Lecture 11: 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?

MOA?

Areas innervated by orexin/hypocretin Ns?

Excitatory or inhibitory?

Types and origin thereof?

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

Evolution of orexins and similarity to other pepties

Answer 3

preprohypocretin (precursor) in puffer fish and various frog species–> 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

Hypocretins/orexins and the hypothalamus

Answer 4

Hypothalamus specific peptides with neuroexcitatory activity.

Particularly in the lateral hypothalamus.

Hypocretins found in both cell bodies and terminal vesicles

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

Question 5

Orexin orexin-R discovery

Answer 5

Family of hypothalamic neuropeptides and GPCR that regulate feeding behaviour

Lateral hypothalamus again

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

fasted rat has high orexin –> reduced post meal (stimulates appetite)

Question 6

Orexin expression and projection

Answer 6

Only lateral hypothalamus, small number but extensive and diffuse projections

(hypo, basal forebrain, cortex, hippocampus, spinal cord)

Question 7

Orexin at the synapse

Answer 7

Excitatory, stimulates postsynaptic cell.

Orexin-ir (immunoreactive) 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.

Low concentrations of glucose activate orexin neurons (high inhibit… response to metabolic challenge)

Question 9

Leptin and ghrelin on orexin

Answer 9

Leptin inhibits orexin neurons (indicates fat reserves)

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

Question 10

How do mice lacking orexin neurons respond to fasting?

Answer 10

Super weirdly !!!!

Question 11

Orexin KO mice + sleep

Answer 11

WT: transitions smoothly between wake/SWS/REM.

KO: rapid transitions to REM from wake… behaviourally unstable

Have more transitions between all behavioural states, both light/dark compared 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 (+OX1R symptoms)

both contribute to narcolepsy-cataplexy syndrome but O2XR more important

Question 13

Other narcoleptic models

3

Answer 13

Doberman Pinschers:
Narcolepsy inherited as autosomal-recessive fully penetrant phenotype; 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 absent in lateral hypothalamus.

MCH mRNA (cocalised w OX) same in narcoleptic and controls.

Thus selective loss of orexin.

Question 15

Progression of narcolepsy

Answer 15

Onset is usually 15-30 –> OX-Ns lost over time

Maybe autoimmune

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

(similar to PD)

Question 16

Orexin wrt activity during REM and waking

triggers what?

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 return into wakefulness.

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

(LH injection with lentiviral vector!!)

Answer 17

channelrhodopsin (blue-R)-
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, fibre optic into brain –>no transition between wake/REM.

Halorhodopsin inhibition of OX-Ns (hyperpol) reduces daytime wakefulness

Question 18

orexin receptor antagonists

Answer 18

Almorexant: blocks both types of OX-Rs: 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.