mTOR-Dependent Synapse Formation
Underlies the Rapid Antidepressant
Effects of NMDA Antagonists
Nanxin Li, et al.
Science 329, 959 (2010)
R1 黃泰翰 V.S. 洪成志
2010.09.21
Ketamine
• Antagonist of Glutamate NMDA receptor
• analgesia, anesthesia, sedation
• Psychological effects similar to
phencyclidine (PCP)
– Dissociative state
– Hallucination
• Schizophrenia like symptoms
Behavior model of depression
• Despair
– Forced Swim Test (FST)
– Learned hopelessness(LH)
• Anxiety
– Novelty-Suppressed Feeding Test (NSFT)
Forced Swim Test (FST)
•
•
•
•
1st swim for 15 mins - 24 hrs later
drug treatment - 24hrs later
2nd swim for 5 mins
Duration of Immobility
Learned-Helplessness (LH)
• Inescapable footshock (IES)
– 60 footshocks,
– duration: 15 s,
– intershock interval: 60 s
• -24hrs- drug treatment -24hrs• Active avoidance testing
– 30 trials of escapable footshock
– duration: 35s
– Number of escape failure
recorded
Novelty-Suppressed Feeding
Test (NSFT)
•
•
•
•
•
food-deprived for 24hrs
placed in an open field
76.5 cm X 76.5 cm X 40 cm
food in the center
latency to feed
A Randomized Trial of an NMDA Antagonist in
Treatment-Resistant Major Depression
• Placebo controlled, double-blinded trial
• 18 subjects with treatment refractory MDD
• A single low dose of Ketamine (0.5mg/Kg)
C. A. Zarate Jr. et al., Arch. Gen. Psychiatry 63, 856 (2006).
Ketamine
Signaling Pathway?
Anti-depression
effects
mTOR Signaling Pathway
• Mammalian target of rapamycin
– Rapamycin = Sirolimus
• Immunosuppresant
• Ubiquitous
• Protein kinase
• translation regulation
– S6K
– 4E-BP
Translation Regulation
• Protein Synthesis
• Translation from mRNA
– Initiation, elongation, termination
• Regulation
– eIF (eukaryotic initiation factor)
– eEF
• Ribosome
• S6K
– S6K phosphorylates ribosomal protein S6,
– component of the small, 40S ribosomal
subunit.
• 4E-BP
– eIF4E binding protein
Methods
• Ketamine
– intraperitoneal injection
• Measurement
• Synaptoneurosome in Prefrontal Cortex
Ketamine (ip) induce transient, dose-dependent
mTOR signaling in synaptoneurosome of PFC
10 mg/Kg
1 hr
ECS
Electroconvulsive seizure,
imipramine, or fluoxetine
did not significantly influence
mTOR signaling
Acute (1hr)
Chronic (21D)
Ketamine
4E-BP
mTOR
S6K
mTOR and growth
factor signaling pathway
• MAPK/ERK cascade
– Extracellular signal–regulated kinase
– Mitogen-activated protein kinase
– MAP3K -> MAP2K -> MAPK
• Akt/PKB pathway
– PI3K -> PDK -> Akt
C. A. Hoeffer, E. Klann, Trends Neurosci. 33, 67 (2010)
10 mg/Kg
1 hr
Ketamine transiently and dosedependently increases pERK & pAkt
Pretreatment (30 mins before Ketamine):
U0126 (20 nmol, ICV): inhibitors of ERK
LY294002 (20 nmol, ICV): inhibitor of PI-3k/Akt
Ketamine
ERK, Akt
4E-BP
mTOR
S6K
Glutamate receptor
• Ionotropic Glutamate receptor
– NMDA receptor
– AMPA receptor
– Kainate receptor
• Metabotropic Glutamate receptor
Antidepressant actions of
ketamine & AMPA receptor
• Glutamate AMPA receptor
• NBQX
– a selective AMPA receptor inhibitor
– it attenuate the reduction in immobility
time induced by ketamine
– Pretreatment (10 min before Ketamine)
with NBQX (10 mg/kg, ip)
S. Maeng et al., Biol. Psychiatry 63, 349 (2008).
NBQX blocked ketamine activation of
mTOR signaling and upstream ERK & Akt
Ketamine
AMPA
ERK, Akt
4E-BP
mTOR
S6K
mTOR and synaptic protein synthesis
• Presynaptic protein:
– Synapsin I
• Postsynaptic proteins:
– PSD95, GluR1
• Arc:
– activity-regulated
cytoskeletal-associated
protein
C. A. Hoeffer, E. Klann, Trends Neurosci. 33, 67 (2010).
• Ketamine induces intermediate (1-2 hr) but transient
increase of Arc
• Ketamine induces delayed (2-72 hr) increase of
synaptic proteins
• Pretreatment (30 min) with a selective mTOR inhibitor,
rapamycin (0.2 nmol, ICV) block the effect
Synape Formation ?
Dendritic spine
formation
Y. Yoshihara, M. De Roo, D. Muller, Curr. Opin. Neurobiol. 19, 146 (2009).
Spine density analysis
• Layer V Pyramidal Cell in PFC
• Tips of tuft branches
approaching the pial
membrane
• Proximal tuft dendrites just
distal to the bifurcation
• Density
• Head diameter
• Length
ketamine increased spine density in distal
and proximal segments of the apical tuft
Head diameter
Spine length
Excitatory postsynaptic
current (EPSC)
• Apical dendrites of Layer V pyramidal cells in
mPFC
– 5-HT and Hypocretin Increase EPSCs
• Restraint stress
– 5-HT and hypocretin-induced EPSCs decreased
– Apical tuft dendritic branch length and spine
density decreased
R. J. Liu, G. K. Aghajanian, Proc. Natl. Acad. Sci. U.S.A. 105, 359 (2008).
EPSC measurement
• Rapamycin was infused (0.2 nmol, ICV) 30
min before ketamine (10 mg/kg, ip)
• Rapid behavioral actions of ketamine require
mTOR signaling
• Infusion of rapamycin (0.01 nmol) into the
mPFC blocked the antidepressant actions of
ketamine (10 mg/kg, ip) in the FST and NSFT
• Pretreatment with inhibitors of
– ERK (U0126, 20 nmol, ICV) or
– PI3 kinase / Akt (LY294002, 20 nmol, ICV)
• block of ketamine effects in FST and NSFT
• Learned helpless with Inescapable Shock (IES)
– Synapsin I, PSD95 and GluR1
• Ketamine given 24hr after IES
• Tissue collected 24 hr after ketamine
• Single dose of ketamine reverse this effect
• Pretreatment with rapamycin (ICV, 30 min before) block ketamine effect
Dose-dependent ketamine
antidepressant action
• FST
• Low dose
– 10 mg/kg
• high anesthetic dose
– 80 mg/kg
• Similar with dose of
ERK, Akt, and
mTOR induction
Ketamine for Depression ?
• Risk of abuse and adverse effects
• Ro 25-6981
– selective NMDA receptor subunit 2B
(NR2B) antagonists
• NMDA Receptor = 2 NR1 + 2 NR2
– NR1: 8 subtype
– NR2: 4 subtype
• NR2A, NR2B, NR2C, NR2D
• Ro 25-6981 produced rapid (24 hr before),
dose-dependent antidepressant action in the
FST
• Pretreatment with rapamycin (0.2 nmol, ICV)
abolished the actions of Ro 25-6981 (10
mg/kg, ip) in FST and NSFT
• mTOR signaling
– 4E-BP1, p70S6K, mTOR
• ERK and Akt signaling
• synaptic proteins
– Arc,
– PSD95, GluR1, synapsin I
Conclusion
• Rapid antidepressant
actions of ketamine
• Fast activation of mTOR
signaling in PFC
• Rapid and sustained
elevation of synapse
associated proteins and
spine number
• Elevated 5-HT
neurotransmission
Discussion
• Rapid antidepressant therapy
– mTOR signal pathway
Thank you for listening !