Antidepressant action of ketamine via mTOR is mediated by inhibition of nitrergic Rheb degradation

Abstract

As traditional antidepressants act only after weeks/months, the discovery that ketamine, an antagonist of glutamate/N-methyl-D-aspartate (NMDA) receptors, elicits antidepressant actions in hours has been transformative. Its mechanism of action has been elusive, though enhanced mammalian target of rapamycin (mTOR) signaling is a major feature. We report a novel signaling pathway wherein NMDA receptor activation stimulates generation of nitric oxide (NO), which S-nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH complexes with the ubiquitin-E3-ligase Siah1 and Rheb, a small G protein that activates mTOR. Siah1 degrades Rheb leading to reduced mTOR signaling, while ketamine, conversely, stabilizes Rheb that enhances mTOR signaling. Drugs selectively targeting components of this pathway may offer novel approaches to the treatment of depression.

Conflict of interest statement

Conflict of interest: The authors declare none.

Figures

Fig. 1. NMDA signaling induces proteasome-mediated Rheb degradation

Means ± SEM are shown. a, NMDA dose-response in cortical cultures showing a dose-dependent decrease in Rheb levels and mTOR activity. b, Injecting mice with 10 mg/kg ketamine i.p. rapidly upregulates Rheb and activates mTOR in the hippocampus. Graphs show quantification of western blot band intensities normalized to loading control. n = 6–8 mice/group. [* P < 0.05, ** P < 0.01; two-tailed t test]. c, Immunoprecipitation of Rheb from cortical cultures treated with the proteasome inhibitor MG132 (2 μM) shows mono- and polyubiquitylation of Rheb. MG132 treatment leads to accumulation of polyubiquitylated Rheb. d, WB and quantification of Rheb and phospho-p70S6 kinase levels following treatment of cortical culture with the proteasome inhibitor bortezomib [* P < 0.05; two-tailed t test]. e, NMDA stimulation of cortical cultures for 15 min decreases Rheb and phospho-p70S6 kinase levels, which is reversed by treatment with the proteasome inhibitor clasto-lactacystin β-lactone (CLβL) as demonstrated by WB. f, Graph shows quantification of band intensities normalized to loading control from 3 replicates [* P < 0.05; two-tailed t test].

Fig. 2. Nitric oxide and NMDA receptor regulate GAPDH-Rheb binding

Representative western blots (WB) are shown. Means ± SEM are shown. a, GAPDH co-immunoprecipitation with Rheb in cortical cultures is increased after 5 min treatment with NO donor CysNO and NMDA determined by western blot (WB). NMDA-induced increase in GAPDH-Rheb binding is blunted by the selective potent GAPDH nitrosylation inhibitor CGP3466B (2 nM) and by NOS inhibitors L-NAME & 7-NI (10 μM). b, Graph shows quantification of band intensities normalized to loading control from 3 replicates [ * P < 0.05; *** P < 0.001; ANOVA (Fisher’s LSD)]. c, WB showing GST pull down assay in HEK 293 cells. HA-C150S mutant of GAPDH binds to GST-Rheb with less affinity compared to HA-WT GAPDH. d, Graph shows quantification of band intensities normalized to loading control from 3 replicates [* P < 0.05; two-tailed t test]. e, WB showing increased levels of GST Rheb expression in HEK 293 cells co-transfected with increasing levels of GAPDH shRNA plasmid. f, WB showing decreased levels of GST Rheb expression in HEK 293 cells co-transfected with WT but not C150S mutant of HA-GAPDH. g, Graph shows quantification of band intensities normalized to loading control from 3 replicates [* P < 0.05; two-tailed t test]. h, NMDA stimulation of cortical cultures elicits a time-dependent decrease in Rheb levels in WT and iNOS KO but not in nNOS KO mice-derived neurons.

Fig. 3. Siah1 directly binds Rheb and acts as its E3 ubiquitin ligase

Means ± SEM are shown. a, NMDA treatment time-course in cortical cultures shows Siah1 and GAPDH co-immunoprecipitation with Rheb increases following NMDA stimulation as determined by WB. b, Graph shows quantification of band intensities normalized to loading control from 3 replicates [ * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ANOVA (Fisher’s LSD test). c, Western blot showing GST pull down assay using purified proteins. GST-Siah1 binds directly to Rheb and GAPDH. CysNO increases the binding between GST-Siah1, Rheb and GAPDH. e, Knockdown of Siah1 in cortical cultures using lentiviral shRNA leads to up-regulation of Rheb protein levels. f, Graph shows quantification of band intensities normalized to loading control from 5 replicates [* P < 0.05; two-tailed t test].

Fig. 4. Rapid antidepressant-like behavioral effects of the GAPDH nitrosylation inhibitor CGP3466B require mTOR

Means ± SEM are shown. Behavioral experiments were scored in a blinded fashion. a, Schematic diagram of experimental time line. FST forced swimming test; NSFT, novelty suppressed feeding test. b, FST in mice showing reduction in immobility or freezing time 30 min after 0.6 mg/kg of CGP3466B reversed by 5 mg/kg rapamycin. Control n = 36; Rapamycin n = 15, CGP3466B n = 35; Rapamycin-CGP3466B n = 20; ANOVA; P = 0.0003; Newman-Keuls multiple comparisons test: *** P < 0.001; * P < 0.05. c, NSFT in mice showing reduction in latency to feed 30 min after 0.6 mg/kg of CGP3466B reversed by 5 mg/kg rapamycin. Control n = 31; Rapamycin n = 15, CGP3466B n = 20; Rapamycin-CGP3466B n = 15; ANOVA; P = 0.008; Newman-Keuls multiple comparisons test: * P < 0.05; ** P < 0.01.