The Molecular Mechanisms Underlying Prostaglandin D2-Induced Neuritogenesis in Motor Neuron-Like NSC-34 Cells

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Article The Molecular Mechanisms Underlying Prostaglandin D2-Induced Neuritogenesis in Motor Neuron-Like NSC-34 Cells

1, 1, , 1 1 Hiroshi Nango † , Yasuhiro Kosuge * † , Nana Yoshimura , Hiroko Miyagishi , Takanori Kanazawa 2, Kaname Hashizaki 3, Toyofumi Suzuki 2 and Kumiko Ishige 1

1 Laboratory of Pharmacology, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan; [email protected] (H.N.); [email protected] (N.Y.); [email protected] (H.M.); [email protected] (K.I.) 2 Laboratory of Pharmaceutics, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan; [email protected] (T.K.); [email protected] (T.S.) 3 Laboratory of Physical Chemistry, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-47-465-4027 These authors contributed equally to this work. † Received: 6 February 2020; Accepted: 8 April 2020; Published: 10 April 2020

Abstract: Prostaglandins are a group of physiologically active lipid compounds derived from arachidonic acid. Our previous study has found that prostaglandin E2 promotes neurite outgrowth in NSC-34 cells, which are a model for motor neuron development. However, the effects of other prostaglandins on neuronal differentiation are poorly understood. The present study investigated the effect of prostaglandin D2 (PGD2) on neuritogenesis in NSC-34 cells. Exposure to PGD2 resulted in increased percentages of neurite-bearing cells and neurite length. Although D-prostanoid receptor (DP) 1 and DP2 were dominantly expressed in the cells, BW245C (a DP1 agonist) and 15(R)-15-methyl PGD2 (a DP2 agonist) had no effect on neurite outgrowth. Enzyme-linked immunosorbent assay 12,14 demonstrated that PGD2 was converted to 15-deoxy-∆ -prostaglandin J2 (15d-PGJ2) under cell-free conditions. Exogenously applied 15d-PGJ2 mimicked the effect of PGD2 on neurite outgrowth. GW9662, a peroxisome proliferator-activated receptor–gamma (PPARγ) antagonist, suppressed PGD2-induced neurite outgrowth. Moreover, PGD2 and 15d-PGJ2 increased the protein expression of Islet-1 (the earliest marker of developing motor neurons), and these increases were suppressed by co-treatment with GW9662. These results suggest that PGD2 induces neuritogenesis in NSC-34 cells and that PGD2-induced neurite outgrowth was mediated by the activation of PPARγ through the metabolite 15d-PGJ2.

12,14 Keywords: prostaglandin D2; 15-deoxy-∆ -prostaglandin J2; peroxisome proliferator-activated receptor γ; motor neurons; neurite outgrowth; NSC-34 cells

1. Introduction Neuritogenesis is an early event in the diﬀerentiation of neural progenitor cells into neurons and enables neurons to develop axons and dendrites with which they connect to other cells and receive and transmit electrical signals [1]. Spinal motor neurons innervate muscle contraction by extending long axons that project from the ventral horn of the spinal cord gray matter directly to peripheral skeletal muscle [2]. Degeneration and loss of spinal motor neurons cause progressive and fatal motor neuron diseases such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and spinal muscular atrophy. More recently, induced pluripotent stem cell-derived motor neurons

Cells 2020, 9, 934; doi:10.3390/cells9040934 www.mdpi.com/journal/cells Cells 2020, 9, 934 2 of 16 have been expected to elucidate the pathogenesis and facilitate the identification of specific drugs for the treatment of such neurodegenerative diseases [3]. Understanding the mechanism involved in neuritogenesis and differentiation of spinal motor neurons could pave the way for the development of regenerative therapies for motor neuron diseases. NSC-34 cells are produced by the fusion of mouse neuroblastoma cells with motor neuron-enriched mouse spinal cord cells [4]. Differentiated NSC-34 cells induced by serum deprivation and additional treatment with all-trans retinoic acid exhibit the unique morphological and physiological characteristics of motor neurons, including neurite outgrowth, expression of motor neuron-specific marker proteins HB9 and Islet-1, and acetylcholine synthesis and storage [5,6]. Furthermore, undifferentiated NSC-34 cells are widely used as motor neuron progenitor cells in the search for small molecular compounds that induce motor neuron differentiation [7–10]. Prostaglandins are small lipid inflammatory mediators derived from arachidonic acid by multiple enzymatic reactions and are involved in a wide array of physiological and pathophysiological responses [11]. In particular, prostaglandin E2 (PGE2) and D2 (PGD2) are the major products of prostaglandins. Arachidonic acid is liberated from cellular membranes by phospholipases A2 and is converted to prostaglandin H2 (PGH2) by cyclooxygenase-1 and -2. Subsequently, PGH2 is converted to PGE2 by PGE synthase [11] or PGD2 by PGD synthase (PGDS), respectively. [12]. Prostaglandins exert their actions by acting on a group of G-protein-coupled receptors. For example, PGE2 mainly binds to four subtypes of E-prostanoid receptor (EP1–4) [13]. PGE2 promotes neuritogenesis in dorsal root ganglion neurons via EP2 (coupled to Gs protein) [14] and in sensory neuron-like ND7/23 cells via EP4 (coupled to Gs protein) [15]. Recently, our research has demonstrated that PGE2 induces neurite outgrowth by activating EP2 in NSC-34 cells [16]. This suggests that PGE2 is involved in neuritogenesis and the differentiation of various neurons including motor neurons. However, the role of prostaglandins other than PGE2 on neuronal differentiation has not been investigated in neurons or their model cells. So far, two isoforms of PGDS are known, lipocalin-type and hematopoietic PGDS [17]. Lipocalin-type PGDS mRNA has been reported to be abundantly expressed in the thalamus, brainstem, cerebellum, and spinal cord [18]. Hematopoietic PGDS is expressed in microglial cells of the mouse cerebral cortex during early postnatal development [19] and in the adult rat cerebellum [20]. In the human brain, the amount of PGD2 is high in the pineal body, pituitary gland, olfactory bulb, and hypothalamus [21]. It is noteworthy that PGD2 is the most abundant eicosanoid in the mouse spinal cord [22] and is present in several regions of the central nervous system (CNS), including the spinal cord. Synthesized PGD2 elicits its downstream effects by activating two G-protein-coupled receptors, D-prostanoid receptor (DP) 1 and DP2. DP1 is coupled to adenylate cyclase via a Gs protein, while DP2 inhibits adenylate cyclase via Gi protein [12]. DP1 and DP2 proteins have been detected in motor neurons of adult mouse spinal cords with fluorescent immunohistochemistry [23]. Moreover, PGD2 12 12,14 are nonenzymatically metabolized to prostaglandin J2 (PGJ2), ∆ -PGJ2, and 15-deoxy-∆ -PGJ2 (15d-PGJ2)[12]. It has been reported that 15d-PGJ2 acts as an agonist of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ)[24]. Indeed, 15d-PGJ2 plays an important role in neurite outgrowth of rat embryonic midbrain cells in a PPARγ-dependent manner [25]. However, unlike PGE2, it is unknown whether PGD2 and/or 15d-PGJ2 exert neurite outgrowth in motor neurons. In this study, we sought to elucidate the effect of PGD2 on neurite outgrowth in motor neurons using NSC-34 cells. We found that exogenously applied PGD2 was converted to 15d-PGJ2 and subsequently induced neurite outgrowth, which was mediated by PPARγ but not by DP in motor neuron-like cells.

2. Materials and Methods

2.1. Materials

All chemicals were purchased from Wako (Osaka, Japan) unless stated otherwise. PGD2, 15d-PGJ2, BW 245C, 15(R)-15-methyl PGD2, MK0524, CAY10471, and GW9662 were obtained from Cayman Chemical (Ann Arbor, MI, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA). The primary antibodies used were against DP1 (Cayman Cells 2020, 9, 934 3 of 16

Chemical, Ann Arbor, MI, USA, diluted 1:1000), DP2 (Novus Biologicals, Centennial CO, USA, diluted 1:1000), Islet-1 (Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000), and β-actin (Sigma-Aldrich, St. Louis, MO, USA, diluted 1:1000).

2.2. Cell Culture NSC-34 cells (provided by Dr. Neil Cashman, University of Toronto, ON, Canada) were cultured to a maximum of 15 passages in a medium consisting of Dulbecco’s Modiﬁed Eagle Medium (DMEM; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies Corporation, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Life Technologies Corporation, Carlsbad, CA, USA). Cultures were incubated at 37 ◦C and 5% CO2 in a humidiﬁed atmosphere. For experimentation, NSC-34 cells were seeded onto Corning BioCoat™ Poly-L-Lysine TC-Treated Culture Dishes (Corning Inc., Corning, NY, USA) at a density of 12,500 cells/cm2 and incubated for 24 h according to the method described previously [16].

2.3. Western Blotting The Western blot analysis was performed as described previously [26,27]. Briefly, NSC-34 cells were lysed in a radioimmunoprecipitation buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 5 mM EDTA, phosphatase inhibitor (Sigma–Aldrich, St. Louis, MO, USA), and protease inhibitor cocktail (Roche, Basel, Switzerland). The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto the membranes, and probed with the antibodies. The following primary antibodies were used: polyclonal anti-DP1 (1:1000), polyclonal anti-CRTH2/GRP44 (DP2) (1:1000), monoclonal anti-Islet-1 (1:1000), and monoclonal anti-β-actin (Sigma–Aldrich, St. Louis, MO, USA). An HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used. Immunoreactive bands were detected by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK). The optical density of the bands was quantified using Scion imaging software 4.0.3.2 (Scion, Frederick, MD, USA).

2.4. Neurite Outgrowth Assay Phase-contrast micrographs were captured using an inverted microscope (IX70, Olympus, Tokyo, Japan) with i-NTER LENS (Microscope Network Co. Ltd., Saitama, Japan). Afterwards, 50 cells per condition were randomly chosen to estimate the number of neurite-bearing cells using ImageJ (National Institute of Health, Bethesda, MD, USA) [28]. The percentage of neurite-bearing cells was calculated as the ratio of cells bearing neurite processes >1 cell diameter in length according to the method described previously [16]. The neurite length on each neurite-bearing cell was measured as the distance from the soma to the end of the neurite using NeuronJ, an ImageJ plugin originally developed for neurite tracing and analysis [29].

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Quantification of PGD2 and 15d-PGJ2 in cell-free culture media was performed according to previous studies [30,31]. To detect PGD2 and 15d-PGJ2 in the culture medium, freshly prepared 15 µM PGD2 was made by diluting a stock solution in cell-free DMEM containing 10% FBS and 1% penicillin–streptomycin, which was incubated in a humidified atmosphere containing 5% CO2 at 37 ◦C. The culture medium was collected at 0, 0.5, 1, 2, 4, and 24 h, homogenized in methyl acetate, and centrifuged at 10,000 g for 10 min at 4 C. The supernatant was evaporated, and then the residue × ◦ was reconstituted in the assay buffer. PGD2 was measured using the Prostaglandin D2–MOX EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The absorbance of samples was measured using the microplate reader SH-1000Lab (Corona Electric, Ibaraki, Japan) at a test wavelength of 412 nm. Similarly, 15d-PGJ2 was measured according to the protocol of the 12,14 15-deoxy-∆ -PGJ2 ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA). The absorbance of samples Cells 2020, 9, 934 4 of 16 was measured using SH-1000Lab (Corona Electric, Ibaraki, Japan) at test and reference wavelengths of 405 and 570 nm, respectively.

2.6. Live/Dead Assay The amount of viable and dead cells was measured by LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Eugene, OR, USA) according to a previously described method [27]. The images were collected with an inversed ﬂuorescence microscope (IX70, Olympus, Tokyo, Japan) and cell mortality was determined by calculating the percentage of EthD-1-positive cells of the total number of cells (the sum of calcein-positive live cells and EthD-1-positive dead cells).

2.7. Statistical Analyses Data analyses were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego CA, USA). Data are expressed as mean standard error of the mean (SEM) or standard deviation ± (SD). Statistical significance was assessed by Student’s t-test or one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple tests. Differences at p < 0.05 were considered statistically significant.

3. Results

3.1. PGD2 Aﬀects Neurite Outgrowth

First, we investigated the effect of exogenously applied PGD2 on neuritogenesis in undifferentiated NSC-34 cells. Neurite-bearing cells were observed in cells treated with 10 µM PGD for 24 h, whereas ≥ 2 0 µM PGD2 (vehicle)-treated cells were mostly round in shape (Figure1A). Moreover, we analyzed the percentage of neurite-bearing cells and the neurite length in each treatment group. Exposure of undifferentiated NSC-34 cells to PGD (1 20 µM) for 24 h resulted in a significant increase in the 2 − percentage of neurite-bearing cells at PGD concentrations of 10 µM (23.7% 1.4%, p = 0.0000000506), 2 ± 15 µM (33.3% 2.7%, p = 0.00000000114), and 20 µM (20.9% 2.1%, p = 0.00000543) compared to ± ± vehicle-treated cells (0.5% 0.3%) (Figure1B). The maximal e ffect of PGD was observed at 15 µM, ± 2 with an increase of up to approximately 30%. Moreover, the average neurite length was significantly increased in PGD (15 µM)-treated cells (78.1 7.9 µm, p = 0.0218) compared to vehicle-treated cells 2 ± (43.6 8.7 µm) (Figure1B). Moreover, we investigated the cytotoxicity of PGD for undifferentiated ± 2 NSC-34 cells. As shown in Figure1C, exposure of di fferentiated NSC-34 cells to 15 µM PGD2 for 24 h did not change the percentage of dead cells stained by EthD-1.

3.2. DP1 and DP2 Agonists Do Not Affect Neurite Outgrowth Next, we tested whether DP1 and DP2 were expressed in undifferentiated NSC-34 cells by Western blotting. Similar to the reported DP1 and DP2 expression in the mouse spinal cord [23], we detected protein expression of both DP1 and DP2 in undifferentiated NSC-34 cells (Figure2). Cells 2020, 9, x FOR PEER REVIEW 4 of 16

2.6. Live/Dead Assay

The amount of viable and dead cells was measured by LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Eugene, OR, USA) according to a previously described method [27]. The images were collected with an inversed fluorescence microscope (IX70, Olympus, Tokyo, Japan) and cell mortality was determined by calculating the percentage of EthD-1-positive cells of the total number of cells (the sum of calcein-positive live cells and EthD-1-positive dead cells).

2.7. Statistical Analyses Data analyses were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego CA, USA). Data are expressed as mean ± standard error of the mean (SEM) or standard deviation (SD). Statistical significance was assessed by Student’s t-test or one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple tests. Differences at p < 0.05 were considered statistically significant.

3. Results

3.1. PGD2 Affects Neurite Outgrowth

First, we investigated the effect of exogenously applied PGD2 on neuritogenesis in undifferentiated NSC-34 cells. Neurite-bearing cells were observed in cells treated with ≥10 μM PGD2 for 24 h, whereas 0 μM PGD2 (vehicle)-treated cells were mostly round in shape (Figure 1A). Moreover, we analyzed the percentage of neurite-bearing cells and the neurite length in each treatment group. Exposure of undifferentiated NSC-34 cells to PGD2 (1−20 μM) for 24 h resulted in a significant increase in the percentage of neurite-bearing cells at PGD2 concentrations of 10 μM (23.7% ± 1.4%, p = 0.0000000506), 15 μM (33.3% ± 2.7%, p = 0.00000000114), and 20 μM (20.9% ± 2.1%, p = 0.00000543) compared to vehicle-treated cells (0.5% ± 0.3%) (Figure 1B). The maximal effect of PGD2 was observed at 15 μM, with an increase of up to approximately 30%. Moreover, the average neurite length was significantly increased in PGD2 (15 μM)-treated cells (78.1 ± 7.9 μm, p = 0.0218) compared to vehicle-treated cells (43.6 ± 8.7 μm) (Figure 1B). Moreover, we investigated the cytotoxicity of CellsPGD20202 for, 9 ,undifferentiated 934 NSC-34 cells. As shown in Figure 1C, exposure of differentiated NSC-345 of 16 cells to 15 μM PGD2 for 24 h did not change the percentage of dead cells stained by EthD-1.

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10 Cells 2020, 9, x FOR PEER REVIEW 5 of 16 5 BthD-1 positive cells

microscopy0 μM images of 15 NSC-34 μM cells treated(% of total cell number) with various concentrations of PGD2. Scale bar: 50 μm. (B) 0 The percentage of neurite-bearing cells (left panel)015 and the average neurite length (right panel) in PGD (μM) each treatment group. Each value represents the mean ±2 SEM of four different experiments. *p < 0.05, ***Figurep < 0.001. 1. (TheC) Photographs eﬀect of prostaglandin show typical D fluorescence(PGD ) on images neurite double outgrowth. staining Undi of calcein-AMﬀerentiated (green, NSC-34 Figure 1. The effect of prostaglandin D2 (PGD2 2) on2 neurite outgrowth. Undifferentiated NSC-34 cells livecells cells) were and exposed EthD-1 to(red, various dead concentrationscells) in each treatment of PGD forgroup. 24 h.Scale (A) bar: Representative 50 μm. Graphs phase-contrast show the were exposed to various concentrations of PGD2 for2 24 h. (A) Representative phase-contrast percentage of EthD-1-positive dead cells in these cells. Each value represents the mean ± SEM of four microscopy images of NSC-34 cells treated with various concentrations of PGD2. Scale bar: 50 µm.

different(B) The experiments. percentage of neurite-bearing cells (left panel) and the average neurite length (right panel) in each treatment group. Each value represents the mean SEM of four different experiments. * p < 0.05, ± 3.2. DP1*** andp < 0.001DP2 .(AgonistsC) Photographs do Not Affect show typicalNeurite fluorescence Outgrowth images double staining of calcein-AM (green, Next,live cells)we tested and EthD-1 whether (red, DP1 dead cells)and DP2 in each were treatment expressed group. in Scale undifferentiated bar: 50 µm. Graphs NSC-34 show cells the by percentage of EthD-1-positive dead cells in these cells. Each value represents the mean SEM of four Western blotting. Similar to the reported DP1 and DP2 expression in the mouse spinal± cord [23], we detecteddiff proteinerent experiments. expression of both DP1 and DP2 in undifferentiated NSC-34 cells (Figure 2). Mouse spinal NSC-34 cord

DP1 (47 kDa)

DP2 (60 kDa)

β-actin

FigureFigure 2. 2.WesternWestern blots blots of ofD-prostanoid D-prostanoid receptor receptor (DP) (DP) expression expression in inundifferentiated undiﬀerentiated NSC-34 NSC-34 cells. cells. MouseMouse spinal spinal cord cord lysate lysate was was used used as as a positive a positive control. control. β-actinβ-actin was was used used as as an an internal internal control. control. Data Data fromfrom three three separate separate expe experimentsriments are are presented. presented.

ToTo characterize characterize the the subtype(s) subtype(s) of of DP DP responsible responsible for for PGD PGD2-induced2-induced neurite neurite outgrowth outgrowth in in undifferentiatedundifferentiated NSC-34 NSC-34 cells, cells, we we investigated investigated the the effects effects of oftwo two well-characterized well-characterized DP DP agonists, agonists, BW245CBW245C (a (aDP1 DP1 agonist) agonist) and and 15(R)-15-methyl 15(R)-15-methyl PGD PGD2 2(a(a DP2 DP2 agonist). agonist). Phase-contrast Phase-contrast microscopy microscopy revealedrevealed no no neurite neurite outgrowth outgrowth in in these these agonist agonist (15 (15 μM)-treatedµM)-treated cells cells for for 24 24 h h(Figure (Figure 3A,B).3A,B). Exposure Exposure toto BW245C BW245C oror 15(R)-15-methyl 15(R)-15-methyl PGD PGD2 exerted2 exerted no significant no significant effect on effect the percentage on the ofpercentage neurite-bearing of neurite-bearingcells and the average cells and neurite the average length neurite within length the concentration within the concentration range tested (1range15 µtestedM) (Figure (1−15 3μA,B).M) − (FigureMoreover, 3A,B). co-treatment Moreover, co-treatment with MK0524 with (a DP1MK0524 antagonist, (a DP1 antagonist, 10 µM) or CAY1047110 μM) or CAY10471 (a DP2 antagonist, (a DP2 antagonist,10 µM) and 10 PGDμM)2 and(15 PGDµM) had2 (15 noμM) eff ecthad on no PGD effect2-induced on PGD2 neurite-induced outgrowth neurite outgrowth (Figure4A). (Figure There 4A). were Thereno significant were no disignificantfferences indifferences the percentage in the of percentage neurite-bearing of neurite-bearing cells or neurite cells length or betweenneurite length the cells betweenco-treated the withcells MK0524co-treated or with CAY10471 MK0524 and or PGD CAY104712, and the and cells PGD treated2, and with the cells PGD 2treatedalone (Figurewith PGD4B).2 alone (Figure 4B).

Cells 2020, 9, 934 6 of 16

Cells 2020, 9, x FOR PEER REVIEW 6 of 16

Cells 2020, 9, x FOR PEER REVIEW 6 of 16 (A) (B)

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60 60

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BW245C (μM) 15(R)-15-methyl PGD2 (μM) m) m) μ 10075 μ 10075 m) m) μ 7550 μ 7550

5025 5025 Neurite length( Neurite length ( 250 250 0 1 10 15 0 1 10 15 Neurite length( BW245C (μM) Neurite length ( 15(R)-15-methyl PGD (μM) 0 0 2 0 1 10 15 0 1 10 15 Figure 3. The effects of BW245CDP agonists (μM) on neurite outgrowth. Undifferentiated15(R)-15-methyl PGD NSC-34(μM) cells were Figure 3. The effects of DP agonists on neurite outgrowth. Undifferentiated2 NSC-34 cells were exposed to various concentrations of BW245C (a DP1 agonist) or 15(R)-15-methyl PGD2 (a DP2 Figure 3. The effects of DP agonists on neurite outgrowth. Undifferentiated NSC-34 cells were exposedagonist) to various for 24 concentrationsh. (A) Representative of BW245Cphase-contrast (a DP1microsco agonist)py images or of 15(R)-15-methyl NSC-34 cells treated PGD with2 (a DP2 exposed to various concentrations of BW245C (a DP1 agonist) or 15(R)-15-methyl PGD2 (a DP2 agonist) forvarious 24h. concentrations (A) Representative of BW245C phase-contrast (upper panel). Scale microscopy bar: 50 μm. imagesGraphs show of NSC-34 the percentage cells treated of with agonist) for 24 h. (A) Representative phase-contrast microscopy images of NSC-34 cells treated with various concentrationsneurite-bearing cells of BW245C(middle panel) (upper and the panel). average Scale neurite bar: length 50 µ (lowerm. Graphs panel) showin each thetreatment percentage of various concentrations of BW245C (upper panel). Scale bar: 50 μm. Graphs show the percentage of group. (B) Representative phase-contrast microscopy images of NSC-34 cells treated with various neurite-bearingneurite-bearing cells (middlecells (middle panel) panel) and and the the averageaverage neurite neurite length length (lower (lower panel)panel) in each intreatment each treatment concentrations of 15(R)-15-methyl PGD2 (upper panel). Scale bar: 50 μm. Graphs show the percentage group. (Bgroup.) Representative (B) Representative phase-contrast phase-contrast microscopymicroscopy im imagesages of NSC-34 of NSC-34 cells treated cells treatedwith various with various of neurite-bearing cells (middle panel) and the average neurite length (lower panel) in each concentrations of 15(R)-15-methyl PGD2 (upper panel). Scale bar: 50 μm. µGraphs show the percentage concentrationstreatment of 15(R)-15-methylgroup. Each value re PGDpresents2 (upper the mean panel). ± SEM Scaleof four bar: different 50 m.experiments. Graphs show the percentage of neurite-bearingof neurite-bearing cells (middle cells (middle panel) panel) and theand averagethe average neurite neurite length length (lower (lower panel) panel) in in each eachtreatment treatment group. Each value represents the mean ± SEM of four different experiments. group. Each value represents the(A) mean SEM of four different experiments. ± MK0524 CAY10471 vehicle (10 μM) (10 μM) (A) MK0524 CAY10471 vehicle (10 μM) (10 μM) (-) M) μ (-) (15 (15 2 M) μ PGD (15 (15

2 (+) PGD (+)

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Neurite-bearing cells Neurite-bearing 20 (% of total cell number) 0 0 vehicle MK0524 CAY10471 25 vehicle MK0524 CAY10471 Neurite length ( Neurite-bearing cells Neurite-bearing (% of total cell number) 0 0 vehicle MK0524 CAY10471 vehicle MK0524 CAY10471

Figure 4. The effects of DP antagonists on PGD -induced neurite outgrowth. Undifferentiated NSC-34 2 cells were treated with 15 µM PGD2 in the presence or absence of 10 µM MK0524 or 10 µM CAY10471 for 24 h. (A) Representative phase-contrast microscopy images in each treatment group. Scale bar: 50 µm. (B) Graphs show the percentage of neurite-bearing cells (left panel) and the average neurite length (right panel) in each treatment group. Each value represents the mean SEM of four different ± experiments. ** p < 0.01, *** p < 0.001. Cells 2020, 9, x FOR PEER REVIEW 7 of 16

Figure 4. The effects of DP antagonists on PGD2-induced neurite outgrowth. Undifferentiated

NSC-34 cells were treated with 15 μM PGD2 in the presence or absence of 10 μM MK0524 or 10 μM CAY10471 for 24 h. (A) Representative phase-contrast microscopy images in each treatment group. Scale bar: 50 μm. (B) Graphs show the percentage of neurite-bearing cells (left panel) and the average Cells 2020, 9, 934 7 of 16 neurite length (right panel) in each treatment group. Each value represents the mean ± SEM of four different experiments. **p < 0.01, ***p < 0.001. 3.3. 15d-PGJ2 Affects Neurite Outgrowth 3.3.15. d-PGJ2 Affects Neurite Outgrowth PGD2 is unstable and readily undergoes dehydration to yield the PPARγ natural ligand 15d-PGJPGD2 2[ 32is ].unstable Therefore, and we readily sought undergoes to identify dehydration the conversion to yield of PGD the2 toPPAR 15d-PGJγ natural2 in cell-free ligand 15d-PGJmedia. PGD2 [32].2 (15 Therefore,µM) was we incubated sought underto identify cell-free the conditions, conversion and of thePGD concentrations2 to 15d-PGJ2 of in PGD cell-free2 and media.15d-PGJ PGDwere2 (15 examined μM) was byincubated ELISA. Theunder initial cell-free concentration conditions, of and PGD thewas concentrations 13.2 0.9 µ ofM PGD at 0 h2 and 2 2 ± 15d-PGJsignificantly2 were decreased examined in aby time-dependent ELISA. The initial manner concentration after incubation of PGD for2 2was h (8.4 13.2 ±0.4 0.9µ M,μMp at= 0.00751),0 h and ± significantly4 h (6.8 1.9 decreasedµM, p = 0.000640), in a time-dependent and 24 h (1.5 manner0.6 µM, afterp = incubation0.00000146) for (Figure 2 h 5(8.4). In ± contrast,0.4 μM, thep = ± ± 0.00751),concentration 4 h (6.8 of ± 15d-PGJ 1.9 μM, psignificantly = 0.000640), increasedand 24 h (1.5 in a ±time-dependent 0.6 μM, p = 0.00000146) manner (Figure from 32.2 5). In 2.1contrast, nM at 2 ± the0 h concentration to 0.6 0.2 µM( of p15d-PGJ= 0.0103)2 significantly after 4 h and increased 1.6 0.2 inµM( a time-dependentp = 0.000000717) manner after 24 from h (Figure 32.2 5±). 2.1 Next, nM ± ± atwe 0 investigatedh to 0.6 ± 0.2 the μM eff (ectp = of0.0103) exogenously after 4 h applied and 1.6 15d-PGJ ± 0.2 μ2Mon (p neurite = 0.000000717) outgrowth after in undi24 hff (Figureerentiated 5). Next,NSC-34 we cells. investigated Neurite-bearing the effect cells wereof exogenously observed after applied 24 h with 15d-PGJ 15d-PGJ2 onconcentrations neurite outgrowth of 5 µinM 2 ≥ undifferentiated(Figure6A). Significantly NSC-34 higher cells. increasesNeurite-bearing in the percentage cells were of neurite-bearingobserved after cells24 h were with observed 15d-PGJ at2 concentrationsconcentrations ofof 5 µ≥M5 (9.4%μM (Figure0.8%, p6A).= 0.000913), Significantly 8 µM (28.4%higher increases2.5%, p = 0.000000000000843),in the percentage andof ± ± neurite-bearing10 µM (13.3% 2.7%,cells werep = 0.00000414), observed at than concentrations in vehicle-treated of 5 cellsμM (0.7%(9.4% ± 0.2%)0.8%, (Figurep = 0.000913),6B). Moreover, 8 μM ± ± (28.4%15d-PGJ ± (82.5%,µM)-treated p = 0.000000000000843), cells exhibited significantly and 10 μ longerM (13.3% neurites ± 2.7%, (71.6 p =4.4 0.00000414),µm, p = 0.0199 than) than in 2 ± vehicle-treated cells cells (0.7% (46.5 ± 6.30.2%)µm) (Figure (Figure 6B).6B). Moreover, Consistent 15d-PGJ with results2 (8 μM)-treated from PGD2-treated cells exhibited cells, ± significantlyexposure to 8longerµM 15d-PGJ neurites2 for (71.6 24 h ± had 4.4 no μm, significant p = 0.0199) effect than on thevehicle-treated percentage of cells dead (46.5 cells ± stained 6.3 μm) by (FigureEthD-1 6B). (Figure Consistent6C). with results from PGD2-treated cells, exposure to 8 μM 15d-PGJ2 for 24 h had no significant effect on the percentage of dead cells stained by EthD-1 (Figure 6C).

PGD2 15d-PGJ2 16 2

12 1.5 M)

### μ ( M) 2 μ ** ( ***

2 8 1

PGD 4 0.5 # *** 15d-PGJ 0 0 -30024 00.5 3060901201 150180210240270300330360390420450480 24510 Incubation time (hours)

12,14 Figure 5. Conversion of PGD2 to 15-deoxy-∆ -prostaglandin J2 (15d-PGJ2) under cell-free conditions. Figure 5. Conversion of PGD2 to 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) under cell-free Freshly prepared 15 µM PGD2 was made by diluting a stock solution in cell-free culture medium. conditions. Freshly prepared 15 μM PGD2 was made by diluting a stock solution in cell-free culture Time-dependent concentrations of PGD2 and 15d-PGJ2 were measured at 0, 0.5, 1, 2, 4, and 24 h. The medium. Time-dependent concentrations of PGD2 and 15d-PGJ2 were measured at 0, 0.5, 1, 2, 4, and left and right axes represent the concentration of PGD2 (black circles) and 15d-PGJ2 (white circles), 24 h. The left and right axes represent the concentration of PGD2 (black circles) and 15d-PGJ2 (white respectively. Each value represents the mean SEM of three diﬀerent experiments. ** p < 0.01, circles), respectively. Each value# represents### the mean± ± SEM of three different experiments. **p < 0.01, *** p < 0.001 vs. PGD2 at 0 h. p < 0.05, p < 0.001 vs. 15d-PGJ2 at 0 h. ***p < 0.001 vs. PGD2 at 0 h. #p < 0.05, ###p < 0.001 vs. 15d-PGJ2 at 0 h.

Cells 2020, 9, x FOR PEER REVIEW 8 of 16 Cells 2020, 9, 934 8 of 16

(A) (B)

0 μM5 μM 60 100 *

*** m) μ 75 40 50 8 μM10 μM 20 25 Neurite length( Neurite-bearing cells (% oftotal cellnumber) 0 0 011.5235810 011.5235810 15d-PGJ (μM) 2 15d-PGJ2 (μM) (C) 20

5 BthD-1 positive cells

0 μM8 μM (% oftotal cell number) 0 08

15d-PGJ2 (μM)

Figure 6. The effect effect of 15d-PGJ2 on neurite outgrowth. ( (AA)) Representative Representative phase-contrast phase-contrast microscopy microscopy µ images of NSC-34NSC-34 cellscells treatedtreated withwith variousvarious concentrationsconcentrations ofof 15d-PGJ15d-PGJ22.. ScaleScale bar:bar: 50 μm. ( B) Graphs show thethe percentagepercentage ofof neurite-bearingneurite-bearing cells cells (left (left panel) panel) and and the the average average neurite neurit lengthe length (right (right panel) panel) in each treatment group. Each value represents the mean SEM of four different experiments. * p < 0.05, in each treatment group. Each value represents the mean± ± SEM of four different experiments. *p < ***0.05,p ***< 0.001.p < 0.001. (C )(C Photographs) Photographs show show typical typical fluorescence fluorescence images images of of calcein-AM calcein-AM (green,(green, livelive cells) µ and EthD-1 (red, (red, dead dead cells) cells) double double staining staining in ineach each treatment treatment group. group. Scale Scale bar: bar:50 μm. 50 Graphsm. Graphs show show the percentage of EthD-1-positive dead cells. Each value represents the mean SEM of four the percentage of EthD-1-positive dead cells. Each value represents the mean ± SEM of± four different diexperiments.fferent experiments.

3.4. The PPARγ Antagonist Affects PGD2-Induced Neurite Outgrowth 3.4. The PPARγ Antagonist Affects PGD2-Induced Neurite Outgrowth After confirming the conversion of PGD2 to 15d-PGJ2 and the effects of 15d-PGJ2 on neurite outgrowth, After confirming the conversion of PGD2 to 15d-PGJ2 and the effects of 15d-PGJ2 on neurite we examined whether treatment with GW9662 (a PPARγ antagonist) prevents PGD2-induced neurite outgrowth, we examined whether treatment with GW9662 (a PPARγ antagonist) prevents outgrowth. The combination of GW9662 (10 µM) and PGD2 (15 µM) eliminated almost all the neurite PGD2-induced neurite outgrowth. The combination of GW9662 (10 μM) and PGD2 (15 μM) outgrowth observed with PGD2 (15 µM) treatment alone (Figure7A). The morphological change was eliminated almost all the neurite outgrowth observed with PGD2 (15 μM) treatment alone (Figure not observed in the cells treated with GW9662 (10 µM) alone. Neurite outgrowth assays showed that 7A). The morphological change was not observed in the cells treated with GW9662 (10 μM) alone. simultaneous treatment with GW9662 (10 µM) and PGD2 (15 µM) for 24 h significantly suppressed Neurite outgrowth assays showed that simultaneous treatment with GW9662 (10 μM) and PGD2 (15 the PGD2 (15 µM)-induced increase in the percentage of neurite-bearing cells from 38.5% 6.3% to μM) for 24 h significantly suppressed the PGD2 (15 μM)-induced increase in the percentage± of 2.5% 0.8% (p = 0.000000542) (Figure7B (left panel)) and resulted in significantly shorter neurites neurite-bearing± cells from 38.5% ± 6.3% to 2.5% ± 0.8% (p = 0.000000542) (Figure 7B) and resulted in (53.3 3.0 µm, p = 0.0116) than the cells treated with PGD2 (15 µM) alone (69.4 2.5 µm) (Figure7B significantly± shorter neurites (53.3 ± 3.0 μm, p = 0.0116) than the cells treated ±with PGD2 (15 μM) (right panel)). Moreover, GW9662 (1 µM) tended to suppress the PGD2 (15 µM)-induced increase in the alone (69.4 ± 2.5 μm) (Figure 7C). Moreover, GW9662 (1 μM) tended to suppress the PGD2 (15 percentage of neurite-bearing cells (34.0% 0.8%, p = 0.872) and elongation of neurites (60.5 4.8 µm, μM)-induced increase in the percentage± of neurite-bearing cells (34.0% ± 0.8%, p = 0.872)± and p = 0.251) (Figure7B (left panel)). Treatment with GW9662 (1 and 10 µM) alone had no significant elongation of neurites (60.5 ± 4.8 μm, p = 0.251) (Figure 7B). Treatment with GW9662 (1 and 10 μM) effect on the percentage of neurite-bearing cells and the average neurite length (Figure7B). alone had no significant effect on the percentage of neurite-bearing cells and the average neurite length (Figure 7B,C).

Cells 2020, 9, 934 9 of 16

(A) Cells 2020, 9, x FOR PEER REVIEW (B) 9 of 16 GW9662 (μM)

0 10 PGD (-) PGD (+) (A) (B) 2 2 PGD2 (-) PGD2 (+) GW9662 (μM) 60 *** *** 100 010 ) *** * PGD2 (-) PGD2 (+) PGD2 (-) PGD2 (+)

(-) μm 75 ) 4060 *** *** 100 μM *** * m) 50

(-) μ 75

bearing cells bearing

(15 -

M) 40 2

μ 20 50 25

(15 PGD

2 Neurite length ( length Neurite (+) Neurite 20 (% of total cell number) cell total of (% 0 25 0 PGD 0 1 10

Neurite length ( 0 1 10 (+) cells Neurite-bearing (% oftotal cell number) 0 GW9662 (μM) 0 GW9662 (μM) 0110 0110 GW9662 (μM) GW9662 (μM) Figure 7. The eﬀect of the peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662 on

PGD2-induced neurite outgrowth. Undiﬀerentiated NSC-34 cells were treated with 15 µM PGD2 in the Figure 7. The effect of the peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662 presence or absence of various concentrations of GW9662 for 24 h. (A) Representative phase-contrast on PGD2-induced neurite outgrowth. Undifferentiated NSC-34 cells were treated with 15 μM PGD2 microscopy images in each treatment group. Scale bar: 50 µm. (B) Graphs show the percentage of in the presence or absence of various concentrations of GW9662 for 24 h. (A) Representative neurite-bearingphase-contrast cells microscopy (left panel) images and thein each average treatment neurite group. length Scale (right bar: 50 panel) μm. ( inB) eachGraphs treatment show thegroup. Each value represents the mean SEM of four diﬀerent experiments. * p < 0.05, *** p < 0.001. percentage of neurite-bearing± cells (left panel) and the average neurite length (right panel) in each treatment group. Each value represents the mean ± SEM of four different experiments. *p < 0.05, ***p 3.5. PGD2

Lastly, we examined the effects of PGD2 and 15d-PGJ2 on the expression of the motor 3.5. PGD2 and 15d-PGJ2 Increase the Expression of Motor Neuron-Specific Protein Marker Islet-1 neuron-specific protein marker Islet-1 using Western blotting (Figure8). Undi fferentiated NSC-34 Lastly, we examined the effects of PGD2 and 15d-PGJ2 on the expression of the motor cells were treated with vehicle, 15 µM PGD2, or 8 µM 15d-PGJ2 with or without 10 µM GW9662 for 24 h. Theneuron-specific levels of Islet-1, protein calculated marker Islet-1 assuming using aWester 100%n intensityblotting (Figure of Islet-1 8). Un expressiondifferentiated in vehicle-treated NSC-34 cells were treated with vehicle, 15 μM PGD2, or 8 μM 15d-PGJ2 with or without 10 μM GW9662 for cells, were significantly increased in PGD -treated cells (201.1% 12.3%, p = 0.000598) and 15d-PGJ 24 h. The levels of Islet-1, calculated2 assuming a 100% intensity± of Islet-1 expression in 2 (8 µM)-treated cells (223.7% 24.9%, p = 0.0000887). The expression of Islet-1 in the cells treated with vehicle-treated cells, were± significantly increased in PGD2-treated cells (201.1% ± 12.3%, p = 0.000598) GW9662 alone remained unchanged (113.0% 7.7%, p = 0.965). Simultaneous treatment with GW9662 and 15d-PGJ2 (8 μM)-treated cells (223.7% ± 24.9%, p = 0.0000887). The expression of Islet-1 in the for 24cells h significantly treated with suppressed GW9662 alone the remained increased unchanged Islet-1 expression (113.0% ± caused7.7%, p by= 0.965). PGD Simultaneous(144.7% 13.3%, 2 ± p = 0.0468)treatment and with 15d-PGJ GW9662(148.1% for 24 h significantly37.6%, p = suppr0.00676).essed the increased Islet-1 expression caused by Figure 7 2 ± PGD2 (144.7% ± 13.3%, p = 0.0468) and 15d-PGJ2 (148.1% ± 37.6%, p = 0.00676).

GW9662 (10 μM) - + - + - +

PGD2 (15 μM) - - + + - -

15d-PGJ2 (15 μM) - - - - + +

Islet-1 (42 kDa)

β-actin

300 *** ** *** * 200 GW9662 (-) GW9662 (+) 100 Relative intensity

(% of vehicle-treated cells) vehicle-treated of (% 0 VehicleNone PGD2PGD 15d-PGJ PGJ2 2 2 Figure 8. The effects of PGD2 and 15d-PGJ2 on the expression of the motor neuron-specific protein Figure 8. The effects of PGD2 and 15d-PGJ2 on the expression of the motor neuron-specific protein marker Islet-1. Undifferentiated NSC-34 cells were treated with vehicle (0.04% DMSO), 15 µM PGD , marker Islet-1. Undifferentiated NSC-34 cells were treated with vehicle (0.04% DMSO), 15 μM PGD2, 2 µ µ γ or 8 Mor 15d-PGJ8 μM 15d-PGJ2 with2 with or without or without 10 10M μ GW9662M GW9662 (a (a PPAR PPARγantagonist) antagonist) for 24 h. h. Equal Equal amounts amounts of of cell lysatecell (10 lysateµg) were (10 μg) analyzed were analyzed by Western by Western blotting blotting and and the the Islet-1 Islet-1 antibody antibody and ββ-actin-actin antibody antibody as as an internalan controlinternal control (upper (upper panel). panel). The The relative relative band band density density was estimated estimated quantitatively quantitatively using using Scion Scion imaging software and is expressed as the ratio of the Islet-1 band intensity relative to the β-actin band intensity. Each value represents the mean SD of three different experiments. * p < 0.05, ** p < 0.01, ± *** p < 0.001. Cells 2020, 9, 934 10 of 16

4. Discussion

PGE2 plays a role in the differentiation of various cell types, such as regulatory T cells [33], osteoblasts [34], and neurons [35]. Our previous study reported that PGE2, which is increased in the spinal cord of ALS patients [36] and model mice [30], induced morphological differentiation of undifferentiated NSC-34 cells [16]. Lipocalin-type PGDS is a major brain-derived protein abundant in human cerebrospinal fluid, second only to albumin [37]. Although PGD2 may be present in the CNS in amounts equal to or greater than PGE2, the influence of PGD2 in motor neuron differentiation remains unclear. In this study, we demonstrated that exposure of undifferentiated NSC-34 cells to PGD2 not only increased the percentage of neurite-bearing cells but also elongated neurite length without affecting the number of EthD-1 cells PI-positive dead cells. This suggests that PGD2, similar to PGE2, induces neuritogenesis in undifferentiated NSC-34 cells without affecting the cell viability. A previous study reported that DP1 and DP2 are expressed in motor neurons of the adult mouse spinal cord [23]. Consistent with this report, our study demonstrated that DP1 and DP2 are expressed in undifferentiated NSC-34 cells. These results suggest that the characteristics of the model cell used in this study are comparable to those of the motor neurons in mouse spinal cords. Therefore, we investigated whether DP1 and/or DP2 contribute to the effect of PGD2 on neurite outgrowth using subtype-specific agonists, BW245C (a DP1 agonist) and 15(R)-15-methyl PGD2 (a DP2 agonist). Neither of these two agonists affected neurite outgrowth. Moreover, we showed that a DP1-selective antagonist (MK0524) and a DP2-selective antagonist (CAY10471) were unable to suppress PGD2-induced neuritogenesis. These results suggest that PGD2-induced neuritogenesis is not mediated by DP1 or DP2 in NSC-34 cells. Our previous study reported that PGE2 induced neurite outgrowth by activating EP2 that is coupled to Gs protein [16]. However, our present study indicated that the activation of DP1 did not induce neuritogenesis despite DP1 being the same Gs protein-coupled receptor as EP2. A previous study demonstrated that PGD2 (1 µM) caused approximately 25% of DP1 expressed at the membrane level to be internalized in HEK293 cells, reaching a plateau after 120 240 min [38]. However, PGE − 2 (1 µM) caused approximately 40% of EP4 to be rapidly internalized, whereas EP2 was not internalized after 60 min in HEK293 cells [39]. These results imply that one possible explanation for the difference between the effect of DP1 and EP2 on neuritogenesis is that DP1 is internalized and desensitized, whereas EP2 is not. 12 PGD2 is unstable and is spontaneously metabolized to J2 prostaglandins including PGJ2, ∆ -PGJ2, and 15d-PGJ2 [32]. It has been reported that PGD2 is converted to J2 prostaglandins in culture medium in the presence of mouse primary cortical neurons, and that a similar pattern of PGD2 metabolism is observed in the absence of neurons [40]. In particular, 15d-PGJ2 plays an important role in neurite outgrowth of the human neuroblastoma cell line LA–N–5 [41] and rat embryonic midbrain cells [25]. We demonstrated the conversion of PGD2 to 15d-PGJ2 in culture medium in the absence of NSC-34 cells. Moreover, exposure of NSC-34 cells to 15d-PGJ2 resulted in significant neurite outgrowth without affecting the number of EthD-1 cells PI-positive dead cells. These results suggest that the conversion of PGD2 to 15d-PGJ2 induces neuritogenesis, and also show that PGD2 and 15d-PGJ2 do not affect the cell survival at the concentration used as a neuritogenesis inducer. The concentration of PGD2 in the CNS is approximately 500 pmol/g tissue (500 pM) in rat hippocampus, 250 pmol/g tissue (250 pM) in rat cerebral cortex [42], and 40 pg/mg tissue (113 pM) in mouse spinal cord [43]. Although the concentration of 15d-PGJ2 in the spinal cord remains unclear, the concentration of 15d-PGJ2 in the hippocampus was demonstrated to be approximately 8 pmol/g tissue (8 pM) in juvenile rats [42]. These in vivo studies suggest that the concentrations of PGD2 and 15d-PGJ2 found in the CNS and spinal cord are in the pM range. However, our findings indicated that the effective concentrations of PGD2 and 15d-PGJ2 on neurite outgrowth in NSC-34 cells were in the µM range. Although the effective concentration of PGD2 on neurite outgrowth has not been reported in other in vitro models, 15d-PGJ2 has been reported to induce neurite outgrowth in rat embryonic midbrain cells at concentrations of 0.5 µM[25]. These results suggest that the necessary concentrations of PGD ≥ 2 and 15d-PGJ2 in in vitro studies are higher than physiological concentrations in vivo. In addition, the Cells 2020, 9, 934 11 of 16 measured concentrations in in vivo studies represent average tissue concentrations. Considering that prostaglandins act as autocrine or paracrine factors for their target cells, local cellular concentration is more important than average tissue concentration. However, to the best of our knowledge, the local cellular concentrations of PGD2 and 15d-PGJ2 in the spinal cord are unknown, and thus further studies are needed. Moreover, it is also important to understand how exogenously applied 15d-PGJ2 was transported into the cells. Although a previous study reported that exogenously applied 15d-PGJ2 was transported into the cells and accumulated in the nucleus [44], the underlying mechanism remains unknown. One possibility is that intracellular uptake was mediated by prostaglandin transporters, similar to that observed with other prostaglandins [43]. Further studies are needed to determine the underlying mechanism of transportation in neurons and its model cells. 15d-PGJ2 binds to DP2 with an affinity similar to PGD2 [45]. Furthermore, 15d-PGJ2 enhancement of nerve growth factor-induced neurite outgrowth is inhibited by CAY10471 (a DP2 antagonist) in PC12 cells [46]. In this study, 15(R)-15-methyl PGD2 (a DP2 agonist) did not induce neurite outgrowth, suggesting that DP2 activation is not involved in 15d-PGJ2-induced neuritogenesis in NSC-34 cells. Moreover, 15d-PGJ2 is commonly known as an endogenous ligand of PPARγ [44]. Indeed, 15d-PGJ2 induces differentiation of rat embryonic midbrain cells into dopaminergic neuronal cells in a PPARγ-dependent manner [25]. Nevertheless, 15d-PGJ2 is also involved in PPARγ-independent pathways via direct covalent binding to proteins other than PPARγ [47]; 15d-PGJ2 enhances nerve growth factor-induced neurite outgrowth via covalent binding to activator protein-1 [48] or the transient receptor potential vanilloid 1 [49] in PC12 cells. These studies suggest that 15d-PGJ2-induced neuritogenesis and neuronal differentiation involve pathways that are dependent or independent of PPARγ, according to the cell type. We demonstrated that simultaneous treatment with GW9662 (a PPARγ antagonist) and PGD2 for 24 h suppressed PGD2-induced neuritogenesis, suggesting that 15d-PGJ2 converted from PGD2 induces neurite outgrowth via a PPARγ-dependent pathway in undifferentiated NSC-34 cells. 15d-PGJ2 contains two electrophilic carbons: the 13-carbon position reacts with the sulfur atom of the cysteine residue in PPARγ, whereas the 9-carbon position within the cyclopentenone ring binds to the cysteine residue in other proteins including nuclear factor-κB, IκB kinase, and activator protein-1 [50]. Further studies are needed to determine how 15d-PGJ2 targets proteins such as PPARγ. Islet-1 is a homeodomain transcription factor that is expressed from the early stages of motor neuron differentiation [51]. The expression of Islet-1 is upregulated during the differentiation of human pluripotent stem cells into motor neurons, and Islet-1 depletion by shRNA prevents Islet-1 expression and motor neuron differentiation [52]. Islet-1 is also expressed in retinoic acid-induced differentiated NSC-34 cells [6]. Therefore, Islet-1 expression is a critical regulator of mature motor neuron formation. In this study, we evaluated Islet-1 expression to determine whether PGD2 and 15d-PGJ2 promote the maturation of NSC-34 cells into motor neurons. We demonstrated that PGD2 and 15d-PGJ2 significantly upregulate Islet-1 expression, suggesting that PGD2 and 15d-PGJ2 can potentiate neural conversion of mature motor neurons. Moreover, we observed that GW9662 (a selective PPARγ antagonist) dramatically inhibited the PGD2- and 15d-PGJ2-induced increase in Islet-1 expression in differentiated NSC-34 cells. Treatment with 15d-PGJ2, retinoic acid, and ciglitazone (a selective PPARγ agonist) suppressed Islet-1 gene expression in neural stem cells generated by culturing dissociated brain cells from newborn mice [53]. In contrast, ciglitazone induced Islet-1 gene expression in human brain tumor stem cell cultures [54]. These results suggest that PPARγ regulates Islet-1 expression at the gene level depending on the type of cell. Although the mechanism underlying the upregulation of Islet-1 warrants further investigation, our results indicate that the PGD2- and 15d-PGJ2-induced increase in Islet-1 expression was mediated by PPARγ activation. Several studies have shown that the differentiation of NSC-34 cells leads to neurite outgrowth and the expression of motor neuron-specific proteins [5,6]; as such, these cells can be employed as an in vitro experimental model to study motor neuron dysfunction in response to neurotoxins such as staurosporine, thapsigargin, hydrogen peroxide, homocysteine [55], glutamate [56,57], and Cells 2020, 9, x FOR PEER REVIEW 12 of 16

Several studies have shown that the differentiation of NSC-34 cells leads to neurite outgrowth Cells 2020 9 and the, expression, 934 of motor neuron-specific proteins [5,6]; as such, these cells can be employed12 of 16as an in vitro experimental model to study motor neuron dysfunction in response to neurotoxins such cerebrospinalas staurosporine, fluid fromthapsigargin, sporadic ALShydrogen patients peroxide, [58]. However, homocysteine as pointed [55], out byglutamate a previous [56,57], study [and59], thecerebrospinal usefulness offluid diff erentiatedfrom sporadic NSC-34 ALS cells patients has been [58]. questioned However, as as these pointed cells exhibitout by lessa previous cell death study and a[59], small the unsustained usefulness increaseof differentiated in the concentration NSC-34 cells of has intracellular been questi calciumoned as after these exposure cells exhibit to glutamate less cell compareddeath and to a primarysmall unsustained motor neurons. increase Our in previous the concentration study has demonstrated of intracellular that calcium EP2 and after EP3, exposure but not EP1to glutamate and EP4, arecompared dominantly to primary expressed motor in dineurons.fferentiated Our NSC-34 previous cells study as well has asdemonstrated motor neurons that in EP2 the and EP3, but not EP1 and EP4, are dominantly expressed in differentiated NSC-34 cells as well as mouse spinal cord [60]. Similar to the increase in EP2 expression in PGE2-treated differentiated NSC-34 cells,motor EP2 neurons upregulation in the in mouse motor neuronsspinal cord of ALS [60]. model Similar mice to was the observed increase at thein EP2 early expression symptomatic in agePGE [226-treated]. Therefore, differentiated these cells NSC-34 are believedcells, EP2 to upre be agulation suitable in model motor for neur assessingons of ALS the responsemodel mice to prostaglandinswas observed at in the motor early neurons. symptomatic age [26]. Therefore, these cells are believed to be a suitable model for assessing the response to prostaglandins in motor neurons. In conclusion, we demonstrated for the first time that exogenously applied PGD2 induces neuritogenesisIn conclusion, in NSC-34 we demonstrated cells. We suggest for the that first the underlyingtime that exogenously mechanism involvesapplied nonenzymaticPGD2 induces neuritogenesis in NSC-34 cells. We suggest that the underlying mechanism involves nonenzymatic conversion of PGD2 into 15d-PGJ2, which then activates PPARγ (Figure9). As such, both PGD 2 and conversion of PGD2 into 15d-PGJ2, which then activates PPARγ (Figure 9). As such, both PGD2 and 15d-PGJ2 (converted from PGD2) promote the differentiation of progenitor cells into motor neurons 15d-PGJ2 (converted from PGD2) promote the differentiation of progenitor cells into motor neurons and could thus be useful for establishing a motor neuron model. This novel effect of PGD2 on the diandfferentiation could thus of be motor useful neuron-like for establishing cells may a motor provide neuron a potential model. target This for novel regenerative effect of therapiesPGD2 on forthe motordifferentiation neuron diseases. of motor neuron-like cells may provide a potential target for regenerative therapies for motor neuron diseases.

PGD2 Non enzymatic conversion

15d-PGJ2 DP1 DP2

Gs Gi

Islet-1 protein

Neuritogenesis

FigureFigure 9.9. AA proposedproposed mechanismmechanism underlyingunderlying PGDPGD22-induced-induced neuritogenesisneuritogenesis inin motormotor neuron-likeneuron-like NSC-34NSC-34 cells.cells.

AuthorAuthor Contributions:Contributions:Conceptualization, Conceptualization, H.N. H.N. and and Y.K.; Y.K.; Methodology, Methodology, H.N. H.N. and and Y.K.; Y.K.; Validation, Validation, H.N., H.N., Y.K., Y.K., and N.Y.;and N.Y.; Formal Formal analysis, analysis, H.N. andH.N. N.Y.; and Investigation, N.Y.; Investigation, H.N., Y.K., H.N., N.Y., Y.K., T.K., N.Y., and T.K., K.H.; and Resources, K.H.; Resources, Y.K., T.K., Y.K., K.H., T.K., and K.I.;K.H., Data and curation, K.I.; Data H.N., curation, H.M., andH.N., N.Y.; H.M., Writing—original and N.Y.; Writing—original draft preparation, draft H.N. prep andaration, H.M.; Writing—reviewH.N. and H.M.; and editing, Y.K.; Visualization, H.N. and Y.K.; Supervision, Y.K. and T.K.; Project administration, Y.K. and Writing—review and editing, Y.K.; Visualization, H.N. and Y.K.; Supervision, Y.K. and T.K.; Project T.S.; Funding acquisition, Y.K., T.K., and T.S. All authors have read and agreed to the published version of theadministration, manuscript. Y.K. and T.S.; Funding acquisition, Y.K., T.K., and T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by a grant to encourage and promote Research Projects in the School ofFunding: Pharmacy, This Nihon research University was funded (Y.K.), in part by aby grant a grant for to cooperative encourage researchand promote in the Research School Projects of Pharmacy, in the NihonSchool Universityof Pharmacy, (Y.K., Nihon K.H., University and T.K.), by(Y.K.), a Nihon by a University grant for co Multidisciplinaryoperative research Research in the GrantSchool for of 2019 Pharmacy, (K.I. and Nihon T.S.), and by a Nihon University Chairman of the Board of Trustees Grant. The funding bodies had no role in the design of the study or in the writing of the manuscript. Cells 2020, 9, 934 13 of 16

Acknowledgments: We are grateful to Neil Cashman for providing the NSC-34 cell line. We thank all the members of our laboratories, especially Sonoko Horikoshi and Rika Kashihara for excellent technical help. The author would like to thank Editage (www.editage.com) for English language editing. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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