Lecture 13: Orexins and Narcolepsy

Question 1

what is narcolepsy?

Answer 1

Sleep disorder incidence of 1 in 2000, appears between ages of 15 and 30 years, with 4 main symptoms:

Excessive daytime sleepiness with irresistible sleep attacks during the day.

Cataplexy (brief episodes of muscle weakness/paralysis precipitated by strong emotions such as laughter or surprise).

Sleep paralysis–abnormal episode of REM sleep atonia.

Hypnagogic hallucinations or dream-like images that occur at sleep onset.

Question 2

what are orexins/hypocretins?

Answer 2

Two carboxy-terminally amidated neuropeptides of related sequence.

Synthesized by a few 1000 cells in the brain.

Function via two G-protein-coupled receptors.
Differentially distributed throughout the brain.

Fibres containing these neuropeptides innervate many brain areas including those implicated in the control of cardiovascular function, ingestive behaviour, and sleep-wake cycles.

Peptides primarily excitatory and central injection stimulates food intake, blood pressure, locomotor activity, increases wakefulness, and suppressing REM sleep.

2 types,
Hcrt1, Hcrt2 or OX1R OX2R both from same precursor.

Question 3

precursor of orexins?

Answer 3

preprohypocretin. found in puffer fish and various frog species.

indicates that gene arose early in chordate lineage.

Sequence similarities with various members of incretin family. Secretin gene, indicate that Hcrt gene formed from secretin gene by three genetic arrangements.

3-D structure determined by NMR show similarities between secretins and hypocretins.

Question 4

USA scientists and hypocretin discovery?

Answer 4

found hypothalamus specific peptides with neuroexcitatory activity.

particularly in the lateral hypothalamus.

hypocretins found in both cell bodies and also in vesicles in terminals.
involved in transmission of info.

hypocretin increases the frequency of postsynaptic events in most cultured hypothalamic neurons.

Question 5

Orexin discovery?

Answer 5

family of hypothalamic neuropeptides and GPCR that regulate feeding behaviour.

lateral hypothalamus again.

rats.
found injection of orexin into the brain stimulates food intake, both OX1/2R. even in full satisfied rats.

fasted rat has high orexin, once ate reduced.

stimulates appetite.

Question 6

orexins found where?

Answer 6

only lateral hypothalamus. small number but extensive projections throughout the brain.

hypo, basal forebrain, cortex, hippocampus, spinal cord.

Question 7

orexin role?

Answer 7

excitatory, stimulates post synaptic cell.

Orexin-ir vesicles found
in presynaptic terminals
forming presumed
excitatory synapses.

Question 8

orexin neurons with green fluorescent protein (GFP) mice.

Answer 8

GFP expressed by the cells with orexin gene.
can make specific recordings.

low concentrations of glucose activate orexin neurones.

can determine metabolic state of animal since glucose crosses BBB.

Question 9

Leptin and Ghren effects on orexin?

Answer 9

sensitive to metabolic cues

Leptin inhibits orexin neurones, indicates fat reserves.

Ghrelin (metabolically comproised, peripheral hormone gets in brain)
activates orexin neurons.

Question 10

how do mice lacking orexin neurons respond to fasting?

Answer 10

unusual.

Question 11

orexin KO mice + sleep?

Answer 11

unusual sleep/wake cycles.

normal mice: transitions smoothly between wake/SWS/REM.

KO: rapid transitions to REM frmo wake.

EMG/EEG:
???

Narcoptic like bout:
EEG/EMG
straight into REM from wake, cannot move.
cannot maintain a behavioural state (behavioural state instability).

Have more transitions between all behavioural states, both light/dark comapred to WT.
also have cataplexy which WT don’t.

Question 12

mice with Ox1/2R KO

Answer 12

OX1R KO:
wake/REM & non-rem/REM transitions.
cataplexy, paralysis, SOREM periods.

OX2R KO:
wake/non-rem transitions.
non-REM sleep attacks, excessive sleepiness.

both contribute to narcolepsy-cataplexy syndrome.

slightly different roles.

Question 13

other narcoleptic models?

Answer 13

Doberman Pinschers:
Narcolepsy inherited as autosomal-recessive fully penetrant phenotype.

once orexin discovered, found out that defective canine narcolepsy gene encodes Hctr2/Ox2R receptor.

Truncated receptor protein which does not localize to membrane and does not bind ligands ie orexin.

Labradors: their Hcrtr2/Ox2R gene has a distinct mutation skipping exon 6 leading to truncated receptor protein.

Dachshunds: point mutation in Hcrtr2/Ox2R gene results in receptor protein that reaches membrane but cannot bind the hypocretin.

Question 14

humans narcoleptic vs controls

Answer 14

orexin mRNA in narcoleptics non existant in lateral hypothalamus.

MCH mRNA (other peptide found in hypothalamus) same in narcoleptic and controls.

therefore selective loss of orexin.

Question 15

model of narcolepsy?

Answer 15

not fully understood.
but since onset is usually 15-30 must have normal amount but lose them over time.

maybe auto immune.

similar to psarkinsons, loss of dopamine over time.

don’t show symptoms until lost a certain amount of orexin/dopamine

Question 16

orexin and behavioural states?

Answer 16

Orexin neurons silent during REM (PS) along with muscle atonia. They become active at end of REM sleep and precede return to waking and muscle tone.

trigger you to come back into wakefulness.

another Indicates Orexin/Hypocretin Neurons Active in Exploratory behaviour, Grooming, Eating, and Quiet Waking. Inactive in Slow Wave Sleep

Question 17

using optogenetics to selective activate orexin neurons with light

Answer 17

made orexin express channelrhodopsin/halorhodopsin.

blue light during REM sleep, rat wakes up.

can show that blue light activates channelrhodopsin and orexin have increased AP in vitro.

mice in vivo, fibreoptic into brain, stimualted via blue light and channel.
stimulated with blue light no transition between wake/REM.

Hallorhodopsin inhibits orexin activity via orange light.
orange light during the day reduces wakefulness.

Question 18

orexin receptor antagonists

Answer 18

Almorexant: blocks both types of orexin receptors: reduces time spent in awake states and facilitates maintenance of sleep state in many animals including humans. With people with insomnia, almorexant improved sleep efficiency (time spent asleep
when confined to a bed for 8h). Also decreased latency to sleep onset and decreased number of wake bouts. Also improved subjective reporting of sleep quality.

In rat, recently developed Ox2R antagonists more effective at reducing latency to sleep and increased NREM more potently
than almorexant. So, targeting Ox2R could be the route to follow.