Plasmalogens rescue neuronal cell death through an activation of AKT and ERK survival signaling.

Neuronal cells are susceptible to many stresses, which will cause the apoptosis and neurodegenerative diseases. The precise molecular mechanism behind the neuronal protection against these apoptotic stimuli is necessary for drug discovery. In the present study, we have found that plasmalogens (Pls), which are glycerophospholipids containing vinyl ether linkage at sn-1 position, can protect the neuronal cell death upon serum deprivation. Interestingly, caspse-9, but not caspase-8 and caspase-12, was cleaved upon the serum starvation in Neuro-2A cells. Pls treatments effectively reduced the activation of caspase-9. Furthermore, cellular signaling experiments showed that Pls enhanced phosphorylation of the phosphoinositide 3-kinase (PI3K)-dependent serine/threonine-specific protein kinase AKT and extracellular-signal-regulated kinases ERK1/2. PI3K/AKT inhibitor LY294002 and MAPK/ERK kinase (MEK) inhibitor U0126 treatments study clearly indicated that Pls-mediated cell survival was dependent on the activation of these kinases. In addition, Pls also inhibited primary mouse hippocampal neuronal cell death induced by nutrient deprivation, which was associated with the inhibition of caspase-9 and caspase-3 cleavages. It was reported that Pls content decreased in the brain of the Alzheimer's patients, which indicated that the reduction of Pls content could endanger neurons. The present findings, taken together, suggest that Pls have an anti-apoptotic action in the brain. Further studies on precise mechanisms of Pls-mediated protection against cell death may lead us to establish a novel therapeutic approach to cure neurodegenerative disorders

Plasmalogens enhance phosphorylation of AKT and ERK1/2.

<p><b>A</b>) To see the signaling pathways, Neuro-2A cells were cultured in 10 % FBS (Control) and 0% FBS with or without Pls (5 µg/ml) for 24 hours. Whole cell extract (50 µg protein) was subjected to western blotting (left). All the data represents more than three independent experiments. <b>B</b>) Quantification of the data showed that Pls treatments significantly increased the phosphorylation of AKT and ERK1/2 compared with the serum free group (each group, n=4, <i>P</i><0.01, Bonferroni’s test). <b>C</b>) Pls significantly increase phosphorylation of Akt and ERK in the increasing time points (each group, n=4, <i>P</i><0.01, Bonferroni’s test). Neuro-2A cells were cultured in serum free medium for 12 hours followed by treatments with Pls (5 µg/ml) for the indicated time periods. Western blotting data showed a time dependent increase in the phosphorylation of AKT and ERK1/2. </p

Retinoic acid-induced apoptosis of Neuro-2A cells was inhibited by Plasmalogens.

<p>Neuro-2A cells were cultured with 5 µM of Retinoic acid (RA) with or without Pls (5 and 20 µg/ml) for 72 hours. TUNEL assays were employed to quantify the apoptotic cells. A significant apoptosis inhibitory effect of Pls was obtained by both the concentrations of 5 and 20 µg/ml (n=4, <i>P</i><0.001, Bonferroni’s test). </p

Plasmalogens reduce sub-G1 population and TUNEL positive Neuro-2A cells.

<p><b>A</b>) Neuro-2A cells were cultured in 10% FBS (control group), 0% FBS (serum starvation) and 0% FBS plus Pls of 5 and 20 µg/ml for 36 hours. FACS assays was performed by PI staining. <b>B</b>) Quantification of the FACS histograms showed a significant reduction of sub-G1 cells population by Pls compared with the control group upon the serum starvation (n=3, <i>P</i><0.001, Bonferroni’s test). <b>C</b>) TUNEL staining shows the reduction of TUNEL positive cells by Pls in the serum starved groups. The experimental condition was similar with that of panel A. Scale bar, 50 µm. <b>D</b>) Pls treatments significantly inhibited TUNEL positive cells (n=5, <i>P</i><0.01, Bonferroni’s test). Histograms show the average number of TUNEL positive cells in percentage of each group. </p

Primary hippocampal neurons were stained by the neuronal marker NeuN.

<p><b>A</b>) To confirm the purity of <i>in </i><i>vitro</i> primary hippocampal neuron culture, cells of DIV6 were stained by NeuN. DAPI was used to stain the cellular nucleus. Almost all the cells of DIV6 were stained by NeuN. Scale bar, 50 µm<b>. B</b>) Quantification of the DIV6 primary cultures cells stained by NeuN (neuron), GFAP (astrocytes), and Iba-1 (microglia). The data represents four independent experiments. </p

Plasmalogens inhibit hippocampal neuronal cell death <i>in</i><i>vitro</i>.

<p><b>A</b>) Pls treatments increase the survivability of primary hippocampal neurons. On DIV 3, hippocampal neurons were cultured with nutrient free medium and treated with vehicle (control) and Pls (5 µg/ml) for 72 hours. On DIV6, neuronal cells were stained by Dil (red color) and imaged by the fluorescence lifetime imaging microscope. Scale bar, 50 µm. <b>B</b>) The bars show the number of primary neurons in the specific area of randomly selected 12 different locations from each dish. The plotted data (average number ± SD) represents four independent experiments. Bonferroni’s test showed a significant difference between the two groups (each group, n=4, <i>P</i><0.001). <b>C</b>) DNA fragmentation assays showed a significant reduction of DNA smearing in the Pls group compared with the vehicle control group (each group, n=4, <i>P</i><0.001, Bonferroni’s test). <b>D</b>) Neuronal cell lysates (50 µg protein) of DIV6 were analyzed by western blotting. Pls treatments showed a reduction in the cleaved caspase-9 and caspase-3. Increased phosphorylation of AKT and ERK was also observed in the Pls treated primary neurons. These data represents three independent experiments. </p

Plasmalogens inhibit cleavage of caspase-9 and caspase-3.

<p><b>A</b>) Serum starvation increases cleavage of caspase-9 but not of caspase-8 and caspase-12. Neuro-2A cells were cultured in the culture medium supplemented with 10% FBS (control group), 0% FBS (serum starvation) and 0% FBS plus Pls of 5 and 20 µg/ml for 72 hours. Cells extracts were then subjected to western blotting with the specific antibodies against caspases. Fifty microgram proteins of each group were analyzed. <b>B</b>) Pls significantly inhibit cleavage of caspase-9 and caspase-3 (each group, n=4, <i>P</i><0.001, Bonferroni’s test). Image-J software was used to quantify the signals. </p