Ketamine’s antidepressant effect is mediated by energy metabolism and antioxidant defense system

This study (2017) examined the hippocampi of mice treated with ketamine in order to ascertain which pathways the drug affected. The researchers found, among other things, that ketamine tended to downregulate the ATP/ADP metabolite ratio.

Abstract

“Fewer than 50% of all patients with major depressive disorder (MDD) treated with currently available antidepressants (ADs) show full remission. Moreover, about one third of the patients suffering from MDD does not respond to conventional ADs and develop treatment-resistant depression (TRD). Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect, especially in patients suffering from TRD. Hippocampi of ketamine-treated mice were analysed by metabolome and proteome profiling to delineate ketamine treatment-affected molecular pathways and biosignatures. Our data implicate mitochondrial energy metabolism and the antioxidant defense system as downstream effectors of the ketamine response. Specifically, ketamine tended to downregulate the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) metabolite ratio which strongly correlated with forced swim test (FST) floating time. Furthermore, we found increased levels of enzymes that are part of the ‘oxidative phosphorylation’ (OXPHOS) pathway. Our study also suggests that ketamine causes less protein damage by rapidly decreasing reactive oxygen species (ROS) production and lend further support to the hypothesis that mitochondria have a critical role for mediating antidepressant action including the rapid ketamine response.”

Authors: Katja Weckmann, Michael J. Deery, Julie A. Howard, Renata Feret, John M. Asara, Frederik Dethloff, Michaela D. Filiou, Jamie Iannace, Christiana Labermaier, Giuseppina Maccarrone, Christian Webhofer, Larysa Teplytska, Kathryn Lilley, Marianne B. Müller & Christoph W. Turck

Summary

Mood disorders including major depressive disorder are a leading cause of disability worldwide. About one third of patients suffering from MDD do not respond to conventional antidepressants and suffer from treatment-resistant depression.

Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been demonstrated to have an antidepressant effect in patients suffering from TRD.

Ketamine was initially reported to act through the activation of the mammalian target of rapamycin complex 1 (mTORC1), but more recent studies implicate the ketamine metabolite (2 R, 6 R)-hydroxynorketamine (HNK) as the actual active compound.

Ketamine’s antidepressant activity is dependent on mitochondrial energy metabolism and cellular oxidative stress defense mechanisms.

Results

Ketamine’s fast antidepressant-like effects are reflected by metabolite ratios that are part of the ‘citrate cycle’ and the connected OXPHOS pathway, including ATP/ADP, NADH/NAD and GTP/GDP ratios. In addition, these metabolites significantly and strongly correlate with the FST floating time at 24 h. Ketamine treatment increases the levels of GTP, a by-product of succinate-CoA to succinate conversion in the citrate cycle. GTP levels correlate with FST floating time at both time points.

Ketamine treatment increased the activity of AMP-activated protein Kinase (AMPK), which leads to an increased energy demand and decreased energy supply. This resulted in an increased pAMPK/AMPK ratio and decreased ATP levels at the 24 h time point.

We next investigated whether the observed energy status alterations upon ketamine treatment were associated with changes in the expression of proteins related to cellular energy metabolism.

After proteomics data processing and missing value imputation, 889 MF and 1173 CF proteins were used for further statistical analyses. PLS-DA models were good for the 2 h time point, but weak for the 24 h time point. We combined the VIP-score of the PLS-DA with SAM analyses to obtain a reliable list of MF and CF proteins that significantly contribute to ketamine’s antidepressant effect. We found that 61 MF and 95 CF proteins were statistically significant altered at the 2 h time point.

Ketamine treatment affected mitochondrial energy metabolism, specifically OXPHOS, as indicated by increased levels of assembly factor 7 (Ndufaf7).

Ketamine reduced the levels of antioxidant molecules and enzymes like peroxiredoxins (Prdxs) that contribute to the antioxidant capacity of the cell. This resulted in decreased total antioxidant capacity and a decrease in protein carbonylation 72 h after ketamine injection.

Figure 1 shows the time-dependent hippocampal analysis of ATP/ADP, NADH/NAD, GTP/GDP, and guanosine triphosphate/guanosine diphosphate metabolite ratios after a single injection of a low dose of ketamine with the forced swim test (FST) floating time.

Discussion

Ketamine treatment reduced ATP and ADP levels and increased AMPK activation, suggesting an energy deficit. In the current study, we observed several metabolite level and metabolite ratio changes in the hippocampal mitochondria as early as 2 h after ketamine administration, reflecting the fast antidepressant effect of the drug. These alterations are part of the citrate cycle and glycolysis pathways.

Ketamine affects glutamate neurotransmission and is tightly linked to mitochondrial energy metabolism. Additionally, ketamine stimulates glucose utilization and ‘glycolysis’, the upstream pathway of the OXPHOS pathway, and is often comorbid with psychiatric disorders including MDD. Previous studies have connected antidepressant response with drugs elevating ATP levels. Ketamine treatment efficacy for TRD patients might be due to the drug’s beneficial effects on mitochondrial energy metabolism.

Ketamine plays a critical role in the development of MDD as well as AD treatment response, at least in a subset of patients with a defined symptomatology. A time-dependent proteomics profiling analysis was also performed.

Figure 3 shows the changes in membrane-associated and cytoplasmic proteins after a single injection of ketamine or vehicle.

Pathway enrichment analysis of significantly altered hippocampal proteins 2 h after a single injection of a low dose of ketamine.

Ketamine treatment affects the OXPHOS pathway, as evident by several complex I protein levels that correlate with FST floating time in a treatment time-dependent manner.

Ketamine stimulates glutamatergic neurotransmission and neuronal activity, ultimately resulting in a long-term potentiation (LTP)-like process. This LTP-like process requires energy in the form of ATP followed by AMPK activation, which we found in the present study. We found that the protein carbonylation decreases over time in ketamine-treated mice, and that the total antioxidant capacity and cytoplasmic Prdx1 and mitochondrial Prdx3 levels are lower at the 2 h and 72 h time points, suggesting an increased protein quality control process.

Ketamine decreased the floating time in the FST, and increased the OXPHOS complex I, cytochrome c1, heme protein, and complex I assembly factor levels. Additional behavioral analyses like the learned helplessness, chronic mild stress, and novelty were not carried out.

Figure 5 shows that ketamine treatment affects several antioxidant defense systems in the hippocampus, including peroxiredoxin 1 and peroxiredoxin 3. The data may be skewed by using several consecutive behavioral stress assays.

The experiments were conducted in accordance with European Communities Council Directive 86/609/EEC.

Methods

Isolation of cytoplasmic protein fraction (CF) and membrane-associated protein fraction (MF) was performed by repeated tissue homogenization and extraction of non-MF proteins and solubilization of MF proteins with sodium dodecyl sulfate (SDS). Pellets were rehomogenized in 0.1 M Na2CO3, 1 mM EDTA, 1 mM EDTA, phosphatase inhibitor cocktail 2 and 3, protease inhibitor cocktail tablets ‘cOmplete’, pH 11.3, and extracted with 5 M urea, 100 mM NaCl, 10 mM Hepes, pH 7.4 and 1 mM EDTA.

Western blot analysis of hippocampal MF and CF proteins was performed on 8-week-old male C57BL/6 mice treated with ketamine for 2 h, 14 h, 24 h and 72 h.

Hippocampal CF and MF proteins were mixed with 15N-labeled protein standards and separated by SDS-PAGE. The separated proteins were stained with Coomassie Brilliant Blue for 20 min and destained overnight.

The gel pieces were washed twice with 25 mM Na4HCO3/50% ACN, reduced with 1x DTT/25 mM NH4HCO3, alkylated with 100 l IAM, digested with 50 l trypsin solution, and extracted with 50 l 2% FA/50% ACN.

Hippocampal MF and CF proteins were identified and quantified using a Dionex Ultimate 3000 RSLC nanoUPLC coupled to a QExactiveTM OrbitrapTM mass spectrometer. Peptides were separated by reversed-phase chromatography and analyzed by high energy collisional dissociation.

Orbitrap raw files were converted to mzXML files using MSConvert software, and then iSPY was used to identify and quantify peptides. Mascot was used to perform a search against the SwissProt Mouse database and a decoy database. The intensities of isotopomeric peaks were calculated for each peptide using retention time and sequence information from MS1 spectra and Mascot search, respectively.

Metabolite intensities and protein ratios were median-normalised and auto-scaled for statistical analysis. Significant protein level changes 2 h and 24 h after ketamine treatment were identified using multivariate partial least squares-discriminant analyses and high-dimensional feature selection significance analysis.

Pathway analyses were performed using MetaboAnalyst64 and String. Proteomic pathway enrichment was assessed with an FDR of 0.1067,68.

K.W., M.D., J.H., R.F., F.D., C.W., M.F., J.I., C.L., G.M., L.T., J.M.A. and K.L. calculated metabolite pair ratios and wrote the paper.

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