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, microtubule-associated 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 significantly 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 significantly 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 significantly 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 significantly increased these signal areas. On the other hand, 18:0 LPE significantly 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 significantly 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). 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. The cultured cortical neurons were incubated with 0.5 µM to 10 µM 16:0 LPE or 18:0 LPE, and the length of the longest neurites was measured at DIV3. Both 16:0 LPE and 18:0 LPE dose-dependently increased the length of neurites (Fig. 4a and b). 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 significant 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.
Differential effects of PLC and PKC inhibitors on 16:0 LPE and 18:0 LPE-stimulated neurite outgrowth
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 LPE-stimulated 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-tri-sphosphate and diacylglycerol, the activator for PKC. To examine whether PKC is involved in LPEs-stimulated 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 signal-regulated 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 significantly increased those signals in a dose dependent manner (Fig. 6a and b). These results suggest that both 16:0 LPE and 18:0 LPE activate MAKP/ERK1/2 in cultured cortical neurons.
Differential effects of MAPK inhibitor on 16:0 LPE- and 18:0 LPE-stimulated neurite outgrowth
The finding 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. This concentration of U0126 completely inhibited activation of MAPK/ERK1/2 (Fig. 7a and b). In the control cultures at DIV3, application of U0126 significantly decreased the length of neurites. In cultures treated with 18:0 LPE, U0126 significantly 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.