A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease

Abstract

Deficiency in docosahexaenoic acid (DHA), a brain-essential omega-3 fatty acid, is associated with cognitive decline. Here we report that, in cytokine-stressed human neural cells, DHA attenuates amyloid-beta (Abeta) secretion, an effect accompanied by the formation of NPD1, a novel, DHA-derived 10,17S-docosatriene. DHA and NPD1 were reduced in Alzheimer disease (AD) hippocampal cornu ammonis region 1, but not in the thalamus or occipital lobes from the same brains. The expression of key enzymes in NPD1 biosynthesis, cytosolic phospholipase A2 and 15-lipoxygenase, was altered in AD hippocampus. NPD1 repressed Abeta42-triggered activation of proinflammatory genes while upregulating the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1). Soluble amyloid precursor protein-alpha stimulated NPD1 biosynthesis from DHA. These results indicate that NPD1 promotes brain cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Abeta42-induced neurotoxicity.

Figure 1

DHA attenuates Aβ peptide secretion and serves as the precursor for NPD1 biosynthesis; meanwhile, sAPPα activates NPD1 formation. (A) HN cells were grown for up to 8 weeks. (B and C) After 4 weeks of culture, aging HN cells displayed approximately equal populations of neurons and glia and stained positive with the (red fluorescent) neuron-specific marker βIII tubulin (6) (B) and the (green fluorescent) glia-specific marker GFAP (C). (D and E) HN cells in culture normally release Aβ40 and Aβ42 peptides over 8 weeks of aging. Secretion by HN cells of Aβ42 peptide was approximately one-tenth that of Aβ40 peptide; IL-1β (10 ng/ml in modified HNMM; see ref. 3) increased, and DHA decreased, the release of both Aβ40 and Aβ42 peptides into the cell culture medium. CON, control. (F) DHA (100 nM) induced NPD1 biosynthesis in HN cells, and this induction was age-dependent. (G–I) HN cells incubated in the presence of 10, 20, 50, and 100 ng/ml of sAPPα showed dose-dependent upregulation of NPD1 formation (G); HN cells incubated with sAPPα (at 20 and 100 ng/ml) and/or DHA (at 50 nM; even in the presence of Aβ42) also displayed upregulated production of NPD1 (H and I). *P < 0.05 (ANOVA).

Figure 2

Aβ42-induced apoptosis is inhibited by NPD1 in HN cells. After 3 weeks in culture, HN cells were treated with DMEM/F12 (control), Aβ42 (8 μM) in DMEM/F12 (Aβ42), or Aβ42 (8 μM) plus NPD1 (50 nM) (Aβ42+NPD1) in DMEM/F12 for 3.5 days, then stained with Hoechst 33258 (Hoechst), anti–βIII tubulin (βIII tubulin), and anti-GFAP (GFAP), and all 3 stains were imaged together (merge A). As revealed by Hoechst staining, in controls, relatively few compact nuclei were observed; in Aβ42-treated cells, many compact nuclei were seen (white arrows). In Aβ42-treated cells, neurons and glia clumped together with condensed cytoplasm and damaged nuclei, and both βIII tubulin and GFAP staining was reduced (compare merges of control, Aβ42, and Aβ42+NPD1-treated cells). When NPD1 was added to Aβ42-treated cells, Hoechst staining revealed many fewer compacted nuclei. Each field under columns marked βIII tubulin, GFAP, and merge A represents approximately 0.1 mm². A comparison of control, Aβ42-treated, and Aβ42+NPD1-treated HN cells at higher magnification, stained with Hoechst 33258 (blue), anti–βIII tubulin (red), and anti-GFAP (green), shows that Aβ42-induced apoptosis is associated with both neuronal and glial nuclei (merge B), and that Aβ42+NPD1-treated HN cells show significantly decreased numbers of compacted apoptotic nuclei (yellow arrows).

Figure 3

Aβ42-induced apoptosis is associated with both neurons and glia. (A) Compacted nuclei were associated with both neurons (white arrows) and glia (pink arrows), and with non–βIII tubulin– or non–GFAP-staining cells (non-neural cells; yellow arrows). (B) Apoptosis, as monitored by the presence of compacted nuclei of no more than 0.5 μm diameter (5, 6) after Hoechst 33258 staining, was significantly suppressed by NPD1. Analysis of numbers of compacted nuclei per field in both neurons (red bars) and glia (green bars) shows that these were significantly attenuated in the presence of Aβ42+NPD1 when compared with Aβ42 alone; black bars indicate abundance of compacted nuclei associated with non–βIII tubulin–staining/non–GFAP-staining cells. n = 16. *P < 0.01 and **P < 0.05 versus controls (ANOVA).

Figure 4

Changes in gene expression in HN cells in the presence of Aβ42 (A), DHA (B), and NPD1 (C). Truncated volcano plots display gene-expression patterns as a function of fold change over age-matched controls, against P (ANOVA). “Nonsignificant genes” that would normally appear in the region of P < 0.05 and fold change of less than 1.0 (either up- or downregulated) have been omitted for clarity. Genes of highest statistical significance are sequestered into the lower left (blue, downregulated) and lower right (red, upregulated) quadrants; further data for a select group of 10 highly significant up- and downregulated genes appear in Table 1 and in Supplemental Table 1. (D) Western immunoblot analysis confirmed upregulation of Bcl-2 and Bfl-1(A1) antiapoptotic proteins over actin controls in DHA- and NPD1-treated cells. Gene transcripts have been classified according to their known major functions, although most of these RNAs may have multiple cellular functions (33). (E) Quantified intensities of Bcl-2 and Bfl-1(A1) bands normalized to constitutively expressed actin in the same sample are shown as bar graphs. *P < 0.02 versus controls.

Figure 5

NPD1 and DHA are reduced in AD brain. (A) When compared with age-matched controls, NPD1 and unesterified DHA were significantly reduced in AD hippocampus (HIP) and temporal lobe (TEM) but not in the thalamus (THA) or occipital lobe (OCC) of the same AD brains. In AD, thalamic and occipital regions were relatively spared AD neuropathology. Signals for NPD1 and unesterified DHA in AD hippocampus averaged about one-twentieth and one-half, respectively, of those values seen in age-matched controls. LC-PDA-ESI-MS-MS–based lipidomic analysis (sensitivity 0.05 pmol/mg total protein). n = 6. *P < 0.01 (ANOVA). (B) Characterization of NPD1 using LC-PDA-ESI-MS-MS–based lipidomic analysis (5, 6). (C) Mass spectrographic identification of 10,17S-docosatriene (NPD1) in human hippocampus. (D) Proposed biosynthetic pathways from DHA to NPD1 and bioactivity. DHA is highly enriched as an acyl side chain of brain and retinal membrane phospholipids, suggesting its importance as an essential component of brain and retinal function (2, 29). Esterified DHA is liberated by PLA₂ action upon membrane phospholipids, whereupon it is oxygenated, initially via a 15-LOX–like enzyme, into 10,17S-docosatriene (NPD1). sAPPα, a secreted neurotrophic peptide, stimulates NPD1 biosynthesis. NPD1 exhibits neuroprotective activity against Aβ42 action, represses apoptosis, and promotes the expression of antiapoptotic genes encoding Bcl-2 and Bfl-1(A1) (37).