Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Abstract

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

Introduction

Motor neurons are neural cells that control muscle movements, and they are divided into two main types based on the location of their cell bodies in the central nervous system: corticospinal upper motor neurons and spinal lower motor neurons (Stifani, 2014). As lower motor neurons receive glutamate from upper motor neurons, the spinal motor neurons fire action potentials and then release acetylcholine (ACh). In the developing spinal cord, motor neurons are generated from the progenitor cell pool in the ventral neural tube. The dividing progenitor cells are stimulated by several triggering factors responsible for the subsequent differentiation process. These signals lead to the division of progenitor domains (Davis-Dusenbery et al., 2014), particularly retinoic acid (RA) and sonic hedgehog (SHH), which tightly regulate the exit from the cell cycle and entry into the differentiation of motor neuron progenitors (Novitch et al., 2003; Lee et al. 2009). As motor neuron progenitor cells deviate from the cell cycle, the expression of transcription factors in the motor neuron, pancreas homeobox 1 (HB9), and Islet-1, begins to increase (Lee et al., 2005). These transcription factors induce a switch toward motor neuron specification and the expression of genes involved in cholinergic neurotransmissions, such as vesicular acetylcholine transporter and choline acetyltransferase (ChAT) (Arber et al. 1999; Lee et al. 2012).

Recently, motor neurons derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proposed as potential ways to facilitate understanding of the pathogenesis of motor neuron diseases and progress drug discovery for therapies to treat these conditions (Jaiswal, 2017). To date, numerous protocols have been devised for the differentiation of stem cells into populations derived from specialized subtypes of motor neurons, most of which use growth factors and small molecules for differentiation due to their reasonable price and cost-effectiveness (Hu & Zhang, 2009). Morizane et al. reported that purmorphamine, a small molecule activator of the SHH signal pathway, can ventralize neural precursors (Morizane et al., 2011), and that LY294002, a phosphatidylinositol 3-kinase inhibitor, enhances the viability and differentiation of stem cells into motor neuron-like cells (Ebrahimi-Barough et al., 2017). The combination of AMP-kinase inhibitor dorsomorphin, β-catenin-mediated transcription inhibitor XAV939, and TGFβ signaling pathway inhibitor A8301 has enhanced the ability to direct human ES cells into the motor neuron lineage, with 39% of total cells differentiating into HB9/Islet-1 positive motor neurons (Valizadeh-Arshad et al., 2018). However, given that small molecule-induced motor neurons have not been shown to exhibit sufficient electrophysiological maturation, there is still a need to improve and accelerate the differentiation and functional maturation of such motor neurons. We have summarized several publications on the generally used protocols for the differentiation from human stem cells into motor neurons (Table 1).

Table 1.

Protocols for the differentiation from human stem cells into motor neurons

Starting cells	Neural induction	Motor neuron differentiation	Motor neuron maturation	Cell features		Total induction period	Differentiation rate	References
				Motor neuron-specific marker protein	Action potential
Endometrial stem cells	LY294002 and RA	BDNF	None	ChAT, ISL1, NF-H, TUJ1	Non-tested	15 days	Approximately 80% (ISL1 positive)	Ebrahimi-Barough et al. 2017
ESCs and iPSCs	Dorsomorphin, SB431542, BIO, RA and purmorphamine	RA, purmorphamine, cAMP,BDNF, GDNF, IGF-1 and ascorbic acid	RA, purmorphamine, cAMP, BDNF, GDNF, IGF-1 and ascorbic acid	HB9, ISL1, ChAT	Non-tested	42 days	Approximately 50% (HB9 or ISL1 positive)	Shimojo et al. 2015
iPSCs	SB431542, LDN-193189, CHIR99021, Y-27632, RA, SAG,BDNF and GDNF	SB431542, LDN-193189, CHIR99021, Y-27632, RA, SAG, BDNF, GDNF and DAPT	BDNF, GDNF, and CNTF	HB9, SMI32, TUJ1, ChAT	Spontaneous repetitive action potentials Spontaneous postsynaptic currents	38 days	Approximately 70–95% (ISL1, SMI32 or ChAT-positive) Approximately 30–60% (cells with spontaneous repetitive action potentials)	Guo et al. 2017
ESCs	Dorsomorphin, XAV939, A8301, RA, and bFGF	Dorsomorphin, XAV939, A8301, ascorbic acid, purmorphamine and RA	Dorsomorphin, XAV939, A8301, bFGF, purmorphamine, RA and BDNF	TUJ1, MAP2, HB9, ISL1	Single action potential	32 days	39% (HB9/ISL1 double-positive)	Valizadeh-Arshad et al. 2018

^{BDNF brain-derived neurotrophic factor, bFGF basic fibroblast growth factor, cAMP cyclic adenosine monophosphate, ChAT choline acetyltransferase, CNTF ciliary neurotrophic factor, ESCs embryonic stem cells, GDNF glial cell-derived neurotrophic factor, IGF-1 insulin-like growth factor-1, iPSCs induced pluripotent stem cells, ISL1 islet-1, MAP2 microtubule-associated protein 2, RA retinoic acid}

Prostaglandins are potent eicosanoid lipid mediators derived from the arachidonic acid cascade (Phillis et al., 2006) (Fig. 1). Arachidonic acid is specifically released by cytosolic phospholipase A₂ and is metabolized by cyclooxygenase (COX) into prostaglandin H₂ (PGH₂). Subsequently, specific synthetases convert PGH₂ into a variety of eicosanoids such as prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), prostaglandin F₂, prostaglandin I₂, and thromboxane A₂ (Ricciotti & Fitzgerald, 2011; Peebles, 2019). Their biological functions have been attributed to interactions with specific G-protein-coupled receptors (GPCRs) at the plasma membrane, termed E-prostanoid (EP) receptor-1 to 4, D-prostanoid (DP) receptor-1 and 2, F-prostanoid (FP) receptor, I-prostanoid (IP) receptor, and thromboxane-prostanoid (TP) receptor (Ricciotti & Fitzgerald, 2011; Peebles, 2019). Of these, EP2, EP4, IP, and DP1 increase intracellular levels of cyclic adenosine monophosphate (cAMP) via Gαs; EP1 and FP increase intracellular levels of calcium via Gαq; DP2 decreases intracellular levels of cAMP via Gαi; and EP3 and TP increase or decrease intracellular cAMP and Ca²⁺ via Gαs, Gαi, or Gαq (Mohan et al., 2012).

Fig. 1.

The biosynthesis and signaling pathway of prostaglandins. PLA₂ phospholipase A₂, ARA arachidonic acid, COX cyclooxygenase, PGG₂ prostaglandin G₂, PGH₂ prostaglandin H₂, PGI₂ prostaglandin I₂, PGF_2α prostaglandin F_2α, PGD₂ prostaglandin D₂, PGE₂ prostaglandin E₂, TXA₂ thromboxane A₂, PGIS prostaglandin I synthase, PGFS prostaglandin F synthase, PGDS prostaglandin D synthase, PGES prostaglandin E synthase, DP1 D-prostanoid receptor-1, DP2 D-prostanoid receptor-2, EP1 E-prostanoid receptor-1, EP2 E-prostanoid receptor-2, EP3 E-prostanoid receptor-3, EP4 E-prostanoid receptor-4

It has been demonstrated that activated Gαs translocated from the plasma membrane promotes neurite outgrowth in PC12 cells (derived from a rat pheochromocytoma) and rat primary hippocampal neurons (Sarma et al., 2015), and that Gq-dependent Ca²⁺ mobilization promotes neurite outgrowth in primary mouse cortical neurons (Peterson et al., 2013). Prior in vivo studies have reported that the COX-2 inhibitors meloxicam and nimesulide inhibit progenitor cell proliferation in the subventricular zone and hippocampus of young adult mice (Goncalves et al., 2010) and that the brain isolated from embryonic day 18.5 embryos in phospholipase A₂-activating protein homozygous null mice had decreased tissue PGE₂ levels and less mature and differentiated neurons (Falik Zaccai et al., 2017). This suggests that prostaglandins play an important role in neuritogenesis and neurogenesis in the central nervous system through GPCRs. Thus, it is possible that a prostaglandin-based agent targeting neuritogenesis and neurogenesis in the spinal cord could provide a potential therapeutic approach for motor neuron degeneration and motor neuron diseases.

This article summarizes our investigations that have focused on the effect of prostaglandins on motor neuron differentiation and reviews previous original research and review articles that have investigated the effect of prostaglandins and signal transduction pathways possibly regulated by prostaglandins on neurite outgrowth and neural differentiation. Keeping in mind these results from previous studies and reviews, we discuss the prospects of the prostaglandin signaling cascade as a target for therapeutic regeneration in motor neuron diseases.

PGE₂ Accelerated Conversion and Maturation Into Motor Neurons

In vitro studies have reported that PGE₂ promotes neurite growth in cultured mouse dorsal root ganglion neurons via EP2 (Hiruma et al., 2000) and ND7/23 mouse sensory neuron-like cells via EP4 (Mitani et al., 2016). PGE₂ has also been reported to retract neurites of mouse neuroblastoma Neuro-2 cells by an unknown mechanism (Tamiji & Crawford, 2010). These reports suggest that PGE₂ has opposing effects on neurite outgrowth and different cell types may be involved in different EP subtypes. ​Therefore, it is necessary to investigate the effect of PGE₂ on neurite outgrowth and neuronal differentiation in each target cell.

Neural differentiation of NSC-34 mouse neuroblastoma and the spinal motor neuron fusion cell line has been commonly used as a motor neuron model in previous research. Exposure to PGE₂ results in inhibited cell proliferation and increases the percentage of neurite-bearing cells in a concentration-dependent manner without affecting cell viability in NSC-34 cells (Nango et al., 2017). We have previously reported that EP2 and EP3 are expressed in NSC-34 cells and mouse spinal motor neurons (Miyagishi et al., 2013). In this study, an EP2-selective agonist (butaprost) replicated the effect of PGE₂ on cell proliferation and neuritogenesis, whereas an EP1 and EP3 agonist (sulprostone) did not. Moreover, co-treatment with an EP2-selective antagonist (PF-04418948) suppressed PGE₂-induced neurite outgrowth. It has been reported that EP2 mediates its actions via cAMP signaling. Interestingly, a membrane-permeable cAMP analog (dibutyryl-cAMP) has been shown to mimic PGE₂- and butaprost-induced neurite outgrowth. These results suggest that PGE₂ suppresses cell proliferation and induces morphological differentiation through EP2 activation; moreover, the Gαs protein-coupled cAMP signaling axis is at least partly involved in the molecular mechanisms of NSC-34 cells.

cAMP signaling is involved in the development of tissues derived from the endoderm, ectoderm, and mesoderm, and thus is highly pleiotropic for differentiation. Although the combination of vascular endothelial growth factor and cAMP synergistically enhances differentiation of human iPS cells into endothelial cells (Ikuno et al., 2017), cAMP along with other small molecules is used to induce motor neurons from human ES cells and human iPS cells (Boulting et al., 2011; Takazawa et al., 2012). cAMP signaling is known to play a role in neural differentiation through the activation of cAMP response element-binding protein in neural stem/progenitor cells (Mantamadiotis et al., 2012). Moreover, an elevation of intracellular cAMP has been shown to promote neurite outgrowth in cultured rat motor neurons (Aglah et al., 2008) as well as survival and neural differentiation of neural stem and progenitor cells in rats with spinal cord injury (Kim et al., 2011). These reports suggest that cAMP stimulates the maturation of lineage-determined progenitor cells rather than regulating the cell lineage. Therefore, PGE₂ is presumed to function as an endogenous cAMP activator during motor neuron maturation in vivo. Indeed, we have recently shown that PGE₂ induces NSC-34 cells to differentiate into functional motor neuron-like cells, supporting the hypothesis that PGE₂ is an endogenous maturation inducer for motor neurons (Nango et al., 2020a).

RA has been shown to inhibit proliferation, promote neuritogenesis, and convert into motor neuron-like cells from proliferating NSC-34 cells (Johann et al., 2011; Maier et al., 2013; Petrozziello et al., 2017) and into motor neurons from iPS cells (Qu et al., 2014; Shimojo et al., 2015). In one study, the percentage of neurite-bearing cells in NSC-34 cells treated with PGE₂ for 2 days was significantly higher than that in NSC-34 cells treated with RA for 2 days and was similar to that in NSC-34 cells treated with RA for 7 days; this suggests that PGE₂ is more efficient for neural differentiation and neuritogenesis than RA (Nango et al., 2020a). Moreover, we evaluated neuronal electrical properties by whole-cell patch-clamp recordings and showed that the threshold currents for action potential generation were significantly lower in PGE₂-treated cells than in RA-treated cells (Nango et al., 2020a). Interestingly, the threshold currents of motor neurons in adult mice lumbar spinal cord slices have been reported to be smaller than those of motor neurons at an early postnatal stage (Mitra & Brownstone 2012). These findings suggest that PGE₂-evoked neuron-like cells exhibit higher neuronal maturation than RA-evoked neuron-like cells.

Among the physiological properties of mature neurons, the generation of action potentials is one of the most important features (Davis-Dusenbery et al., 2014). Surprisingly, in NSC-34 cells, the threshold potential, peak potential, and amplitude of the first spike in PGE₂-induced neuron-like cells for 2 days were the same as those of RA-induced neuron-like cells for 7 days (Nango et al., 2020a). On the other hand, treatment with dibutyryl-cAMP alone requires at least 21 days to differentiate from NG108-15 cells into action potential-producing cells (Liu et al., 2012). Likewise, the combination of small molecules takes 11 days to generate an action potential in human ES cell-derived neurons (Valizadeh-Arshad et al., 2018). Although differences in cell types and other aspects cannot be straightforwardly compared, PGE₂ more rapidly induces differentiation into cells with action potentials. We advocate the possibility that PGE₂ is a highly agile and efficient differentiation-inducing factor for mature neurons.

We have also previously assessed the expression of neural differentiation markers using western blot analysis (Nango et al., 2020a). This study showed that the levels of neuronal marker proteins microtubule-associated protein (MAP) 2c and synaptophysin, and motor neuron-specific markers HB9 and Islet-1, were upregulated to the same extent in PGE₂-treated cells and RA-treated cells. However, the protein level of ChAT in PGE₂-treated cells for 2 days was higher than that in RA-treated cells for 7 days. Consistent with this result, the amount of ACh released from PGE₂-treated cells for 2 days was significantly higher than that from RA-treated cells for 7 days. Taken together, these results strongly suggest that PGE₂ promoted the conversion of NSC-34 cells into mature motor neuron-like cells (Fig. 2). Hounoum et al. have argued that the differentiated NSC-34 cells induced by RA are unsuitable as experimental models for glutamate excitotoxicity in motor neuron diseases (Hounoum et al., 2016). Although further in vitro studies should be carried out to evaluate glutamate excitotoxicity in PGE₂-differentiated NSC-34 cells, PGE₂-differentiated cells may be an equal or superior model to unravel the pathological mechanism of motor neuron death compared to RA-differentiated cells.

Fig. 2.

A schematic diagram showing the comparison of PGE₂- and RA-induced cell differentiation. PGE₂ prostaglandin E₂, RA retinoic acid, EP2 E-prostanoid receptor-2, EP3 E-prostanoid receptor-3

PGD₂ had Promising Effects on Motor Neuron Differentiation

It is worth noting that PGD₂ is the prevailing prostaglandin in the murine spinal cord (Kihara et al., 2009); moreover, DP1 and DP2 are located in adult mouse spinal cord motor neurons (Grill et al., 2008). Although it has been reported that PGD₂ reverses serum starvation-induced neurite outgrowth in hypothalamic cell lines (N37 cells)  (Tsuchiya et al., 2016), it has not been shown to induce neurite outgrowth in motor neurons. We previously explored whether PGD₂ can induce neurite outgrowth in undifferentiated NSC-34 cells (Nango et al., 2020b).

In our study, PGD₂ increased the proportion of cells bearing neurites without affecting cell viability and extended neurite length (Nango et al., 2020b). We also investigated whether BW245C and 15(R)-15-methyl PGD₂, which are specific agonists of DP1 and DP2, could regulate neurite outgrowth. Neither BW245C nor 15(R)-15-methyl PGD₂-induced neurite outgrowth (Nango et al., 2020b). In agreement with this result, MK0524 and CAY10471, which are specific antagonists of DP1 and DP2, did not inhibit PGD₂-induced neurite outgrowth (Nango et al., 2020b). These results imply that neither DP1 nor DP2 contribute to PGD₂-evoked neuritogenesis. On the other hand, PGD₂ is non-enzymatically metabolized to J₂ prostaglandins (Liu et al., 2013a). We have recently demonstrated the conversion of PGD₂ into 15d-PGJ₂ in a cell-free culture medium (Nango et al., 2020b). 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a major metabolite of PGD₂ (Straus & Glass 2001), is known to enhance neuritogenesis and synaptogenesis in LA-N-5 human neuroblastoma cells (Han et al., 2001) and rat embryonic midbrain cells in a peroxisome proliferator-activated receptor γ (PPARγ)-dependent manner (Park et al., 2004). We have also recently demonstrated that the exogenous application of 15d-PGJ₂ leads to significant neurite outgrowth with no influence on cell viability in undifferentiated NSC-34 cells (Nango et al., 2020b). Subsequently, GW9662 (a PPARγ antagonist) suppressed PGD₂-induced neuritogenesis in NSC-34 cells (Nango et al., 2020b). Furthermore, we have shown that PGD₂ and 15d-PGJ₂ significantly upregulate Islet-1 protein expression and that these increases in Islet-1 protein were suppressed by GW9662 in PGD₂-treated cells and 15d-PGJ₂-treated cells (Nango et al., 2020b). Taken together, these results indicate that PGD₂, through conversion to 15d-PGJ₂, induces neuritogenesis via a PPARγ-dependent pathway; the results also show that PGD₂ and 15d-PGJ_2, as well as PGE₂, may contribute to the preparation of functionally mature motor neurons from undifferentiated NSC-34 cells.

To the best of our knowledge, no published studies examined the relationship between PPARγ and motor neuron differentiation. The only relevant evidence so far is that of the regulation of motor neuron-specific transcription Islet-1 expression by PPARγ. PPARγ-mediated regulation of Islet-1 gene expression depends on the type of cell. In neural stem cells derived from the neonatal mouse brain, a selective PPARγ agonist (ciglitazone), RA, and 15d-PGJ₂ abrogated Islet-1 gene expression (Kanakasabai et al., 2012). In human brain tumor stem cells, ciglitazone has been shown to upregulate the gene expression of Islet-1 (Pestereva et al., 2012). Importantly, Liu et al. predicted transcription factor-target gene interactions between PPARγ and Islet-1 in human extravillous and villous cytotrophoblasts (Liu et al., 2020). In contrast, PPARγ activation promotes differentiation of newborn mouse brain-derived neural stem cells into oligodendrocytes (Kanakasabai et al., 2012). Further studies are needed to determine how PPARγ activation triggers the conversion of mature motor neurons.

Limitations of this Review due to the Lack of In Vivo Studies

Through this review, we explained the effects of PGE₂ and PGD₂ on motor neuron differentiation and discussed their usefulness. However, one of the main limitations worth noting is that no clear link has yet been reported between prostaglandins and motor neuron differentiation in vivo. To the best of our knowledge, null mutations of prostaglandin-related genes have not been reported to induce motor neuron hypoplasia and subsequent lethal progression. Studies of neonatal lethal phenotypes in mutant mice have shown that deletions of EP4, prostaglandin dehydrogenase, and COX lead to a patent ductus arteriosus (Ushikubi et al., 2000; Turgeon & Meloche 2009). This is consistent with the result of a previous report showing that PGE₂ induces mesoderm differentiation in human embryonic cells (Zhang et al., 2018). Moreover, PPARγ-deficiency in neonatal mice is not lethal (O’Donnell et al., 2016). As shown in Fig. 3, PGE₂-dependent signaling and the PGD₂-15d-PGJ₂-mediated cascade are at least partially different from each other in NSC-34 cells. This evidence suggests that a single deficiency of prostaglandin in the central nervous system might be compensated for by other prostaglandin signals and that motor neuron development, even if PGE₂ or PGD₂ is lost, can still occur. Another explanation for the role of PGE₂ and PGD₂ in motor neuron development in vivo relates to the induction of differentiation into motor neurons from adult neural progenitor cells but not stem cells.

Fig. 3.

A proposed mechanism underlying PGE₂- and PGD₂-induced neuritogenesis in motor neuron-like NSC-34 cells. cAMP cyclic adenosine monophosphate, 15d-PGJ₂15-deoxy-Δ^12,14-prostaglandin J₂, PGE₂ prostaglandin E₂, PGD₂ prostaglandin D₂, RA retinoic acid, DP1 D-prostanoid receptor-1, DP2 D-prostanoid receptor-2, EP2 E-prostanoid receptor-2, EP3 E-prostanoid receptor-3

The second major limitation is the in vivo concentration of prostaglandins. The concentration of PGE₂ and PGD₂ in the spinal cord is approximately 68 ng/g tissue (193 nM) in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) expressing a mutant form of SOD1 detectable in familial ALS patients (Miyagishi et al., 2017) and 130 pg/mg tissue (369 nM) in spinal cord injury rat model (Redensek et al., 2011), respectively. In addition, although the concentration of 15d-PGJ₂ found in the ischemic hippocampus is approximately 18 pmol/g tissue (18 pM) (Liu et al., 2013b), the concentration of 15d-PGJ₂ in the spinal cord remains unclear. These in vivo studies suggest that the concentrations of PGE₂, PGD_2, and 15d-PGJ₂ found in the spinal cord and the central nervous system are in the nM and pM range even under pathological conditions. However, the effective concentrations of PGE₂, PGD_2, and 15d-PGJ₂ on neurite outgrowth were in the μM range in NSC-34 cells. It is worth noting that the measured concentrations in in vivo studies represent whole tissue concentrations. Considering that prostaglandins act as autocrine or paracrine factors for their target cells, local tissue concentration is more important than whole tissue concentration. The report that neural progenitor cells migrated to the vicinity of degenerated motor neurons and then differentiated into neurons in the spinal cord of ALS model mouse during disease onset and progression supports the importance of local tissue concentrations of prostaglandin for motor neuron differentiation (Chi et al., 2006).

Kondo et al. reported that co-localization of COX-2 and 15d-PGJ₂ in neuron and glia in the anterior spinal cords of sporadic ALS patients (Kondo et al., 2002). Moreover, we had reported previously that protein expression of microsomal prostaglandin E synthase (mPGES)-1, the enzyme that controls the final step of PGE₂ synthesis, is increased in motor neurons and microglia in lumbar spinal cords of G93A mice at the pre-symptomatic and early symptomatic stage (Miyagishi et al., 2012). Although the signaling pathways involved in the upregulation of mPGES-1 are poorly understood and further investigations are needed, interleukin-1β, which is increased in the spinal cord of ALS patients, G93A mice and spinal cord injured rats (Papadimitriou et al., 2010; Wang et al., 1997), has been reported to induce COX-2 expression through protein kinase C and mitogen-activated protein kinases (MAPKs) in mouse astrocytes (Molina-Holgado et al., 2000) and through the extracellular signal-regulated kinase/p38 MAPK pathway in cultured rat dorsal root ganglia neurons (Amaya et al., 2009). These studies suggest that the proinflammatory cytokine-induced local upregulation of COX-2 and mPGES-1 under pathological conditions contributes to increased local concentrations of prostaglandins. However, to the best of our knowledge, the local cellular concentrations of PGE_2, PGD_2, and 15d-PGJ₂ in the spinal cord are unknown, and thus further studies are needed.

Possible Involvement of Other Prostaglandins in Motor Neuron Differentiation

Previous studies reported an increase in COX-2 protein expression in spinal cord motor neurons of spinal cord injury rat (Kim et al., 2017) and ALS patient (Kondo et al., 2002; Maihöfner et al., 2003), and therefore it is assumed that prostaglandins other than PGE₂ and PGD₂ are also increased in the spinal cord. Hence, this section provides investigations into the effects of prostaglandins other than PGE₂, PGD₂, and 15d-PGJ₂ on neurite outgrowth and discusses their possible involvement in motor neuron differentiation.

Thromboxane A₂, which is synthesized by the metabolism of PGH₂ by thromboxane synthetase, has been reported to be involved in neurite outgrowth via MAPKs in cultured rat cerebral cortical neurons (Sumimoto et al., 2015). Moreover, PGE₁, which is synthesized from dihomo-γ-linolenic acid metabolized by COX, induced neurite outgrowth in mouse neuroblastoma NB2a cell line (Gurwitz & Cunningham. 1988). Although the mechanism of PGE₁-induced neurite outgrowth has not been mentioned, the affinity of PGE₁ and PGE₂ for EP2 is similar (Kiriyama et al., 1997), suggesting that EP2 could be involved in PGE₁-induced neurite outgrowth. Conversely, PGA₂, PGC₂, and PGB₂ are non-enzymatic dehydration metabolites of PGE₂, similar to the metabolism from PGD₂ to 15d-PGJ₂ (Straus & Glass, 2001). These metabolites of PGE₂ have been reported to be agonists of PPARα or PPARδ, a PPAR family (Forman et al., 1997). The activation of PPARα has been reported to promote neural differentiation in cultured rat forebrain neurons (Bento-Abreu et al., 2007), thus suggesting that these metabolites could also be involved in neural differentiation. These findings suggest that thromboxane A₂, PGE₁, and metabolites of PGE₂ are involved in neural differentiation in brain-derived neurons. However, no study has suggested a relationship between these prostaglandins and the differentiation of motor neurons and their presence in the spinal cord, and further studies are needed.

Conclusion and Future Perspectives

PGE₂ levels are increased in various areas in neurodegenerative disorders and not just amyotrophic lateral sclerosis, such as in the cerebrospinal fluid in Alzheimer’s disease (Ho et al., 2000), the substantia nigra in Parkinson’s disease (Mattammal et al., 1995), and the brain in stroke (Yokota et al., 2004). Similar to PGE₂, an increase in PGD₂ is detected in the post-mortem cerebral cortex of patients with Alzheimer's disease (Iwamoto et al., 1989) and animal models of stroke (Gaudet et al., 1980). Interestingly, activation of EP2 exhibits neuroprotective effects in models of Alzheimer's disease and stroke (Yagami et al., 2016). Combined with reports that COX-2 inhibitors suppress neurogenesis (Goncalves et al., 2010) and PGE₂ promotes neural differentiation in neuroectodermal stem cells derived from mouse brains (Wong et al., 2016), prostaglandins may be increased in these neurodegenerative disorders to induce differentiation from endogenous neural stem/progenitor cells into neural cells. Although further in vivo studies using adult neural progenitor cells will be needed to define the role of prostaglandins in the conversion of motor neurons, the prostaglandin signaling cascade can now be identified as a target for the treatment of patients with neurodegenerative disorders. This article may provide new insights into the potency of prostaglandins as differentiation inducers or enhancers, and help develop novel treatment strategies for patients with motor neuron disorders.

Author Contributions

HN and YK designed the paper; HN wrote the paper and designed the figures; and YK reviewed the manuscript.

Funding

This work was funded in part by a grant to encourage and promote research projects in the School of Pharmacy, Nihon University (Y.K.), 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 the writing of the manuscript.

Declarations

Conflicts of interest

The authors declare no conflicts of interest.