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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Mol Cell Neurosci. 2020 Sep 26;109:103562. doi: 10.1016/j.mcn.2020.103562

Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis

Christen M Crosta 1,2,#, Kristina Hernandez 1,3,#, Atul K Bhattiprolu 1,#, Allen Y Fu 1, Jennifer C Moore 4, Stephen G Clarke 1, Natasha R Dudzinski 1, Linda M Brzustowicz 4, Kenneth G Paradiso 1, Bonnie L Firestein 1,*
PMCID: PMC7736313  NIHMSID: NIHMS1632954  PMID: 32987141

Abstract

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with D-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, D-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Keywords: hiPSC-derived neurons, NOS1AP, Antipsychotic medication, D-serine, Morphology, Arborization

Graphical abstract

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INTRODUCTION

Induced pluripotent stem cell (iPSC) involves the reprogramming of somatic cells into a pluripotent state by the overexpression of key transcription factors, such as OCT4, c-Myc, SOX2, and KLF4, which maintain pluripotency in embryonic stem cells (ESCs; (Takahashi & Yamanaka, 2006)), and iPSCs can give rise to any cell type from the three germ layers. The study of neurodevelopmental disorders can particularly benefit from iPSC technology due to the lack of or inadequacy of existing animal models and the difficulty of accessing human neural stem cells and neurons for in vitro studies. Disease phenotypes cannot be recapitulated faithfully in animal models, especially in the case of neuropsychiatric disorders, such as schizophrenia, which involve higher order cognitive function. In addition, despite the genetic similarities between humans and rodents, there are differences in the downstream effects of genetic alterations, and animals do not always demonstrate disease symptoms seen in humans (Inoue & Yamanaka, 2011).

Drug development relies on cell lines for proof- of- concept studies and toxicity screenings. In the case of neurons, cultured cells are limited in that they do not develop in a natural environment, often do not contain a full complement of cell types, such as astrocytes and microglia in the brain, lack two-way communication with other parts of the brain and sensory systems, and do not integrate into a network that includes local and extended network level activity. Additionally, rodent cell lines do not fully mimic human biological processes, and this is due, in part, to the fact that the type and/or distribution of ion channels and receptors on the surface of the cell may be different from those in human cells (Inoue & Yamanaka, 2011; Paradiso, Zhang, & Steinbach, 2001). Immortalized human cells lines are also not ideal for disease studies because disease-relevant cell types are often not available, and the immortalization process alters native cellular responses (Ebert & Svendsen, 2010). In fact, compounds that have shown efficacy in cell lines and animals have not shown therapeutic effects in humans, and in the worst case scenario, have shown toxic effects in humans (Inoue & Yamanaka, 2011). Therefore, the use of human neural cell cultures can greatly complement the use of cell lines and animal models for disease studies. With the availability of iPSCs, the study of the differentiation process of human neural stem cells into mature neurons in a more physiologically relevant manner is now possible.

iPSC technology can be used to ectopically express a gene that has been linked to a neuropsychiatric disorder, such as schizophrenia, in the disease-relevant cell type, the neuron. For polygenic-based diseases, such as schizophrenia, ectopic expression or knockdown of proteins of interest for disease modeling aids in the understanding of how each gene contributes to the disease phenotype. Postmortem studies show that neurons from patients with cognitive and neurological disorders display aberrant neuronal morphology, such as a reduction in dendrite number or branching (reviewed in (Kulkarni & Firestein, 2012; Zoghbi, 2003)). Specifically, in patients with schizophrenia, the dendritic arbor of cortical layers III and V pyramidal neurons in the prefrontal cortex is less complex than arbors in neurons from control patients (Black et al., 2004; Broadbelt, Byne, & Jones, 2002). Importantly, many of the genes that have been linked to schizophrenia are involved in determining dendrite morphology. Specifically, we have reported that there is increased expression of the long and short isoforms of Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP-L and NOS1AP-S) in postmortem BA46 tissue from patients with schizophrenia (Hadzimichalis et al., 2010) and that NOS1AP-L plays a major role in the regulation of dendrite branching (Carrel et al., 2009; Carrel et al., 2015). Overexpression of NOS1AP-L results in altered dendrite patterning (Carrel et al., 2009; Carrel et al., 2015), increases formation of immature spines (Hernandez et al., 2016), and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs) (Hernandez et al., 2016). In contrast, overexpression of NOS1AP-S regulates dendrite number only when overexpressed between day in vitro (DIV) 5-7 (Carrel et al., 2009), increases the number of both immature and mature spines (Hernandez et al., 2016), and increases the frequency of mEPSCs (Hernandez et al., 2016). Thus, overexpression of NOS1AP isoforms in human iPSC (hiPSC)-derived neurons may serve as a model for understanding the role of NOS1AP in development of schizophrenia.

Here, we used human induced pluripotent stem cell (iPSC) technology to generate human neural progenitor cells (NPCs) and neurons and characterize dendrite development. We determined how development affects expression of NOS1AP isoforms and investigated the role that NOS1AP plays in shaping the dendritic arbor. We overexpressed either NOS1AP-L or NOS1AP-S in human neurons and observed decreased dendrite branching. Furthermore, we treated the human neurons with antipsychotic agents, such as clozapine, haloperidol, fluphenazine, the NMDA partial agonist GLYX-13, or the NMDA co-agonist D-serine and found that only treatment with D-serine significantly decreases expression of NOS1AP-L. NOS1AP-S expression was unaffected by all treatments. However, treatment with D-serine does not rescue decreased dendrite branching in neurons overexpressing NOS1AP isoforms. Our studies demonstrate that hiPSC-derived neurons can be used to study perturbations in protein expression that occur in schizophrenia and can help elucidate the mechanisms by which schizophrenia develops as neurons mature.

MATERIALS AND METHODS

Ethics approval

This study was determined to be exempt (category 4) by the Rutgers Institutional Review Board.

Antibodies

Chicken polyclonal green fluorescent protein (GFP) antibody (cat. #PA1-9533; 1:500) was purchased from ThermoFisher (Omelchenko et al., 2020). Mouse monoclonal GAPDH (cat# MAB374; 1:1000), mouse monoclonal OCT4 (cat# MAB4401; 0.5 μg/ml), rabbit polyclonal Nanog (cat# AB5731; 0.5 μg/ml), rabbit polyclonal SOX2 (cat# AB5603; 0.5 μg/ml), mouse monoclonal Tra-1-60 (cat# MAB4360; 0.5 μg/ml), and rabbit polyclonal synaptophysin antibodies (cat# 041019; clone YE269; 1:200) were from Millipore. Mouse monoclonal PAX6 (cat# MAB5554; 1:100) and rabbit polyclonal Musashil (cat# AB59977; 1:50) were also from Millipore. These antibodies have been used to characterize iPSCs and NSCs (i.e. (Scarnati, Boreland, Joel, Hart, & Pang, 2020)). Chicken polyclonal microtubule associated protein 2 (MAP2; cat# 102130-230; 1:1000) was from VWR, and rabbit polyclonal Tbr2 antibodies (cat# 23345) were from Abeam. Rabbit polyclonal NOS1AP (cat#sc-9138) antibody was from Santa Cruz Biotechnology (Hernandez et al., 2016; Svane et al., 2018). Rabbit polyclonal vesicular glutamate transporter 1 (VGLUT1) antibody (cat# 135302; 1:1000) was from Synaptic Systems. All secondary antibodies were purchased from ThermoFisher.

DNA constructs

cDNAs encoding long and short isoforms of human NOS1AP (NOS1AP-L and NOS1AP-S), NOS1AP-L-214-end (NOS1AP-L-APTB), and NOS1AP-L-181-307 (NOS1AP-M) were subcloned into pCAG-GFP as described previously (Carrel et al., 2009; Hernandez et al., 2016). cDNA encoding NOS1AP-L-1-487 (NOS1AP-L-APDZ) and NOS1AP-L were subcloned into in pCAG-IRES-EGFP and pCAG-IRES-TagRFP plasmid, respectively.

Human induced pluripotent stem cell (hiPSC) Derivation

Human foreskin fibroblasts (HFFs) were infected with retroviruses expressing OCT4, SOX2, KLF4, and c-MYC. Five days after transduction, cells were plated onto Matrigel (BD Biosciences)-coated 10 cm dishes in mTESR medium (StemCell Technologies). Medium changes were performed daily with fresh mTESR, and the cells were grown for 23 days. hiPSC colonies were picked manually and plated onto Matrigel-coated 6 well plates in mTESR. hiPSCs were passaged after dissociation using Dispase (BD Biosciences) and a cell scraper and replating into mTESR. Human iPSCs were not used for experiments until passage 13, when the Sendai virus can no longer be detected.

hiPSC differentiation into Neural Progenitor Cells (NPCs) and neurons

hiPSCs were grown in mTeSR/Neurobasal medium (Invitrogen) supplemented with 500 ng/ml Noggin. After six days, the medium was changed to Neurobasal medium supplemented with 500 ng/ml Noggin. On day 10, the cells were dissociated and plated in Neurobasal medium onto 20 μg/ml laminin (Sigma)-coated dishes. Eight days later, the medium was changed to DMEM/F12 with Glutamax/Neurobasal supplemented with 20ng/ml Fibroblast Growth Factor (FGF; Peprotech), 0.5x N2 supplement (Invitrogen), and 0.5x B27 (−) Vitamin A supplement (Invitrogen), which we refer to as Neural Progenitor Medium (NPM). NPCs were grown and passaged in NPM onto ¼ diluted Matrigel-coated plates. For differentiation into neurons, NPCs were plated in NPM onto a ¼ diluted Matrigel-coated 10 cm dish. One day after plating, the medium was changed to Neurobasal medium supplemented with 1x B27 (−) Vitamin A, and 10ng/ml of Brain Derived Neurotrophic Factor (BDNF; Peprotech), which we refer to as Neural Differentiation Medium (NDM). Four days later, the cells were dissociated using Accutase (Stem Cell Technologies) and plated in NDM onto substrates that were previously coated with 10 μg/ml poly-D-lysine (PDL; Sigma) for 2 hours followed by 10 μg/ml laminin for 2 hours.

The complete differentiation (iPSC to NSC and then to neurons) was performed three independent times. Percentage of cells expressing an iPSC or NSC marker was computed by dividing the number of cells that are immunopositive for the marker by the total number of cells as determined by Hoechst 33342 staining for nuclei.

Neuronal culture and drug administration

hiPSC-derived neurons grown for 4 days post-differentiation (DD 4; DD is days post-differentiation, and DD0 is the day of differentiation) were plated at 210,000 cells/cm2 (158,000 cells/cm2 for neurons differentiated from NPCs generated using Gibco® PSC Neural Induction Medium) onto PDL/laminin-coated substrates. Substrates were coated with 10 μg/ml PDL for 2 hours, followed by a 2 hour coating with 10 μg/ml laminin. Medium changes were performed three times per week with NDM supplemented with 1 μg/ml laminin. Flupenazine, clozapine, haloperidol, D-serine, and GLYX-13 (all from Sigma) were dissolved in sterile vehicle containing dimethyl sulfoxide (DMSO). The final concentration of DMSO in treatments was 0.1%. The hiPSC-derived neurons were treated on DD 19 by adding drugs or vehicle to the medium. Concentrations of antipsychotic drugs were chosen based on the therapeutic plasma level concentrations, with 10- to 30-fold higher levels in brain tissue (G. Zhang, Terry, & Bartlett, 2007).

Immunocytochemistry

Cells were fixed for 15 min in 4% paraformaldehyde (PFA) in 1 x phosphate-buffered saline (PBS) for neuronal marker immunostaining or for 15 min in methanol at −20°C for 15 min stem cell marker immunostaining. Cells were permeabilized and blocked in 2% normal goat serum and 0.1% Triton-X-100 in 1 x PBS at room temperature for 1 hour. The cells were incubated with primary antibodies for 2 hours, washed twice with 1 x PBS, and then incubated with secondary antibodies for 1hour.

Western Blot Analysis

Twelve micrograms of protein from cell lysates in TEE buffer (25 mM Tris-HCl, pH 7.4, 1mM EDTA, 1mM EGTA) were loaded and resolved on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to Immobilon P membrane (Millipore) in transfer buffer lacking SDS. The blot was probed with a rabbit antibody to NOS1AP (Santa Cruz, #R-300) and visualized using ECL Plus (Amerisham Biosciences) with a secondary antibody coupled to horseradish peroxidase. For GAPDH normalization, the blot was probed with a mouse antibody to GAPDH (Millipore).

Electrophysiology

DD 28 neurons were patch-clamped in the whole cell configuration using thick walled glass pipettes with a resistance of 4-6 mega Ohms. To activate voltage-gated ion channels, neurons were voltage-clamped at −70mV and a series of 200ms voltage jumps from −40 to +10 mV were applied at 10 mV increments. A series of current injections were applied at 20 pA increments to activate voltage-gated sodium channels and generate action potential activity. Internal solution contained (in mM) 125 K-gluconate, 20 KCl, 10 Na2-phosphocreatine, 4 MgATP, 0.3 GTP, 10 HEPES, 5 EGTA, and was adjusted to pH 7.2 using KOH. The external recording solution contained (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 glucose, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, 2 sodium pyruvate and 25 NaHCO3, and was adjusted to pH 7.4 by bubbling with carbogen (95% O2, 5% CO2).

Transfection and Cell Imaging for Dendrite Branching Analysis

At DD19, human neurons were transfected with pCAG-GFP, pCAG-GFP-NOS1AP-L, or pCAG-GFP-NOS1AP-S using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were fixed with 4% PFA in PBS for 15 min and immunostained for GFP and MAP2. For dendrite branching analysis, we used the Bonfire program (Kutzing, Langhammer, Luo, Lakdawala, & Firestein, 2010; Langhammer et al., 2010). Neurons were imaged at 20x using an Olympus Optical (Tokyo, Japan) IX50 microscope with a Cooke Sensicam CCD cooled camera, fluorescence imaging system, and ImagePro software (MediaCybernetics, Silver Spring, MD). Only neurons with clear, non-condensed nuclei, as assessed by Hoechst 33225 dye staining were included in the analysis. Neurons were traced with the experimenter blinded to the condition using NeuronJ (NIH, Bethesda, MD) and NeuronStudio (Mt. Sinai Medical School, NYC, NY). Data were analyzed using MatLab (MathWorks, Natick, MA). Sholl analyses were performed at 6 μm intervals, and dendrites were counted if >3 μm.

Statistics

All statistics were calculated using the Prism 5.0 and Prism 7.0 software from GraphPad (La Jolla, CA). Tests used are noted in figure legends.

RESULTS

Successful generation of human iPSCs and NPCs.

To assess the role of NOS1AP in human neuronal development, we first set up our human culture model. Human induced pluripotent stem cells (hiPSCs) were generated using somatic cell reprogramming, and they were differentiated into neuronal progenitor cells (NPCs) using conditions that favor the generation of forebrain NPCs. To characterize whether our procedures were successful, we performed immunocytochemical analysis. As expected, hiPSCs generated are positive for the pluripotency markers OCT4, Tra-1-60, Nanog, and SOX2 (Figure 1A). The NPCs are positive for the early neural progenitor cell markers MusashiI and PAX6, the NPC marker SOX2, and the cortical progenitor marker Tbr2 (Figure 1B). Furthermore, the immunostaining shows that almost all cells in the appropriate culture are iPSCs and NPCs as evidenced by overlap with Hoechst 33342 staining for total nuclei (Figure 1C,D).

Fig. 1. Characterization of hiPSCs and hiPSC-derived NPCs.

Fig. 1.

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Validation of hiPSCs and hiPSC-derived human neural progenitor cells in culture. A. hiPSCs express pluripotency markers OCT4, Tra-1-60, Nanog, and SOX2. Representative images are shown. B. hiPSC-derived NPCs express NPC markers MusashiI, SOX2, PAX6 and the cortical progenitor marker, Tbr2. C. Quantitation of percentage of total cells expressing iPSC and NPC markers represented in panels A and B. Hoechst 33342 staining is shown to identify nuclei of all cells in the culture. The complete differentiation (iPSC to NSC and then to neurons) was performed three independent times. Scale bar = 50 μm.

Human iPSC-derived neurons mature in culture and have functional ion channels

Our goal was to produce a glutamatergic population of neurons by withdrawing FGF from and adding BDNF to the medium. To confirm that we did indeed produce these neurons, we immunostained the iPSC-derived neurons and found that approximately 50-70% of the cells in the cultures are immunoreactive for the neuronal markers, microtubule-associated protein 2 (MAP2) and synaptophysin, and the glutamatergic marker, vesicular glutamate transporter 1 (VGLUT1) at days post-differentiation (DD) 14 (Figure 2A,B). To test that these neurons are indeed functional, we performed whole cell patch-clamp analysis. The hiPSC-derived neurons show normal sodium and potassium currents when voltage-clamped and normal induced action potentials when current-clamped (Figure 2C,D). Of the 34 neurons recorded, 32 neurons demonstrated voltage-gated sodium and potassium channels, and 24 out of 34 neurons demonstrated action potential firing (i.e. 71% had the ability to fire action potentials). Taken together, our data support the idea that we generated functional glutamatergic neurons.

Fig. 2. hiPSC-derived neurons are glutamatergic and show functional ion channels.

Fig. 2.

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A. hiPSC-derived neurons express neuronal markers MAP2, synaptophysin, and VGLUT1 at days post-differentiation (DD) 14. Scale bar = 50 μm. Hoechst 33342 staining is shown to identify nuclei of all cells in the culture. Representative images are shown. B. Quantitation of percentage of total cells expressing neuronal markers represented in panel A. C, D. DD 28 neurons were patch-clamped in the whole cell configuration using thick walled glass pipettes with a resistance of 4-6 mega Ohms. To activate voltage-gated ion channels, neurons were voltage-clamped at −70mV, with a jump to −40 mV, and returned to −70 mV. Additional jumps were made at 10 mV increments, with the final jump to +10 mV. In current clamp mode, a series of depolarizing current injections were applied at 20pA intervals to activate voltage-gated sodium channels to generate action potential activity. C. hiPSC-derived neurons show normal sodium and potassium currents when voltage-clamped on DD 28. D. hiPSC-derived neurons show normal induced action potentials when current-clamped on DD 28. n=34 neurons. Of the 34 neurons recorded, 32 neurons demonstrated voltage-gated sodium and potassium channels, and 24 out of 34 neurons demonstrated action potential firing (i.e. 71% had the ability to fire action potentials). The complete differentiation (iPSC to NSC and then to neurons) was performed three independent times.

To assess maturation of the human neurons in our culture system, we quantified the number of MAP2-positive cells in the population of cells over three different time points during differentiation (Figure 3A,B). The percentage of MAP2-positive cells significantly increases from 58.5% to 94% from DD 14 to DD 42. The increase in MAP2-positive cells in our neuronal cultures over time is indicative of neuronal maturation.

Fig. 3. hiPSC-derived Neurons mature in vitro.

Fig. 3.

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Neuronal cultures were grown for the indicated number of days post-differentiation (DD), fixed, and immunostained for microtubule-associated protein 2 (MAP2), a marker of neuronal maturity. A. MAP2 immunoreactivity increases from DD14 to DD42. Representative images are shown. B. Percentage of MAP2-immunopositive cells for neurons on DD 14, DD 28, and DD 42. Hoechst 33342 staining was used to identify nuclei of all cells in the culture. Scale bar = 50 μm. ***p< 0.001 and ****p< 0.0001 versus neurons on DD 14. p values were determined by one-way ANOVA followed by Bonferroni multiple comparisons test. Error bars indicate ± s.e.m. n = 59 neurons, DD14; n = 56, DD28, n = 56, DD42.

NOS1AP protein expression increases during hiPSC-derived neuronal maturation.

Since NOS1AP plays a role in neuronal development, including dendritogenesis (Carrel et al., 2009; Carrel et al., 2015) and cortical neuron migration (Carrel et al., 2015), we determined the stages in development when NOS1AP is expressed. As such, we isolated protein from cell lysates from hiPSCs, hiPSC-derived NPCs, hiPSC-derived neurons on DD14 and hiPSC-derived neurons on DD28. We performed Western blot analysis to detect NOS1AP protein expression (Figure 4A,B). Interestingly, NOS1AP-L protein levels stay relatively consistent throughout neural differentiation. In contrast, NOS1AP-S protein expression significantly increases from both the iPSC state and the NPC state to neurons on DD28.

Fig. 4. NOS1AP-S, but not NOS1AP-L, protein levels increase during neuronal development.

Fig. 4.

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A. Densitometry analysis of immunoblotting for two isoforms of NOS1AP, long (L) and short (S) in cell lysates from hiPSC-derived cell types. NOS1AP-S increases during neural differentiation, whereas NOS1AP-L expression does not change. **p< 0.01 and ***p< 0.001 versus neurons on DD14. p values were determined by one-way ANOVA followed by Bonferroni multiple comparisons test. Error bars indicate s.e.m. n = 6 for each cell type. B. Representative Western blot analyzed in panel A. C. Inverted GFP images of representative hiPSC-derived neurons. hiPSC-derived NPCs were transfected with peGFP and differentiated. Active primary and secondary dendrite branching occurs at DD21 in developing hiPSC-derived neurons. Scale bar = 50 μm.

Overexpression of both NOS1AP isoforms decreases dendrite branching in hiPSC-derived neurons.

To investigate how NOS1AP affects the arborization of developing human neurons, we first determined the developmental time period when hiPSC-derived neurons undergo active dendritogenesis. We transfected differentiating NPCs at different time points with a plasmid that allows for GFP expression to monitor changes in cellular morphology (Figure 4C). The cells undergo neurite extension from DD 1 to DD 9, with axon specification occurring on DD12. Secondary dendrite extension begins on approximately DD17, with active primary and secondary branching occurring by DD21. Taken together, our data suggest that our in vitro human neuronal culture system exhibits the hallmark stages of dendritogenesis, similar to stages seen in rat neuronal cultures (Arimura & Kaibuchi, 2007).

We then asked whether NOS1AP isoforms play a role in dendrite branching in human neurons. We chose a time point when active dendrite branching is occurring so that we could compare our data to those we previously published in rodent neurons (Carrel et al., 2009; Svane et al., 2018). We transfected human neurons with constructs encoding GFP or GFP fusions of either NOS1AP-L or NOS1AP-S on DD19. After 48 hours of expression, neurons were fixed and immunostained for GFP and MAP2 (Figure 5A). For dendrite branching analysis, only GFP-positive neurons with non-condensed nuclei were included, and neurons were traced and analyzed using an automated Sholl analysis (Kutzing et al., 2010; Langhammer et al., 2010) with the experimenter blinded to the condition. Although overexpression of NOS1AP-L in rat neurons shows a more robust decrease in overall dendrites as evidenced by Sholl analysis (Svane et al., 2018), overexpression of either NOS1AP- L or NOS1AP- S significantly decreases dendrite branching to the same degree in human neurons during the time point when active branching is occurring (Figure 5B). These data demonstrate that NOS1AP-S plays a role in dendritogenesis in humans that is distinct from that in rat, and when expression is upregulated too early in development, overexpression of either NOS1AP isoform results in a reduction in dendritic complexity.

Fig. 5. Overexpression of NOS1AP-L or NOS1AP-S decreases dendrite branching in hiPSC-derived neurons.

Fig. 5.

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A. Inverted GFP images of representative hiPSC-derived neurons transfected on DD19 with constructs encoding GFP (control), GFP-NOS1AP-L, or GFP-NOS1AP-S, and fixed and immunostained for GFP for dendrite counting on DD21. Scale bar = 50 pm. B. Proximal Sholl analysis within the first 120 μm from the soma. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed to compare overexpression of NOS1AP-L or NOS1AP-S versus control. p values are shown in table. Error bars indicate s.e.m. n = 57 neurons, Control; n = 46, NOS1AP-L; n= 47, NOS1AP-S. Neurons are from three independent cultures for GFP (control) and NOS1AP-L conditions and two independent cultures for NOS1AP-S condition.

Overexpression of NOS1AP-L or NOS1AP-S decreases secondary and tertiary dendrite number.

To better understand how NOS1AP-L and NOS1AP-S overexpression alters dendrite patterning of human neurons, we analyzed changes in dendrite length and number when either isoform is overexpressed. Overexpression of either isoform of NOS1AP has no effect on the number of primary dendrites (Figure 6A). In contrast to our findings in rat hippocampal cultures where overexpression of NOS1AP-L decreases dendrite branching between DIV 0-12 and overexpression of NOS1AP-S only has an effect on dendrite number during DIV 5-7 (Carrel et al., 2009), overexpression of either NOS1AP-L or NOS1AP-S decreases the number of secondary and tertiary dendrites compared to control (GFP) neurons in the hiPSC derived neurons at DD21. Overexpression of either isoform did not change the length of primary, secondary, or tertiary dendrites (Figure 6B). Taken together, our results indicate that overexpression of NOS1AP isoforms decreases the number of higher order (> secondary) dendrites but does not alter dendrite length.

Fig. 6. Overexpression of NOS1AP isoforms decreases secondary and tertiary dendrite number.

Fig. 6.

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A. Quantification of average number of primary, secondary, and tertiary dendrite branches from DD21 hiPSC-derived neurons overexpressing GFP (control), GFP-NOS1AP-L, or GFP-N0S1AP-S. **p< 0.01, ***p< 0.001 and 0.0001 versus control, p values were determined by one-way ANOVA followed by Bonferroni multiple comparisons test. Error bars indicate ± s.e.m. n = 57 neurons, Control; n = 46, NOS1AP-L, n = 47, NOS1AP-S. B. Quantification of average length of primary, secondary, and tertiary branches from DD21 hiPSC-derived neurons overexpressing GFP (control), GFP-NOS1AP-L, or GFP-NOS1AP-S. Error bars indicate ± s.e.m. n = 57 neurons, Control primary length; n = 54 neurons, Control secondary length; n = 51 neurons, Control tertiary length; n = 46, NOS1AP-L primary length; n = 37, NOS1AP-L secondary length; n = 21, NOS1AP-L tertiary length; n = 47, NOS1AP-S primary length; n = 44, NOS1AP-S secondary length; n = 34, NOS1AP-S tertiary length. Neurons are from three independent cultures for GFP (control) and NOS1AP-L conditions and two independent cultures for NOS1AP-S condition.

D-serine treatment decreases NOS1AP-L protein levels.

We previously reported that d-serine reduces expression of NOS1AP-L in the cortex when administered to male rats, but not female rats (Svane et al., 2018). Furthermore, haloperidol, an antipsychotic medication that acts on the dopaminergic system had no effect of NOS1AP-L, regardless of sex (Svane et al., 2018). Thus, to determine whether endogenous NOS1AP-L and NOS1AP-S protein expression can be reduced in our male human neurons (derived from foreskin fibroblasts) in a manner similar to those we observed in rats, we treated hiPSC-derived neurons on DD19 with three commonly prescribed antipsychotic agents, fluphenazine, clozapine, and haloperidol, and two NMDA receptor agonists, D-serine and GLYX-13. Given the therapeutic plasma levels of fluphenazine (0.4 ng/ml), clozapine (100-400 ng/ml), and haloperidol (5- 14 ng/ml), with 10- to 30-fold higher levels in brain tissue, neurons were treated with vehicle (DMSO), 12 ng/ml fluphenazine, 3 ug/ml clozapine, or 250 ng/ml haloperidol for 48 hours, from DD19-21. D-serine is a potent agonist of the glycine co-agonist site of NMDA receptors, whereas GLYX-13 is a partial agonist of the glycine site. To maximally activate NMDAR glycine binding sites, neurons were treated for 48 hours, from DD19-21, with 10 μM D-serine or 1 μM GLYX-13, concentrations that we have previously used in our studies (Svane et al., 2018). Protein extracts from treated neurons were subjected to Western blot analysis for NOS1AP isoform protein expression (Figure 7A). Consistent with our report in rats, treatment with antipsychotics has no effect on NOS1AP protein expression (Figure 7B). In contrast, treatment with D-serine, but not GLYX-13, significantly decreases NOS1AP-L protein expression (Figure 7B). It is important to note that all treatments affected the range of NOS1AP-S expression levels. These data suggest that D-serine may be a viable treatment for patients with NMDA hypofunction as it decreases NOS1AP-L in both the rodent and human neurons.

Fig. 7. D-serine treatment decreases NOS1AP-L protein levels.

Fig. 7.

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A. hiPSC-derived neurons were treated with antipsychotics or NMDA receptor agonists on DD 19 for 48 hours. Extracts from cultures were analyzed by SDS-PAGE and Western blotting using antibodies that recognize both isoforms of NOS1AP or GAPDH. Representative blot is shown. B. Densitometry analysis of NOS1AP normalized to GAPDH expression. Error bars indicate ± s.e.m. n = 6, DMSO; n = 5, clozapine, n = 6, Haloperidol; n = 6, Fluphenazine; n = 6, D-Serine; n = 6, GLYX-13 from three experimental replicates. All analyses were performed by first normalizing to GAPDH as an internal loading control and then comparing experimental condition to DMSO. *p< 0.05 versus DMSO. p values were determined by one-way ANOVA followed by Bonferroni multiple comparisons test.

D-serine treatment does not reverse NOS1AP isoform-mediated changes to dendrites.

Since we found that treatment with D-serine significantly decreases NOS1AP-L protein expression in the hiPSC-derived neurons, we performed experiments to determine whether D-serine treatment can reverse decreased dendrite branching as a result of NOS1AP isoform overexpression. We dissolved D-serine in a vehicle that includes 0.1% DMSO that is commonly used for administration of antipsychotics, including our previous work in rat hippocampal neurons (Svane et al., 2018). Unexpectedly, treatment with vehicle alone altered baseline dendrite branching and the effects of overexpression of NOS1AP isoforms on dendrite branching (Supplementary Figure 1A,B). Most importantly, treatment with D-serine decreased dendrite branching (Figure 8B) and did not reverse the effects of NOS1AP isoform overexpression on dendrites (Figure 8B). Taken together, our data suggest that regardless of the effect of vehicle, treatment with D-serine decreases both endogenous NOS1AP-L protein (Fig. 7) and dendrites but does not restore dendritic arborization when NOS1AP isoforms are overexpressed.

Fig. 8. Treatment with D-serine does not rescue decreased dendrite number as a result of GFP-NOS1AP isoform overexpression.

Fig. 8.

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A. Inverted GFP images of representative hiPSC-derived neurons transfected on DD19 with constructs encoding GFP (control), GFP-NOS1AP-L, or GFP-NOS1AP-S, treated with vehicle (0.1% DMSO) or D-serine (10μM) and fixed and immunostained for GFP for dendrite counting on DD21. Scale bar = 50 μm. B. Proximal Sholl analysis within the first 120 μm from the soma. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed to determine statistical significance between groups, p values are shown in table. Error bars indicate s.e.m. n = 199 neurons, GFP-Control, Vehicle; n=77, GFP-Control, D-serine; n=77, GFP-NOS1AP-L, Vehicle; n=60, GFP-NOS1AP-L, D-serine; n=150, GFP-NOS1AP-S, Vehicle; n=38, GFP-NOS1AP-S, D-serine. Neurons are from at least three independent cultures.

DISCUSSION

Schizophrenia is believed to be a developmental disorder (Benes, 1991; Brent, Seidman, Thermenos, Holt, & Keshavan, 2014; Bunney, Potkin, & Bunney, 1995; Murray, Jones, & O’Callaghan, 1991), and as such, our laboratory has studied the role of isoforms of NOS1AP, a protein encoded by a schizophrenia susceptibility gene, in neuronal development using both in vitro and in vivo rodent models (Carrel et al., 2009; Carrel et al., 2015; Hernandez et al., 2016). However, there are limitations to animal models of schizophrenia, such as differences in biological substrates and metabolic pathways (Tordjman et al., 2007), lack of relevance to humans of effects of prenatal manipulations, such as stress, in mice (Tordjman et al., 2003), and non-similarity of behaviors (reviewed in (Tordjman et al., 2007)). In the current study, we developed a human iPSC-derived neuron platform in which to study the effects of NOS1AP in dendritogenesis. We did observe similar decreases in endogenous NOS1AP-L expression with D-serine treatment (Svane et al., 2018) and in dendrite arborization with NOS1AP-L overexpression (Carrel et al., 2009) in both the rodent and human systems. However, we found important differences between the two species. Specifically, D-serine treatment decreases NOS1AP-S protein expression in cultured rat neurons (Svane et al., 2018), but no statistically significant decrease was observed in human neurons. Second, although overexpression of NOS1AP-S shows a less robust decrease in dendritogenesis than does overexpression of NOS1AP-L in cultured rat neurons (Carrel et al., 2009; Svane et al., 2018), in human neurons, overexpression of either isoform yields similar decreases in dendrites. Thus, rodent neurons may be good model system for studying the roles of proteins and genes related to neurodevelopmental disorders since they recapitulate most characteristics of human neurons.

We found that NOS1AP-S protein levels increase during neuronal development from NPCs to neurons on DD28. However, NOS1AP-L expression maintains relatively constant expression throughout development. Our results are consistent with NOS1AP isoform expression in the developing rat brain, with NOS1AP-S expression increasing dramatically during development from E15 to P14 and NOS1AP-L expression increasing only moderately (Carrel et al., 2009). These data point to distinct roles for NOS1AP isoforms during neuronal development.

We used our human neuron system to overexpress isoforms of NOS1AP to compare neurons that are genetically identical. We study NOS1AP for the following reasons. A subset of patients with schizophrenia displays single nucleotide polymorphisms (SNPs) within NOS1AP that are significantly associated with schizophrenia (Brzustowicz et al., 2004; Kremeyer et al., 2008; Zheng et al., 2005). In particular, one SNP in NOS1AP was identified that increases gene expression by enhancing transcription factor binding (Wratten et al., 2009). Taken together with the finding that both mRNA and protein expression of NOS1AP isoforms are increased in postmortem brain samples of subjects with schizophrenia (Hadzimichalis et al., 2010; Xu et al., 2005), it is important to study functional implications of increased levels of NOS1AP in neuronal culture systems.

In vitro models of human neuronal development afford the ability to screen drugs for their potential therapeutic or harmful effects on human neurodevelopment. As such, we tested antipsychotic agents, clozapine, haloperidol, and fluphenazine, and two NMDA receptor agonists, D-serine and GLYX-13, for their ability to alter NOS1AP protein expression in human neurons. This is relevant as there is a correlation between chronic treatment with antipsychotic medications and reduced NOS1AP mRNA in postmortem cortical tissue from patients with schizophrenia (Xu et al., 2005). Furthermore, since antipsychotic medications, which target dopamine receptors, do not reduce all symptoms of schizophrenia, an NMDA receptor hypofunction hypothesis has been proposed for schizophrenia (Coyle, 1996; Howes, McCutcheon, & Stone, 2015). The ability of NOS1AP isoforms to compete with PSD-95 for binding to NOS1 suggests that increased expression of isoforms of NOS1AP, as seen in schizophrenia, sequesters NOS1 from NMDA receptors (Jaffrey, Snowman, Eliasson, Cohen, & Snyder, 1998; Li et al., 2015), attenuating downstream signaling pathways (Jaffrey et al., 1998; Li et al., 2013; Li et al., 2015). Thus, the action of D-serine in reducing NOS1AP-L protein expression may act to remedy the NMDAR hypofunction in schizophrenia by increasing the interaction of NOS1 with the NMDAR complex, thereby increasing downstream NMDAR signaling.

It is also possible that NOS1AP binding to NOS1 would not compete with NOS1 binding to PSD-95, but instead, would form a ternary complex and alter NMDA receptor signaling. In fact, it has been reported that an internal motif adjacent to the PDZ domain of NOS1 binds to the second PDZ domain of PSD-95 (Christopherson, Hillier, Lim, & Bredt, 1999; Li et al., 2015), which would allow the binding of NOS1AP directly to the PDZ domain of NOS1. Furthermore, glutamate-induced enhanced interaction between NOS1 and NOS1AP activates p38MAPK (Li et al., 2013) and glutamate-induced excitotoxicity decreases O-Linked N-acetylglucosamine modification of NOS1AP (Zhu et al., 2015), both of which result in neuronal death. Similarly, the interaction between NOS1 and NOS1AP mediates neuronal death in response to amyloid beta (Y. Zhang et al., 2018) and neuropathic pain (W. H. Lee et al., 2018). These studies point to a different model of NOS1AP where increases in NOS1AP, and hence the NOS1:NOS1AP interaction, act to regulate dendritogenesis in a manner distinct from NOS1AP competition with PSD-95 for NOS1 binding.

NMDA signaling pathways shape dendrite arborization (Bustos et al., 2014; Candemir et al., 2016; Previtera, Langhammer, Langrana, & Firestein, 2010; Sceniak et al., 2019; Xiong, Mojsilovic-Petrovic, Perez, & Kalb, 2007). Our data suggest that treatment with D-serine decreases dendrite number when used as a treatment alone. There are conflicting studies that show a role for NMDA receptors in either increasing or decreasing dendrites. For example, antagonism of the NMDA receptor results in reduced dendritic arborization of Purkinje cells and spinal motor neurons (Kalb, 1994; Vogel & Prittie, 1995). A mutation in the GluN2B/NR2B subunit of the NMDAR that prevents trafficking of the NMDAR to the surface of the neuron results in underdeveloped dendrites (Sceniak et al., 2019). In contrast, trigeminal principal nucleus barrelette neurons from NMDA receptor subunit 1 knockout mice show more complex dendritic arbors and longer dendrites (L. J. Lee, Lo, & Erzurumlu, 2005). NMDA receptors play a role in pruning, and knockout of NMDA receptor subunit 2B results in extra primary dendrites in barrel cortex layer 4 spiny stellate cells (Espinosa, Wheeler, Tsien, & Luo, 2009). However, we cannot rule out the possibilities that 1) our results are due to occlusion of D-serine action at baseline due to the action of DMSO in the vehicle on NMDA receptors (Penazzi et al., 2017), 2) that our human neurons are immature and that D-serine may act on young human neurons differently than in rodent systems due to the fact that D-serine has different effects depending on the NMDA receptor subunits expressed (Martina, Krasteniakov, & Bergeron, 2003), and 3) lack of astrocytes in our cultures, versus mixed rodent cultures and in vivo conditions in the studies above, results in altered modulation of NMDA receptors by D-serine (Panatier et al., 2006).

We observed that treatment with vehicle containing 0.1% DMSO, which is a DMSO concentration that does not affect neuronal viability (C. Zhang et al., 2017), reversed the effects of NOS1AP isoform overexpression on dendrite branching. This finding, although unexpected, is consistent with literature on modulation of neuronal activity and morphology by DMSO. In a mouse model of Alzheimer’s Disease, ingestion of low amounts of DMSO administered in drinking water results in enhanced hippocampal-dependent spatial memory (Penazzi et al., 2017). Furthermore, DMSO treatment rescues altered spine density via NMDA receptors both in vivo and when hippocampal slice cultures derived from these animals are treated with DMSO (Penazzi et al., 2017). Additionally, treatment of hippocampal slices with 0.05% DMSO alters the intrinsic excitability of pyramidal neurons, including membrane resistance and impedance (Tamagnini, Scullion, Brown, & Randall, 2014). DMSO acts as a differentiation agent for stem cells (Pal, Mamidi, Das, & Bhonde, 2012). However, since treatment with D-serine in vehicle containing 0.1% DMSO did not reverse the effects of NOS1AP overexpression, we can conclude that the effects of D-serine are not mediated by the vehicle.

Importantly, treatment with D-serine did not reverse the deficits observed with overexpression of either NOS1AP isoform. Although we previously demonstrated that D-serine treatment reverses NOS1AP-L overexpression-mediated decreases in dendrites, only proximal dendrites were rescued. Additionally, since human neurons develop more slowly than do rodent neurons, both in culture and in vivo, it is possible that activation of NMDA receptors by D-serine also differs between the culture types at the same timepoint (i.e. DIV21 for rat cultures and DD21 for human cultures). Furthermore, it should be noted that D-serine treatment decreases endogenous NOS1AP-L levels in our human cultures and that exogenous NOS1AP isoforms are expressed from plasmids containing the cytomegalovirus promoter, and thus exogenous expression is subject to different regulation than endogenous expression.

Taken together, our data demonstrate that the use of a human model system for neuronal development uncovers important information about how NOS1AP, a protein linked to the development of schizophrenia, regulates dendritic morphology. Future studies will include the use of the human iPSC-derived neuron platform to study other schizophrenia-related proteins and genes and for drug screening.

Supplementary Material

Supplemental Figure 1

Highlights.

  • Increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons in vitro.

  • Treatment of human iPSC-derived neurons with D-serine results in a reduction in NOS1AP-L, but not NOS1AP-S, protein expression.

  • Treatment of human iPSC-derived neurons with clozapine, haloperidol, or fluphenazine, does not alter NOS1AP isoform protein expression.

  • NOS1AP overexpression decreases dendrite branching in hiPSC-derived neurons.

  • D-serine reduces NOS1AP-L but not exogenous NOS1AP-promoted decreased dendrites.

Acknowledgments

We thank Dr. Gabriella D’Arcangelo and the imaging core facility of the Human Genetics Institute of New Jersey at Rutgers University for use of their confocal microscopes. We thank Drs. Ron Hart and Jay Tischfield for insightful discussions about the project. We thank Anton Omelchenko for his assistance in data analysis and editing the manuscript.

Funding

This work was supported by National Institutes of Mental Health grant R01 MH062440 (to LB), a National Alliance for Research on Schizophrenia and Depression 2012 Marion G. Nicholson Distinguished Investigator Award (to BLF), a grant from the Human Genetics Institute of New Jersey (to BLF), and in part, by National Science Foundation grants IBN-0919747 and IBN-1353724 (to BLF). KH was supported in part by National Institutes of Health Initiative for Maximizing Student Development Grant 2R25 GM55145 and NSF DGE 0801620, and KH and CMC were supported in part by National Institutes of Health Biotechnology Training Grant T32 GM008339-20. KGP and SGC were supported by NIH R00 award NS051401-42 (to KGP). AKB, AYF, and ND received funding from the Aresty Undergraduate Research Fellowship and AKB and ND were awarded Rutgers University School of Arts and Sciences Undergraduate Research Fellowships.

Footnotes

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Conflicts of interest/Competing interests

Christen M. Crosta, Kristina Hernandez, Atul K. Bhattiprolu, Allen Y. Fu, Steven G. Clarke, Dr. Jennifer C. Moore, and Dr. Kenneth G. Paradiso declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Linda M. Brzustowicz serves as a consultant for the Janssen Pharmaceutical Companies of Johnson & Johnson. Drs. Bonnie L. Firestein and Linda M. Brzustowicz reported patent US 12/263,939 titled “Methods and compositions for the diagnosis and treatment of schizophrenia.”

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