A role for docosahexaenoic acid–derived neuroprotectin D1 in neural
cell survival and Alzheimer disease (original) (raw)
DHA downregulates secretion of A β peptides from aging HN cells and is the precursor of NPD1. HN cells, a primary coculture of human neurons and glia, are a useful in vitro test system to study stress mechanisms during human brain cell development, aging, and AD (3) (Figure 1A). As indicated by βIII tubulin and glial fibrillary acidic protein (GFAP) immunostaining, these cultures are mixtures of approximately equal numbers of neurons and glia under our growth conditions at 4 weeks of development (Figure 1, B and C). Interestingly, amyloidogenic Aβ peptides were progressively secreted from HN cells into the incubation medium throughout 8 weeks of culture (Figure 1, D and E). The ratio of Aβ40, a resident peptide of AD blood vessels, to Aβ42, which aggregates at lower concentrations than Aβ40 and is enriched within the amyloid plaque of AD (23, 24), was about 10:1 throughout this 8-week period. To determine the effect of cytokine-mediated stress on aging HN cells, Aβ40 and Aβ42 peptide release was assayed in the incubation medium after addition of IL-1β, a potent inducer of reactive oxygen species and a promoter of oxidative stress (3, 25). We found a time-dependent release of both Aβ40 and Aβ42 peptides as a function of number of weeks in culture. While soluble Aβ peptide secretion from HN cells was enhanced in the presence of IL-1β, parallel experiments with DHA in the culture medium showed attenuation of Aβ peptide release (Figure 1, D and E).
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).
We next found that in HN cells, DHA was used as a precursor of NPD1 biosynthesis (Figure 1F). We observed an approximately 5-fold increase in NPD1 appearance at 4 weeks of culture; at 8 weeks, the concentration of this oxygenated DHA derivative was about half that observed at 4 weeks (Figure 1F). Notably, during oxidative stress in human retinal cells and ischemia/reperfusion in brain, NPD1 elicits neuroprotection (5, 6). These observations suggest that in aging HN cells, attenuation of the potentially neurotoxic Aβ peptide release by DHA could be mediated, at least in part, by NPD1.
sAPP α induces NPD1 biosynthesis. Because DHA mediated the downregulation of Aβ40 and Aβ42 release and stimulated NPD1 production in HN cells, we next explored the possibility that NPD1 biosynthesis might be affected by the neurotrophic peptide sAPPα, a 612–amino acid fragment derived from α-secretase–mediated cleavage of βAPP, which appears to be neurotrophic (23, 24). sAPPα promotes neuritogenesis and long-term survival of hippocampal and cortical neurons in culture and protects brain cells against the toxicity of Aβ40 and Aβ42 peptides and excitotoxic and ischemic injury both in cell cultures and in vivo (23, 26, 27). It is important to note that the sAPPα generated via the α-secretase pathway does not give rise to the shorter amyloidogenic Aβ peptides; hence, the shunting of βAPP into the α-secretase pathway may have a beneficial effect by the relative lowering of Aβ peptide levels (24, 26, 28, 29). We observed a dose-dependent NPD1 induction by sAPPα (Figure 1G). For the additivity experiments with DHA and sAPPα (Figure 1, H and I), we selected concentrations of sAPPα that elicited a small (20 ng/ml) and a larger (100 ng/ml) NPD1 induction. We next found that sAPPα at 20 and 100 ng/ml, in the presence of 50 nM added DHA, induced NPD1 abundance 2.3- and 5-fold, respectively, over that in controls treated with DHA alone (Figure 1, H and I). sAPPα at 20 ng/ml in the absence of added DHA elicited negligible NPD1 synthesis; however, at 100 ng/ml, sAPPα stimulation strongly promoted NPD1 synthesis in the absence of added DHA. These results indicate that some of the neurotrophic activity of sAPPα may be elicited, at least in part, by an upregulation in the biosynthesis of DHA-derived NPD1. This may be a complementary cell-survival mechanism activated early in AD pathogenesis. sAPPα may activate NPD1 biosynthetic enzymes PLA2 and/or a 15-LOX–like enzyme integral to NPD1 biosynthesis from DHA (5). It is interesting that muscarine, a positive regulator of PLA2, is also a potent inducer of sAPPα in human neuroblastoma SH-SY5Y cells (30, 31); therefore, the enzymatic pathways involving PLA2-mediated DHA and NPD1 biosynthesis may exhibit positive feedback regulation through sAPPα. sAPPα also appears to protect neural cells against the proapoptotic action of thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, and the adverse effect of thapsigargin can be abolished in cells overexpressing antiapoptotic Bcl-2 (23). Aβ42 elevated NPD1 levels in the presence of added DHA (Figure 1H). This action of Aβ42 peptide may represent a cytoprotective response of brain cells when confronted with a peptide that triggers oxidative stress. The unesterified DHA pool size assessed by liquid chromatography–photodiode array–electrospray ionization–tandem mass spectrometry–based (LC-PDA-ESI-MS-MS–based) lipidomic analysis (5, 6) (Figure 1I) showed that neither Aβ42 nor sAPPα was able to promote unesterified DHA pool-size changes, indicating that a tight regulation of unesterified DHA may take place when sAPPα (100 μM) activates NPD1 production in the absence of added exogenous DHA (Figure 1I). Note that in instances where DHA was added to the HN cell culture medium, the presence of Aβ42 peptide did not modify its cellular pool size.
Neuroprotective activity of NPD1 in HN cells. Because Aβ42 peptides promote apoptosis and cell death in both neurons and glia (1, 32), we next investigated the ability of NPD1 to protect HN cells against Aβ42-induced cytotoxicity. For this purpose, 3-week-old HN cells were incubated for an additional 3.5 days in serum-free HN cell maintenance medium (HNMM) made 8 μM in Aβ42 peptide. Except for the experiments depicted in Figure 1, D–F, HN cells used in these studies were used at a developmental stage of 3–4 weeks in culture, at which time there were approximately equal populations of neuronal and glial HN cells (Figure 1, A–C). Because selective cell loss may take place in older HN cell cultures (when neuronal cells drop out), the use of HN cells at a fixed age (and 50:50 neuronal/glial populations) was selected to minimize this possibility. Apoptosis was found to occur in both neurons and glia (Figures 2 and 3). When NPD1 (50 nM) was added to this test system, NPD1 protected both neurons and glia from Aβ42-directed apoptosis, as evidenced by quantification of Hoechst 33258 staining of compacted nuclei in control, Aβ42-treated, and Aβ42+NPD1-treated cell fields (Figure 3). Unlike control HN cells, Aβ-treated HN cells also exhibited retracted neurites; however, when treated with NPD1, cells assumed extended neurites and an overall morphology resembling that of control cells (Figure 3).
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).
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).
DHA and NPD1 activate a neuroprotective gene-expression program. We next explored proinflammatory and apoptosis-related gene-expression patterns in 4-week-old HN cells after exposure to Aβ42, DHA, and NPD1 using DNA array–based human genome expression profiling (Affymetrix). We chose to focus on the inducible expression of the proinflammatory cytokines IL-1β and chemokine exodus protein 1 (CEX-1), the prostaglandin synthase COX-2, the TNF-α–inducible proinflammatory element B94 (33, 34), and TNF-α, whose RNA levels are upregulated in the brains of AD patients (33), and 5 members of the Bcl-2 gene family, 3 of which are antiapoptotic [Bcl-xl, Bcl-2, and Bfl-1(A1)] and 2 of which are proapoptotic (Bax and Bik; refs. 23, 35). In the experiments presented in Figure 4, HN cells were treated with Aβ42 (25 μM) and DHA or NPD1 (each 50 nM ambient) for 18 hours. We used experimental cutoff parameters combining 2 stringent criteria for up- or downregulated genes: (a) changes in gene expression of 2-fold or greater difference over controls that (b) achieved significance of P less than 0.05 (ANOVA). Results were graphed as truncated “volcano plot” representations (Figure 4, A–C) using GeneSpring 7.2 algorithms (Silicon Genetics). As shown in Table 1, Aβ42 markedly upregulated a complex proapoptotic and proinflammatory gene-expression program. These analyses indicated significant Aβ42-mediated upregulation in the expression of a family of genes encoding the cytokines IL-1β, CEX-1, and TNF-α, COX-2, B94, and the proapoptotic Bax and Bik proteins (35–37). DHA and NPD1 each showed upregulation of Bcl-xl, Bcl-2, and Bfl-1(A1), neuroprotective members of the Bcl-2 gene family, and relative downregulation of Bax and Bik, proapoptotic members of the Bcl-2 gene family (Table 1). Bax and Bik were upregulated 3.2- and 2.8-fold (each P < 0.05), respectively, in Aβ42-treated cells over age-matched control cells, and these enhanced RNA levels were driven to the status of “no significant change” after treatment with DHA or NPD1 (Table 1). The antiapoptotic Bcl-2 family member Bfl-1(A1) was upregulated by DHA and NPD1 to about 4- and 6-fold over controls, respectively, and Bfl-1(A1) reached the highest significance of any upregulated gene in NPD1-treated HN cells (Figure 3). Subtraction of DHA from NPD1 DNA-array signals revealed an additional 56 genes that were upregulated 2-fold or greater over controls (P < 0.05). These genes included those encoding 6 membrane receptors, 5 transcription factors, 5 kinase/phosphatase/phosphodiesterases, 2 oxygenase/oxidoreductases, and 12 novel expressed sequence tags of unknown function (see Supplemental Table 1; available online with this article; doi:10.1172/JCI25420DS1). To confirm changes in Bcl-2 family-member proteins, Western immunoblot analysis was performed using anti–Bcl-2, anti–Bfl-1(A1), and anti-actin primary antibodies (Figure 4D). After DHA treatment, Bcl-2 protein was upregulated almost 2-fold over controls, and after NPD1 treatment, Bcl-2 and Bfl-1(A1) showed highly significant upregulation, averaging 2.3-fold and 3.4-fold increases, respectively, over untreated controls (Figure 4E; each P < 0.05). Taken together, the results suggest that DHA and NPD1 induce a gene-expression program that is neuroprotective through downregulation of proapoptotic and proinflammatory factors and upregulation of the Bcl-2 family of antiapoptotic proteins that are critical integral regulators of cell survival.
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.
Changes in gene-expression patterns in Aβ42-, DHA-, or NPD1-stressed HN cells
DHA and NPD1 content is decreased in the cornu ammonis region 1 of the hippocampus from AD patients. To further investigate the possible significance of DHA-derived NPD1, we examined the levels of these bioactive lipids in AD hippocampal cornu ammonis region 1 of hippocampus (CA1), a brain region specifically targeted by AD neuropathology (28, 34, 38). According to the plaque and tangle count (Table 2), all except 1 AD brain sample were from AD patients at a moderate stage of disease development. While there were no significant differences in the age or postmortem sampling interval between the AD and control brain groups, and no significant differences in the RNA yields or spectral quality between AD and control groups (Table 2; ref. 34; Methods), unesterified DHA pool sizes in controls were 2-fold higher than in AD hippocampus, and NPD1 levels in AD were on average about one-twentieth of those in age-matched controls (Figure 5, A–C). Quantitative morphometric analysis of AD brains shows a dropout of neurons; depending on brain region and stage of disease development, the population of neurons remaining in AD brain has been estimated to range from 59% (39), to 77% (40), to 89% (41) of age-matched controls for the same brain region. Thus, we would argue that the loss of 11–41% of neurons is insufficient to account for the observed 20-fold reduction in the amount of NPD1 in AD hippocampus when compared with age-matched controls. The results indicate that in AD brain, despite modestly decreased availability of unesterified DHA, NPD1 levels were dramatically reduced, perhaps as the result of excessive oxidative stress, and NPD1’s neuroprotective bioactivity during brain cell degeneration may be effectively lost. In these same human CA1 hippocampal samples, we also examined the levels of expression of a cytosolic PLA2 (cPLA2) (GenBank D38178; encoding an 82.5-kDa, calcium-dependent cPLA2) and 15-LOX (GenBank M23892; encoding 15-lipoxygenase), 2 key enzymes in the mobilization of DHA and NPD1 biosynthesis (Figure 5D). In AD brain, when compared with age-matched controls, cPLA2 abundance was increased 4.6-fold (P < 0.02) and 15-LOX decreased almost 2-fold (P < 0.05) (Table 2). Decreased abundance of NPD1 in AD brain may be explained, at least in part, by a disruption in the expression and regulation of the PLA2 and/or 15-LOX–like enzymes essential for NPD1 biosynthesis (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 10,17_S_-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 10,17_S_-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).
cPLA2 and 15-LOX gene expression in control and AD hippocampal CA1