2022 Volume 45 Issue 5 Pages 643-648
Plasmalogens are a group of glycerophospholipids containing a vinyl-ether bond at the sn-1 position in the glycerol backbone. Cellular membrane plasmalogens are considered to have important roles in homeostasis as endogenous antioxidants, differentiation, and intracellular signal transduction pathways including neural transmission. Therefore, reduced levels of plasmalogens have been suggested to be associated with neurodegenerative diseases such as Alzheimer’s disease. Interestingly, although arachidonic acid is considered to be involved in learning and memory, it could be liberated and excessively activate neuronal activity to the excitotoxic levels seen in Alzheimer’s disease patients. Here, we examined the protective effects of several kinds of plasmalogens against cellular toxicity caused by arachidonic acid in human neuroblastoma SH-SY5Y cells. As a result, only phosphatidylcholine-plasmalogen-oleic acid (PC-PLS-18) showed protective effects against arachidonic acid-induced cytotoxicity based on the results of lactate dehydrogenase release and ATP depletion assays, as well as cellular morphological changes in SH-SY5Y cells. These results indicate that PC-PLS-18 protects against arachidonic acid-induced cytotoxicity, possibly via improving the stability of the cellular membrane in SH-SY5Y cells.
Plasmalogens are a group of glycerophospholipids containing a vinyl-ether bond at the sn-1 position in the glycerol backbone. The sn-3 position of plasmalogens often binds to phosphatidylcholine (PC) or phosphatidylethanolamine (PE) and the sn-2 position usually binds to poly-unsaturated fatty acid, such as arachidonic acid or oleic acid, etc., by an ester bond. It has been reported that the cell membrane contains approximately 10 to 20 mol% of plasmalogens out of the total phospholipids of all mammalians including humans.1–3) Membrane plasmalogens are considered to have important roles in homeostasis as endogenous antioxidants, differentiation, and intracellular signal transduction pathways including neural transmission.1–3) Indeed, reduced levels of plasmalogens have been suggested to be associated with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, although the mechanisms remain unclear.3) Therefore, if plasmalogens are appropriately provided, they would be good candidates for ameliorating these diseases.
In the brain, essential fatty acids such as arachidonic acid and docosahexaenoic acid (DHA) are incorporated and enriched in phospholipids, which are known to play important roles in normal neuronal functions.4) Free arachidonic acid in the brain is known to increase neuronal firing and long-term potentiation so that arachidonic acid is considered to be involved in learning and memory.4) However, arachidonic acid could be liberated and excessively activate neuronal activity to excitotoxic levels, e.g., by elevation of amyloid β, a well-known biomarker peptide in Alzheimer’s disease.4) Arachidonic acid also exhibits cytotoxic effects in most cells at concentrations of 50–100 µM.5)
As described above, the arachidonic acid-induced excitotoxic activity in Alzheimer’s disease could be ameliorated by treatment with plasmalogens. Here, we examined the protective effects of several kind of plasmalogens on cellular toxicity caused by arachidonic acid in human neuroblastoma SH-SY5Y cells.
SH-SY5Y human neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/Ham’s F-12; Invitrogen, Carlsbad, CA, U.S.A.) containing 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, U.S.A.), 100 µg/mL streptomycin, and 100 IU/mL penicillin (Meiji Seika, Japan) at 37 °C. Unless otherwise stated, all materials were obtained from Wako Pure Chemical Corporation (Osaka, Japan).
Lactate Dehydrogenase (LDH) AssaySH-SY5Y cells were cultured in 96-well plates and, prior to the experiments, mediums were switched to Opti-MEM (Thermo Scientific) for 24 h at 37 °C containing antibiotics as stated above. Cells were pretreated with either vehicle (ethanol), 1 µg/mL of plasmalogens, or corresponding glycerophospholipids for 24 h at 37 °C. Then, cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. The cytotoxicity of the arachidonic acid-treated cells was determined using the Cytotoxicity LDH assay kit (Dojindo Laboratories, Kumamoto, Japan), measuring LDH activity released from damaged cells. The cytotoxicity was determined by measuring the amount of formazan dye, which is proportional to that of LDH released into the medium. Briefly, 50 µL of the supernatant of each 96-well was used, prepared, and determined as the test substance in culture medium by measuring absorbance at 490 nm according to the manufacturer’s instructions with TECAN infinite M200 (TECAN, Mannedorf, Switzerland), along with measuring high control, low control, and background control.
ATP AssaySH-SY5Y cells were cultured in 96-well plates and, prior to the experiments, mediums were switched to Opti-MEM for 24 h at 37 °C containing antibiotics as stated above. Cells were pretreated with either vehicle (ethanol), 1 µg/mL of plasmalogens, or corresponding glycerophospholipids for 24 h at 37 °C. Then, cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. Cell viability was determined by measuring amounts of ATP using ATPlite (PerkinElmer, Inc., Waltham, MA, U.S.A.), an ATP-monitoring system based on firefly luciferase. Briefly, we added 50 µL of lysis buffer to each well and shook for 5 min at room temperature. Then, we added 50 µL of ATP substrate buffer to each well and shook for another 5 min at room temperature. A total of 160 µL of each sample obtained from each well was then transferred to the new 96-well plate and we measured luciferase activity according to the manufacturer’s instructions with TECAN infinite M200 (TECAN).
Phase Contrast Microscopy ImagingSH-SY5Y cells were cultured in 6-well plates and, prior to the experiments, mediums were switched to Opti-MEM for 24 h at 37 °C containing antibiotics as stated above. Cells were pretreated with either vehicle (ethanol), 1 µg/mL of plasmalogens, or corresponding glycerophospholipids for 24 h at 37 °C. Then, cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. Cells were fixed with 500 µL of cold methanol for 5 min and washed with phosphate buffered saline (PBS). Cells were stained with Mayer’s Hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 10 min and then stained with 1% eosin in water solution (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 10 min. The pictures of the cells were obtained using a Nikon eclipse TS100 microscope (Nikon, Tokyo, Japan).
Statistical AnalysisData are expressed as the mean ± standard error of the mean (S.E.M.), and statistical analysis was performed using Prism 5 for Mac OS X software (GraphPad Software, La Jolla, CA, U.S.A.). Unpaired t-tests and Multiple comparison tests in ANOVA when the control value was 100% were used to evaluate three or more independent experiments.
To evaluate the neuroprotective effects of plasmalogens, 3 species of PC-plasmalogens (PC-PLS) and 3 corresponding species of PC-diacylglycerophospholipids (PC-DPL), as well as 3 species of PE-plasmalogens (PE-PLS) and 3 corresponding species of PE-diacylglycerophospholipids (PE-DPL) (Fig. 1A) were pretreated before arachidonic acid-induced cytotoxicity in human SH-SY5Y neuroblastoma cells. Note that we have used undifferentiated SH-SY5Y cells since retinoic acid-differentiated SH-SY5Y cells are reported to show the higher tolerance by up-regulating survival signaling; the study concluded that undifferentiated SH-SY5Y cells are appropriate for evaluating neurotoxicity and/or neuroprotection.6)
(A) The basic structures of diacylglycerophospholipid (DPL) or plasmalogen (PLS), with ester-bonded (DPL) or vinyl-ether bonded (PLS) stearic acid (C18:0) at the sn-1 position. The sn-2 position (at position R) of DPL or PLS was ester-bonded with oleic acid (C18:1), arachidonic acid (C20:4), or DHA (C22:6). The sn-3 position (at position X) of DPL or PLS was bonded with phosphatidylcholine (PC) or phosphatidylethanolamine (PE). (B) The structure of PC-PLS-18; stearic acid at the sn-1 position, oleic acid at the sn-2, and PC at sn-3 are bonded to PLS.
PC-PLS showed choline at the sn-3 position, with vinyl-ether-bonded stearic acid (C18:0) at the sn-1 position, and were ester-bonded at the sn-2 position with oleic acid (C18:1, PC-PLS-18) (Fig. 1B), arachidonic acid (C20:4, PC-PLS-20), or DHA (C22:6, PC-PLS-22). PC-DPL showed with choline at the sn-3 position, with ester-bonded stearic acid at the sn-1 position, and were ester-bonded at the sn-2 position with oleic acid (PC-DPL-18), arachidonic acid (PC-DPL-20), or DHA (PC-DPL-22). The evaluated ethanolamine-plasmalogens showed ethanolamine at the sn-3 position, with vinyl-ether bonded stearic acid at the sn-1 position, and were ester-bonded at the sn-2 position with oleic acid (PE-PLS-18), arachidonic acid (PE-PLS-20), or DHA (PE-PLS-22). The corresponding ethanolamine-glycerophospholipids showed ethanolamine at the sn-3 position, with ester-bonded stearic acid at the sn-1 position, and were ester-bonded at the sn-2 position with oleic acid (PE-DPL-18), arachidonic acid (PE-DPL-20), or DHA (PE-DPL-22). For the experiments, SH-SY5Y cells were treated with 1 µg/mL of each choline-plasmalogen or choline-glycerophospholipid for 24 h, and were then treated with 10 µg/mL of arachidonic acid for 6 h. The protective effects were evaluated by LDH assay, as described in Materials and Methods.
Treatment with arachidonic acid alone for 6 h of SH-SY5Y cells had cytotoxic effects, which were measured and estimated by released LDH (% of control, set as 100%) (Fig. 2A). In the case of arachidonic acid-untreated but vehicle-treated SH-SY5Y cells, leakage of LDH was around 26% (Fig. 2A). Therefore, treatment with arachidonic acid alone of the cells caused significant cytotoxic effects compared with the vehicle-treated control. As shown in Fig. 2B, although it was not significant, pretreatment with PC-DPL-18 for 24 h slightly increased LDH release to approximately 120% when compared with the control. However, pretreatment with PC-PLS-18 for 24 h significantly reduced arachidonic acid-induced LDH release to approximately 80% when compared with the control and/or PC-DPL-18-pretreated cells. Similar to pretreatment with PC-DPL-18, when cells were pretreated with PC-DPL-20 (Fig. 2C) and/or PC-DPL-22 (Fig. 2D), there was a slight increase in LDH release to approximately 120% when compared with the control. Interestingly, in the case of other plasmalogens examined, LDH release levels were similar to those of the control, so that reduced LDH release was not observed when cells were pretreated with PC-PLS-20 (Fig. 2C) and/or PC-PLS-22 (Fig. 2D) for 24 h. On the other hand, however, no pretreatment with any kind of PE-PLS or PE-DPL for 24 h altered the arachidonic acid-treated increased release of LDH when compared with the arachidonic acid-alone treated control cells (Figs. 2E–G).
(A) SH-SY5Y cells were treated either with vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. (B–G) Cells were pretreated with either vehicle (ethanol), or 1 µg/mL of plasmalogens, or corresponding glycerophospholipids for 24 h at 37 °C; (B) pretreated with PC-DPL-18 or PC-PLS-18 (C18:1 oleic acid), (C) with PC-DPL-20 or PC-PLS-20 (C20:4 arachidonic acid), (D) with PC-DPL-22 or PC-PLS-22 (C22:6 DHA), (E) with PE-DPL-18 or PE-PLS-18 (C18:1 oleic acid), (F) with PE-DPL-20 or PE-PLS-20 (C20:4 arachidonic acid), (G) with PE-DPL-22 or PE-PLS-22 (C22:6 DHA), for 24 h, and then cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. The arachidonic acid-induced LDH release was then measured and normalized as 100% (cont (AA)). Data are shown as the mean ± S.E.M. * p < 0.05, unpaired t-test (A), or † p<0.05, multiple comparison tests in ANOVA (B–G), a control value of 100% was used to evaluate three or more independent experiments.
To confirm that the reduced effects of PC-PLS-18 on arachidonic acid-induced cytotoxicity were due to increased cellular viability, the amounts of its marker, ATP, were then estimated by ATP assay. The amounts of cellular ATP will rapidly decline when cells undergo apoptosis and/or necrosis. We evaluated the neuroprotective effects of PC-PLS-18, PC-PLS-20, or PC-PLS-22, and corresponding PC-DPL-18, PC-DPL-20, or PC-DPL-22 pretreatment before arachidonic acid-induced cytotoxicity in human SH-SY5Y neuroblastoma cells. The amounts of ATP from cellular lysate prepared from arachidonic acid-treated SH-SY5Y cells were plotted as 100% (Fig. 3A). In the case of arachidonic acid-untreated but vehicle-treated SH-SY5Y cells, the amount of ATP from cellular lysate was around 370% (Fig. 3A). Thus, treatment with arachidonic acid alone for 6 h of SH-SY5Y cells caused cytotoxic effects, so that the amounts of ATP were significantly reduced to approximately one-fourth (370 to 100%). Pretreatment with PC-DPL-18 for 24 h did not alter the arachidonic acid-treated decrease in amounts of ATP when compared with the arachidonic acid alone-treated control cells (Fig. 3B). Pretreatment with PC-PLS-18 for 24 h significantly inhibited the arachidonic acid-induced reduction in ATP to less than half (370 to 150%), when compared with the control and/or PC-DPL-18-pretreated cells (Fig. 3B). When cells were pretreated with PC-DPL-20 (Fig. 3C) and/or PC-DPL-22 (Fig. 3D), there was a slight further reduction in the ATP to approximately 20% (100 to 80%). In the case of pretreatment with PC-PLS-20 (Fig. 3C) and/or PC-PLS-22 (Fig. 3D), the amounts of ATP were similar or even a little less than those of the control cells. As similarly shown in Figs. 2E to G, SH-SY5Y cells were treated with 1 µg/mL of PE-PLS or PE-DPL for 24 h, and then treated with 10 µg/mL of arachidonic acid for 6 h. Then, the protective effects were also evaluated by ATP assay. However, again, no pretreatment with any kind of PE-PLS or PE-DPL for 24 h altered the arachidonic acid-treated decreased amounts of ATP when compared with the arachidonic acid alone-treated control cells (Figs. 3E–G). These results strongly indicate that only PC-PLS-18 may have an ability to protect against cellular cytotoxicity induced by arachidonic acid among the examined PC-PLS and PC-DPL, in terms of the LDH assay as well as ATP assay, as shown in Figs. 2 and 3, respectively.
(A) SH-SY5Y cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. (B–G) Cells underwent pretreatment with either vehicle (ethanol), or 1 µg/mL of plasmalogens, or corresponding glycerophospholipids for 24 h at 37 °C; (B) pretreated with PC-DPL-18 or PC-PLS-18 (C18:1 oleic acid), (C) with PC-DPL-20 or PC-PLS-20 (C20:4 arachidonic acid), (D) with PC-DPL-22 or PC-PLS-22 (C22:6 DHA), (E) with PE-DPL-18 or PE-PLS-18 (C18:1 oleic acid), (F) with PE-DPL-20 or PE-PLS-20 (C20:4 arachidonic acid), (G) with PE-DPL-22 or PE-PLS-22 (C22:6 DHA), for 24 h, and then cells were treated with either vehicle (ethanol) or 10 µg/mL arachidonic acid for 6 h at 37 °C. The cellular ATP amounts were measured, and the arachidonic acid-induced ATP content in SH-SY5Y cells was normalized as 100% (cont (AA)). Data are shown as the mean ± S.E.M. * p < 0.05, unpaired t-test (A), or † p < 0.05, multiple comparison tests in ANOVA (B–G), a control value of 100% was used to evaluate three or more independent experiments.
As shown in Figs. 2 and 3, among the glycerophospholipids tested, only PC-PLS-18 showed protective effects against arachidonic acid-induced cytotoxicity in SH-SY5Y cells. As described above, LDH is known to be released from damaged cells and cellular ATP amounts are considered to be rapidly reduced when cells undergo necrosis and/or apoptosis. Thus, the cytoprotective effect of PC-PLS-18 on cellular morphology was then examined under the influence of arachidonic acid treatment in SH-SY5Y cells. As shown in Fig. 4, treatment with arachidonic acid for 6 h induced cellular morphological changes, such as shrinkage and/or destruction of the cells (panel b) when compared with vehicle-treated control cells (panel a). These arachidonic acid-treated cell shape changes were not prevented when PC-DPL-18 (panel c) and/or PE-DPL-18 (panel e) was applied for pretreatment before treatment with arachidonic acid. When cells were pretreated with PC-PLS-18 (panel d), but not PE-PLS-18 (panel f), large numbers of cells evaded arachidonic acid-induced cellular shape changes and retained their morphology, to a certain degree, similar to arachidonic acid-untreated control cells. Thus, the protective effects of PC-PLS-18 against arachidonic acid-induced cytotoxicity are possibly due to the preventive effect of this plasmalogen on cellular morphological changes to some extent.
(A) SH-SY5Y cells were treated either with vehicle (ethanol, panel a) or 10 µg/mL arachidonic acids (panel b) for 6 h at 37 °C. (B) Cells were treated with 10 µg/mL arachidonic acid for 6 h following pretreatment with 1 µg/mL of plasmalogens or corresponding glycerophospholipids for 24 h at 37 °C; pretreated with PC-DPL-18 (panel c) or PC-PLS-18 (panel d) (C18:1 oleic acid), with PE-DPL-18 (panel e) or PE-PLS-18 (panel f) (C18:1 oleic acid). Cells were stained with hematoxylin and eosin, and then images were captured. These images are representative of three or more independent experiments.
Plasmalogens are generally considered to be endogenous antioxidants that protect other phospholipids from oxidative stresses.7) As described in Introduction, the 6 plasmalogens tested in this study are not special, but common and/or general types.1–3) However, among them, only PC-PLS-18 has shown protective effects against arachidonic acid-induced cytotoxicity in SH-SY5Y cells. Therefore, according to the results obtained in this study, the protective effects of PC-PLS-18 against arachidonic acid-induced cytotoxicity may not solely result from its general antioxidant activity.
PC-PLS-18 but Not PC-PLS-20 or PC-PLS-22Although its mechanism is currently unknown, the only difference among the three PC-PLS tested is the poly-unsaturated fatty acid at position sn-2. We demonstrated that the addition of C18 oleic acid reduced the cytotoxicity of PC-PLS induced by exogenous arachidonic acid, but the addition of C20 arachidonic acid or C22 DHA did not. Arachidonic acid is a well-known precursor of numerous mediators such as prostanoids and leukotriens; the eicosanoids, including both pro- and anti-inflammatory mediators.8) However, as described in Introduction, arachidonic acid is known to show cytotoxic effects in most cells at high concentrations,5) and is also known to be liberated and excessively activate neuronal activity to excitotoxic levels, e.g., by elevation of amyloid b, a well-known biomarker peptide in Alzheimer’s disease.4)
Instead of arachidonic acid, DHA is widely known to have protective effects on neuronal cells in the presence of amyloid β-peptide- and/or methylmercury-induced neurotoxicity.4) On the other hand, DHA has also been reported to have cytotoxic effects on cells, such as neuroblastoma cell lines including SH-SY5Y cells9); therefore, the effects of DHA are considered to differ among cell types and/or disease states.4,9) Although the neuroprotective effects of DHA are mainly due to it promoting cell resistance to exogenous cytotoxic stimuli, the cytotoxic effects of DHA itself remain unclear. While oleic acid has been reported to be able to induce apoptosis and/or necrosis,10) or mild central nervous system depression, oleic acid is widely considered and/or reported to be practically non-toxic.5,11) Again, the only difference among the three PC-PLS tested is the poly-unsaturated fatty acid at the sn-2 position; C18 may be less toxic than C20 and C22. One possible explanation why only PC-PLS-18 showed protective effects on cells is that exogenous arachidonic acid may be replaced by PC-PLS by e.g., the activity of phospholipase A2 (PLA2); possibly the plasmalogen-selective membrane-associated Ca2+-independent PLA2.12–14) Thus, among the fatty acids cleaved by PLA2 from tested PC-PLS, C18 oleic acid may be less toxic than exogenous arachidonic acid.
PC-PLS-18 but Not PE-PLS-18It is well-recognized that the cellular membrane shows polarity: positively charged outside, and negatively charged on the cytosolic side of the bilayer membrane. Since PC-glycerophospholipid is known to have a positive charge and PE-glycerophospholipid has a negative charge, PC-lipid tends to locate outside of the cellular membrane while PE-lipid locates on the cytosolic side.15)
Asymmetrical distributions of plasmalogens are also known at the mitochondrial membranes. Thus, PE-PLSs are reported to be the highest tendency for the stabilization in an inner leaflet of the cristae membrane ultrastructure.16) Thus, certain amounts of exogenously added PE-PLS-18 may re-locate their distribution from cell surface membrane to mitochondrial membrane. Although the detailed functions of PC-PLSs in mitochondria membrane are not well known, PC-PLSs including PC-PLS-18 are reported to be presented at very low abundance in mitochondria,17) so that most of PC-PLSs may be distributed at the cell surface membrane rather than mitochondrial membrane.
Thus, one possible reason why PC-PLS-18, but not PE-PLS-18 showed a protective effect against arachidonic acid-induced cytotoxicity is the differential localization of both PLS. Thus, exogenous arachidonic acid would have effect outside rather than inside and/or mitochondrial membrane; hence, PC-PLS-18 showed more protective effects than PE-PLS-18.
PC-PLS-18 but Not PC-DPL-18The only difference between PC-PLS-18 and PC-DPL-18 is the bonding form at the sn-1 position: a vinyl-ether bond in PC-PLS-18 and an ester bond in PC-DPL-18. Thus, PC-PLS-18 can release oleic acid from the sn-2 position by the action of PLA2, while PC-DPL-18 can also release stearic acid from the sn-1 position by the action of PLA1 in addition to oleic acid at the sn-2 position. Therefore, if exogenous arachidonic acid could activate PLAs, including PLA1 and PLA2, it is possible that PC-DPL-18 would be cleaved by both stearic acid and oleic acid so that PC-DPL-18 incorporated in the cellular membrane would be more vulnerable than a PC-PLS-18-incorporated membrane. This could be one possible reason why PC-PLS-18 showed protective effects when compared with PC-DPL-18.
PC-PLS-18, but Not Other Phospholipids Tested, Showed Protective Effects against Cellular Shape ChangeAs shown in Fig. 4, the cytotoxic effects of arachidonic acid were accompanied by cellular morphological change. It is possible to speculate that the factor causing the cellular shape change is LDH release and/or depletion of cellular ATP induced by arachidonic acid. However, the release of LDH and depletion of ATP from cells were also likely to be the results of cellular shape changes following membrane collapse caused by exogenous arachidonic acid treatment. In either case, PC-PLS-18 may protect against arachidonic acid-induced cytotoxicity via improving the stability of the cellular membrane. Unfortunately, the reason why only PC-PLS-18 showed cytoprotective effects and the molecular target(s) of PC-PLS-18 are currently unknown, which should be investigated by verifying the points discussed above in the future.
This research was supported in part by Marudai Food Co., Ltd. We would like to thank all the lab members, old, current, and new, of Department of Pharmacology for Life Sciences for kind suggestions and discussions.
NY, HK, HO and MY carried out LDH, ATP assays and Microscopy. JK, SK and KF participated in coordination of the experiments and draft manuscript writing. HF conceived, coordinated, designed and analyzed the experiments as well as manuscript writing.
The authors declare no conflict of interest.
