Acute Amino Acid d-Serine Administration, Similar to Ketamine

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Acute amino acid D-serine administration, similar to ketamine, produces antidepressant-like effects through the identical mechanisms I-Hua Wei, Kuang-Ti Chen, Mang-Hung Tsai, Ching-Hsiang Wu, Hsien-Yuan Lane, and Chih-Chia Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04217 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 23, 2017

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Journal of Agricultural and Food Chemistry

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Acute amino acid D-serine administration, similar to ketamine, produces

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antidepressant-like effects through the identical mechanisms Title Page I-Hua Wei†, Kuang-Ti Chen‡, Mang-Hung Tsai†, Ching-Hsiang Wu§, Hsien-Yuan Lane‡,∥, ⊥,

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Chih-Chia Huang*,‡,∥,⊥

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Author Affiliations:

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†

Department of Anatomy, China Medical University, Taichung, Taiwan

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‡

Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan

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§

Department of Anatomy, College of Medicine, Taipei Medical University, Taipei, Taiwan Brain Disease Research Center & Department of Psychiatry, China Medical University

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Hospital, Taichung, Taiwan ⊥ Department of Psychiatry, China Medical University, Taichung, Taiwan

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∥

*

Corresponding author. Dr Chih-Chia Huang, Department of Psychiatry, China Medical University Hospital, Taichung, Taiwan, No. 2, Yuh-Der Road, Taichung, Taiwan. Tel:886-4-22052121 ext 1015, Fax:886-4-22361230, E-mail: [email protected]

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ACS Paragon Plus Environment

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Abstract

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D-serine is an amino acid and can work as an agonist at the glycine sites of

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N-methyl-D-aspartate receptor (NMDAR). Interestingly, both types of glutamatergic

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modulators, NMDAR enhancers and blockers, can improve depression through common

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targets, namely alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionaic acid receptors

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(AMPARs) and mammalian target of rapamycin (mTOR). To elucidate the cellular signaling

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pathway underlying this counterintuitive observation, we activated NMDARs in rats by using

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D-serine. Saline, ketamine (NMDAR antagonist), and desipramine (tricyclic antidepressant)

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were used as controls. The antidepressant-like effects of all agents were evaluated using the

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forced swim test. The activation of the AMPAR–mTOR signaling pathway, release of

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brain-derived neurotrophic factor (BDNF), and alteration of AMPAR and NMDAR

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trafficking in the hippocampus of rats were examined. A single high dose of D-serine exerted

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an antidepressant-like effect that was mediated by rapid AMPAR-induced mTOR signaling

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pathway and increased BDNF proteins, identical to that of ketamine. Furthermore, in addition

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to the increased protein kinase A phosphorylation of the AMPAR subunit GluR1 (an

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indicator of AMPAR insertion in neurons), treatment with individual optimal doses of

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D-serine and ketamine also increased adaptin β2–NMDAR association (an indicator of the

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intracellular endocytic machinery and subsequent internalization of NMDARs). Desipramine

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did not influence these processes. Our study is the first to demonstrate an association between


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D-serine and ketamine; following adaptative regulation of AMPAR and NMDAR may lead to

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common changes of them. These findings provide novel targets for safer antidepressant

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agents with mechanisms similar to those of ketamine.

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Keywords: Amino acid, D-serine, ketamine, Depression, NMDA, AMPA, trafficking

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INTRODUCTION

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Increasing evidence suggests that the glutamatergic system plays a key role in the

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pathophysiology of psychiatric disorders including depression and schizophrenia.1-6

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Consequently, focusing on the glutamatergic system is a new target for treating depression

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and schizophrenia. For example, the noncompetitive N-methyl-D-aspartate receptor

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(NMDAR) antagonist ketamine at subanesthetic doses has been reported to cause mood

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enhancement in healthy individuals7 and rapidly ameliorate depressive symptoms in

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depressed patients.8-9 Subsequently, other drugs that antagonize NMDARs, such as

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CGP39551, CGP37849, AP7, and MK-801 also have antidepressant-like actions in

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preclinical

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alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated

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activated mammalian target of rapamycin (mTOR) signaling pathway has been found to be a

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key mechanism underlying the fast-acting antidepressant effects of NMDAR antagonist

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ketamine.11-12 In addition, NMDAR antagonists such as ketamine or phencyclidine have been

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reported to transiently induce schizophrenia-like symptoms.7, 13-15 Based on the prediction

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that the enhancement of NMDA function will improve the pathological state induced by

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NMDAR antagonists, the administration of several amino acids, such as glycine, D-serine

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(NMDAR coagonist), or sarcosine (glycine transporter 1 inhibitor), which activate NMDAR,

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can alleviate psychotic symptoms in patients with schizophrenia.16-19 NMDAR blockers and

studies.10

Furthermore,


the

rapid

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enhancers are thought to exhibit opposite effects. Paradoxically, preclinical and clinical

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studies have also found that the directly or indirectly enhanced NMDAR-mediated

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transmission by using NMDAR modulators, such as D-cycloserine (DCS), GLYX-13,

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sarcosine, SSR504734, D-serine, benzoate, and N,N-dimethylglycine, has antidepressant

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effects.20-27 Notably, the antidepressant-like actions of sarcosine, an NMDAR co-agonist28

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and a competitive GlyT1 inhibitor,29 are exerted through the activated AMPAR–mTOR

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signaling pathway,30 similar to those of ketamine, an NMDAR antagonist. In addition, a

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recent study also indicated that the baseline plasma D-serine level can be used to predict an

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antidepressant response to ketamine in patients with treatment-resistant depression.31-32 The

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reason that both NMDAR blockers and enhancers alleviate the depressive symptoms of

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through common targets remains unclear. Comparing NMDAR enhancers and blockers is

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essential for elucidating the cellular signaling mechanism underlying this counterintuitive

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observation, and the comparison may provide insights into the development of

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antidepressants. But, until now, the effects of NMDAR enhancers is not as well investigated

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as that of NMDAR blockers.

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The antidepressant action of some NMDAR enhancers, such as DCS or GLYX-13, may

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result from their potentially antagonist properties at high doses, which is consistent with the

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action of antagonists, such as ketamine.33-34 D-serine is a non-essential amino acid involved

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in glia-synapse interactions that has unique neurotransmitter characteristics.35 Unlike DCS,


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D-serine is a selective and potent co-agonist at the glycine site on NMDAR and coactivates

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the receptor along with glutamate, which does not act as an antagonist at high doses.36-38 This

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study investigated the acute effect of D-serine on immobility during the forced swim test

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(FST) for assessing the potential antidepressant response and activation of the mTOR

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signaling pathway and release of BDNF. Saline, ketamine (NMDAR antagonist), and

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desipramine (tricyclic antidepressant) were used as controls. We also examined whether the

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antidepressant-like effects of D-serine are mediated through the AMPAR–mTOR signal

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pathway.

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In addition, our previous study found that sarcosine increased AMPAR membrane

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insertion, leading to a hypothesis that sarcosine facilitates AMPAR insertion, amplifies the

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ratio of AMPAR:NMDAR in the postsynaptic membrane, and finally activates the mTOR

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signaling pathway.30 These effects are identical to those of ketamine. The activation of

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AMPAR is dependent on NMDAR and vice versa.39 Therefore, in addition to the

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investigation of the AMPAR insertion, this study also examined the effects of D-serine,

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ketamine, and desipramine on the NMDAR trafficking to further disclosure why NMDAR

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enhancers and blockers have the same antidepressant properties.

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MATERIALS AND METHODS Rats and Experimental design


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Male Wistar male rats aged 6-8 weeks old, weighing 250–350 gram were used in this

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study. The Experimental rats were supplied food and water ad libitum. All animal use and

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procedures conformed to the guide for the care and use of laboratory animals and was

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approved by the Institutional Animal Care and Use Committee of China Medical University,

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Taiwan (permit No. 104-263).

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We dissolved D-serine (Sigma, St. Louis, MO, USA), ketamine (Pfizer, New Taipei

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City, Taiwan), desipramine (Sigma, St. Louis, MO, USA), NBQX (Tocris, Bristol, UK), and

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rapamycin (Toku-E, Bellingham, WA, USA) in saline and all drugs were intraperitoneally

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injected. Experimental rats were treated with either saline or D-serine at different doses (560,

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1000, and 2000 mg/kg) randomly. Additionally, the NMDA antagonist ketamine (10 mg/kg)40

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and the tricyclic antidepressant desipramine (20 mg/kg)41-42 were used as positive controls.

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These rats were first subjected to a 15 minutes preswim 24 hours before the FST (Figure 1A).

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Next day, after 30 minutes of administration, the FST was conducted. Each group comprised

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10 rats. In another study, the rats were intraperitoneally injected with saline, D-serine,

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ketamine, and desipramine43 and at 30 minutes later, rats were screened in the elevated plus

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maze test (EPM) to assess the general locomotor activity (Figure 1B). Each experimental

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group consisted of 8–11 rats. After the EPM test, four rats per group were sacrificed

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immediately to remove hippocampus for further biochemical analysis.

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In addition, the 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, a


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AMPAR inhibitor) or rapamycin (an mTOR pathway inhibitor), was used to validate whether

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D-serine might provide antidepressant-like actions mediating by these signaling pathways

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directly (Figure 1C). Each group comprised 10 experimental rats. Saline, NBQX at 10

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mg/kg12 or rapamycin at 20 mg/kg44 was intraperitoneally injected 30 minutes before

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treatment with D-serine at 1000 mg/kg or saline. The treated-rats were assessed in the FST 30

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minutes later. In a separate study, sixteen rats were divided to four groups randomly, with

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four rats for each group (Figure 1D). Thirty minutes before intraperitoneally administration

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of D-serine at 1000 mg/kg, saline, NBQX at 10 mg/kg or rapamycin at 20 mg/kg was

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intraperitoneally injected. Thirty minutes after the final administration, these experimental

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rats were sacrificed to remove hippocampus for biochemical analysis.

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Besides, in this study, the assessment of the toxicity of D-serine was also carried out.

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Sixteen naïve rats were divided to four groups with four rats for each group randomly (Figure

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1E). The saline or D-serine at different doses (560, 1000, and 2000 mg/kg) was

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intraperitoneally injected. Thirty minutes following the injection, these experimental rats

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were killed to remove liver, kidney, hippocampus, and cortex and collect plasma from blood

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for toxicity assessment.

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FST and EPM test The FST and EPM procedures were performed as previously described.30 On the EPM,


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the total number of closed arm entries and distances travel were quantitatively measured as a

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index of general locomotor activity to assess the possibility of false-positive result in the

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FST.45-46

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Western blot analysis and Immunoprecipitation of AP2 with NMDARs

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The tissue processing and Western blotting were performed as previously described.30, 47

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The phosphorylated forms of mTOR (pmTOR), Akt (pAkt), extracellular signal-regulated

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protein kinase (pERK), (mTOR upstream kinases), BDNF protein, and PKA phosphorylation

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of AMPAR subunit GluR1 were analyzed through Western blotting. The endocytotic

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machinery of the NMDARs was analyzed through an immunoprecipitation assay of anti-AP2

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and NMDARs, which was adapted from a previous report.48

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Evaluation of Liver and Kidney Function Using

commercial

kits

(Wako,

Osaka,

Japan),

we

measured

the

alanine

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aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and

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creatinine levels in the plasma to detect liver and kidney function.

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In Situ Apoptosis Assay and Histological Analysis of the Liver, Kidney and Brain

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Tissues


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In situ apoptosis and histology were determined by fixing the liver, kidney and brain in

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buffered formalin, processing, embedding in paraffin, and then staining with hematoxylin and

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eosin (H&E). Stained slides were analyzed and captured on a Zeiss microscope (Zeiss,

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Oberkochen, Germany).

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Statistical analysis

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All data were reported as mean ± s.e.m. The Kolmogorov-Smirnov test and Levenes’s

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test were applied to test the assumption of normality and homogeneity of variance

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respectively. The behavioral experiment data showed a parametric distribution. In addition,

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biochemical experiment data about the expression of Western blot analysis and

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immunoprecipitation were quantified as fold changes in each band relative to saline-treated

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group, that were taken in triplicate readings. The level of saline-treated group set at 100 %.

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The parameters of biochemical data were tested concerning normality (Kolmogorov-Smirnov

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test) and the data showed a parametric distribution. Finally, data of the behavioral and

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biochemical experiments were evaluated using the one-way analysis of variance followed by

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Tukey post-hoc test (SPSS 12.0) for multiple comparisons. All statistical tests were 2-tailed.

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A p-value of less than 0.05 was regarded statistically significant. Each behavioral

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experimental group consisted of 8–11 rats. Each biochemical experimental group consisted of

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4 rats.


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RESULTS AND DISCUSSION

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Acute anti-depressant Effects of D-serine

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A dose-dependent reduction in the immobility time was observed when rats were

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injected with D-serine during the FST (Figure 2A). Compared with the saline-treated control

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rats, the rats treated with D-serine at high doses (1000 mg/kg and 2000 mg/kg) showed a

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significantly reduced immobility time. Ketamine and desipramine also significantly reduced

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immobility. Furthermore, to examine whether these compounds yielded a pseudo-positive

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effect during the FST, we measured the total number of closed arm entries and distances

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traveled in the EPM as indicators of general locomotor activity.45-46 At the doses tested,

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neither D-serine nor ketamine and desipramine increased locomotor activity; however, a high

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dose (2000 mg/kg) of D-serine significantly reduced the general activity.

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To evaluate whether the antidepressant-like effects is accompanied by an increase in the

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activated mTOR signaling and BDNF release, the levels of the phosphorylation of mTOR,

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Akt, ERK, and BNDF proteins were measured following D-serine treatment. The rats treated

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with D-serine at high doses (1000 mg/kg and 2000 mg/kg) exhibited significant increases of

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immunoreactivity of pmTOR, pAkt, and pERK (Figure 2B). The levels of total mTOR, Akt,

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and ERK remained unaltered. The increased pmTOR activity of the D-serine-treated rats was

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also confirmed in statistic analysis (Figure 2C). The similar trends were also noted in both


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mTOR upstream regulators (pAkt and pERK) (Figure 2C). Furthermore, compared with the

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saline-treated group, the rats in the D-serine group treated with all doses 560, 1000, and 2000

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mg/kg exhibited a significant increase in the BDNF level (Figure 2C). Similar effects of

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increased activation of mTOR signaling and BDNF protein level were observed in rats

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treated with the NMDAR antagonist ketamine at a subanesthetic dose but not in

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desipramine-treated ones.

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Role of AMPAR and mTOR signaling on the D-serine-induced antidepressant-like

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actions

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To investigate the involvement of AMPAR and mTOR signaling on the D-serine-induced

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antidepressant-like actions; NBQX (10 mg/kg), an AMPAR antagonist, and rapamycin (20

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mg/kg), an mTOR inhibitor, were applied. Figure 3A shows the NBQX administration 30

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min before D-serine treatment abolished the D-serine-induced antidepressant-like actions of

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decreased immobility. Similarly, rapamycin completely reversed the reduced immobility in

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the FST (Figure 3A). These findings indicate that the antidepressant-like properties of

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D-serine required the activations of mTOR signaling and AMPAR.

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Role of glutamatergic system on the D-serine-induced antidepressant-like actions and

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BDNF release


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We future studied whether the activity of AMPAR-mTOR signaling pathway and

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release of BDNF caused by D-serine treatment can be affected by pretreatment with NBQX

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or rapamycin. Similarly, acute D-serine treatment at 1000 mg/kg increased the

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immunoreactivity of pmTOR, pAkt, pERK, and BDNF significantly (Figure 3B). There were

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no changes for total mTOR, Akt, and ERK levels. Pretreatment with NBQX abolished the

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D-serine-induced increase in the immunoreactivity of pmTOR, pAkt, pERK, and BDNF

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proteins (Figure 3B). The increases of immunoreactivity of pmTOR and BDNF following

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D-serine treatment was completely reversed by rapamycin administration (Figure 3B), but

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increased pAkt and pERK levels induced by D-serine was not abolished (Figure 3B). These

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results suggest that D-serine stimulates the mTOR signaling pathway and BDNF release,

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which is dependent on AMPAR activation.

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Alteration of AMPAR membrane insertion and NMDAR membrane internalization

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following acute D-serine or ketamine treatment

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We determined whether D-serine regulates the phosphorylation of AMPAR subunit

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GluR1ser845 and co-precipitation of NMDARs together with AP2 after an acute in vivo

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treatment in rats to investigate the influence of D-serine on the AMPAR membrane insertion

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and NMDAR membrane internalization.49-53 As shown in Figure 4A, 4B, a dose-dependent

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increase in the phosphorylation of hippocampal GluR1ser845 and the level of NMDARs


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precipitating tightly with AP2 were observed in the rats treated with D-serine. When

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compared with the saline-treated controls, D-serine at high doses significantly increased the

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phosphorylation of GluR1ser845 in a dose-dependent manner (Figure 4A) and the association

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of AP2 and NMDARs (Figure 4A). Furthermore, ketamine at a subanesthetic dose as a

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positive control also significantly enhanced the phosphorylated GluR1ser845 and

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AP2–NMDAR association, which was not observed in desipramine-treated rats (Figure 4A,

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4B). These results are significantly consistent with those of mTOR activation and BDNF

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release.

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Pretreatment with NBQX abolished the D-serine-induced increased activity of

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pGluR1ser845. But, rapamycin pretreatment did not reverse the D-serine-induced increase

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(Figure 4C). Pretreatment with NBQX or rapamycin did not block the D-serine-induced

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increase in the AP2–NMDAR association (Figure 4D). The total GluR1 and AP2 levels

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remained unchanged (Figure 4). These data indicated that D-serine in a dose-dependent

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manner and ketamine at a subanesthetic dose enhanced AMPAR membrane insertion and

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NMDAR membrane internalization. The AMPAR activation is required for D-serine-induced

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AMPAR membrane insertion but not for the NMDAR membrane internalization.

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Effects of D-serine Exposure on the Levels of ALT, AST, BUN, and creatinine ALT and AST are indices of cellular necrosis and tissue damage in liver. The rats treated


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with D-serine at 560 mg/kg, 1000 mg/kg, and 2000 mg/kg showed no significant difference

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in mean values of the serum ALT (Figure 5A) and AST (Figure 5B) in comparison to those of

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saline-treated rats. In addition, BUN and creatinine are indices of cellular necrosis and tissue

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damage in kidney. The rats treated with D-serine at 560 mg/kg, 1000 mg/kg, and 2000 mg/kg

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showed no significant difference in mean values of the serum BUN (Figure 5C) and

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creatinine (Figure 5D) compared to saline-treated rats.

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Histological Analysis and TUNEL staining of the Liver, Kidney, Hippocampus and

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Cortex.

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Histological of Hematoxylin-eosin (H&E) staining and apoptosis TUNEL assay were

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performed to observe the histological change and dying cells of acute administration of

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D-serine at 560 mg/kg, 1000 mg/kg, and 2000 mg/kg on liver (Figure 6A), kidney (Figure

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6B), hippocampus (Figure 6C) and cortex (Figure 6D) in experimental rats. Analysis of H&E

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-stained liver slices of rats treated with normal saline, D-serine 560 mg/kg, 1000 mg/kg, and

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2000 mg/kg (Figure 6A) showed a general preservation of liver histoarchitecture, hepatocytes

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with a normal cytoplasmic eosinophilic aspect, and one or two nuclei with loose chromatin

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and evident nucleolus. No other visual signs of hepatotoxicity being observed after D-serine

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560 mg/kg, 1000 mg/kg, and 2000 mg/kg treatment.


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In kidney tissues, no histological architecture change was observed after administration

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of normal saline and D-serine (560 mg/kg, 1000 mg/kg, and 2000 mg/kg) (Figure 6B). The

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well-designated glomeruli have numerous capillary loops with surrounding proximal and

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distal convoluted tubules in the cortical region of kidney in control or different doses of

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D-serine treated groups (Figure 6B).

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H&E staining of hippocampal tissues in normal saline, D-serine 560 mg/kg, 1000 mg/kg,

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and 2000 mg/kg-treated rats showed normal histological features (Figure 6C). The

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hippocampal pyramidal neurons are regularly arranged, the nucleus is big and round, nucleoli

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are clear. There were no significant atrophy and histopathological changes in the

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hippocampal region of rats treated with normal saline or D-serine (Figure 6C).

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The results of H&E staining showed a normal distribution of neurons in the cerebral

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cortex of the normal saline group. The rounded nuclei, small neurites, pyramidal cells with

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elliptic neuronal bodies, well-defined nucleoli and a basophilic cytoplasm were observed.

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Compared with the saline-treated rats group, there were no morphological changes in the

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D-serine treated groups (Figure 6D). There were no TUNEL -positive cells were detected in

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the tissues of all groups (Figure 6).

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The present study provides convincing evidence that a single injection of amino acid

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D-serine, an NMDAR co-agonist, at high doses provides antidepressant-like effects during ACS Paragon Plus Environment

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the FST, and elicits its action, at the molecular level, in a manner identical to that of ketamine,

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an NMDAR antagonist, at subanesthetic dose. In addition, similar to ketamine, D-serine at

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high doses causes the rapid AMPAR-mediated mTOR signaling pathway activation.

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Interestingly, both of them increase the PKA phosphorylation of AMPAR subunit GluR1 and

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AP2–NMDAR association. Notably, acute treatment with the conventional antidepressant,

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desipramine, did not exhibit these effects. These findings are the first to demonstrate an

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association between D-serine and ketamine; following adaptative regulation of AMPAR and

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NMDAR at their individual optimal doses may lead to common changes of them.

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We observed that high doses of D-serine reduced immobility during the FST without an

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increase in general activity, thus demonstrating its antidepressant-like effect. These results are

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consistent with the reported antidepressant-like responses of other NMDAR enhancers.20-22, 30,

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a dose-dependent manner. Pretreatment with mTOR or AMPAR inhibitor significantly

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blocked the antidepressant-like effects in FST and the increases levels of activated form of

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mTOR signaling pathways proteins caused by D-serine. The present evidence clearly

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confirmed that the activated AMPA–mTOR signaling pathway is necessary for the

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antidepressant-like actions of the NMDAR co-agonist D-serine and, essentially, identical

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mechanisms underlie the antidepressant-like effects of ketamine at a subanesthetic dose.11 In

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addition to activation of mTOR, BDNF release also involves in the fast-acting

Moreover, a single injection of D-serine activated the mTOR signaling pathway rapidly in


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antidepressant-like effect of ketamine.55 Therefore, we evaluated whether D-serine altered the

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BDNF level. As expected, ketamine instantly increased the BDNF protein level. Interestingly,

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the rats treated acutely with a single dose of D-serine, similar to ketamine, produced an

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identical effect in a dose-dependent manner. The similar effects of D-serine and ketamine

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extend to the BDNF level. In this study, both pretreatment with NBQX and rapamycin

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reversed the D-serine-induced increase in the hippocampal BDNF protein level, thereby

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suggesting that mTOR activation leads to an increased BDNF release. But this is different;

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previous studies have indicated that the ketamine-induced mTOR activation lies downstream

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from increased BDNF mediated by AMPAR.56 Although the exact mechanism remains

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uncertain, the activated BDNF/mTOR signaling pathway also increased the number of cells

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in the hippocampal dentate gyrus and established a positive feedback loop of BDNF

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productions following the activation of mTOR.57-58 Rapamycin may deactivate the positive

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feedback loop, which in turn reduced the upstream BDNF.

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Furthermore, to study the effects of D-serine on both AMPAR and NMDAR trafficking,

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here, we measured the PKA site of AMPAR subunit GluR1 phosphorylation, which is an

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indicator for insertion of GluR1 membrane in neurons, and the AP2–NMDAR association,

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which is an indicator of NMDAR membrane internalization. The results demonstrated that

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D-serine rapidly increases AMPAR membrane insertion in dose-dependent manner. Moreover,

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our study is the first to demonstrate that a single dose of D-serine treatment caused the


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recruitment of the endocytic machinery to the NMDARs in vivo, thus leading to NMDAR

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internalization in a dose-dependent manner. These findings are consistent with those of a

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previous in vitro study, which showed that NMDAR internalization can be primed by an

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increase in glycine or D-serine for subsequent internalization on stimulation of glutamate and

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glycine sites.48 Notably, we observed that ketamine also exhibited increased effects on

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AMPAR membrane insertion and recruitment of the endocytic machinery to the NMDARs,

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which were identical to the change observed following the administration of D-serine at high

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dose. Thus, the enhancement of AMPAR insertion and NMDAR internalization represent

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common features of the treatment of both the NMDAR coagonist D-serine and antagonist

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ketamine at their optimal doses.

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The current findings clearly demonstrate that the activated AMPAR-mTOR signaling

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pathway is required for the behavioral actions of D-serine in the FST, importantly, showing a

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convergence with the mechanisms underlying the action of ketamine. When assessing the

341

AMPAR and NMDAR trafficking, we observed that the NMDAR coagonist D-serine

342

enhanced AMPAR insertion in a dose-dependent manner and theoretically caused the

343

subsequent upregulation of AMPARs. Furthermore, in this in vivo study, we demonstrated for

344

the first time that acute administration of D-serine could have led to the recruitment of AP2

345

for priming the internalization of NMDAR in a dose-dependent manner, consistent with a

346

previous in vitro study48 and theoretically have caused the subsequent downregulation of


19


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347

NMDARs. Following D-serine administration, particularly at high doses, the combined effect

348

of AMPR insertion and NMDAR internalization resulted in the high AMPAR:NMDAR ratio

349

in the postsynaptic membrane may favor AMPAR throughput and thereby contribute to an

350

activated mTOR signaling pathway, which in turn lead to antidepressant action. Notably, we

351

also found that ketamine at subanesthetic doses acted as a positive control increased AMPAR

352

insertion and NMDAR internalization, similar to the action of D-serine at higher doses.

353

Ketamine-primed NMDAR internalization is not entirely unexpected because ketamine not

354

only acts as an antagonist of NMDAR but also increases extracellular glutamate levels at low

355

subanesthetic doses, whereas anesthetic doses reduce these levels.59 The stimulation of

356

metabotropic glutamate receptors (mGluR) with glutamate can activate NMDAR

357

internalization.52,

358

extracellular glutamate levels, followed by the mGluR stimulation active NMDAR

359

internalization. The consequence results from a combined effect of AMPAR and NMDAR

360

adaptative trafficking caused by NMDAR co-agonist D-serine and NMDAR antagonist

361

ketamine at their respective optimal doses, which in turn lead to identical changes at the

362

behavioral and molecular levels.

60

Therefore, ketamine at a low subanesthetic dose may increase the

363

Ketamine blocks NMDAR, whereas D-serine acts as an NMDAR co-agonist. In an

364

intuitive manner, the effects of D-serine and ketamine can be easily considered to be the

365

opposite. However, acutely increased levels of NMDAR co-agonist D-serine can lead to an


20

Page 21 of 46


366

adaptive downregulation and desensitization of postsynaptic NMDARs.48 Moreover, repeated

367

administration of NMDAR antagonist ketamine at high doses upregulates the NMDARs.61-63

368

Based on these observations, negative feedback for the regulation of NMDAR homeostasis

369

occurs in response to glutamatergic NMDAR modulators including both enhancers and

370

blockers. Hence, the combined effect of adaptive changes in AMPAR and NMDAR numbers

371

or sensitivity following ketamine or D-serine administration at their individual optimal doses

372

may provide a final common AMPAR:NMDAR ratio in the postsynaptic membrane, thereby

373

yielding convergent outcomes in their downstream mTOR signal pathway for antidepressant

374

action (Figure 7). Such mechanisms may apply broadly to the absence of the effect of

375

ketamine at high anesthetic dose. The current study and other works on the mechanisms

376

underlying the antidepressant action of ketamine are complementary and not contradictory to

377

each other. Our data indicated a novel mechanism in which the feedback induced by

378

glutamatergic modulators at their individual optimal doses results in increased NMDAR

379

internalization and AMPAR insertion finally, this combined effect lead to antidepressant

380

action. Clearly, additional studies are necessary to causally link the action of ketamine at

381

wide-range doses to the potential change in AMPAR and NMDAR trafficking.

382

Behavior analysis using FST revealed that D-serine at high doses, ketamine, and

383

desipramine exerted antidepressant-like effects. However, only D-serine and ketamine, but

384

not desipramine, increased activation of the mTOR signaling and release of BDNF following


21


Page 22 of 46

385

a single dose. The rapid induction of mTOR signaling and the increased BDNF level mediate

386

the fast-acting antidepressant effects of ketamine, which may represent a mechanism

387

common to other putative rapid antidepressants.11,

388

antidepressants do not rapidly increase the mTOR signaling and BDNF levels, elevated

389

BDNF and mTOR levels are only evident following chronic treatment, which corresponds to

390

the time course of the clinical effect.11, 55, 65 Therefore, the rapidly increased mTOR signaling

391

and BNDF level is proposed to be crucial for the development of antidepressants with

392

fast-acting onset. Our results from this series of experiments suggest that D-serine at high

393

dose might have rapid antidepressant effect in human, similar to that of ketamine.11 In this

394

preclinical study, these rats treated with D-serine at 560 to 2000 mg/kg once did not have

395

histological change and did not have impaired liver and kidney function. A clinical study also

396

reports doses of D-serine ranging from 30 to 120 mg/kg/d following a 4-week adjunctive

397

treatment were well tolerated in patients with schizophrenia; but, one patient did show a

398

nephrotoxic-like pattern at 120 mg/kg/day.66 The rats treated with D-serine at 1000 mg/kg

399

and 2000 mg/kg showed antidepressant-like responses, the equivalent doses in humans would

400

be predicted to be 135.7 mg/kg and 271.4 mg/kg respectively.67 Currently, clinical application

401

with D-serine at high dose remains lacking, but, present results are encouraging of further

402

study of higher-dose D-serine. Although much work is required for clarifying the actual

403

therapeutic potential of NMDAR co-agonist, taken together, the use of NMDAR co-agonists

55,


64

By contrast, traditional

22

Page 23 of 46

404


may be a novel therapeutic strategy to develop fast-acting antidepressants.

405

In conclusion, the amino acid D-serine has been demonstrated to play crucial roles in

406

brain development, synaptic plasticity, depression, and cognition.68-71 Our results provide

407

direct evidence that the NMDAR coagonist D-serine at high dose exerts antidepressant-like

408

effect via the rapidly activated AMPA–mTOR signaling pathway, which is identical to the

409

effects of ketamine at subanesthetic doses. Not just simple NMDAR inhibition or enhanced

410

AMPAR throughput, we hypothesize that the combined effect of adaptative regulation in

411

AMPAR and NMDAR induced by NMDAR co-agonist D-serine or antagonist ketamine

412

treatment may result in a common change to possess identical antidepressant-like action. The

413

results provide a novel opportunity for investigating the mechanisms that mediate rapid

414

antidepressant effects. Furthermore, our results may provide new insights into the

415

pathogenesis of depression. Most importantly, our behavioral and biochemical findings raise

416

the possibility that D-serine has the potential of being highly efficacious and safer to treat

417

depression, with mechanisms similar to those of ketamine.

418 419

ABBREVIATIONS USED

420

N-methyl-D-aspartate receptor, NMDAR;

421

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionaic acid receptors, AMPAR;

422

mammalian target of rapamycin, mTOR;


23


423

extracellular signal-regulated protein kinase, ERK;

424

AMPA GluR1 serine845, GluR1ser845;

425

brain-derived neurotrophic factor, BDNF;

426

phospho-mTOR, pmTOR;

427

phospho-extracellular signal-regulated protein kinase, pERK;

428

phospho-Akt, pAkt;

429

phospho-AMPA GluR1 serine845, pGluR1ser845;

430

alanine aminotransferase, ALT;

431

aspartate aminotransferase, AST;

432

blood urea nitrogen, BUN;

433

Hematoxylin-eosin, H&E;

434

glycine transporter 1, GlyT1;

435

D-cycloserine, DCS;

436

forced swim test, FST;

437

elevated plus maze test, EPM;

438

metabotropic glutamate receptors, mGluR;

Page 24 of 46

439 440 441

Acknowledgements This work was supported by the Ministry of Science and Technology, Taiwan (MOST


24

Page 25 of 46


442

105-2314-B-039-027, MOST 105-2320-B-039-013-MY3, and MOST 106 - 2314 - B - 039 -

443

029 - MY3), China Medical University Hospital, Taiwan (DMR-105-075, DMR-106-096).

444 445

Notes

446

The authors declare no competing financial interest.

447 448

ASSOCIATED CONTENT

449

* S Supporting Information:

450

The supporting Information is available free of charge on the ACS Publications website at

451

DOI:

452

Composition of Materials and methods

453 454

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human. J Basic Clin Pharm 2016, 7 (2), 27-31. 68. Avellar, M.; Scoriels, L.; Madeira, C.; Vargas-Lopes, C.; Marques, P.; Dantas, C.;

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Manhaes, A. C.; Leite, H.; Panizzutti, R., The effect of D-serine administration on cognition

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and mood in older adults. Oncotarget 2016, 7 (11), 11881-8. 69. Nomura, J.; Jaaro-Peled, H.; Lewis, E.; Nunez-Abades, P.; Huppe-Gourgues, F.;

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Cash-Padgett, T.; Emiliani, F.; Kondo, M. A.; Furuya, A.; Landek-Salgado, M. A.; Ayhan, Y.;


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Kamiya, A.; Takumi, T.; Huganir, R.; Pletnikov, M.; O'Donnell, P.; Sawa, A., Role for

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neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult

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Pick1 knockout mice. Mol Psychiatry 2016, 21 (3), 386-93. 70. Gomez-Galan, M.; De Bundel, D.; Van Eeckhaut, A.; Smolders, I.; Lindskog, M.,

661

Dysfunctional astrocytic regulation of glutamate transmission in a rat model of depression.

662 663

Mol Psychiatry 2013, 18 (5), 582-94. 71. Lai, C. H.; Lane, H. Y.; Tsai, G. E., Clinical and cerebral volumetric effects of sodium

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benzoate, a D-amino acid oxidase inhibitor, in a drug-naive patient with major depression.

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Biol Psychiatry 2012, 71 (4), e9-e10.

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669

Figures Legends

670

Figure 1. Protocol for experiments with D-serine administration and using the inhibitors

671

NBQX and rapamycin. (A) The experimental rats were administered a single intraperitoneal

672

injection with D-serine (560, 1000, and 2000 mg/kg). Saline, ketamine (10 mg/kg), and

673

desipramine (20 mg/kg) were used as controls. A 15 minutes preswim exposure was used at

674

24 hours before the forced swim test (FST) session. The (FST was conducted 30 minutes

675

after injection. (B) To determine the possibility of false-positive result in FST, we separately

676

assess general locomotor activity in a elevated plus maze test (EPM), the rats were

677

administered saline, D-serine (560, 1000, or 2000 mg/kg), ketamine (10 mg/kg), or

678

desipramine (20 mg/kg), once during the experiment. Thirty minuets later, EPM was

679

conducted. Then, the rats were sacrificed immediately and rapidly decapitated for

680

biochemical analysis. For acute D-serine treatment with or without inhibitors of AMPAR or

681

mTOR, intraperitoneally pretreratment with NBQX at 10 mg/kg (a AMPA inhibitor) or

682

rapamycin at 20 mg/kg (an mTOR pathway inhibitor) was applied (C) Rats were treated with

683

either NBQX rapamycin administered 30 min before intraperitoneal D-serine at 1000 mg/kg

684

treatment and immobility in the FST was determined 30 min after the last injection.

685

Experimental rats were administered saline, NBQX, rapamycin, or D-serine at the doses

686

indicated and immobility in the FST was determined 30 min after the last injection. A

687

separate study was conducted for biochemical analysis (D), rats were administered saline,


32

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688

NBQX, rapamycin, or D-serine at the doses indicated and were sacrificed and then rapidly

689

decapitated 30 min to remove hippocampus after last injection. (E) To investigate the

690

possibility of toxicity, rats were injected with saline or D-serine at the dose indicated and

691

were killed to remove plasma, liver, kidney, hippocampus, and cortex.

692 693

Figure 2. Percentage of immobility duration in forced swim test (FST) and representative

694

Western blots of rats after acute treatment with saline, ketamine (10 mg/kg), desipramine (20

695

mg/kg), or D-serine (560, 1000, and 2000 mg/kg). (A) A dose-dependent effect of D-serine

696

on reduced immobility duration was observed. The rats that received a single injection of

697

D-serine at 1000 mg/kg and 2000 mg/kg, ketamine at 10 mg/kg, and desipramine at 20 mg/kg

698

exhibited a significantly reduced immobility duration in the FST [main effect: F (5,54) =

699

8.663, p < .001; n = 10 per group]. (B) Representative Western blots revealed a notably

700

increase of pmTOR, pAkt and pERK in the hippocampus of rat after treatment with ketamine

701

(10 mg/kg) and acute treatment with D-serine in a dose-dependent manner. (C) Densitometric

702

analysis of pmTOR, pAkt, pERK, and BDNF protein (normalized to β-actin) in the

703

hippocampus after treatments verified the increased immunoreaction of pmTOR, pAkt, pERK,

704

and BDNF protein in each experimental group [main effect: mTOR, F (5,18) = 11.751, p

705

< .001; pAkt, F (5,18) = 20.022, p < .001; pERK, F (5,18) = 44.878, p < .001; BDNF, F (5,18)

706

= 57.893, p < .001; n = 4 per group; * p < .05, **p < .01, ***p < .001 compared with the


33


707

Page 34 of 46

saline-treated group with the Tukey post-hoc analysis]. Values are expressed as mean ± s.e.m.

708 709

Figure 3. The percentage of immobility duration in forced swim test (FST) and

710

representative Western blots of the rat hippocampal slices after acute intraperitoneal

711

administration of D-serine at 1000 mg/kg and intraperitoneal pretreatment with NBQX at 10

712

mg/kg or rapamycin at 20 mg/kg. (A) Note that pretreatment of rats with NBQX at 10 mg/kg

713

completely blocks the antidepressant-like effect of D-serine on decreased immobility.

714

Similarly, pretreatment with rapamycin (20 mg/kg) also attenuated the decreased immobility

715

elicited by D-serine in the FST [main effect: F (3,36) = 5.346; p < .01, n = 10 per group]. (B)

716

Representative Western blots revealed a notable increase of pmTOR, pAkt, pERK, and

717

BDNF protein in the rat hippocampal slices after acute D-serine treatment. Notably, when

718

rats were

719

ERK, and BDNF protein resulting from acute D-serine treatment is abolished [main effect:

720

mTOR, F (3,12) = 20.484, p < .001; pAkt, F (3,12) = 31.754, p < .001; pERK, F (3,12) =

721

33.868, p < .001; BDNF, F (3,12) = 6.242, p < .01; n = 4 per group]. The increased

722

expression of pmTOR and release of BDNF resulting from acute D-serine treatment is

723

blocked on pretreatment with rapamycin, but the increases of pAkt and pERK resulting from

724

acute D-serine treatment is not reversed (* p < .05, ** p < .01, ***p < .001compared with the

725

saline+saline-treated group;

intraperitoneal preadministrated with NBQX, the increases of pmTOR, pAkt,

#

p < .05,

##

p < .01,

###

p < .001 compared with the


34

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726

saline+D-serine-treated group with the Tukey post-hoc analysis). Values are expressed as

727

mean ± s.e.m.

728 729

Figure 4. Representative Western blots of (A) pGluR1ser845 and (B) immunoexpression of

730

NMDAR2A/B and adaptin β2 from the rat hippocampal slices intraperitoneally treated with

731

saline, D-serine (560, 1000, and 2000 mg/kg), ketamine (10 mg/kg), or desipramine (20

732

mg/kg) and after D-serine at 1000 mg/kg intraperitoneally preadministration with NBQX at

733

10 mg/kg or rapamycin at 20 mg/kg (C,D). Acute D-serine treatment increased (A) the

734

expression of pGluR1ser845 and (B) the association of NMDAR2A/B and adaptin β2

735

significantly in a dose-dependent manner [main effect: F (5,18) = 16.783, p < .001; F (5,18) =

736

13.484, p < .001; respectively]. Ketamine (10 mg/kg) also significantly enhanced

737

phosphorylated GluR1ser845 and AP2–NMDAR association, which was not observed in

738

desipramine-treated (20 mg/kg) rats (*p < .05, **p < .01, ***p < .001 compared with

739

saline-treated group with the Tukey post-hoc analysis; n = 4 per group). When rats were

740

pretreated with NBQX, the increase of pGluR1ser845 caused by acute D-serine treatment

741

was reversed (c) [main effect: F(3,12) = 10.796, p = .001]. However, the effects do not

742

diminished by rapamycin pretreatment (C). The increase of the association of adaptin

743

β2-NMDAR resulting from acute D-serine treatment was not abolished when rats were

744

pretreated with NBQX or rapamycin (D) [main effect: F(3,12) = 25.300, p < .001, **p < .01,


35


##

Page 36 of 46

745

***p < .001 compared with saline+saline-treated group;

p < 0.01 compared with

746

saline+D-serine-treated group with the Tukey post-hoc analysis; n = 4 per group] Values are

747

expressed as mean ± s.e.m.

748 749

Figure 5. Effect of D-serine at different doses (560, 1000, and 2000 mg/kg) on serum marker

750

enzymes such as (A) aspartate aminotransferase (AST), (B) alanine aminotransferase (ALT),

751

(C) blood urea nitrogen (BUN), and (D) creatinine [main effect: AST, F (3,12) = 2.235, p

752

> .05; AST, F (3,12) = 0.295, p > .05; BUN, F (3,12) = 1.102, p > .05; Creatinine F (3,12) =

753

2.000, p > .05; n = 4 per group]. Values are expressed as mean ± s.e.m.

754 755

Figure 6. Bright-field (H&E) (a-h) and fluorescence (i-p) photomicrographs in the (A) liver,

756

(B) kidney, (C) hippocampus, and (D) cortex of rats being treated with normal saline (a, e, i,

757

m), D-serine 560 mg/kg (b, f, j, n), 1000 mg/kg (c, g, k, o), and 2000 mg/kg (d, h ,l, p). After

758

staining with H&E, histopathological examinations were performed under light microscopy

759

at 100X (a-d, Scale bar, 100 mm) and 200X (e-p, Scale bar, 50 mm) magnifications. At higher

760

magnified liver slices demonstrated well-organized hepatocytes radiating from the central

761

vein (arrows in A) with intact liver plates and portal triads were observed in normal saline,

762

D-serine 560, 1000, and 2000 mg/kg groups (A, e-h). Examination of kidney sections in all

763

groups revealed that the kidney tissues retained its normal architecture in glomeruli (arrows


36

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764

in B) and surrounding with proximal and distal convoluted tubules convoluted tubules (B,

765

a-h). H&E staining of hippocampal neurons (arrows in C) arranged closely, neuronal

766

structure is complete and clear. There are no degeneration and atrophy changes in

767

pathological patterns between control and D-serine treated rats(C, a-h). Photomicrographs of

768

the Inner pyramidal layer of the cerebral cortex in Control or D-serine treated groups (D, a-h)

769

exhibiting a normal morphology, the organisational structure in the pyramidal cells (arrows in

770

D) were no morphologically altered. In rats treated with normal saline, D560, D1000, D2000

771

mg/kg, no TUNEL -positive cells were revealed in the tissues of Liver, kidney, hippocampus

772

and cortex (A-D, m-p). All cells were morphologically determined using

773

colocalized DAPI staining (A-D, i-l).

774 775

Figure 7. Proposed cellular signaling pathways underlying the common antidepressant-like

776

properties for both NMDAR coagonist D-serine and NMDAR antagonist ketamine. The

777

activation of synaptic NMDARs by the co-agonist D-serine at high doses induce AMPAR

778

insertion into the postsynaptic membrane and NMDAR internalization at postsynaptic sites,

779

which amplify the postsynaptic AMPAR:NMDAR ratio, leading to enhanced glutamatergic

780

AMPAR to NMDAR stimulation. The increased glutamatergic throughput of AMPARs

781

caused the activation of mTOR and release of BDNF. Ultimately, D-serine exhibits

782

antidepressant-like actions. Meanwhile, ketamine at subanesthetic dose can induce


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783

extracellular glutamate. Increased glutamate release activates AMPARs and stimulates

784

mGluR, resulting in NMDAR internalization. This will also increase AMPAR throughput,

785

thereby contributing to an activated mTOR signaling pathway that results in antidepressant

786

action. By contrast, a high anesthetic dose of ketamine reduces glutamate levels and may

787

upregulate NMDARs, which may also explain why an anesthetic dose of ketamine cannot

788

produce an antidepressant effect.


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789


39



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D-serine dose-dependently

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ketamine ketamine at subanesthetic dose at anesthetic dose + glutamate

NBQX

mGluR

+

↑AMPAR:NMDAR ratio increased AMPAR throughput

+

+

Akt, ERK activation

BDNF release

NMDAR internalization

NMDA-R

Glu

NMDA-R

AMPAR insertion

Glu

NMDA-R NMDA-R

Glu

AMPA-R AMPA-R

AMPA-R AMPA-R

AMPA-R

AMPA-R

+

Glu

NMDA-R NMDA-R

Glu

rapamycin

+ rapamycin

mTOR activation ACS Paragon Plus Environment

Antidepressant effect

Acute Amino Acid d-Serine Administration, Similar to Ketamine

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