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).

Signaling Pathway?

Ketamine

Anti-depressioneffects

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

Electroconvulsive seizure, imipramine, or fluoxetine

did not significantly influence mTOR signaling

ECS

Acute (1hr) Chronic (21D)

Ketamine

mTOR4E-BP 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)

Ketamine transiently and dose-dependently increases pERK & pAkt

10 mg/Kg 1 hr

Pretreatment (30 mins before Ketamine):U0126 (20 nmol, ICV): inhibitors of ERK

LY294002 (20 nmol, ICV): inhibitor of PI-3k/Akt

Ketamine

mTOR4E-BP S6K

ERK, Akt

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

mTOR4E-BP S6K

ERK, Akt

AMPA

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 !