mTOR-Dependent Synapse Formation Underlies the Rapid Antidepressant Effects of NMDA Antagonists

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mTOR-Dependent Synapse Formation Underlies the
Rapid Antidepressant Effects of NMDA Antagonists

• Nanxin Li, et al.
• Science 329, 959 (2010)
• R1 ??? V.S. ???2010.09.21

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Ketamine

• Antagonist of Glutamate NMDA receptor
• analgesia, anesthesia, sedation
• Psychological effects similar to phencyclidine
  (PCP)
• Dissociative state
• Hallucination
• Schizophrenia like symptoms

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Behavior model of depression

• Despair
• Forced Swim Test (FST)
• Learned hopelessness(LH)
• Anxiety
• Novelty-Suppressed Feeding Test (NSFT)

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Forced Swim Test (FST)

• 1st swim for 15 mins - 24 hrs later
• drug treatment - 24hrs later
• 2nd swim for 5 mins
• Duration of Immobility

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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

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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

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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).
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Signaling Pathway?
Ketamine
Anti-depression effects
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mTOR Signaling Pathway

• Mammalian target of rapamycin
• Rapamycin Sirolimus
• Immunosuppresant
• Ubiquitous
• Protein kinase
• translation regulation
• S6K
• 4E-BP

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Translation Regulation

• Protein Synthesis
• Translation from mRNA
• Initiation, elongation, termination
• Regulation
• eIF (eukaryotic initiation factor)
• eEF
• Ribosome

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• S6K
• S6K phosphorylates ribosomal protein S6,
• component of the small, 40S ribosomal subunit.
• 4E-BP
• eIF4E binding protein

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Methods

• Ketamine
• intraperitoneal injection
• Measurement
• Synaptoneurosome in Prefrontal Cortex

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Ketamine (ip) induce transient, dose-dependent
mTOR signaling in synaptoneurosome of PFC
10 mg/Kg
1 hr
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Electroconvulsive seizure, imipramine, or
fluoxetine did not significantly influence mTOR
signaling
ECS
Chronic (21D)
Acute (1hr)
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Ketamine
mTOR
4E-BP
S6K
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mTOR and growth factor signaling pathway

• MAPK/ERK cascade
• Extracellular signalregulated kinase
• Mitogen-activated protein kinase
• MAP3K -gt MAP2K -gt MAPK
• Akt/PKB pathway
• PI3K -gt PDK -gt Akt

C. A. Hoeffer, E. Klann, Trends Neurosci. 33, 67
(2010)
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Ketamine transiently and dose-dependently
increases pERK pAkt
1 hr
10 mg/Kg
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Pretreatment (30 mins before Ketamine)U0126 (20
nmol, ICV) inhibitors of ERKLY294002 (20 nmol,
ICV) inhibitor of PI-3k/Akt
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Ketamine
ERK, Akt
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Glutamate receptor

• Ionotropic Glutamate receptor
• NMDA receptor
• AMPA receptor
• Kainate receptor
• Metabotropic Glutamate receptor

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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).
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NBQX blocked ketamine activation of mTOR
signaling and upstream ERK Akt
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Ketamine
AMPA
ERK, Akt
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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).
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• 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

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Synape Formation ?
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Dendritic spine formation
Y. Yoshihara, M. De Roo, D. Muller, Curr. Opin.
Neurobiol. 19, 146 (2009).
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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

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ketamine increased spine density in distal and
proximal segments of the apical tuft
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Head diameter
Spine length
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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).
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EPSC measurement
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• Rapamycin was infused (0.2 nmol, ICV) 30 min
  before ketamine (10 mg/kg, ip)
• Rapid behavioral actions of ketamine require mTOR
  signaling

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• Infusion of rapamycin (0.01 nmol) into the mPFC
  blocked the antidepressant actions of ketamine
  (10 mg/kg, ip) in the FST and NSFT

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• 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

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• 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

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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

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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

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• Ro 25-6981 produced rapid (24 hr before),
  dose-dependent antidepressant action in the FST

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• Pretreatment with rapamycin (0.2 nmol, ICV)
  abolished the actions of Ro 25-6981 (10 mg/kg,
  ip) in FST and NSFT

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• mTOR signaling
• 4E-BP1, p70S6K, mTOR
• ERK and Akt signaling
• synaptic proteins
• Arc,
• PSD95, GluR1, synapsin I

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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

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Discussion

• Rapid antidepressant therapy
• mTOR signal pathway

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