Antipsychotics promote GABAergic interneuron genesis in the adult rat brain: Role of heat-shock protein production

Accepted Manuscript Antipsychotics promote GABAergic interneuron genesis in the adult rat brain: Role of heat-shock protein production Hiroo Kaneta, Wataru Ukai, Hanako Tsujino, Kengo Furuse, Yoshiyasu Kigawa, Masaya Tayama, Takao Ishii, Eri Hashimoto, Chiaki Kawanishi PII:

S0022-3956(16)30385-5

DOI:

10.1016/j.jpsychires.2017.03.008

Reference:

PIAT 3090

To appear in:

Journal of Psychiatric Research

Received Date: 15 September 2016 Revised Date:

7 March 2017

Accepted Date: 8 March 2017

Please cite this article as: Kaneta H, Ukai W, Tsujino H, Furuse K, Kigawa Y, Tayama M, Ishii T, Hashimoto E, Kawanishi C, Antipsychotics promote GABAergic interneuron genesis in the adult rat brain: Role of heat-shock protein production, Journal of Psychiatric Research (2017), doi: 10.1016/ j.jpsychires.2017.03.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antipsychotics promote GABAergic interneuron genesis in the adult rat brain: role of heat-shock protein production

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Hiroo Kaneta, Wataru Ukai*, Hanako Tsujino, Kengo Furuse, Yoshiyasu, Kigawa, Masaya Tayama, Takao Ishii, Eri Hashimoto, Chiaki Kawanishi

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Department of Neuropsychiatry, Sapporo Medical University, School of Medicine, S-1,

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W-16, Chuo-ku, Sapporo, 0608543, Japan

*Corresponding author: Department of Neuropsychiatry, Sapporo Medical University, School of Medicine, S-1, W-16, Chuo-ku, Sapporo, 0608543, Japan

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Tel.: +81116112111; Fax: +81116443041

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E-mail address: [email protected]

E-mail addresses of other authors;

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Hiroo Kaneta: [email protected]; Hanako Tsujino: [email protected]; Kengo Furuse:

[email protected];

Yoshiyasu,

Kigawa:

[email protected];

Masaya Tayama: [email protected]; Takao Ishii: [email protected]; Eri Hashimoto:

[email protected];

[email protected]

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Chiaki

Kawanishi:

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ABSTRACT Current antipsychotics reduce positive symptoms and reverse negative symptoms in conjunction with cognitive behavioral issues with the goal of restoring impaired

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occupational and social functioning. However, limited information is available on their influence on gliogenesis or their neurogenic properties in adult schizophrenia brains, particularly on GABAergic interneuron production. In the present study, we used young

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adult subventricular zone (SVZ)-derived progenitor cells expressing proteoglycan NG2 cultures to examine the oligodendrocyte and GABAergic interneuron genesis effects of

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several kinds of antipsychotics on changes in differentiation function induced by exposure to the NMDA receptor antagonist MK-801. We herein demonstrated that antipsychotics promoted or restored changes in the oligodendrocyte/GABAergic interneuron differentiation functions of NG2(+) cells induced by the exposure to

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MK-801, which was considered to be one of the drug-induced schizophrenia model. We also demonstrated that antipsychotics restored heat-shock protein (HSP) production in NG2(+) cells with differentiation impairment. The antipsychotics olanzapine,

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aripiprazole, and blonanserin, but not haloperidol increased HSP90 levels, which were reduced by the exposure to MK-801. Our results showed that antipsychotics,

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particularly those recently synthesized, exerted similar GABAergic interneuron genesis effects on NG2(+) neuronal/glial progenitor cells in the adult rat brain by increasing cellular HSP production, and also suggest that HSP90 may play a crucial role in the pathophysiology of schizophrenia and is a key target for next drug development.

Keywords: Antipsychotic, GABA, Interneuron, NG2, Parvalbumin, Heat shock protein (HSP)

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1.

Introduction

Schizophrenia is a complex and severe brain disorder that is characterized by

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disturbances in perception, emotion, social functioning, and cognition. The dysfunction of appropriate GABAergic inhibition, and the consequent imbalance between excitation and inhibition in the corticolimbic neural network, underlies at least part of the

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pathophysiological processes of psychiatric disorders (Belmonte et al., 2004; Guidotti et al., 2005; Rocco et al., 2015). Previous studies have reported changes in several

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presynaptic and postsynaptic components of the GABAergic system, in particular the decreased expression of the rate-limiting synthetic enzyme glutamic-acid-decarboxylase 67 (GAD67), as observed in human and animal samples of schizophrenia (Benes et al., 2007; Curley et al., 2011; Rocco et al., 2015). Furthermore, parvalbumin (PV)-positive

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interneurons, many of which display a fast-spiking firing pattern, were found to be severely disrupted in schizophrenia brains (Curley et al., 2011; Gonzalez-Burgos et al., 2010; Rocco et al., 2015). Since the ability of PV-containing fast-spiking interneurons to

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drive synchronous oscillatory activity at gamma frequencies is recognized as a cellular basis for cognitive and executive brain function (Fries et al., 2009), PV(+) interneuron

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dysfunctions may induce social/cognitive behavioral abnormalities (Gonzalez-Burgos et al., 2015; Lewis et al., 2012; Nakazawa et al., 2011). The effects of antipsychotics on the number and functions of PV(+) interneurons remain unknown. In the central nervous system, NG2 proteoglycan-expressing progenitor cells are responsible for the generation of mature oligodendrocytes during development and adulthood (Vigano et al., 2016). However, as recently demonstrated, postnatal hippocampal and neocortical cells expressing oligodendrocyte markers (CNPase and

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NG2) also express neuronal markers (CRMP-4, class Ш β-tubulin, NeuN, and GAD67), have spontaneous synaptic currents and fire action potentials, which are indicators of a neuronal phenotype (Aguirre et al., 2004; Belachew et al., 2003; Chittajallu et al., 2004).

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These findings provide support for GABAergic cortical neurons, particularly GABAergic interneurons, being derived from NG2(+) cells residing within the cortex during adulthood (Dayer et al., 2005).

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The molecular mechanisms regulating cerebral cortical neurogenesis and gliogenesis are poorly understood. Heat Shock Proteins (HSPs) are the most representative group of

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chaperones, are critical for the folding and regulation of a wide array of cellular proteins, and are being increasingly recognized as crucial factors in several important phenomena in the brain, including neurite outgrowth (Ishima et al., 2012), neuronal differentiation (Loones et al., 2000), polarization (Benitez et al., 2014), and neurodegeneration (Luo et

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al., 2010).

We herein investigated the involvement of oligodendrocyte/GABAergic interneuron differentiation changes of NG2(+) cells with the administration of N-methyl-D-aspartate

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(NMDA) receptor antagonist and antipsychotics. We used a culture method of MK-801 (NMDA blocker, also known as Dizocilpine) treatment protocol as one of the drug

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induced model of schizophrenia, which mimics many of the symptoms of schizophrenia in human (Krystal et al., 1994; Lahti et al., 2001; Olney and Farber, 1995), as well as other NMDA modulating agents such as ketamine or phencyclidine. We showed that some aspects of MK801-induced GABAergic interneuron genesis impairments were reversed by the antipsychotics olanzapine, aripiprazole, and blonanserin and also that these antipsychotic treatments were associated with increases in the expression levels of HSP90 in differentiated NG2(+) cells from the adult rat subventricular zone (SVZ). Our

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results also indicate that the up-regulation of HSP90 by the antipsychotic treatments tested is critical for recovery efficacy in this culture model. Our results suggest for the first time GABAergic interneuron genesis impairments in neural circuit changes in the

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adult rat brain being associated with pathophysiology of schizophrenia and new therapeutic mechanisms of action for antipsychotic treatments to restore the GABAergic

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interneuron system.

2.1. Animals and ethical statements

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2. Materials and methods

Timed pregnant Jcl:Wistar rats were purchased from CLEA Japan (Tokyo, Japan), and

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housed at 24°C on a 12:12-hr light:dark cycle with free access to food and water until experiments were conducted. All experimental procedures were approved by the Institutional Animal Care Committee and performed following the Sapporo Medical

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University Guidelines for the Care and Use of Laboratory Animals in accordance with the guide for the Care and Use of Laboratory Animals as adopted and promulgated by

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the United States National Institutes of Health.

2.2. Progenitor cell cultures

Progenitor cell cultures were prepared from 14- to 30-day-old Wister rats. The SVZ was isolated and minced with a rat brain slicer. SVZ tissue from 2-3 brains was digested at 37°C for 10 min in 5 ml of phosphate-buffered saline (PBS) containing 0.25% trypsin

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(Invitrogen, Carlsbad, USA). Digestion was stopped by adding an equal volume of Neurobasal Medium (NBM) (Invitrogen) containing 50% Fetal Bovine Serum (FBS) (Invitrogen) and 0.01% DNase Ι (Sigma-Aldrich, Saint Louis, USA) at room

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temperature for 5 min. The cell suspension was centrifuged for 5 min at 300×g. The pellet obtained was mechanically dissociated 25 times in NBM supplemented with 2% B27 supplement, 0.5 mM L-glutamine (Wako, Tokyo, Japan), and 1% gentamicin

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(Invitrogen) using a glass Pasteur pipette and 20 times using 1-ml pipette tips. Undissociated cells were decanted, and single cells were seeded on a 100-mm Tissue

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Culture Dish (Iwaki, Tokyo, Japan) in NBM supplemented with 2% B27 supplement, 0.5 mM L-glutamine, 1% gentamicin, 20 ng/ml epidermal growth factor (EGF) (PeproTech, Rocky Hill, USA), 10 ng/ml fibroblast growth factor 2 (FGF2) (PeproTech), and 10 ng/ml pigment epithelium-derived factor (PEDF) (PeproTech) at a density of 104

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cells/cm2 and then cultured in suspension at 37°C for 7 days, 5% CO2. EGF, FGF2, and PEDF were added fresh every 2–3 days.

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2.3. Oligodendrocyte/Neuronal

differentiation

and

induction

of

abnormal

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differentiation by MK-801

After 7 days in vitro (DIV), floating progenitor cells (neurospheres) were completely dissociated by trypsinization and trituration. Dissociated cells in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 2% B27 supplement (Invitrogen) and 20 ng/ml brain-derived neurotrophic factor (BDNF) (PeproTech) were seeded onto glass chamber slides (Asahi Glass, Tokyo, Japan) coated with poly-L-ornithine (Sigma) and fibronectin (Invitrogen) at a density of 1.0×105 cells

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per well. DMEM was supplemented with 20 mM glucose (Kanto chemical, Tokyo, Japan), 2 mM L-glutamine, 1 mM sodium bicarbonate (Invitrogen), and 100 U/ml gentamicin regent. Dissociated cells were cultured for three days. Abnormal

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differentiation was induced by exposing cells to MK-801 (Sigma). MK-801 was dissolved in dimethyl sulfoxide (DMSO) (Wako) and added to the medium with

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progenitor cells. The final concentration of DMSO was less than 0.4%.

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2.4. Treatments with antipsychotic agents

In order to measure the effects of antipsychotic agents, olanzapine, blonanserin, aripiprazole, and haloperidol (Wako) were dissolved in DMSO and added to the medium with cultured cells at the same time as their exposure to MK-801. Olanzapine,

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aripiprazole, and blonanserin were kindly gifted form Eli Lilly and Company (Indianapolis, IN, USA), Otsuka Pharmaceutical (Tokyo, Japan), and Dainippon

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Sumitomo Pharma (Osaka, Japan), respectively.

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2.5. Blocking of HSP90 signals with 17-AAG

At the same time as the MK-801 treatment to produce the differentiation function change described above and/or the addition of antipsychotic agents to cultured progenitor cells, the HSP90 inhibitor 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) (Sigma-Aldrich) dissolved in DMSO was added to the medium with cultured cells.

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2.6. Immunocytochemistry and cell counting

An immunocytochemical analysis was performed according to a previously described

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method (Ishii et al., 2008). Cell cultures were fixed in 4% formalin neutral buffer solution (Wako). Cells were treated for 10 min with PBS containing 0.1% Triton X-100 (Wako) and then for 30 min with PBS containing 10% normal goat serum (Vector,

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Burlingame, CA, USA). Cells were stained with the following primary antibodies: an anti-Nestin monoclonal antibody (Millipore, Billerica, MA, USA, 1:200), anti-NG2 Sulfate

Proteoglycan

polyclonal

antibody

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Chondroitin

(Millipore,

1:500),

anti-oligodendrocyte lineage-specific basic helix-loop-helix-2 polyclonal antibody (Santa Cruz Biotechnology, CA, USA, 1:200), anti-β-III tubulin (Tuj-1) monoclonal antibody (Millipore, 1:400), anti-glutamic acid decarboxylase-67 polyclonal antibody

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(Santa Cruz Biotechnology, 1:50), or anti-PV monoclonal antibody (Sigma-Aldrich, 1:2000), and secondary antibodies of an anti-rabbit IgG antibody conjugated with Alexa Fluor 594 (Invitrogen, 1:100) or anti-mouse IgG antibody conjugated with Alexa Fluor

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488 (Invitrogen, 1:100). Nuclei were stained with 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Dojindo, Tokyo, Japan). Cover slips were mounted with

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Mounting Medium (Dako, Carpinteria, USA). Following immunostaining, the representative areas of each well in 8-well glass chamber slides (IWAK) were photographed with a fluorescence microscope and the number of each stained cell in a 0.5-mm square field was counted by three independent experiments (2 fields each). Digital images were obtained with a fluorescence or optical instrument, the Olympus BX52TF microscope (Olympus, Tokyo, Japan). Results were expressed as the mean ± S.E.

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2.7. Western blot analysis

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A Western blot analysis was performed as previously described (Ishii et al., 2008; Tateno et al., 2005; Ukai et al., 2004). Briefly, cells were washed with ice-cold PBS and harvested in RIPA Lysis Buffer (Santa Cruz Biotechnology). After sonication for 30 sec,

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the amount of protein in each sample was estimated using the bicinchoninic acid method, followed by the addition of 1/2 volume of Tris-Glycine SDS Sample Buffer

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(Invitrogen). Samples were boiled for 5 min, and 20-µg aliquots were subjected to SDS-PAGE on 10% Tris-Glycine gels (Invitrogen) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with blocking Reagent (Toyobo, Osaka, Japan) at 4°C overnight, blots were probed with

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anti-HSP90α/β (Abcam, Cambridge, UK, 1:100) and glyceraldehyde 3-phosphate dehydrogenase (rabbit, 1:200; Santa Cruz) at room temperature for 1.5 h, then washed and

incubated

for

1

h

with

horseradish

peroxidase-conjugated

anti-rabbit

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immunoglobulin G (1:2000; Dako Cytomation, Glostrup, Denmark). Immunoreactive bands were detected with an enhanced chemiluminescence system (ECL system; GE

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Healthcare, Buckinghamshire, UK), and quantitatively analyzed by capturing images using a Sensobation Camera (UVP, Upland, CA, USA) in conjugation with the software, Vision Works LS ver.6.1.1 (UVP).

2.8. Statistical analysis

Results are presented as the mean ± S.E. Data were analyzed using a one-way ANOVA

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between subjects. Post hoc comparisons were performed using Tukey’s HSD test. In all cases, p-values of < 0.05 were considered to be significant. Statistical analyses were

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performed using SPSS 12.0 for Windows (SPSS Japan Inc., Japan).

Results

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SVZ-derived NG2(+) oligodendrocyte precursor cells

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3.1. Induction of oligodendrocytes and GABAergic interneurons from adult

The SVZ from young rats (P14-30) was dissociated to obtain proliferating progenitor cells. Cells were maintained for 7 DIV in the presence of the mitogenic factors EGF, FGF2, and PEDF (Fig. 1A). After 7 DIV, progenitor cells (neurospheres)

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(approximately 100% Nestin and NG2-postive, Fig. 1B) were committed to differentiate to the oligodendrocyte and GABAergic neuron phenotypes in differentiation medium in the presence of BDNF for 3 days. Each phenotypic cell differentiation was calculated as

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the percentage of Olig2 (oligodendrocyte), Tuj-1 (neuron), GAD67 (GABAergic neuron), and PV (GABAergic interneuron)-positive cells among the total number of

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cells counterstained with DAPI. We obtained approximately 60% oligodendrocytes and 40% neurons (expressed as total neurons, GABAergic neurons, or GABAergic interneurons) following differentiation for 3 days (Fig. 1C, D, E, F).

3.2. Suppression of GABAergic interneuron genesis from NG2(+) cells by exposure to MK-801

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In order to investigate the effects of MK-801 on oligodendrocyte and GABAergic neuron differentiation, progenitor cells were differentiated for 3 days in the presence of MK-801 (Fig. 2). The proportion of Olig2-positive cells in differentiation medium in the

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absence of MK-801 was approximately 60% (0 µM in Fig. 2A) and the addition of MK-801 (1, 10 µM) to the medium did not affect these differentiation rates. In contrast, MK-801 (10 µM) significantly decreased the number of differentiated Tuj-1-positive

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cells (42% (0 µM) to 18% (10 µM)), GAD67-positive cells (41% (0 µM) to 15% (10 µM)), and PV-positive cells (40% (0 µM) to 20% (10 µM)) in a dose-dependent manner,

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suggesting the specific suppression of GABAergic interneuron genesis from NG2(+) cells by the MK-801 treatment. These decreases are shown in Figures 2B, C, and D with Tuj-1, GAD67, and PV immunostaining, respectively.

after MK-801 exposure

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3.3. Changes in oligodendrocyte genesis from NG2(+) cells by antipsychotic agents

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Recent neuroimaging and postmortem studies have reported abnormalities in the white matter of schizophrenic brains, suggesting the involvement of oligodendrocytes in the

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etiopathology of schizophrenia (Xiao et al., 2008). However, information on oligodendrocyte genesis dysfunctions in the adult schizophrenic brain is limited. In order to investigate the hypothesis that antipsychotic agents with protective/genetic properties on embryonic brain-derived cells have the potential to promote oligodendrocyte genesis from NG2(+) cells in the adult brain, we assessed the effects of various kinds of antipsychotic agents on Olig2-positive oligodendrocyte differentiation in adult rat SVZ-derived NG2(+) cells exposed to MK-801 (Fig. 3A). The treatment

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with MK-801 did not affect Olig2-positive cell differentiation, whereas the co-treatment with the antipsychotic olanzapine (3 µM), and the treatment of olanzapine alone significantly increased Olig2-positive cell differentiation (63% (control), 65% (0 µM) to

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83% (3 µM, co-treatment), 85% (3 µM, treatment alone), respectively (Fig. 3B). In contrast, the co-treatments with the antipsychotics aripiprazole (1, 3 µM), blonanserin

differentiation of Olig2-positive cells (Fig. 3C, D, E).

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(1, 3 µM), haloperidol (1, 3 µM), and their treatments alone did not affect the

agents after MK-801 exposure

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3.4. Changes in GABAergic interneuron genesis from NG2(+) cells by antipsychotic

PV-positive GABAergic interneurons are selectively disrupted in schizophrenia brains

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(Curley AA et al., 2011; Gonzalez-Burgos G et al., 2010). However, until recently, the physiological mechanisms regulating PV-positive GABAergic interneuron density in adult normal and schizophrenia brains remained unclear. In order to establish whether

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antipsychotic agents with neuroprotective and neurogenic properties have the potential to promote GABAergic interneuron genesis from NG2(+) cells in the adult

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schizophrenia brain, we assessed the effects of various kinds of antipsychotics on PV-positive GABAergic interneuron differentiation from adult rat SVZ-derived NG2(+) cells exposed to MK-801 (Fig. 4A). The treatment with MK-801 significantly inhibited PV-positive cell differentiation. The treatment alone or the co-treatments with the antipsychotics olanzapine ((39% (control), (23% (0 µM) to 48% (3 µM, co-treatment), 51% (3 µM, treatment alone) in Fig. 4B)), aripiprazole ((43% (control), (23% (0 µM) to 36% (3 µM, co-treatment), 39% (3 µM, treatment alone) in Fig. 4C)), and blonanserin

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((40% (control), (25% (0 µM) to 42% (3 µM, co-treatment), 45% (3 µM, treatment alone) in Fig. 4D)) significantly suppressed the inhibition of PV-positive cell differentiation by MK-801. In contrast, the co-treatment with the antipsychotic

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haloperidol (1 and 3 µM) did not affect the MK-801-induced inhibition of differentiation ((41% (control), (25% (0 µM) to 27% (1 µM, co-treatment), 27% (3 µM, co-treatment)), and the treatment of haloperidol alone significantly inhibited the

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differentiation (27% (3 µM, treatment alone)) (Fig. 4E).

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3.5. Identification of responsive molecular mechanisms underlying antipsychotic agent effects on the suppression of GABAergic interneuron genesis from NG2(+) cells

HSP90 activity was previously reported to be necessary for controlling the expression,

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activity, and localization of specific kinases and motor proteins during axon specification and elongation processes (Benitez et al., 2014). In order to investigate the involvement of the HSP90 pathway in the differentiation function of NG2(+) progenitor

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cells, we assessed changes in HSP90 activity using a Western blot analysis. HSP90 was detectable in adult SVZ-derived NG2(+) cells maintained under differentiation culture

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conditions. The treatment with MK-801 (0.1-10 µM for 48 h) significantly decreased HSP90α/β levels in a dose-dependent manner (Fig. 5), and the co-treatments with the antipsychotics olanzapine (0.01, 0.1, and 1 µM), aripiprazole (0.01 and 0.1 µM), and blonanserin (0.01, 0.1, and 1 µM) significantly inhibited MK-801-induced decreases in HSP levels (Fig. 6A, B, C). In contrast, the co-treatment with the antipsychotic haloperidol (0.1 µM) significantly reduced the expression of HSP90, which was already decreased by the MK-801 treatment (Fig. 6D).

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To examine whether the HSP90 expression levels alter the inhibitory effects of antipsychotics on GABAergic interneuron genesis disorders after MK-801 exposure, we co-treated with HSP90 inhibitor 17-AAG for MK-801 exposure and antipsychotics.

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17-AAG (0.01 and 0.1 µM) did not exert any additive effects on GABAergic interneuron differentiation in cells treated with MK-801 (Fig. 7A). 17-AAG (0.1 µM) significantly inhibited the recovery of GABAergic interneuron differentiation by the

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antipsychotics olanzapine (3 µM), aripiprazole (3 µM), and blonanserin (3 µM) (Fig. 7

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B, C, D).

Discussion

There is increasing evidence to show that the functional properties of certain

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subtypes of cortical interneurons and cell type-specific changes in the markers of GABAergic signaling have been identified as the key mechanisms underlying the impaired gamma oscillations and cognitive deficits associated with schizophrenia

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(Lewis et al., 2011; Hiyoshi et al., 2014). Neurogenesis is known to continue in the adult mammalian central nervous system (CNS), and as a candidate source of neuronal

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progenitors in the adult brain, we have recently investigated NG2 chondroitin proteoglycan-expressing cells, which are distributed in both neurogenesis and non-neurogenesis regions and form a large pool of proliferating progenitor cells. These NG2(+) progenitor cells have been identified as proliferative precursors, the progeny of which

appears

to

differentiate

into

GABAergic

neurons,

astrocytes,

and

oligodendrocytes, which are capable of propagating action potentials and displaying functional synaptic inputs (Belachew et al., 2003).

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Based on these findings, we performed in vitro studies involving glia-/neuron genesis functional analyses using adult SVZ-derived NG2(+) progenitor cell cultures and found that Tuj-1 and GAD67-positive GABAergic neurons, but not oligodendrocyte

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differentiation, were specifically inhibited by the exposure of NG2(+) progenitor cells to the NMDA receptor antagonist MK-801. Although the precise cellular and molecular mechanisms of this cell fate-specific influence of MK-801 on NG2(+) cell

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differentiation remain unclear, these results indicate that impaired NG2(+) cell differentiation in the adult SVZ plays some roles in the pathophysiology of Furthermore,

we

observed

that

most

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schizophrenia.

of

the

differentiated

GAD67-positive cells reduced by MK-801 were positive for one of the GABAergic interneuron markers, PV. In addition, we focused on oligodendrocyte and neuronal differentiation change and final differentiated cell population is still unclear. Since the

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evidence that NG2(+) cells can give rise to protoplasmic astrocytes was observed in the gray matter of ventrolateral forebrain in a mouse of Cre specifically expressed in NG2(+) cells (Zhu X et al., 2008), further experiments are needed to elucidate the

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alterations of other lineage cell differentiation. Cortical GABAergic interneurons may be a prime target for NMDA receptor

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hypofunction (Lisman et al., 2008; Olney et al., 1995). The acute systemic administration of NMDA receptor antagonists was previously reported to result in hyperactivity in cortical pyramidal neurons (Jackson et al., 2004), and the repeated administration of NMDA receptor antagonists decreased the expression of GAD67 and PV in cortical GABAergic neurons, linking NMDAR hypofunction to dysfunctions in GABAergic neurons (Cochran et al., 2003; Morrow et al., 2007; Rujescu et al., 2006; Braun et al., 2007). Neuropathological findings in human schizophrenia have not yet

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provided evidence for a causal involvement of altered differentiation of interneurons in the onset of schizophrenia or psychotic symptoms. However, our results showing impaired GABAergic interneuron differentiation from SVZ-derived NG2 (+) cells may

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provide new information contributing to this hypothesis. Further in vivo research is needed in order to clarify the relationship between inhibited PV-positive interneuron differentiation and impaired cognitive and social functions using a schizophrenia model.

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The notable results of the present study are that co-treatments with the antipsychotics aripiprazole, olanzapine, and blonanserin recovered MK-801-induced

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impairments in GABAergic interneuron differentiation from NG2(+) cells. Furthermore, the treatment with the typical antipsychotic haloperidol did not suppress MK-801-induced differentiation of NG2(+) cells.

In order to elucidate the molecular mechanisms underlying the recovery of

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GABAergic interneuron genesis by the antipsychotic treatments tested, we evaluated activity changes in HSP signaling in NG2(+) cells because HSPs, particularly HSP90 and HSC70, are known to be constitutively expressed and play a crucial role in neural

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cell fate in the adult nervous system (Aquino et al., 1993; Quraishi et al., 1995). The importance of protein misfolding and aggregation and the expression of HSPs in the

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pathogenesis of neurodegenerative diseases, including Alzheimer’s, Huntington’s, Parkinson’s, and prion diseases has been suggested (Benn et al., 2004; Sherman et al., 2001). Although less information is available on the contribution of these proteins to the pathogenesis of schizophrenia, increases in the expression of HSP70 have been reported in the prefrontal cortex of schizophrenic patients (Schwarz et al., 1999), and cell-invasive pathogenic aggregates of HSP70 with DISC1, which is one of the putative susceptible schizophrenia genes linked to neural development, have been detected (Ottis

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et al., 2011). In the present study, we found that the antipsychotics olanzapine, aripiprazole, and blonanserin increased HSP90 levels in NG2(+) cells, thereby recovering the reductions caused by the exposure to MK-801. In contrast, the treatment

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with haloperidol did not affect the reductions induced in HSP90 levels by MK-801. These results suggest that changes in the protein level of HSP90 induced by antipsychotics contribute to the recovery of GABAergic interneuron genesis from

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NG2(+) cells. In our experiment, we detected reduction of HSP90 levels by MK801 at lower doses which did not affect to the GABAergic interneuron differentiation

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significantly. This may be due to a threshold difference between HSP90 level and differentiation function change in this cell culture method.

If antipsychotic effects are mediated by the regulation of the cellular signaling pathway of HSP90, antagonists of HSP90 may suppress antipsychotic-induced

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GABAergic interneuron differentiation. In order to test this hypothesis, we found that 17-AAG, an inhibitor of HSP90, attenuated antipsychotic-induced GABAergic interneuron differentiation against impairments caused by MK-801. Although 17-AAG

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inhibited the reversing effects of aripiprazole as well as those of olanzapine and blonanserin, we found that aripiprazole exerted its effects at doses that were not parallel

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to the changes observed in HSP90 expression levels. HSP90 is an essential molecular chaperone, ubiquitously active in many signaling and other cellular pathways. It is likely that aripiprazole may act directly and indirectly on endogenous protein expressions, leading to downstream changes in the GABAergic neuron differentiation signaling pathways. This cannot be easily explained and, thus, further studies are needed in order to more completely characterize the activation of HSP pathway by antipsychotics relevant to GABAergic interneuron differentiation followed by the

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analysis of specific GABAergic marker protein expression change, including GAD67 and PV. Aripiprazole acts through the partial agonism of dopamine D2 and 5-HT1A receptors

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and antagonism of 5-HT2A receptors. A recent study indicated that aripiprazole potentiated NGF-induced neurite outgrowth in PC12 cells by increasing HSP90 levels, and also that this potentiation may be partially blocked by the selective 5-HT1A receptor

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antagonist WAY-100635, but not the D2 receptor antagonist sulpiride (Ishima et al., 2012). Thus, the activation of 5-HT1A receptors may play a role in the mechanism of

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action of aripiprazole leading to the enhanced production and differentiation of HSP90 against MK-801.

In our previous study, Olanzapine was shown to potentiate the NGF-induced differentiation of PC12 cells (Lu et al., 2005) and protect cortical neurons exposed to

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MK-801 (Ono et al., 2010) through Akt and ERK pathway, suggesting that similar cellular signaling mechanisms play an important role in the promotion of GABAergic interneuron differentiation induced by olanzapine. In accordance with this result, it is

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reported that olanzapine promotes the differentiation of oligodendrocyte progenitor cells by regulating oligodendrocyte lineage transcription factors Olig1 and olig2 (Fang et al.,

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2013) and proliferation block and differentiation induction of glioma stem-like cells through modulating the Wnt signaling pathway (Guo et al., 2015). Blonanserin is a novel antipsychotic that preferentially interacts with dopamine D2

and serotonin 2A. Although few studies have been conducted on blonanserin, blonanserin acts as an atypical antipsychotic, as recently demonstrated, because it induces little or no EPS expression and enhances Fos expression in the forebrain, and has antagonistic effects on dopamine D3, and releases dopamine and acetylcholine in

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the cortex, thereby improving cognitive functions (Huang et al., 2015). A D3 receptor stimulation has been reported to drive the proliferation of progenitor cells in order to promote murine SVZ neurogenesis (Kim et al., 2010); therefore, we intend to

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investigate the requirement of D3 antagonism in the mechanism of action of blonanserin on NG2(+) cell differentiation function changes. Most typical and atypical antipsychotics possess considerable binding affinity for the D3 receptor in vitro

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(McCormick et al., 2010; Schotte et al., 1996). However, several studies have shown that the antipsychotics haloperidol, aripiprazole, clozapine, olanzapine and risperidone

al., 2011; McCormick et al., 2013).

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exhibit no or very low occupancy of the rat D3 receptor ex vivo or in vivo (Gyertyan et

In the present study, the typical antipsychotic haloperidol did not suppress MK-801-induced impairments in GABAergic interneuron differentiation or decreases in

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HSP90 levels in NG2(+) cells. The different effects on the functions of NG2(+) cells have been elucidated at the molecular level. As recently demonstrated, some of progenitor cells produced in the SVZ region migrate to the cortical and subcortical

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regions, and are involved in the maintenance of the neural network. These findings are as previously reported, i.e., reduction in gray matter volume is suppressed with an

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atypical antipsychotic, but not haloperidol (van Haren et al., 2007; Molina et al., 2005). More recently, it is also reported that that interneuron transcriptional dysregulation caused by loss of PGC-1α alters the effects of haloperidol on hippocampal synaptic transmission and circuit function including a power of hippocampal gamma oscillation (Brady et al., 2016). In the examination of oligodendrocyte differentiation from NG2(+) cells, MK-801 did not influence differentiation function at a dose that suppressed GABAergic

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interneuron differentiation, and olanzapine, but not aripiprazole, blonanserin, or haloperidol increased oligodendrocyte differentiation more in these cells than in control cells. In addition, as previously demonstrated, olanzapine promotes the differentiation

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of oligodendrocyte progenitor cells by regulating transcription factors, olig1 and 2, in the oligodendrocyte system, and inhibits proliferation and induces differentiation by modifying the Wnt signaling in glioma stem cell-like cells. The effects of MK-801 on

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the maturation of neonatal oligodendrocytes should be further investigated in association with the abnormal white matter in schizophrenia.

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We cannot discuss any clinical implication of our in vitro results. However, our results showing the potential promotion of GABAergic interneuron genesis by antipsychotics from adult SVZ-derived NG2(+) cells suggests that enhanced adult progenitor cell activation and/or generation may inhibit or even reverse the

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pathophysiology of schizophrenia, not only by treatments with antipsychotics, but also the possible development of stem cell therapy to modify disease processes, opening the

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Figure captions

Fig. 1. Protocol used to obtain differentiated oligodendrocytes and neurons from

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SVZ-derived progenitor cells. (A) Schematic view of the protocol showing the time course and culture conditions. During proliferation, cells were cultured in suspension in the presence of EGF (20 ng/ml), FGF2 (10 ng/ml), and PEDF (10 ng/ml). After 7 DIV,

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differentiation was induced to the oligodendrocyte and neuron phenotypes in differentiation medium in the presence of BDNF (20 ng/ml) for 3 days. (B) At the

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beginning of differentiation, dissociated neurospheres were immunofluorescently stained with Nestin and NG2, and counterstained with DAPI. Histograms showing the proportion of differentiated oligodendrocytes, neurons, GABAergic neurons, and GABAergic interneurons identified by immunofluorescence with the anti-Olig2 (C),

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anti-Tuj-1 (D), anti-GAD67 (E), and anti-PV antibodies (F) relative to the total number of cells revealed with DAPI counterstaining. Counts were performed after 3 days of differentiation, and data represent means ± S.E. of three independent experiments (3

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fields each). *P<0.05 significantly different from the beginning of differentiation (day

Fig. 2. Quantification of dose responses for neurogenesis suppression of NG2(+) cells induced by MK-801 exposure. (A) NG2(+) cells were treated with (1 and 10 µM) MK-801 for 72 h during the differentiation period. Each cell phenotype proportion was measured by the proportion of cells immunofluorescently stained with anti-Olig2 (A), anti-Tuj-1 (B), anti-GAD67 (C), and anti-PV antibodies (D) relative to the total number of cells revealed by DAPI counterstaining. Data represent the means ± S.E. of three

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independent experiments (3 fields each). *P<0.05 significantly different from the untreated control (MK-801, 0 µM).

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Fig. 3. Effects of antipsychotics on changes in oligodendrocyte genesis from NG2(+) cells induced by an exposure to MK-801. (A) Oligodendrocyte differentiation was evaluated by the proportion of immunofluorescently stained cells with anti-Olig2 (A)

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relative to the total number of cells revealed by DAPI counterstaining. Differentiation into oligodendrocytes, which is not altered by MK-801 alone, was significantly

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promoted by combining olanzapine (B), but not aripiprazole (C), blonanserin (D), or haloperidol (E). Data represent the means ± S.E. of three independent experiments (3 fields each). *P<0.05 significantly different from the untreated control (c); #P<0.05

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significantly different from MK-801 alone (antipsychotic, 0 µM).

Fig. 4. Effects of antipsychotics on the suppression of GABAergic interneuron genesis from NG2(+) cells by an exposure to MK-801. (A) GABAergic interneuron

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differentiation was evaluated by the proportion of immunofluorescently stained cells with anti-PV (A) relative to the total number of cells revealed with DAPI

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counterstaining. The co-treatment with olanzapine (B), aripiprazole (C), and blonanserin (D), but not haloperidol (E) significantly recovered the suppression of PV-positive GABAergic interneuron differentiation induced by MK-801 (10 µM) for 72 h. Data represent the means ± S.E. of three independent experiments (3 fields each). *P<0.05 significantly different from the untreated control (c); #P<0.05 significantly different from the MK-801 treatment alone (antipsychotic 0 µM).

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Fig. 5. Exposure to MK-801 decreased HSP90 protein levels in NG2(+) cells. NG2(+) cells from SVZ were exposed to (0.1 - 10 µM) MK-801 for 48 h. (A) Representative photographs are shown. (B) Results were analyzed by immunoblotting with an antibody

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to HSP90α/β, and presented as the ratio of HSP90α/β to GAPDH. The histogram shows data representing the means ± S.E. of four independent experiments as percent control (MK-801, 0 µM). *P<0.05 significantly different from the untreated control (MK-801, 0

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Fig. 6. Antipsychotics reversed MK-801-induced reductions in HSP90 protein levels in NG2(+) cells. NG2(+) cells from SVZ were exposed to 10 µM MK-801 with or without the antipsychotics olanzapine (A), aripiprazole (B), blonanserin (C), and haloperidol (D) for 48 h. Representative photographs are shown. The results obtained were analyzed by

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immunoblotting with an antibody to HSP90α/β, and presented as the ratio of HSP90α/β to GAPDH. The histogram shows data representing the means ± S.E. of four independent experiments as percent control (c). *P<0.05 significantly different from the

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untreated control (c); #P<0.05 significantly different from the MK-801 exposure alone.

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Fig. 7. Effects of 17-AAG on recovery by antipsychotics for GABAergic interneurogenesis from NG2(+) cells. GABAergic interneuron differentiation was evaluated by the proportion of immunofluorescently stained cells with anti-PV relative to the total number of cells revealed by DAPI counterstaining. NG2(+) cells were exposed to MK-801 (10 µM) with or without 17-AAG (0.01, 0.1 µM) for 72 h. 17-AAG (0.01, 0.1 µM) did not have an additive effect on the suppression of GABAergic interneuron differentiation (A). The co-treatment with olanzapine (B), aripiprazole (C),

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and blonanserin (D) significantly recovered the suppression of PV-positive GABAergic interneuron differentiation induced by MK-801 (10 µM) exposure for 72 h, and all antipsychotic effects were reversed by 17-AAG (0.1 µM). Data represent the means ±

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S.E. of three independent experiments (3 fields each). *P<0.05 significantly different from the untreated control (c); #P<0.05 significantly different from the MK-801 treatment alone; †P<0.05 significantly different from the antipsychotic treatment with

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