2024 Volume 47 Issue 1 Pages 192-195
Plasmalogens are a family of glycerophospholipids containing one vinyl-ether bond at the sn-1 position in the glycerol backbone, and play important roles in cellular homeostasis including neural transmission. Therefore, reductions of plasmalogens have been associated with neurodegenerative disorders, such as Alzheimer’s disease (AD). To evaluate the potential protective effects of plasmalogens against the pathology of AD, protein expression levels of key factors in amyloid precursor protein (APP) metabolic processes were examined using human neuroblastoma SH-SY5Y cells. Here, phosphatidylcholine-plasmalogen-oleic acid (PC-PLS-18) was shown to reduce protein expression levels of β-site APP cleaving enzyme 1 (BACE1), clusterin, and Tau, factors involved in the amyloid β-associated pathogenesis of AD. Thus, PC-PLS-18 may have preventive effects against AD by delaying the onset risk for a certain period.
One family of glycerophospholipids containing a vinyl-ether bond at the sn-1 position in the backbone is classified as plasmalogens.1–3) Approximately 10 to 20 mol/% of plasmalogens are incorporated into the cellular membrane, and play important roles in cellular homeostasis including neural transmission.1–3) For example, reductions of plasmalogens have been associated with neurodegenerative disorder such as Alzheimer’s disease (AD), possibly via reducing amyloid β (Aβ) accumulation.1–3) Among the many species of plasmalogens, phosphatidylcholine (PC)-plasmalogen-oleic acid (PC-PLS-18) has been reported to have protective effects against arachidonic acid-induced cytotoxicity in SH-SY5Y cells.4) Indeed, arachidonic acid could induce elevation of Aβ via excessive activation of neuronal activity.5) Therefore, to evaluate the potential protective effects of PC-PLS-18 against the pathology of AD, protein expression levels of key factors of amyloid precursor protein (APP) metabolic processes potentially involved in the onset risk of AD were examined using SH-SY5Y cells along with comparing other plasmalogen species such as phosphatidylethanolamine (PE)-plasmalogen-oleic acid (PE-PLS-18), and their corresponding diacyl-glycerophospholipid-oleic acids (DPL-18), PC-DPL-18 and PE-DPL-18 (Fig. 1).
Human neuroblastoma SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/Ham’s F-12; Invitrogen, Carlsbad, CA, U.S.A.) with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 IU/mL penicillin (Meiji Seika, Japan), as described previously.4) All materials were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), unless otherwise stated.
Western BlottingSH-SY5Y cells were seeded at 1.6 × 106 cells/well, cultured on 6-well plates. Prior to the experiments, the medium was replaced with Opti-MEM (Thermo Scientific, Waltham, MA, U.S.A.) with antibiotics and cells were treated with either vehicle (2% ethanol), 1 μg/mL of PC-DPL-18, PC-PLS-18, PE-DPL-18, or PE-PLS-18 for 16 to 20 h at 37 °C, and samples were prepared as previously reported.6) Then, 20 to 60 μg of protein were subjected to immunoblot analysis with antibodies against the C-terminal of APP (A8717, Sigma-Aldrich, St. Louis, MO, U.S.A.), C99 of APP (MABN380, Sigma-Aldrich), BACE1 (sc-33711, Santa-Cruz Biotechnology, Santa Cruz, CA, U.S.A.), CLU (sc-5289, Santa-Cruz Biotechnology), or Tau (sc-32274, Santa Cruz Biotechnology). Of note, C83 of APP was estimated according to the protein size of the band detected. After incubating with primary antibodies, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1705046, 1705047, Bio-Rad, Hercules, CA, U.S.A.), and immunoreactivities were visualized using LAS-1000 (FUJIFILM, Tokyo, Japan), as described previously.6) To ensure equal loading of proteins, membranes were stripped and re-probed with anti-β-tublin-antibody (014-25041), as described previously.6)
Enzyme-Linked Immunosorbent Assay (ELISA)Similar to Western blotting preparation, SH-SY5Y cells were seeded and the medium was replaced with Opti-MEM with antibiotics, which were then treated either with vehicle, 1 μg/mL of PC-DPL-18, or PC-PLS-18 for 16 to 20 h at 37 °C. Quantitative measurements of Aβ (1–42) concentrations in cell culture supernatants were performed using the ELISA kit according to the manufacturer's instructions (298-62401), and optical density was assessed using a microplate reader (TECAN, Mannedorf, Switzerland).
It is widely known that reduction of plasmalogens is associated with neurodegenerative disorders such as AD.1–3) To evaluate the effects of plasmalogens on APP metabolic processes, PC-PLS-18 was utilized since this species has been shown to have protective effects against cytotoxicity in SH-SY5Y cells.4) The final metabolic process of intracellular APP is known to be cleavage by γ-secretase. However, before the cleavage by γ-secretase, when APP is cleaved by α-secretase, APP is processed to C83, whereas when cleaved by β-secretase, it will be C99, a precursor of Aβ.7) Therefore, if C99 protein is increased, Aβ protein would also be increased. To examine the effects of PC-DPL-18 and PC-PLS-18 on the processing of APP, the ratio of C83/C99 was evaluated. As shown in Fig. 2A, treatment with 1 μg/mL of PC-PLS-18 overnight significantly increased the ratio of C83/99, possibly because of reduction of the C99 protein level, whereas when PC-DPL-18 was treated, the ratio was not significantly altered compared with the vehicle-treated control.
SH-SY5Y cells were treated either with vehicle (vehi), PC-DPL-18, PC-PLS-18 (A, B, C), PE-DPL-18, or PE-PLS-18 (D) overnight at 37 °C. (A, C, D) Immunoblot analysis with antibodies against C83 (upper panel), and C99 (lower panel) (A), against BACE1 (upper panels), and β-tubulin (β-tub, lower panels) (C, D), and the histograms representing the ratio of upper to lower panels, as assessed with pooled densitometric data (mean ± standard deviation (S.D.)) from three or more than three independent experiments. (B) After overnight treatments with lipids, cell culture media were subjected to ELISA to examine the amounts of released Aβ (1–42). * p < 0.05, † p < 0.05, ANOVA. The vehicle-treated control was set as 1.0 for evaluation.
Next, to examine whether the reduction of intracellular C99 protein is reflected in the extracellular Aβ (1–42) protein levels, PC-PLS-18-treated cell culture supernatants were examined by ELISA. However, as shown in Fig. 2B, the extracellular protein levels of Aβ (1–42) were not significantly altered among the samples treated.
Then, the expression levels of β-site APP cleaving enzyme 1 (BACE1), an important β-secretase to produce C99, was examined. As the results, the BACE1 protein expression (arrow) was slightly but significantly reduced when cells were treated with PC-PLS-18, but not PC-DPL-18 overnight, as shown in Fig. 2C. To further confirm that the reduction of BACE1 protein expression was a PC-PLS-18-specific event, the effects of PE-PLS-18 as well as PE-DPL-18 were evaluated. As shown in Fig. 2D, neither treatment with PE-PLS-18 nor PE-DPL-18 overnight significantly altered the expression levels of BACE1. Of note, expression levels of α-secretases such as ADAM9 were not altered by treatment with PC-PLS-18 or PC-DLP-18 overnight (data not shown).
PC-PLS-18 May Reduce Aβ Protein-Associated Down-stream Signaling Factors in SH-SY5Y CellsReductions of BACE1 expression and C99 protein production may have effects on the down-stream signaling of Aβ protein-associated factors. One such factor is clusterin (CLU), since CLU has been reported to be involved as a mediator of Aβ toxicity.8) Therefore, the effect of PC-PLS-18 on the expression level of CLU was then examined. Similar to the results of BACE1 expression, PC-PLS-18 significantly reduced the expression level of CLU, but PC-DPL-18 did not alter the level that was similar to the vehicle-treated control (Fig. 3A).
SH-SY5Y cells were treated either with vehicle (vehi), PC-DPL-18, or PC-PLS-18 overnight at 37 °C. Immunoblot analysis with antibodies against CLU (upper panel) (A), or against Tau (upper panels) (B), and β-tubulin (β-tub, lower panels), and histograms representing the ratio of upper to lower panels, as assessed with pooled densitometric data (mean ± S.D.) from three or more than three independent experiments. † p < 0.05, ANOVA. The vehicle-treated control was set as 1.0 for evaluation. (C) Treatment with PC-PLS-18 reduces the expression of intracellular C99 but not C83, resulting in the reduction of expressions of CLU and Tau. However, extracellular expression levels of Aβ were not reduced by PC-PLS-18 treatment. Reduction of expression levels of Tau may lead to delaying the onset risk of AD.
Another important factor may be Tau protein because Tau is considered a down-stream factor of Aβ that triggers the conversion of Tau to a toxic state in AD pathogenesis.9) As shown in Fig. 3B, treatment with PC-PLS-18 significantly reduced the expression level of Tau, but such an effect was not caused by PC-DPL-18 treatment.
It has been reported that among the major plasmalogens tested, only PC-PLS-18 has been reported to show protective effects against arachidonic acid-induced cytotoxicity in SH-SY5Y cells.4) Here, PC-PLS-18 was also shown to reduce protein expression levels of BACE1, CLU, and Tau, factors involved in the Aβ-associated pathogenesis of AD.
Interestingly, treatment with PC-PLS-18 reduced expression levels of C99 without altering levels of C83 (Fig. 2A). This may be because of reduced expression levels and activities of BACE1. Moreover, as shown in Figs. 2C, 3A, and 3B, reduced BACE1 expression levels may be responsible for reductions of the expression levels of CLU as well as Tau. Reducing BACE1 would reduce levels of intracellular Aβ, an up-stream factor of CLU and Tau. Indeed, BACE1 knockout mice have been reported to show lower β-secretase activities, so levels of Aβ were reduced.10) In addition, when CLU was injected into the rat hippocampus, increased levels of Tau were observed,11) suggesting that CLU may be another up-stream factor of Tau, as presented in Fig. 3C.
Although the intracellular C99 level was decreased by PC-PLS-18, the extracellular Aβ (1–42) level was not reduced (Fig. 2B). Thus, the levels of Aβ (1–42) were not different regardless of the presence or absence of the lipids tested. As one possible reason why the extracellular level of Aβ was not altered by PC-PLS-18, the metastatic bone tumor-derived SH-SY5Y cells may essentially not have the potential to produce Aβ per se, since it has been reported that tumoral amyloidosis of bone is a rare condition.12)
With respect to the other species of plasmalogens, they have not been evaluated in terms of the Aβ-associated pathogenesis of AD except for PE-PLS-18, which should be examined in the future. Of note, the detailed reason why PC-PLS-18 but not PE-PLS-18 showed the reduced expression level of BACE1 is currently not clear. However, it is possible to speculate that it may be due to their asymmetrical distributions and/or charged headgroups. Thus, PC-PLS-18 may tend to be incorporated into the outer-membrane site, while PE-PLS-18 may be into the inner-membrane site. It may be difficult to explain how outer-membrane-incorporated PC-PLS-18 induces these inhibitory effects, but if PC-PLS-18 is translocated to inner-membrane by flip-flop, its negatively charged headgroup would pull the positively charged protein into its side for inducing the effects. Since PE-PLS-18 does not have charged headgroup, this species did not show the inhibitory effects that PC-PLS-18 showed.
Although the effects of PC-PLS-18 were significant, they were not marked. However, cumulative effects of reducing levels of BACE1, CLU, and Tau would eventually lead to delaying the onset risk of AD for a period. Moreover, the slight but significant effects of PC-PLS-18 on APP metabolic processes may cause less stresses to the cells. Thus, PC-PLS-18 may not have direct therapeutic effects, but would have potential AD-preventive effects. The detailed mechanisms as well as molecular target of PC-PLS-18 are expected soon.
This research was supported in part by Marudai Food Co., Ltd.
The authors declare no conflict of interest.
