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

1. 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

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

3. Behavior model of depression • Despair • Forced Swim Test (FST) • Learned hopelessness(LH) • Anxiety • Novelty-Suppressed Feeding Test (NSFT)

4. Forced Swim Test (FST) • 1st swim for 15 mins - 24 hrs later • drug treatment - 24hrs later • 2nd swim for 5 mins • Duration of Immobility

5. 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

6. 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

7. Placebo controlled, double-blinded trial 18 subjects with treatment refractory MDD A single low dose of Ketamine (0.5mg/Kg) A Randomized Trial of an NMDA Antagonist in Treatment-Resistant Major Depression C. A. Zarate Jr. et al., Arch. Gen. Psychiatry 63, 856 (2006).

8. Ketamine Signaling Pathway? Anti-depression effects

10. mTOR Signaling Pathway • Mammalian target of rapamycin • Rapamycin = Sirolimus • Immunosuppresant • Ubiquitous • Protein kinase • translation regulation • S6K • 4E-BP

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

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

15. Methods • Ketamine • intraperitoneal injection • Measurement • Synaptoneurosome in Prefrontal Cortex

16. Ketamine (ip) induce transient, dose-dependent mTOR signaling in synaptoneurosome of PFC 10 mg/Kg 1 hr

17. ECS Electroconvulsive seizure, imipramine, or fluoxetine did not significantly influence mTOR signaling Chronic (21D) Acute (1hr)

18. Ketamine 4E-BP mTOR S6K

20. 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)

21. Ketamine transiently and dose-dependently increases pERK & pAkt 1 hr 10 mg/Kg

22. Pretreatment (30 mins before Ketamine):U0126 (20 nmol, ICV): inhibitors of ERKLY294002 (20 nmol, ICV): inhibitor of PI-3k/Akt

23. 4E-BP mTOR S6K Ketamine ERK, Akt

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

25. 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. Psychiatry63, 349 (2008).

26. NBQX blocked ketamine activation of mTOR signaling and upstream ERK & Akt

27. 4E-BP mTOR S6K Ketamine AMPA ERK, Akt

29. 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).

30. 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

31. Synape Formation ?

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

33. 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

34. ketamine increased spine density in distal and proximal segments of the apical tuft

36. Head diameter Spine length

38. 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).

39. EPSC measurement

43. Rapamycin was infused (0.2 nmol, ICV) 30 min before ketamine (10 mg/kg, ip) • Rapid behavioral actions of ketamine require mTOR signaling

44. Infusion of rapamycin (0.01 nmol) into the mPFCblocked the antidepressant actions of ketamine (10 mg/kg, ip) in the FST and NSFT

45. 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

46. 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

47. 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

48. 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

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

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