Involves interplay of homeostatic, circadian processes & light

Homeostatic: adenosine build-up/VLPO activation

Circadian: SCN output to DMH/ regulation of LH orexin and VLPO neurons

Light: Effects on SCN and Sleep/wake regulatory centres

Question 7

What causes delayed/advanced phase sleep syndromes? (brief)

Answer 7

involve genetic differences in clockwork

Question 8

What causes Irregular/non-24 sleep syndromes? (brief)

Answer 8

impaired clock function/photoentrainment

Question 9

What causes Insomia/Hypersomnia? (brief)

Answer 9

Causes probably unrelated to clock

Question 10

What is delayed sleep syndrome/advanced?

Answer 10

Normal length of sleep (around 8hr) but genetic differences in clockwork means either delayed/advanced.

Question 11

What is irregular sleep?

Answer 11

Normal total length of sleep but irregular or scattered throughout the day in naps.

(no clock, ie from tumour in pituitary gland compressing SCN)

Question 12

What is non 24hr sleep?

Answer 12

Not a 24hr cycle, occurs in lost eyes or dmg to HPT.

clock works but cant entrain to LD.

Question 13

What is insomnia/hypersomnia?

Answer 13

Not enough or too much sleep.

Question 14

How can you measure sleep?

Answer 14

polysomonography.

The PSG monitors many body functions including brain (EEG), eye movements (EOG), muscle activity or skeletal muscle activation (EMG) and heart rhythm (ECG) during sleep.

Question 15

How can you measure circadian rhythms?

Answer 15

melatonin rhythm, other hormones or body temperature.

Question 16

Problems with measuring sleep (properly vs others)

Answer 16

Long, expensive, easy to disturb, unnatural sleeping environment.

Can get just as good data from questionaires or fitness trackers or apps etc.
can combine with blood samples to relate to circadian genetics.

Question 17

What are the various chronotypes?

Answer 17

morning “lark” 15-20% of population:
midsleep 00:00-03:00

indifferent 60-70%:
midsleep 03:00-08:00

evening “owl” 15-20%:
midsleep 08:00-12:00

(midsleep hours are for free day)

Question 18

What factors influence chronotype?

Answer 18

gender, age, light exposure/latitude within time zone.

genetics!!!

Question 19

Predictor of human chronotypes

Answer 19

through skin biopsy.

Skin samples of early/late chronotypes, infected culture with lentivirus (expresses Bmal1:luciferase)

Circadian period length in fibroblasts predicts larks vs. owls

Question 20

What modulates the clock speed/chronotype?

Answer 20

Degradation rate of Per via CK1ε/δ.

F-Box proteins Fbxl3 and Fbxl21 control CRY degradation

Question 21

PER3 Variable-Number Tandem-Repeat.

Answer 21

(interaction sites for CK1 and CRY)

hPER3 gene has a repeated sequence fo DNA motifs either 4 or 5 (2 copies thus 4/4, 4/5 or 5/5)

PER34/4 is associated with evening preference and delayed sleep phase.

PER35/5 is strongly predictive of extreme morning preference, increased evening sleep drive, increased deep sleep, early arousal; very poor performance if sleep deprived.
~10% of UK “long variant”

Question 22

What is Familial Advanced Sleep Phase Syndrome (FASPS)?

Answer 22

Heritable (& rare) form of extreme early chronotype, autosomal dominant:
Sleep: ~7:30pm
Wake: ~4:00am

Specific mutations: Per2(S662G); CK1δ (T44A, H46R)
All alter phosphorylation of PER protein and speed up clock, 23.3hr vs 24.2hr cycle.

CK1ɛtau (T178C substitution) in mice or hamsters = 20h clock!

Question 23

How does altered PER protein stability impact on the speed of the circadian clock in FASPS?

Answer 23

Increased PER degradation (fast pathway) speeds up clock

Reduced PER degradation -> constant repression of Clock/Bmal1 (Slow clock)

Question 24

What does CK1δ/ε inhibition cause?

Answer 24

Lengthened clock period–>slows down molecular clock (PER2::luc rhythms in SCN slices); slows down behavioural rhythms

High dose up to 7h slower.

Question 25

PER2 phosphorylation

Answer 25

PER2 has 21 phosphorylation sites, role of phosphorylation at sites if complex and interdependent

Some act as priming sites

Some increase speed of nuclear translocation (PER1 moving into the nucleus).

some increase ability of PERCRY to inhibit BMAL/CLOCK.

Others increase PER degredation.

Question 26

FASPS MT (molecular… cycloheximide experiment)

function of cycloheximide

FASPS mimic vs phosphomimic

Cells no expressing CK1

Dead CK1

Conclusions

Answer 26

FASPS mutation reduces ‘FASPS site’ phosphorylation but increases per degradation.

(cycloheximide blocks protein synthesis, so can measure degradation rate)

FASPS mimic Per2 degradation faster than in Phosph-mimic

Cells not expressing CK1 have no difference in PER2 expression –>If add CK1ε FASPS has decreased PER2 so degradation has increased.

Dead CK1ε has no effect.

Thus FASPS enhances the ability of CK1 enzyme to degrade PER2.

Question 27

Degredation of PER2 (pathways)

Answer 27

β-TrCP site has instant degradation by CK1d/e

FASP site takes longer to degrade, phosphorylated by other kinases, but allowed CK1d/e to target other sites further down the pathway.

FASPS mutation can’t phosphorylate the FASP pathway, so uses the faster β-TrCP pathway and accelerates the clock.

Question 28

what is delayed sleep phase syndrome?

risk factors/ mutations

point mutations in mcie

Answer 28

Extreme late chronotype -
Sleep onset insomnia, inability to wake up at a conventional time
Up 16% of late adolescents.

No single mutation like FASPS in humans.

PER3 4/4 increases chances.

Mis-sense mutation (V647G) in Per3 increases risk of developing DSPS.

Mutation in CK1ε (S408N) gene decreases risk of developing DSPS.

Point mutations that dramatically lengthen period have been identified in mice

Afterhours (C358S) and overtime (I364T) mutations in FBXL3 
-clock period >26hr
-block binding to CRY and
subsequent targeting for
degradation

Question 29

gene and phenotype?

Answer 29

TABLE

Question 30

How can we fix circadian related sleep disorders?

Light

Melatonin

Pharmacological intervention

LiCl

Answer 30

1. Using light:
   Regardless of condition (ASPS/DSPS/jet-lag etc.) approach should be:

Up to 2h after desired wake-up time: maximise activation of melanopsin (while making colour resemble daylight i.e. white)

At least 2h before desired sleep time: minimise activation of melanopsin (while making colour resemble ‘night’ i.e. bluer)

2. Melatonin:
   Used alone or in combination with light for many sleep and circadian-related disorders.
   e.g. melatonin agonist
   (Tasimelteon) In totally blind patients with non 24-h sleep syndrome.
3. Direct pharmacological targeting of the clock:
   reveb ligands, CK1 inhibitors, CRY activators. check papers etc.

LiCl used clinically for treatment of bipolar disorder – lengthens period!