Structurally different lysophosphatidylethanolamine species stimulate neurite outgrowth in cultured cortical neurons via distinct signaling pathway

Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identication and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that lysophosphatidylethanolamine (LPE), one of the lysophospholipids, inuences neurite outgrowth in cultured cortical neurons. Extracellular application of either of the structurally different LPE spices, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE) dramatically increased the areas of axon and dendrite without affecting the neuronal viability. Subsequent analysis revealed that both LPEs increased the length of neurite in a dose-dependent manner. Interestingly, inhibition of phospholipase C, one of the effectors for G-protein-coupled receptor-mediated signaling pathways, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C (PKC) inhibitors on neurite outgrowth were also different. Inhibitor against PKCα, β, δ, ε, η, and θ inhibited both 16:0 LPE- and 18:0 LPE-induced neurite outgrowth. In contrast, an inhibitor against PKCα, β, γ, δ, and ζ inhibited the 18:0 LPE effect but not the 16:0 LPE effect. We also found that both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2. There was no substantial difference in the amount of phosphorylated MAPK/ERK1/2 between 16:0 LPE and 18:0 LPE-treated cultures. MAPK inhibitor completely inhibited 18:0 LPE-induced neurite outgrowth and partially inhibited 16:0 LPE-induced neurite outgrowth. Thus, the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through the distinct signaling cascades in cultured cortical neurons.


Introduction
Neurite outgrowth is important in neuronal wiring during development and synaptic plasticity under physiological conditions, and in the regeneration of neuronal wiring following trauma and disease in the brain [1][2][3][4]. Thus, identifying the molecules that regulate neurite outgrowth and elucidating their mechanism are essential for understanding of mechanisms of neuronal wiring and brain functions, and for development of therapeutics to treat trauma or neurological disorders. To date, several extracellular ligands that regulate the neurite outgrowth of central nervous system (CNS) neurons have been identi ed.
Phospholipids are amphiphilic molecules with a hydrophilic head esteri ed by a hydrophobic fatty acid chain. They play crucial roles in various biological processes, ranging from structural integrity of cellular and organelle membranes, to intracellular signal transduction [12]. The brain is one of the most lipid-rich organs, and phospholipids especially play important roles not only in membrane organization but also in brain function [3,13,14]. Among the physiologically active phospholipids, the role of lysophospholipids in biological functions has attracted recent attention. Lysophospholipids consisted of a hydrophilic head group and one hydrophobic fatty acid chain, and many lysophospholipid molecular species exist.
Lysophosphatidylethanolamine (LPE) is one of the lysophospholipids derived from phosphatidylethanolamine (PE) via a phospholipase A-type reaction and is known to be a minor constituent of cell membranes [15,16]. As for other lysophospholipids, LPE presents many structurally different species that differ in fatty acid length and degree of unsaturation [15]. Extracellularly, LPE has been detected in human serum at levels of several hundred nanograms per milliliter [17], but its physiological role remains largely unknown. A few studies suggest the cell-type speci c roles of LPE in cultures [18][19][20][21]. Interestingly, several studies show that cognitive impairment or traumatic brain damage in rodent model is accompanied by changes in LPE revels in the brain [22][23][24]. These suggest the physiological or pathological signi cance of LPE in the brain. However, to date, the role of LPE in CNS neurons remains unclear. In this study, we demonstrate that LPEs stimulate neurite outgrowth in the cultured cortical neurons. Moreover, using several enzyme inhibitors, the structurally different LPE species, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE) were found to stimulate neurite outgrowth through distinct signaling cascades.

Egg yolk LPE increase axon and dendritic area in cultured cortical neurons
To examine the effects of phospholipids on neuronal morphology, a commercially available phospholipid kit containing 10 different phospholipids (see the Methods for detail) was applied to cultured cortical neurons prepared form mice embryos. The phospholipids bovine liver lysophosphatidylcholine (LPC), egg yolk LPE, soybean phosphatidylinositol (PI), bovine heart sphingomyelin (SM), porcine brain phosphatidylserine (PS), bovine heart phosphatidylcholine (PC), egg yolk phosphatidylethanolamine (PE), bovine heart cardiolipin (CL), phosphatidic acid (PA) prepared from egg yolk PC, and porcine brain cerebrosides (CB) were applied to a cultured medium at days in vitro (DIV) 3. At DIV14, microtubuleassociated protein 2 (MAP2)-positive dendrites and microtubule-associated protein tau (tau)-positive axons had spread all over the dish in the control cultures (Fig. 1a). When the cultured cortical neurons were incubated with 1 µM egg yolk LPE for 11 days, the tau and MAP2 signals area were signi cantly increased compared to those in the control cultures ( Fig. 1a-c). These results suggest that egg yolk LPE strongly stimulates both axonal and dendritic outgrowth in cultured cortical neurons. In cultures treated with soybean PI and bovine heart PC, MAP2 and tau signals were slightly increased compared to those in the control cultures. On the other hand, there was no substantial difference in the number of neuronal nuclear antigen (NeuN)-positive neurons between the control and the cultures treated with phospholipids, except for treatment with bovine heart CL ( Fig. 1a and d). In the bovine heart CL-treated cultures, the number of NeuN-positive neurons, the tau and MAP2 signal areas were signi cantly decreased. 16:0 LPE and 18:0 LPE increase axon and dendritic area in cultured cortical neurons Among phospholipids tested, egg yolk LPE was the most effective on the morphological change. To our knowledge, no previous study examined the role of LPE in the CNS neurons. Therefore, LPE effects were examined in this study. The LPE used was derived from egg yolk and contained two structurally different LPE species, 16:0 LPE and 18:0 LPE. To examined whether 16:0 LPE or 18:0 LPE effects the neuronal morphology, cultured cortical neurons were incubation with different concentrations of LPE, 16:0 LPE, and 18:0 LPE for 11 days. Incubation with 0.1 or 1 µM 16:0 LPE signi cantly increased the tau and MAP2 signal areas compared to those in the control cultures (Fig. 2a-c). At a higher concentration of 10 µM, 16:0 LPE did not signi cantly increased these signal areas. On the other hand, 18:0 LPE signi cantly increased the tau and MAP2 signal areas at all concentrations ( Fig. 2a-c). There was no substantial difference in the number of NeuN-positive neurons when comparing the control and the LPEs-treated cultures ( Fig. 2a and d). These results suggest that the structurally distinct LPE species 16:0 LPE and 18:0 LPE both increase axonal and dendritic area in cultured cortical neurons. 16:0 LPE and 18:0 LPE stimulate neurite outgrowth in cultured cortical neurons.
The effects of 16:0 LPE and 18:0 LPE on neurite outgrowth were examined by directly measuring the length of neurites. Either 16:0 LPE or 18:0 LPE was applied at 1 µM to the culture medium at DIV0, and the longest neurites length was measured at DIV1, 2, and 3, respectively. From DIV0 to DIV3, the lengths of neurites were gradually increased in all conditions. In cultures treated with 16:0 LPE and 18:0 LPE, the neurites were signi cantly longer than those of control cultures ( Fig. 3a and b). The number of neurites emerging from the soma was also increased in these cultures at DIV1 and 2 ( Fig. 3a and c). On the other hand, the numbers of branches for the longest neurite in 16:0 LPE-and 18:0 LPE-treated cultures were comparable to those in the control culture ( Fig. 3a and d). Several studies suggest that the one of actions of LPE is mediated by G-protein-coupled receptor (GPCR)activated G protein-coupled phospholipase C (PLC) [19,20,25,26]. Therefore, we examined whether the GPCR-PLC cascade mediates the actions of 16:0 LPE and 18:0 LPE. Cultured cortical neurons were incubated with PLC inhibitor U73122 at 0.5 µM. Application of U73122 decreased the length of 18:0 LPEstimulated neurite to almost the same length as in the inhibitor-treated control cultures; this suggests that 18:0 LPE action is mediated by GPCR coupled to PLC. In contrast, U73122 had little effect on 16:0 LPE-treated cultures ( Fig. 5a and d). It is known that activation of PLC leads to production of inositol 1,4,5-trisphosphate and diacylglycerol, the activator for PKC. To examine whether PKC is involved in LPEsstimulated neurite outgrowth, the PKC inhibitor Go6983, which inhibits PKCα, β, γ, δ, and ζ [27], was applied to the cultures. Application of 1 µM Go6983 inhibited 18:0 LPE-stimulated neurite outgrowth. In contrast, Go6983 had little effect on 16:0 LPE-treated cultures ( Fig. 5b and e). It is known that PKC family proteins consist of PKCα, β, γ, δ, ε, η, θ, ζ, Mζ, and ι/λ [28]. Therefore, a different PKC inhibitor, Sotrastaurin, which inhibits PKCα, β, δ, ε, η, and θ [29], was selected. When Sotrastaurin was applied to the cultures, both 16:0 LPE-and 18:0 LPE-stimulated neurite length changes were completely inhibited ( Fig. 5c and f). These results suggest that 16:0 LPE and 18:0 LPE activate distinct receptors and signaling cascades involved in the neurite outgrowth. 16:0 LPE and 18:0 LPE activate MAPK in cultured cortical neurons It has been reported that LPE activates mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) 1/2 in manner that depends on cell type [18,19,30,31]. However, the response of LPE in neurons remains unknown. It is known that MAPK signal cascade involved in neurite outgrowth [32]. Thus, one possibility is that 16:0 LPE and 18:0 LPE activate MAPK/ERK1/2 in the cultured cortical neurons. To examine whether this takes place, the cultured cortical neurons were incubated with 16:0 LPE or 18:0 LPE for 10 min and subjected to western blot analysis using anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. Anti-ERK1/2 antibody then detected the bands corresponding to the size of ERK1/2 in all conditions (Fig. 6a). In the absence of LPE, anti-phospho-ERK1/2 antibody detected bands corresponding to the size of phosphorylated ERK1/2, and incubation of 16:0 LPE and 18:0 LPE signi cantly increased those signals in a dose dependent manner ( Fig. 6a and b). These results suggest  Fig. 7a and b). In the control cultures at DIV3, application of U0126 signi cantly decreased the length of neurites. In cultures treated with 18:0 LPE, U0126 signi cantly decreased the length of neurite to a degree comparable to that in the U0126-treated control cultures ( Fig. 7c and d). In contrast, U0126 decreased 16:0 LPE-stimulated neurite length, although these were longer than in the control and 18:0 LPE-treated cultures. These results suggest that 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through the activation of MAPK, and that 16:0 LPE also stimulates another signal cascade that stimulates neurite outgrowth.

Discussion
Recently, the role of lysophospholipids in biological functions has attracted attention. However, in the CNS neurons, the cellular response to extracellular lysophospholipids remains largely unclear, although some phospholipids such as lysophosphatidic acid (LPA), sphingosine-1-phosphate, and 2arachidonoylglycerol have been reported to act extracellularly as an agonist on their membrane receptor and to regulate neuronal functions [34][35][36][37]. The present study demonstrates that extracellular application of 16:0 LPE and 18:0 LPE stimulates neurite outgrowth in cultured cortical neurons through a distinct signal cascade.
The effects of 16:0 LPE and 18:0 LPE in stimulating neurite outgrowth in cultured cortical neurons are clearly shown (Figs. 3 and 4). LPE is a minor constituent of cell membranes [15]. Extracellularly, LPE was detected in human serum in the range of several hundred nanograms per milliliter [17], but its physiological role remains largely unknown. A few studies have indicated the cell-type speci c roles of LPE in culture cells, as follows: The LPE extracted from Grifola frondosa, a kind of mushroom, stimulates neurite-like outgrowth of rat pheochromocytoma PC12 cells [18]. The 18:1 LPE stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells [19] and cell proliferation of MDA-MB-231 breast cancer cells [20]. Egg yolk LPE stimulates differentiation and maturation of mouse astrocyte in culture [21]. Thus, LPE evokes various response, depending on the cell type. At this time, whether LPE promotes neurite outgrowth in other types of CNS neurons remains unclear.
In cultured cortical neurons, 18:0 LPE-stimulated neurite outgrowth was inhibited by the application of PLC inhibitor U73122 (Fig. 5a and d), suggesting that the action of 18:0 LPE is mediated by GPCR-PLC cascade. In contrast, U73122 exerted little effect on 16:0 LPE-treated cultures. Therefore, a different type of receptor is likely to exist for 16:0 LPE. In addition to neurite outgrowth, 16:0 LPE and 18:0 LPE may have different physiological functions for cortical neurons. Further investigation is needed to clarify this. To date, receptors for LPE remains unidenti ed, but several studies have suggested the existence of membrane-bound receptors for different LPE species [19,20,[25][26][27]. In MDA-MB-231 cells, SH-SY5Y neuroblastoma cells, and PC12 cells, 18:1 LPE stimulates intracellular Ca 2+ concentration, and this effect is inhibited by pertussis toxin (PTX), an inhibitor of Gi/Go proteins, and antagonist of LPA receptor LPA 1 [20,[25][26][27]. On the other hand, in SK-OV3 cells, the effect of 18:1 LPE is inhibited by PTX, but not by an antagonist of LPA receptor LPA 1 or of LPA 1 and LPA 3 [19; 27]. In addition, in some cells, LPE stimulates intracellular Ca 2+ concentration, but their effect is completely dependent on the fatty acid lengths of the LPE [26,27]. For example, in PC12 cells, intracellular Ca 2+ concentration is stimulated by 18:0 LPE and 14:0 LPE, but not 16:0 LPE [26]. LPA 1 activation has been reported to induce neurite retraction in B103 neuroblastoma cells [38]. Identi cation of receptors for 16:0 LPE and 18:0 LPE will be needed to completely elucidate how LPE induces a signal cascade in the cortical neuron.
Thus far, the roles of LPE in the brain has not been fully elucidated, but several reports suggest the pathophysiological signi cance of LPE. Previous reports show that cognitive impairment or traumatic brain damage is associated with an imbalance in the composition of phospholipids in the brain, such that some studies have reported changes of LPE level in the brain. Aged triple transgenic Alzheimer's model mice harboring PS1M 146V , APP Swe , and tau P301L transgenes with cognitive impairment show increasement of LPE in the hippocampus [39,40]. Postischemic cognitive impairment rat model show increasement of LPE species, 18:1, 20:3, and 22:6 LPE, in the hippocampus [24]. Traumatic brain injury in rat transiently increases 22:6 LPE in the injury area [23]. A change in LPE was reported in the serum of a major depressive disorder patient [40]. These observations imply the importance of LPE in the brain in normal development, as well as in pathological setting. Comprehensive analysis of LPE species in the brain and investigation of their functions are necessary to understand their roles in pathophysiological conditions.

Cell cultures
Primary cortical neuron cultures were prepared from ICR mice at embryonic day 18 as previously described [41]. Brie y, the cerebral cortices were treated with phosphate-buffered saline (PBS) containing 1% trypsin (Sigma-Aldrich) and 0.1% DNase I (Sigma-Aldrich) for 5 min and dissociated by passing through a re-polished Pasteur pipette in PBS containing 0.05% DNaseI, 0.03% trypsin inhibitor (Sigma-Aldrich), and 2 mM MgCl 2 . The cells were placed on 12-diameter coverslips coated with 30 µg/ml poly-Llysine (weight 70-150 kDa; Sigma-Aldrich) and 10 µg/ml mouse laminin (Thermo Fisher Scienti c) at a density of 1.0 × 10 5 cells /well on 24-well culture plates. The cells were maintained in Neurobasal-A (Thermo Fisher Scienti c) supplemented with 2% B-27 supplement (Thermo Fisher Scienti c), 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2 mM GlutaMax-I (Thermo Fisher Scienti c) for 24 and then the medium was changed to the same medium without fetal bovine serum. All animal procedures were approved by the Animal Care and the Use Committee of Shinshu University (Approval No. 290072).

Image Acquisition And Quanti cation
Images of culture experiments were taken with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems) using HC PL APO CS2 20×/0.75 NA multiple immersion lens (Leica Microsystems) or HC PL APO CS 10×/0.40 NA multiple immersion lens (Leica Microsystems) under constant conditions in terms of laser power, pinhole size, gain, z-steps, and zoom setting throughout the experiments. All quantitative measurements were performed with ImageJ 1.52a software [42]. For the quanti cation of MAP2 and tau staining areas, z-series of optical sections were projected by the brightest point method. MAP2 and tau staining areas were de ned as area in which staining signal intensites were 2 and 3 times stronger than those of background signals on the same eld, respectively. For analysis of the time course of neurite outgrowth, z-series of optical sections were projected by the brightest point method. Neurite of cortical neurons was identifed by Tuj1 signals, and the length of the longest neurite, the number of neurites emerging from the soma, and the numbers of branches per longest neurite were measured.

Western Blot Analysis
Western blot analysis Primary cortical neuron cultures were prepared as described above and plated on the 24-well dish at a density of 5.0 × 10 5 cells /well. One to 100 µM 16:0 LPE or 18:0 LPE was applied to the culture medium at DIV7. After 10 min incubation, the cultures were solubilized with SDS sample buffer containing 2% SDS, 50 mM-Tris-HCI, pH6.8, 10% glycerol, 100 mM Dithiothreitol, phosphatase inhibitor cocktail (Nakarai Tesque), and 0.025% Bromophenol Blue, followed by boiling at 95℃ for 10 min. For inhibition of MAPK, 5 µM MEK/ERK inhibitor U0126 was applied to the cell culture medium 30 min before treatment with LPE.
The cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking with PVDF Blocking Reagent for Can Get Signal (TOYOBO), the membranes were incubated with mouse anti-phospho-MAPK/ERK1/2 (Thr202/Tyr204) antibody (E10, 1:2000; Cell Signaling Technology), followed by incubation with horseradish-peroxidase-conjugated secondary antibody. After stripping, the membranes were probed with rabbit anti p44/42 MAPK antibody (1:1000; Cell Signaling Technology), followed by incubation with horseradish-peroxidase-conjugated secondary antibody. Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) was used for dilution of antibodies. The proteins were visualized by ECL Select Western Blotting Detection System (GE Healthcare) and detected by Las-4000 mini luminescent imaging analyzer (GE Healthcare). The quanti cation analysis was performed using ImageQuant TL image analysis software (GE Healthcare).

Statistical analysis
Results of at least 2 independent experiments were subjected to statistical analyses. No statistical method was used to determine sample size. No data were excluded. There was no randomization of samples before analysis. Statistical signi cance was evaluated with two-way or one-way ANOVA followed by Tukey's, Dunnett's post hoc test, or Student's t test, or Kruskal-Wallis test followed by posthoc Steel-Dwass or Steel's test using R software (R Core Team, 2017). Statistical signi cance was assumed when p < 0.05.  Effects of phospholipids on neuronal morphology. a Extracellular application of LPE, PI, or PC increased the MAP2 and tau staining signals in the cultured cortical neurons. The cultures were incubated with 1 μM of indicated phospholipids for 11 days, and immunostained with antibodies against MAP2, tau, and NeuN at DIV14, respectively. Scale bar represents 50 μm. abovine liver LPC; begg yolk LPE; csoybean PI; dbovine heart SM; eporcine brain PS; fbovine liver PC; gegg yolk PE; hbovine heart CL; iPA form egg yolk PC; jporcine brain CB. b Quanti cation of tau signal area in (a). The percentage of tau signal occupied in in the observation eld was measured. c Quanti cation of MAP2 signal area in (a). The percentage of tau signal occupied in in the observation eld was measured. d Quanti cation of number of NeuN positive neurons in (a). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers are 1.5× IQR. Statistical signi cance was evaluated with the Kruskal-Wallis test followed by post hoc Steel test: ** p < 0.01 and * p < 0.05, n = 10.    Western blot analysis using anti-phospo-ERK1/2 and anti-ERK1/2 antibodies. b Quanti cation of phosphorylated ERK1/2 signals in (a). Intensities of phosphorylated ERK1/2 signals were normalized with those of ERK1/2 signals. Red-lled squares and blue-lled triangles represent 16:0 LPE-treatment and 18:0 LPE-treatment cultures, respectively. All values represent mean ± s.e.m. Statistical signi cance was evaluated using two-way ANOVA followed by post hoc Student's t test. n = 3.