Structurally different lysophosphatidylethanolamine species stimulate neurite outgrowth in cultured cortical neurons via distinct G-protein-coupled receptors and signaling cascades

Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identi�cation 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 structurally different lysophosphatidylethanolamine (LPE) species, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Interestingly, YM ‐ 254890, an inhibitor of Gq/11 protein, inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, pertussis toxin, an inhibitor of Gi/Go proteins, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C inhibitors on neurite outgrowth were also different. In addition, both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2, but 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 distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.


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].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 [4].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 [5,6].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 [7,8].As for other lysophospholipids, LPE presents many structurally different species that differ in fatty acid length and degree of unsaturation [7].Extracellularly, LPE has been detected in human serum at levels of several hundred nanograms per milliliter, but its physiological role remains largely unknown [8].A few studies suggest the cell-type speci c roles of LPE in cultures [9][10][11][12].Interestingly, several studies show that cognitive impairment or traumatic brain damage in rodent model is accompanied by changes in LPE revels in the brain [13][14][15], suggesting the physiological or pathological signi cance of LPE.However, to date, the role of LPE in CNS neurons remains unclear.In this study, we demonstrate that both palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE) stimulate neurite outgrowth in the cultured cortical neurons.Moreover, using several G-proteins and enzyme inhibitors, we found that distinct G-protein-coupled receptors (GPCRs) and signaling cascades are involved in 16:0 LPE and 18:0 LPE-induced neurite outgrowth.

Cell cultures
Primary cortical neuron cultures were prepared from ICR mice at embryonic day 18 as described previously [16].The cells were placed on 12-diameter coverslips coated with poly-L-lysine and laminin at a density of 1.0 × 10 5 cells /well on 24-well culture plates.For western blot analysis, primary cortical neuron cultures were plated on the 24-well dish at a density of 5.0 × 10 5 cells /well.All animal procedures were approved by the Animal Care and the Use Committee of Shinshu University (Approval No. 290072).

Morphological analysis
Phospholipid kit including bovine liver lysophosphatidylcholine (LPC), egg yolk LPE, soybean phosphatidylinositol (PI), bovine heart sphingomyelin (SM), porcine brain phosphatidylserine (PS), bovine liver phosphatidylcholine (PC), egg yolk phosphatidylethanolamine (PE), bovine heart cardiolipin (CL), phosphatidic acid (PA), and porcine brain cerebrosides (CB) were purchased from Olbracht Serdary Research Laboratories.At DIV3, each phospholipid was applied to the medium.The cells were xed at DIV14 and immunostained with antibodies.For inhibitor experiments, inhibitors were applied to the culture medium at DIV0, and xed at DIV3.Detail information is shown in Supplementary materials and methods.

Statistical analysis
Results of at least 2 independent experiments were subjected to statistical analyses.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 post-hoc Steel-Dwass or Steel's test using R software (R Core Team, 2017).Statistical signi cance was assumed when p < 0.05.

Egg yolk LPE increases 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 was applied to cultured cortical neurons.In the cultured cortical neurons, microtubule-associated protein 2 (MAP2)-positive dendrites and microtubule-associated protein tau (tau)-positive axons had spread all over the dish (Fig. 1A).When the cultured cortical neurons were incubated with 1 μM egg yolk LPE for 11days, 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.
3.2 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 egg yolk 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. 1E-G).At a higher concentration of 10 μM, 16:0 LPE did not signi cantly increased these signal areas.On the other hand, egg yolk LPE and 18:0 LPE signi cantly increased the tau and MAP2 signal areas at all concentrations (Fig. 1E-G).There was no substantial difference in the number of NeuN-positive neurons when comparing the control and the LPEstreated cultures (Fig. 1E and H).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.From DIV0 to DIV3, the lengths of the longest neurites were gradually increased in all conditions.In cultures treated with 1 μM 16:0 LPE and 18:0 LPE, the neurites were signi cantly longer than those of control cultures (Fig. 2A and C).The number of neurites emerging from the soma was also increased in these cultures at DIV1 and 2 (Fig. 2A and D).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. 2A and E).In terms of morphologies examined, there was no substantial difference between the 16:0 LPE-and 18:0 LPE-treated cultures.The effects of different concentrations of 16:0 LPE and 18:0 LPE were also examined in terms of neurite outgrowth.18:0 LPE dose-dependently increased the length of neurites as reported previously (Fig. 2B and F).At a concentration of 10 μM, both LPEs increased the neurite length to approximately twice as long as that in the control cultures.There was no signi cant difference between 16:0 LPE and 18:0 LPE in the lengths at any concentration.These results suggest that 16:0 LPE and 18:0 LPE stimulate neurite outgrowth in cultured cortical neurons and have similar effects on neuronal morphology.
3.5.Differential effects of MAPK inhibitor on 16:0 LPE-and 18:0 LPE-stimulated neurite outgrowth The nding of activation of MAPK/ERK1/2 in 16:0 LPE-and 18:0 LPE-treated cortical cultures rises the possibility that these LPEs stimulate neurite outgrowth through the activation of MAPK/ERK1/2.To examine this, the cultured cortical neurons were treated with MAPK inhibitor U0126 at 5 μM in the presence or absence of 16:0 LPE or 18:0 LPE for 3 days.In the control cultures at DIV3, application of 5 μM 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. 3E and F).In contrast, U0126 decreased 16:0 LPE-stimulated neurite length, although these were longer than in the control U0126-treated, and 18:0 LPE and U0126-treated cultures.These results suggest that 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through the activation of MAPK.In addition, the difference effect of MAPK inhibitor on 16:0 LPE-and 18:0 LPE-stimulated neurite outgrowth raises the possibility that these LPEs activate distinct membrane receptors and signaling cascades.

Discussion
Recently, the role of lysophospholipids in biological functions has attracted attention.However, in the CNS neurons, the cellular response to extracellular LPE remains unknown.In this study, we found that extracellular application of 16:0 LPE and 18:0 LPE stimulates neurite outgrowth in cultured cortical neurons and that distinct GPCRs and signaling cascades are involved in these processes.
The effects of 16:0 LPE and 18:0 LPE in stimulating neurite outgrowth in cultured cortical neurons are clearly shown (Fig. 2).To date, a few studies have indicated the cell-type speci c roles of LPE in culture cells, but physiological role remains largely unknown.The LPE extracted from Grifola frondosa, a kind of mushroom, stimulates neurite-like outgrowth of rat pheochromocytoma PC12 cells [9].The 18:1 LPE stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells [10] and cell proliferation of human MDA-MB-231 breast cancer cells [11].Egg yolk LPE stimulates differentiation and maturation of mouse astrocyte in culture [12].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.
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 PS1M146V, APPswe, and tau P301L transgenes with cognitive impairment show increasement of LPE in the hippocampus [13].Postischemic cognitive impairment rat model show increasement of LPE species, 18:1, 20:3, and 22:6 LPE, in the hippocampus [15].Traumatic brain injury in rat transiently increases 22:6 LPE in the injury area [14].A change in LPE was reported in the plasma of a major depressive disorder patient [27].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.

Declarations Figures
of phospholipids on neuronal morphology.(A) Extracellular application of egg yolk LPE, soybean PI, or bovine liver PC increased the MAP2 and tau staining signals in the cultured cortical neurons.tau (green), MAP2 (red), and NeuN (magenta) signals are merged on the bottom.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, C) Quanti cation of tau (B) and MAP2 (C) signals area in (A).The percentage of tau and MAP2 signals occupied in in the observation eld were measured.(D) Quanti cation of number of NeuN positive neurons in (A).(E) Extracellular application of 16:0 LPE and 18:0 LPE increased the MAP2 and tau staining signals.tau (green), MAP2 (red), and NeuN (magenta) signals are merged on the bottom.(F, G) Quanti cation of tau (F) and MAP2 (G) signals area in (E).The percentage of tau and MAP2 signals occupied in in the observation eld were measured.(H) Quanti cation of number of NeuN positive neurons in (E).Scale bars represent 50 μm.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 in (B-D) or Steel-Dwass test in (F-H): ** p < 0.01 and * p < 0.05, n = 10 areas.

Figure 2 Effects
Figure 2