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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Mol Cell Neurosci. 2018 Mar 27;89:20–32. doi: 10.1016/j.mcn.2018.03.011

D-serine administration affects Nitric Oxide Synthase 1 Adaptor Protein and DISC1 expression in sex-specific manner

Kirsten C Svane a,b, Ericka-Kate Asis a, Anton Omelchenko a,b, Ansley J Kunnath a, Linda M Brzustowicz c, Steven M Silverstein d, Bonnie L Firestein a
PMCID: PMC5970076  NIHMSID: NIHMS957931  PMID: 29601869

Abstract

Antipsychotic medications are inefficient at treating symptoms of schizophrenia (SCZ), and N-methyl D- aspartate receptor (NMDAR) agonists are potential therapeutic alternatives. As such, these agonists may act on different pathways and proteins altered in the brains of patients with SCZ than do antipsychotic medications. Here, we investigate the effects of administration of the antipsychotic haloperidol and NMDAR agonist D-serine on function and expression of three proteins that play significant roles in SCZ: nitric oxide synthase 1 adaptor protein (NOS1AP), dopamine D2 (D2) receptor, and disrupted in schizophrenia 1 (DISC1). We administered haloperidol or D-serine to male and female Sprague Dawley rats via intraperitoneal injection for 12 days and subsequently examined cortical expression of NOS1AP, D2 receptor, and DISC1. We found sex-specific effects of haloperidol and D-serine treatment on the expression of these proteins. Haloperidol significantly reduced expression of D2 receptor in male, but not female, rats. Conversely, D-serine reduced expression of NOS1AP in male rats and did not affect D2 receptor expression. D-serine treatment also reduced expression of DISC1 in male rats and increased DISC1 expression in female rats. As NOS1AP is overexpressed in the cortex of patients with SCZ and negatively regulates NMDAR signaling, we subsequently examined whether treatment with antipsychotics or NMDAR agonists can reverse the detrimental effects of NOS1AP overexpression in vitro as previously reported by our group. NOS1AP overexpression promotes reduced dendrite branching in vitro, and as such, we treated cortical neurons overexpressing NOS1AP with different antipsychotics (haloperidol, clozapine, fluphenazine) or D-serine for 24 hours and determined the effects of these drugs on NOS1AP expression and dendrite branching. While antipsychotics did not affect NOS1AP protein expression or dendrite branching in vitro, D-serine reduced NOS1AP expression and rescued NOS1AP- mediated reductions in dendrite branching. Taken together, our data suggest that D-serine influences the function and expression of NOS1AP, D2 receptor, and DISC1 in a sex-specific manner and reverses the effects of NOS1AP overexpression on dendrite morphology.

Graphical abstract

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Introduction

Schizophrenia (SCZ) is a debilitating mental illness that affects 1% of the U.S. population (Perälä et al., 2007), and symptoms are categorized as positive, negative, and cognitive (Citrome, 2014). The dopamine hypothesis attributes the psychotic, or positive, symptoms of SCZ to elevated dopamine signaling along the mesolimbic pathway (Citrome, 2014; Howes et al., 2015; Urs et al., 2017). Typical and atypical antipsychotics act as D2 dopamine receptor antagonists (Horacek et al., 2006; Lally and MacCabe, 2015; Lau et al., 2013; Seeman, 2010). Antipsychotics alleviate positive symptoms of SCZ and marginally reduce negative and cognitive symptoms, suggesting the involvement of other signaling pathways. (Citrome, 2014). The N-methyl D-aspartate receptor (NMDAR) hypothesis attributes SCZ to reduced NMDAR functioning, which brings about all symptom domains (Coyle, 1996). NMDAR antagonists reproduce all symptoms of SCZ in healthy individuals, which strengthens the validity of the NMDAR hypothesis (Lahti et al., 2001; Malhotra et al., 1996; Morris et al., 2005; Vincent et al., 1979). NMDARs regulate dopamine signaling (Balla et al., 2003; Breier et al., 1998; Howes et al., 2015; Miller and Abercrombie, 1996; Smith et al., 1998; Vollenweider et al., 2000) and are critical for synaptic plasticity, learning, and memory, which are impaired in SCZ (Brigman et al., 2010; Burgos-Robles et al., 2007; Kantrowitz and Javitt, 2010; Wang and Peng, 2016). NMDAR activation requires the binding of glutamate to the NMDA NR2 subunit (Furukawa et al., 2005) and the binding of glycine or D-serine, as co-agonists, to the glycine modulatory site (GMS) on the NMDA NR1 subunit (Balu and Coyle, 2015). As GMS is not saturated in vivo, it is an ideal pharmacological target to induce elevation of NMDAR signaling (Hashimoto and Oka, 1997). NMDAR co-agonists are currently in clinical trial as therapeutic candidates to treat all symptom domains of SCZ (Heresco-Levy et al., 2005; Kantrowitz et al., 2010; Kantrowitz et al., 2015; Lane et al., 2005; Singh and Singh, 2011; Tsai et al., 1998; Weiser et al., 2012). Additionally, disrupted in schizophrenia 1 (DISC1) mediates glutamate signaling and is encoded by a SCZ susceptibility gene. (Maher and LoTurco, 2012; Millar et al., 2000). DISC1-binding stabilizes serine racemase, which is responsible for D-serine synthesis, and mutant DISC1 promotes degradation of serine racemase and subsequent D-serine deficiency (Ma et al., 2013).

Nitric oxide synthase 1 adaptor protein (NOS1AP) negatively regulates NMDAR signaling and is encoded by a SCZ susceptibility gene (Brzustowicz et al., 2004; Eastwood, 2005; Jaffrey et al., 1998). NOS1AP was identified in rat brain as a binding partner of nitric oxide synthase 1 (NOS1) (Jaffrey et al., 1998). Upon NMDAR activation, NOS1 is recruited to NMDAR by postsynaptic density-95 (PSD-95) (Brenman et al., 1996). NOS1AP competes with PSD-95 for NOS1 binding and subsequently sequesters NOS1 from NMDAR. This disrupts nitric oxide (NO) signaling, and thus, NMDAR signaling (Jaffrey et al., 2002; Jaffrey et al., 1998). We previously reported that NOS1AP long (NOS1AP-L), short (NOS1AP-S), and short’ (NOS1AP-S’) isoforms are upregulated in Brodmann’s area 46 in postmortem tissue of patients with SCZ (Hadzimichalis et al., 2010) and that overexpression of NOS1AP-L and NOS1AP-S reduces dendrite branching (Carrel et al., 2009) and alters dendritic spines in vitro (Hernandez et al., 2016), reproducing neuronal and synaptic abnormalities associated with SCZ (Black et al., 2004; Kolomeets et al., 2005; Kulkarni and Firestein, 2012). Chronic antipsychotic treatment correlates with increased NOS1AP-L mRNA expression in the DLPFC of SCZ patients (Xu et al., 2005). However, it is not yet known how treatment with antipsychotics or NMDAR agonists affects expression or function of proteins linked to SCZ. Here, we investigate the effects of haloperidol and D-serine administration in vivo on expression of NOS1AP, D2 receptor, and DISC1. Using primary neuronal culture as a model for the contribution of NOS1AP to SCZ phenotypes (Carrel et al., 2009; Carrel et al., 2015; Hernandez et al., 2016), we examine how treatment with antipsychotic medications or D-serine affects NOS1AP protein expression and dendrite branching in vitro. Our data indicate that antipsychotic medications and D-serine exert distinct sex-specific effects on protein expression and neuronal development.

Material and methods

Animals

Adult male and female Sprague Dawley rats (Taconic, Hudson, NY) were single-housed with access to food and water ad libitum and maintained under 12-h light/dark cycle (on at 7:00 A.M.) at constant 22°C and relative humidity of 50%. Rats were injected with haloperidol (1.0mg/kg; Henry Schein, Mylan Institutional LLC, Rockford IL, serial #7513033) diluted in sterile water or vehicle (sterile water). Haloperidol dosage and treatment duration for in vivo administration was chosen based on values in literature (Park et al., 2011). This dosage was reported to produce drug plasma levels similar to values associated with therapeutic effect in humans (Sunderland and Cohen, 1987; Weigmann et al., 1999; Zhang et al., 2007). A separate group of rats was injected with D-serine (200mg/kg; Sigma Aldrich, S4250) diluted in sterile water or vehicle (sterile water). D-serine dosage treatment and duration for in vivo administration was based on dosage used in literature (Shimazaki et al., 2010). All drugs were administered via intraperitoneal injection 1×/day for 12 days. Animals were sacrificed on day 13 by CO2 inhalation. Cortices were isolated, stored at −80°C, homogenized, lysed in TEE (containing 1 mM PMSF and protease inhibitor cocktail, and analyzed using Western blotting. All studies were reviewed and approved by Rutgers University Institutional Animal Care and Use Committee in accordance with the guidelines the National Institute of Health Laboratory Animals Resources Commission on Life Sciences’ 1996 Guide for the Care and Use of Laboratory Animals.

Primary cortical neuron cultures

Cortical neuron cultures were prepared from embryonic rats at gestation day 18 as previously described (Hernandez et al., 2016; Kutzing et al., 2011, 2012). Cells were plated at a density of 2 × 105/well on 12- mm glass coverslips (Thermofisher) coated with poly-D-lysine (0.1mg/ml) for dendrite branching analysis or 35mm dishes for protein analysis.

Treatment and lysis of cultured cortical neurons

To analyze NOS1AP protein expression, neurons (DIV21) were treated for 24 hours with antipsychotics, NMDAR agonists, or vehicle. On DIV22, cells were washed with phosphate buffer saline (PBS) and lysed in ice cold TEE buffer (25 mM TrisHCl, 1 mM EDTA, 1 mM EGTA) containing 1 mM phenylmethylsulfonylfluoride (PMSF). Lysates were passed through a 25 ½ G needle five times. Triton X-100 was added to lysates to final concentration of 1%, and lysates were incubated on ice for 30 min and vortexed every 5 min. Lysates were clarified by centrifugation at 12,000 × g, 4°C, for 15 min and stored at −20°C. Concentrations of and treatment duration for clozapine, haloperidol, and fluphenazine for in vitro administration were based on values from literature (Sunderland and Cohen, 1987; Weigmann et al., 1999; Zhang et al., 2007). Concentrations of D-serine for in vitro administration were based on concentrations from literature (Cherubini et al., 1990; Stobart et al., 2013)

Plasmids

EGFP of pEGFP-C1 (Clontech; Mountain View, CA) was subcloned into vector with CMV–actin–β-globin promoter (pCAG) to produce pCAG-GFP. cDNAs of human NOS1AP-L and NOS1AP-S were subcloned into pCAG-GFP (Carrel et al., 2009).

Transfection, treatment, and immunostaining for dendrite analysis

In this study, Lipofectamine-mediated transfection was performed to analyze alterations in dendritic branching as a result of NOS1AP overexpression and drug treatment. Experiments were performed in the same fashion as previously reported by our group (Carrel et al., 2009; Kwon et al., 2011; O’Neill et al., 2015; Previtera et al., 2010). Lipofectamine-mediated transfection typically results in 10% transfection efficiency in our primary cultures, which is ideal for the tracing if individual neurons in order to analyze and quantify alterations in dendritic branching and number. Higher efficiency rates would result in overlapping processes from neighboring neurons.

Cortical neurons on glass coverslips were transfected at day in vitro (DIV) 6 using Lipofectamine LTX and Plus reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. On DIV7, neurons were treated with 3.0 μg/ml clozapine, 0.25 μg/ml haloperidol, 8.4 μg/ml fluphenazine, 10 μM D- serine (all Sigma-Aldrich, St. Louis, Missouri), or 0.1% DMSO vehicle. D-serine concentrations in the cerebrospinal fluid (CSF) are reported to be 2.72μM in control patients and 1.26 μM in patients with SCZ. D-serine is also thought to be taken up and released by astrocytes, and our cultures contain ~50% astrocytes as evidenced by GFAP and vimentin immunostaining (data not shown). It is for this reason that we chose 10 μM D-serine as the treatment dosage, as it is on the same order of concentration found in the brain, taking into account some uptake by astrocytes. Following 24 hours, cells were fixed with 4% Paraformaldehyde and subsequently immunostained with anti-MAP2 and chicken anti-GFP (both 1:200) and incubated with 1× Hoechst 33342 dye for nuclear staining. Coverslips were mounted onto microscope slides with Fluoromount G (Southern Biotechnology; Birmingham, AL).

Western blot analysis

Lysates from cultures and drug-treated rats were analyzed using Western blot analysis as previously described (Carrel et al., 2009; Hadzimichalis et al., 2010; Hernandez et al., 2016). Proteins were resolved on 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to polyvinylidene difluoride membrane without SDS. Blots were incubated with NOS1AP primary antibody (1:500 in 3% bovine serine albumin (BSA) in buffer), and incubated overnight at 4°C. D2, DISC1, and GAPDH primary antibodies were used at 1:1000 in 3% BSA for 1 hour at room temperature. Membranes were washed and probed with either polyclonal anti-mouse IgG (H&L) or anti-rabbit IgG (H&L) peroxidase conjugated secondary antibody (1:5000; Rockland Inc; Limerick, PA) or monoclonal anti-rabbit IgG (γ-chain specific)-peroxidase conjugated secondary antibody (1:500; Sigma-Aldrich; St. Louis, MO) diluted in 5% non-fat dry milk. Further information about antibodies used for Western blot analysis in this study are shown in Supplementary Table 1. Membranes were developed using the enhanced chemiluminescence system (GE Healthcare; Piscataway, NJ) and Syngene G:BOX iChemi XR system and GeneSnap software (Version 7.09.a, Syngene, Frederick, MD). ImageJ was used for quantitation, and NOS1AP, D2, and DISC1 expression was normalized to GAPDH expression. Data from cultures were analyzed by one- way ANOVA followed by Tukey multiple comparisons test using Prism6 (GraphPad). Data from drug-treated rats were analyzed in Prism using two-tailed Student’s t-test comparing animals injected with drug to vehicle group.

Analysis of NOS1AP mRNA using qRT-PCR

mRNA from cortex of D-serine and vehicle-injected rats was extracted by homogenization in Trizol reagent following manufacturer’s instructions (Life Technologies) and stored at −80°C. 0.2 μg mRNA from each sample was reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit following manufacturer’s protocol (Applied Biosystems; Carlsbad, CA). The 2720 Thermal Cycler (Applied Biosystems) was set at the following: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes, 4°C hold. cDNA was stored at −20°C until amplification by qPCR.

Each qPCR reaction was prepared according to manufacturer’s instructions for Taqman Fast Universal PCR Master Mix (Applied Biosystems). Briefly, each reaction included 10 μl Taqman Fast Universal PCR Master Mix (Applied Biosystems), 4 μl cDNA template, 5 μl RNase free water, and 1 μl either NOS1AP (FAM 20X RN01490325, Thermo Fisher, catalog # 4351372) or β-actin (FAM 20X RN00667869, Thermo Fisher, catalog # 4331182) primer. Primers were used at a 1:20 dilution. All cDNA from the previous step was used to allow multiple technical replicates per sample. qPCR was performed using 7900HT real-time PCR system (Applied Biosystems) at the Department of Genetics, Rutgers University. DataAssist V3.01 was used to generate RQ (Fold Change), and p-values were computed using two-tailed Student’s t-test comparing the 2(−ΔCT) values of the two groups. β-actin was internal control, and vehicle was a reference to calculate fold changes. The p-value was adjusted using Benjamini- Hochberg False Discovery Rate (Benjamini and Hochberg, 1995).

Assessment of dendrite number using Sholl analysis

Neurons were imaged at under a 20X objective on an EVOS FL Cell Imaging system. Semi-automated Sholl analysis was used to analyze dendrite branching as we previously described (Kutzing et al., 2010; Langhammer et al., 2010). Images were traced using NeuronJ plugin (Meijering et al., 2004) for ImageJ (NIH, Bethesda, MD). Axons were determined by the experimenter, blinded to condition. Traces were converted to SWC files using MATLAB (Mathworks) and verified using NeuronStudio. Data were exported to Excel using MATLAB. Sholl curves were analyzed by two-way ANOVA followed by Bonferroni multiple comparisons test (Prism6; Graphpad), and dendrite numbers were analyzed by one- way ANOVA followed by Dunn’s multiple comparisons test.

Results

Haloperidol treatment does not affect NOS1AP expression but significantly reduces expression of D2 receptor in male rats

Work from our laboratory has focused on how overexpression of NOS1AP, a protein encoded by a SCZ susceptibility gene (Brzustowicz et al., 2004; Eastwood, 2005; Jaffrey et al., 1998), contributes to dendritic alterations present in patients with SCZ (Carrel et al., 2009; Carrel et al., 2015; Hernandez et al., 2016). To investigate whether antipsychotics affect NOS1AP expression in vivo, we injected adult Sprague Dawley rats of both sexes with the typical antipsychotic haloperidol and assessed effects on NOS1AP-L expression in cortex. We focused on expression of the long isoform of NOS1AP, NOS1AP- L, as mRNA for this isoform may be affected by antipsychotic treatment (Xu et al., 2005). We found that acute haloperidol treatment did not affect cortical expression of NOS1AP-L (Fig. 1a and b). As patients with SCZ show altered dopamine receptor expression (Brisch et al., 2014; Seeman, 2010) and haloperidol acts as a D2 receptor antagonist, we also examined whether haloperidol treatment affects expression of the D2 receptor. Acute haloperidol administration resulted in sex-specific effects. Haloperidol treatment reduced expression of D2 receptor in the cortex of male, but not female, rats. (Fig. 1c and d). Thus, acute treatment with haloperidol does not alter NOS1AP expression but induces sex-specific changes in D2 receptor expression.

Fig. 1. Haloperidol treatment does not affect NOS1AP-L expression but decreases D2 receptor expression in male rats.

Fig. 1

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a and b. Haloperidol administration does not affect cortical NOS1AP-L expression in male or female rats. c. Haloperidol administration significantly reduces cortical D2 receptor expression in male rats. d. Haloperidol treatment does not affect D2 receptor expression in female rats. **p<0.01 two-tailed Student’s t-test. Error bars represent SEM. n = 7 animals per treatment group.

D-serine treatment affects expression of NOS1AP and DISC1, but not D2 receptor in sex-specific manner

Since haloperidol acts as a D2 receptor antagonist, and NOS1AP is part of an NMDAR signaling pathway, we next investigated if acute treatment with the NMDAR agonist D-serine affects expression of NOS1AP in vivo. Administration of D-serine significantly reduced NOS1AP-L expression in the cortex of male, but not female, rats (Fig. 2a and b). This is an interesting finding as SCZ occurs more frequently in males (Iacono and Beiser, 1992), with a male to female rate ratio of 1.4:1 (McGrath et al., 2008), and our data suggest that the effect of D-serine on NOS1AP expression is male-specific. To investigate the mechanism by which D-serine treatment affects NOS1AP expression, we used qRT-PCR to examine NOS1AP mRNA levels in cortices of rats treated with D-serine. D-serine treatment did not affect NOS1AP-L mRNA expression in the cortex (Fig. 2c and d), suggesting that D-serine regulation of NOS1AP expression is transcription-independent.

Fig. 2. D-serine treatment reduces cortical NOS1AP-L protein but not mRNA expression in male rats.

Fig. 2

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a. D-serine administration significantly reduces cortical NOS1AP-L protein expression in male rats. b. D-serine administration does not affect on NOS1AP-L expression in female rats. c and d. D-serine administration does not affect cortical NOS1AP mRNA expression in male rats or female rats. 2^(−ΔCt) values between groups were compared with two-tailed Student’s t-test. Benjamini-Hochberg False Discovery Rate was used to adjust p-values. n = 7 animals per group and 3-4 technical replicates were performed. Beta-actin served as endogenous control. Reference group was vehicle treated control group.

As acute haloperidol treatment significantly reduced expression of D2 receptor in vivo, we next investigated the effect of acute D-serine administration on D2 receptor expression. In contrast to haloperidol, D-serine administration did not change cortical expression of D2 receptor (Fig. 3a and b), supporting the hypothesis that D-serine acts exclusively at the NMDAR (Hashimoto and Oka, 1997).

Fig. 3. D-serine treatment reduces DISC1 but not D2 receptor expression in sex-specific manner.

Fig. 3

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a. D-serine administration decreases cortical DISC1 expression in male rats. b. D-serine administration increases cortical DISC1 expression in female rats. D-serine has no effect on expression of the dopamine D2 receptor in c. male or d. female rats *p <0.05, **p<0.01 by two-tailed Student’s t-test. Error bars represent SEM. n = 7 animals per treatment group.

Since SCZ is a heterogeneous disorder and is linked to multiple risk genes, we investigated whether D- serine treatment affects expression of DISC1, a protein encoded by a SCZ susceptibility gene (Millar et al., 2000). DISC1 binds to and stabilizes serine racemase, the enzyme synthesizing D-serine, and mutant DISC1 leads to degradation of serine racemase and D-serine deficiency (Ma et al., 2013). Additionally, as DISC1 affects glutamatergic transmission, it may be a potential target for treatment of SCZ (Maher and LoTurco, 2012; Millar et al., 2005). We found that D-serine administration reduced DISC1 expression in male rats but increased DISC1 expression in female rats (Fig. 3c and d). These data further underscore the sex-specificity of D-serine action.

Treatment with antipsychotics does not affect NOS1AP expression or rescue NOS1AP-induced dendritic alterations in vitro

Since acute treatment with D-serine in vivo results in changes to NOS1AP and DISC1, we chose our primary culture assay to determine whether D-serine can act to shape the dendritic arbor, and hence, neuronal function. Although we acknowledge that our developing cultures are distinct from the fully developed brains of adult rats, we (i.e. Akum et al., 2004; Carrel et al., 2009; Kwon et al., 2011; Sweet et al., 2011; O’Neill et al., 2015; O’Neill et al., 2016) and others (Chen et al., 2014; de Wit et al., 2013; Liao et al., 1999; Yi et al., 2016)) have used this assay to understand mechanisms by which drugs and molecules shape neurons. Additionally, our culture conditions allow us to determine the effects of treatment directly on neurons as our cultures contain approximately 50% neurons and 50% immature astrocytes as determined by immunostaining for MAP2 and vimentin, respectively (unpublished data). Furthermore, we have used this assay to understand the effect of NOS1AP on the dendritic arbor (Carrel et al., 2009). In contrast to our previous work (Carrel et al., 2009), we chose cortical, rather than hippocampal neurons, as NOS1AP is overexpressed in the DLPFC of patients with SCZ. Furthermore, this model system allows for the study of a greater number of drugs due to ease of treatment, lower costs, and simplicity of culture vs. the intact brain. First, we confirmed that our cultures do indeed express D1 and D2 receptors (Fig. 4a). We then examined whether administration of traditional antipsychotics affects expression of NOS1AP in vitro in rat cortical cultures. We extended our studies to include both atypical (clozapine) and typical (haloperidol and fluphenazine) standardly prescribed antipsychotics and their effects on three isoforms of NOS1AP (NOS1AP-L, NOS1AP-S, and NOS1AP-S’). On day in vitro (DIV) 21, we treated cortical neurons with clozapine, haloperidol, fluphenazine, or vehicle for 24 hours and examined expression of NOS1AP-L, NOS1AP-S, and NOS1AP-S’. We found that acute in vitro treatment of cortical cultures with antipsychotics does not affect NOS1AP protein levels, which are elevated in SCZ (Fig. 4b–f).

Fig. 4. Treatment with clozapine, haloperidol, and fluphenazine does not affect expression of NOS1AP Isoforms in vitro.

Fig. 4

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a. DIV21 cortical neuron cultures show expression of D2 and D1 receptor. b. Representative blots of NOS1AP-L protein expression in cortical neuron cultures treated with vehicle (DMSO), clozapine (3.0 μg/mL), haloperidol (0.25 μg/mL), and fluphenazine (8.4 μg/mL). c. Representative blots of NOS1AP-S and NOS1AP-S’. d-f. Quantitation of NOS1AP isoforms normalized to GAPDH expression. n = 8 (NOS1AP-L), n = 8 (NOS1AP-S), n = 11 (NOS1AP-S’) independent cultures. Antipsychotic treatment did not affect cortical NOS1AP isoform expression in cortical neuron cultures.

Since NOS1AP overexpression reduces dendrite number (Carrel et al., 2009), we examined whether treatment with antipsychotics would rescue NOS1AP-induced reduction in dendrite numbers. Cortical neurons were transfected at DIV6, during the period of active dendrite branching (Charych et al., 2006), with cDNA encoding EGFP (control), NOS1AP-L, or NOS1AP-S. On DIV7, neurons were treated with clozapine, haloperidol, fluphenazine, or vehicle for 24 hours. Cells were fixed on DIV8 and immunostained for GFP and MAP2 (Fig. 5a). Sholl analysis of these neurons suggests that treatment with haloperidol, but not clozapine or fluphenazine, decreases overall branching proximal to the soma (Fig. 5b, e and h); however, no change to the overall arbor was observed with any drug treatment. In neurons overexpressing NOS1AP-S, haloperidol had no effect on primary or secondary dendrites (Fig. 5c and d), clozapine decreased primary and secondary dendrites (Fig. 5f, g) and fluphenazine reduced primary dendrites (Fig. 5i). Overexpression of NOS1AP-L, combined with antipsychotic treatment, led to significant amounts of cell death, thus the relevant Sholl analysis data could not be obtained. Together, our data suggest that antipsychotic treatment does not rescue NOS1AP-mediated reductions in dendrite branching.

Fig. 5. Effect of haloperidol, clozapine, and fluphenazine on cortical neurons overexpressing NOS1AP-S.

Fig. 5

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Haloperidol, clozapine, and fluphenazine do not reverse effects of NOS1AP-S (N-S) overexpression on dendrite branching. Colored significance bars (Supplementary Tables 1-3) represent significance of at least p<0.05. a. Representative images from each treatment. b. Haloperidol treatment of neurons which overexpress N-S does not affect dendrite branching. c. Haloperidol treatment of control (GFP) neurons reduces proximal dendrites. d. Haloperidol treatment of neurons which overexpress N-S does not affect primary dendrites or secondary dendrites. e. Overexpression of N-S (DMSO) reduces proximal branching, but increases distal branching, and clozapine treatment does not affect overall dendritic arbor. f. Clozapine treatment of neurons which overexpress N-S significantly reduces primary dendrites and g. secondary dendrites. h. Treatment of neurons which overexpress N-S with fluphenazine does not affect dendrites. i and j. Treatment of neurons which overexpress N-S with fluphenazine reduces primary but not secondary dendrites. n = 54-79 neurons from 4 independent cultures. Statistics determined with two-way ANOVA followed by Tukey multiple comparisons test. Error bars represent SEM. Scale bar = 50 μm.

D-serine reduces NOS1AP expression and rescues NOS1AP-induced alterations in dendritic branching in vitro

It is possible that antipsychotics, which act primarily via dopamine receptors, cannot help rescue the dendritic phenotypes seen with NOS1AP overexpression as NOS1AP is part of the NMDAR, and not D2 receptor, signaling pathway. Thus, we investigated whether treatment of cortical cultures with D-serine affects expression of NOS1AP. We treated cortical neurons with D-serine or vehicle for 24 hours and subsequently lysed and performed Western blot analysis. We found that treatment with D-serine, a full co-agonist at the glycine modulatory site on the NMDAR, reduced expression of three NOS1AP isoforms (Fig. 6a–e). Thus, we conclude that NMDAR full co-agonism significantly reduces expression of NOS1AP.

Fig. 6. D-serine treatment reduces expression of NOS1AP isoforms in vitro.

Fig. 6

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a. Representative blots showing detection of NOS1AP-L in neurons treated with vehicle (DMSO) and D-serine (10 μM). b. Representative blots of NOS1AP-S and NOS1AP-S’ expression. c-e, Quantitation of NOS1AP-L expression normalized to GAPDH expression following drug treatment. D-serine significantly reduces expression of NOS1AP-L, NOS1AP-S, and NOS1AP-S’ n = 7 (NOS1AP-L), n = 9 (NOS1AP-S), n = 12 (NOS1AP-S’) independent cultures. Lanes shown in each panel are part of the same Western blot although each band is shown separately. *p<0.05 by one-way ANOVA followed by Tukey multiple comparisons test. Error bars represent SEM. Outliers excluded with Grubb’s outlier test.

Since D-serine treatment reduced NOS1AP expression in cortical cultures, we examined whether D- serine would rescue NOS1AP-induced reductions in dendrites. We overexpressed NOS1AP-S or NOS1AP-L in DIV6 cortical neurons, and treated cultures with D-serine or vehicle for 24 hours (Fig. 7). D-serine did not affect branching in neurons that overexpress NOS1AP-S (Fig. 7b). Excitingly, treatment with D-serine rescued alterations in dendrite branching resulting from NOS1AP-L overexpression (Fig. 7e and g). Taken together, our data suggest that the NMDAR full co-agonist D-serine, and not traditional antipsychotics, rescue NOS1AP-induced reductions in dendrite branching.

Fig. 7. Acute treatment with D-Serine reverses NOS1AP-induced reductions in dendrite branching.

Fig. 7

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Neurons overexpressing NOS1AP-L and NOS1AP-S were treated with D-serine or DMSO vehicle. Colored bars (Supplementary Tables 5-8) represent significance of at least p<0.05. a. Representative images of neurons from each treatment condition. b. Treatment of neurons overexpressing NOS1AP-S with D-serine reverses NOS1AP-S-mediated decreases in dendrite number. c. and d. Treatment of neurons overexpressing NOS1AP-S with D-serine does not affect primary and secondary dendrite number. e. Treatment of neurons overexpressing NOS1AP-L with D-serine rescues dendrites. f. Treatment of neurons overexpressing NOS1AP-L with D-serine does not affect primary dendrites. g. Overexpression of NOS1AP-L reduces secondary dendrites, but treatment of neurons overexpressing NOS1AP-L with D-serine is not different than control, indicating that D-serine partially rescues NOS1AP-L-mediated reductions in dendrite branching. n=56-64 total neurons per condition from 4 independent cultures. Statistics by two-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent SEM. Scale bar = 50 μm.

It is important to note that we observed distinct effects of NMDAR agonists on primary and secondary dendrites in primary cortical cultures compared to effects observed in primary hippocampal cultures. We previously reported that NOS1AP-L decreases both primary and secondary dendrites in hippocampal neurons (Carrel et al., 2009). Data here suggest that NOS1AP-L overexpression reduces secondary, but not primary, dendrites in cortical neurons (Fig. 7f and g). This could be due to different regulatory mechanisms and signaling pathways that influence neurite outgrowth in hippocampal and cortical neurons (Ko et al., 2005; Kwon et al., 2011; Lepagnol-Bestel et al., 2013). Importantly, D-serine partially restored secondary dendrites in NOS1AP-L-overexpressing neurons (Fig. 7g). In sum, administration of D-serine reduces expression of NOS1AP in vitro and promotes functional changes to neuronal development.

Discussion

Here, we investigated the effects of acute in vivo administration of haloperidol, a traditional antipsychotic, and D-serine, an NMDAR agonist, on the expression of the SCZ-related proteins NOS1AP, D2 receptor, and DISC1. We provide evidence that D-serine, but not haloperidol, reduces NOS1AP protein expression in a sex-specific manner. We then used developing cultured cortical neurons to assess drug action on the dendritic arbor. Specifically, we examined whether treatment with antipsychotics or D-serine affects NOS1AP expression and whether treatment with antipsychotics or D-serine could rescue NOS1AP- induced alterations in dendritic branching. We found that treatment with the full NMDAR co-agonist D- serine, and not traditional antipsychotics, reduces NOS1AP expression and reverses NOS1AP-induced alterations in dendritic branching.

Our study links D-serine action to expression and function of NOS1AP, a protein encoded by a SCZ risk gene (Brzustowicz et al., 2004). NOS1AP protein is overexpressed in the DLPFC of patients with SCZ (Hadzimichalis et al., 2010), leading to reduced NMDAR signaling (Eastwood, 2005), which is thought to produce symptoms of SCZ (Coyle, 1996; Howes et al., 2015). Additionally, NOS1AP mRNA is overexpressed in postmortem tissue of patients, and chronic treatment with antipsychotic medications correlates with reduced NOS1AP-L mRNA (Xu et al., 2005). NOS1AP overexpression also reduces dendrite branching in vitro (Carrel et al., 2009), reproducing dendrite alterations in postmortem tissue of patients with SCZ (Black et al., 2004; Kolomeets et al., 2005; Kulkarni and Firestein, 2012).

Antipsychotic medications target dopamine receptors and are unable to treat all symptom domains of SCZ for the majority of patients (Patel et al., 2014). Chronic treatment with antipsychotic medications correlates with reduced NOS1AP mRNA in postmortem cortical tissue (Xu et al., 2005); however, causality has not been established. We therefore investigated if acute antipsychotic treatment reduces NOS1AP protein expression in vivo and in vitro. Haloperidol administration did not affect NOS1AP expression in vivo, and administration of haloperidol, clozapine, and fluphenazine did not change NOS1AP expression in vitro. Additionally, antipsychotic medications did not rescue NOS1AP-induced alterations to dendritic branching in vitro. Taken together, our data suggest that while these drugs were reported to correlate with decreased NOS1AP mRNA (Xu et al., 2005), they do not affect NOS1AP protein levels or dendritic branching. It is important to note that most patients with SCZ have been chronically treated with antipsychotic medication and our studies represent an animal model of acute treatment. Thus, this difference may partially account for differences seen in changes in NOS1AP mRNA levels in a previous postmortem report (Xu et al., 2005) compared with our current study.

Due to the failure of antipsychotic medications to reduce all of the symptom types of SCZ, NMDAR agonists have been suggested as potential treatment options (Hashimoto, 2014). The NMDAR hypothesis of SCZ proposes that individuals with SCZ exhibit reduced NMDAR signaling (Coyle, 1996; Howes et al., 2015), which is important for synaptic plasticity, learning and memory, and perception. (Brigman et al., 2010; Burgos-Robles et al., 2007; Kantrowitz and Javitt, 2010; Phillips and Silverstein, 2003; Wang and Peng, 2016). NOS1AP is proposed to play a role in NMDAR signaling by interacting with NOS1 (Jaffrey et al., 1998). Overexpression of NOS1AP, as in SCZ, interferes with NO signaling by sequestering NOS1, which in turn reduces NMDAR function (Eastwood, 2005; Jaffrey et al., 2002). Our data show that administration of D-serine, a full co-agonist at the NMDAR, reduces NOS1AP protein expression in vivo and in vitro and corrects NOS1AP-induced reductions in dendrite branching. Importantly, D-serine (Heresco-Levy et al., 2005; Kantrowitz et al., 2010; Kantrowitz et al., 2015; Lane et al., 2005; Tsai et al., 1998; Weiser et al., 2012) has undergone clinical trials for SCZ and depression. While D-serine clinical studies have produced mixed results (Balu and Coyle, 2015; Möller and Czobor, 2015), D-serine has been reported to alleviate symptoms of SCZ (Heresco-Levy et al., 2005; Kantrowitz et al., 2010; Kantrowitz et al., 2015; Tsai et al., 1998). Additionally, individuals with SCZ show reduced D-serine levels in cerebrospinal fluid, serum, and plasma (Bendikov et al., 2007; Calcia et al., 2012; Hashimoto et al., 2005; Hashimoto et al., 2003; Yamada et al., 2005). Our data suggest that the therapeutic benefit of D-serine may be due to two independent mechanisms: increased NMDAR signaling and reduced NOS1AP protein expression. In fact, presence of these two mechanisms may account for the mixed findings in D-serine studies in patients. Taken together, these data shed light on the shortcomings of antipsychotic treatment and further support the therapeutic potential of D-serine as a treatment option for SCZ.

Our data also suggest that haloperidol and D-serine treatment exerts sex-specific effects on protein expression of NOS1AP, D2 receptor, and DISC1. Haloperidol administration reduces D2 receptor expression in rat male cortex, but does not affect NOS1AP expression. Conversely, D-serine treatment reduces expression of NOS1AP in male rats and promotes differential DISC1 expression in male and female rats, suggesting that the therapeutic effect of D-serine may be restricted to male patients with SCZ. Thus, our data show that haloperidol and D-serine influence expression of proteins associated with SCZ through sex-specific pathways. These differential effects should be taken into account when designing future clinical trials and animal studies for the evaluation of D-serine treatment. Mixed outcomes in D- serine clinical trials may have resulted from contrasting interactions between drug action and the sex of the participants. Additionally, the sex of the patient should be factored in when designing or administering treatments for SCZ. Male and female patients respond differently to drugs used to treat those with SCZ, and different side effects occur in males and females (Patel et al., 2014), providing evidence to further justify a more personalized approach to SCZ treatment.

More in-depth investigation of sex differences in SCZ pathophysiology and pharmacology is a priority. SCZ is more prevalent in men (Iacono and Beiser, 1992), with a median rate ratio of 1.4 to that of females (McGrath et al., 2008). Additionally, the age-of-onset of SCZ is earlier in males compared to that of females (Hafner et al., 1993). The operationalized definitions of onset (first sign of mental illness, earliest SCZ symptom, start of index episode, and index admission) are reported to have a mean age of 24.3, 26.5, 27.8, 28.5, for males and 27.5, 30.6, 31.7, 32.4, for women, respectively (Hafner et al., 1993). There are a multitude of studies that focus on sex differences in SCZ, and the most recent findings include hormone, genetic, and lipid studies. For example, there is evidence that estradiol affects dopamine (Hafner et al., 1991) and that estrogen modulates the expression of brain-derived neurotrophic factor (reviewed in (Wu et al., 2013)), both of which provide neuroprotection from SCZ in females. Moreover, there is evidence for X-linked psychosis (Carrera et al., 2009; Crow, 2008; Feng et al., 2009; Goldstein et al., 2011; Philibert et al., 2007; Piton et al., 2011; Roser and Kawohl, 2010; Wei and Hemmings, 2006) and that reduced penetrance and/or X inactivation of the risk chromosome may be responsible for the lower ratio of females affected with SCZ (Goldstein et al., 2011). Moreover, males have higher levels of erythrocyte membrane lipid peroxidation, a measure of oxidative damage, than do females, and males with SCZ have even higher levels (Ramos-Loyo et al., 2013). Thus, mechanisms underlying sex differences in SCZ are complex.

Antipsychotic medications show greater efficacy in women (Cotton et al., 2009; da Silva and Ravindran, 2015; Ochoa et al., 2012; Seeman, 1986; Szymanski et al., 1995). Our data suggest that haloperidol exerts sex-specific effects on expression of D2 receptor, the receptor to which most antipsychotic medications bind. Updated versions of the dopamine hypothesis propose that positive symptoms of SCZ are due to elevated dopamine release along the mesolimbic pathway, while negative and cognitive symptoms are the consequence of reduced dopamine release in the mesocortical pathway (Citrome, 2014). Thus, a reduction in cortical D2 receptor expression, as observed in male rats, may further reduce dopamine release along the mesocortical pathway, and consequently, exacerbate negative and cognitive symptoms of SCZ. Our results underscore the fact that sex is a critical factor to consider when evaluating treatment for a patient with SCZ. While D-serine treatment may promote a therapeutic effect in males through reduction of NOS1AP expression, haloperidol treatment may show better efficacy in females as it does not reduce D2 receptor expression in the cortex.

In addition, it is necessary to further investigate the effects of D-serine and haloperidol administration on NOS1AP, D2 receptor, and DISC1 expression during different phases of the estrous cycle, as female rat estrous cycles were not synchronized in this study. The estrous cycle time points during which drug administration occurs may interact with the main effects of these drugs on the expression of these proteins. Estrogen fluctuations throughout the estrous cycle influence expression of various synaptic proteins in vivo (Ramos-Ortolaza et al., 2017; Sarkar and Kabbaj, 2016; Tada et al., 2015). Hormones, such as estrogen, may be involved in the regulation of treatment response and thus subsequently affect treatment-mediated expression of NOS1AP, DISC1, and D2 receptor in female rats. Additionally, further clinical and animal studies are needed to investigate potential interactions between patient intake of antipsychotics or NMDAR agonists and patient hormone levels or phase in the menstrual cycle.

One important factor to consider in the translation of D-serine to the clinic is potential differences in the metabolism of D-serine based on administration method. In this study, we administered D-serine via intraperitoneal injection to remain consistent with previous animal studies of D-serine action (Andersen and Pouzet, 2004; Hashimoto and Chiba, 2004; Karasawa et al., 2008; Wolosker, 2007). It is reported that oral administration of D-serine to animals results in significant D-serine metabolism by D-amino acid oxidase, which can lead to confounding results between different studies based on the administration method used (Hashimoto et al., 2009). This facet should be considered when determining D-serine dosages in clinical studies, as oral D-serine administration may also require the administration of D- amino acid oxidase inhibitors in tandem (Hashimoto et al., 2009).

Taken together, our data provide critical insight into the effects of haloperidol and D-serine administration on the expression of NOS1AP, D2 receptor, and DISC1. We provide evidence that drug action is sex-dependent, and we advance a molecular basis for observed sex differences in drug efficacy in patients with SCZ. Lastly, we find that D-serine treatment reverses the detrimental effects of increased NOS1AP expression on dendrites, and our data promote further investigation into D-serine as a potential therapeutic agent for SCZ.

Supplementary Material

1

Highlights.

  • Haloperidol treatment does not affect NOS1AP expression in vivo and in vitro.

  • Haloperidol affects D2 receptor expression in vivo in sex-specific manner.

  • D-serine treatment reduces NOS1AP expression in vivo in male rat cortex.

  • D-serine affects DISC1 expression in sex-specific manner but does not affect D2R.

  • D-serine rescues NOS1AP-induced reductions in dendrite branching in vitro.

Acknowledgments

This work was supported by National Alliance for Research on Schizophrenia and Depression 2012 Marion G. Nicholson Distinguished Investigator Award (to B.L.F.) and National Science Foundation (grant number IBN-1353724) to B.L.F. K.C.S. and A.O. were supported by National Institutes of Health Biotechnology Training Grant (grant number T32 GM008339-20). E.K.A. was awarded Aresty Research Funding from Rutgers University.

Footnotes

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Declarations of interest: none

Author Contributions:

KCS planned and performed experiments, analyzed data, and wrote manuscript.

EKA performed experiments and analyzed data.

AO analyzed data and wrote manuscript.

AJK performed experiments and analyzed data.

LMB and SMS wrote manuscript.

BLF planned experiments, analyzed data, and wrote manuscript.

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