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, 53 (4), 2733-49

Pro-Resolving Lipid Mediators Improve Neuronal Survival and Increase Aβ42 Phagocytosis

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Pro-Resolving Lipid Mediators Improve Neuronal Survival and Increase Aβ42 Phagocytosis

Mingqin Zhu et al. Mol Neurobiol.

Abstract

Inflammation in the brain is a prominent feature in Alzheimer's disease (AD). Recent studies suggest that chronic inflammation can be a consequence of failure to resolve the inflammation. Resolution of inflammation is mediated by a family of lipid mediators (LMs), and the levels of these specialized pro-resolving mediators (SPMs) are reduced in the hippocampus of those with AD. In the present study, we combined analysis of LMs in the entorhinal cortex (ENT) from AD patients with in vitro analysis of their direct effects on neurons and microglia. We probed ENT, an area affected early in AD pathogenesis, by liquid chromatography-tandem mass spectrometry (LC-MS-MS), and found that the levels of the SPMs maresin 1 (MaR1), protectin D1 (PD1), and resolvin (Rv) D5, were lower in ENT of AD patients as compared to age-matched controls, while levels of the pro-inflammatory prostaglandin D2 (PGD2) were higher in AD. In vitro studies showed that lipoxin A4 (LXA4), MaR1, resolvin D1 (RvD1), and protectin DX (PDX) exerted neuroprotective activity, and that MaR1 and RvD1 down-regulated β-amyloid (Aβ)42-induced inflammation in human microglia. MaR1 exerted a stimulatory effect on microglial uptake of Aβ42. Our findings give further evidence for a disturbance of the resolution pathway in AD, and indicate that stimulating this pathway is a promising treatment strategy for AD.

Keywords: Alzheimer; Human; Inflammation; Lipoxin; Maresin; Microglia; Neuroprotection; Omega-3; Resolvin; SPMs.

Conflict of interest statement

Conflict of interest

C.N.S. has the following disclosures: as inventor on patents [resolvins] assigned to BWH and licensed to Resolvyx Pharmaceuticals; scientific founder of Resolvyx Pharmaceuticals with equity ownership in the company; and has interests reviewed and managed by the Brigham and Women’s Hospital and Partners Health Care in accordance with their conflict of interest policies.

Figures

Fig. 1
Fig. 1
a–b. Lipid mediator (LM) identification in post mortem entorhinal cortex (ENT) tissue from controls and Alzheimer’s disease (AD) patients. Tissues were extracted and subjected to LM-metabololipidomics. (a) Representative multiple reaction monitoring (MRM) chromatograms for the identified LMs in the ENT. (b) Related MS-MS spectra employed for identification of LMs, based on analysis of n = 14 ENT tissues. RvD5 = 7S,17S–dihydroxy-docosa-5Z,8E,10Z,13Z,15E,19Z–hexaenoic acid. PD1 = 10R,17S–dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z–hexaenoic acid. MaR1 = 7R,14S–dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z–hexaenoic acid. LXA4 = (5S,6R,7E,9E,11Z,13E,15S)-5,6,15-trihydroxyicosa-7,9,11,13-tetraenoic acid. LXB4 = (5S,6E,8Z,10E,12E,14R,15S)-5,14,15-trihydroxyicosa-6,8,10,12-tetraenoic acid. RvE1 = 5S,12R,18R–trihydroxy-6Z,8E,10E,14Z,16E–eicosapentaenoic acid.
Fig. 2
Fig. 2
a–b. Lipid mediator (LM) levels in the human entorhinal cortex (ENT). LMs were quantified by LC-MS-MS. The levels of resolvin D5 (RvD5) (a), maresin 1 (MaR1) (b) and protectin D1 (PD1) (c) were significantly lower in post mortem ENT tissue from Alzheimer’s disease (AD) patients (n = 7) than in tissue from controls (n = 7), while prostaglandin D2 (PGD2) (d) levels were higher in AD. * indicates p < 0.05. Results are expressed in pg/g tissue.
Fig. 3
Fig. 3
a–c. Staurosphorine (STS)-induced neurotoxicity in differentiated human SH-SY5Y neuroblastoma cells. A significant decrease in viability by 100 nM STS is seen as assessed by resazurin assay (a). Analysis with LDH assay shows increased cell death by STS (b). As an index of cell survival the ratio of data from the resazurin and LDH assays was calculated, showing a significant reduction by STS (c). * indicates p < 0.01 compared to vehicle, ** indicates p < 0.001 compared to vehicle. LDH = lactate dehydrogenase.
Fig. 4
Fig. 4
a–c. SPMs reduce STS-induced neurotoxicity in differentiated human SH-SY5Y neuroblastoma cells. The cells were incubated with 0 – 0.5 µM lipoxin A4 (LXA4), resolvin D1 (RvD1) and protectin DX (PDX), an isomer of protectin D1 (PD1), or maresin 1 (MaR1). STS at 100 nM was added immediately afterwards. Incubation with SPMs was repeated at 6 and 24 h, and cell viability and cytotoxicity were assessed at 48 h by resazurin (a) and LDH (b) assay, respectively. As an index of cell survival, the ratio between data from the resazurin and LDH assays was calculated, showing a significant effect by all SPMs tested (c). The data were normalized to the average of each individual experiment. The data are presented as median ± non-outlier range (n = 7). * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.005, all compared to STS treatment alone. K-W = Kruskal-Wallis analysis of variance, LDH = lactate dehydrogenase, SPMs = specialized pro-resolving mediators, STS = staurosporine
Fig. 5
Fig. 5
a–c. Actions of SPMs on Aβ42-induced microglial activation. Human CHME-3 microglia were incubated with 10 µg/ml Aβ42 in combination with 0 – 0.5 µM of the SPMs LXA4, RvD1, MaR1 and PDX. Flow-cytometry analysis showed an up-regulation of CD11b by Aβ42 at both 1 and 6 h (a, b). Both RvD1 and MaR1 counteracted this at 1 h (a), and RvD1 at 6 h (b). CD40 expression was increased at 6 h (c), and this was down-regulated by MaR1 at 0.1 µM. # indicates p < 0.01 compared with vehicle, * indicates p < 0.05 and *** indicates p < 0.005 compared to Aβ42 treatment. The data were normalized to the average of each individual experiment and are presented as median ± non-outlier range (n = 8). K-W = Kruskal-Wallis analysis of variance, LXA4 = lipoxin A4, MaR1 = maresin 1, PDX = protectin DX, RvD1 = resolvin D1, SPMs = specialized pro-resolving mediators.
Fig. 6
Fig. 6
MaR1 stimulates Aβ42 phagocytosis by human microglia. Human microglial cells (CHME3) were co-incubated with 0 – 100 nM MaR1 and 1 µg/ml Hilight-488-conjugated Aβ42 for 1 and 6 h. Phagocytosis was analyzed by flow-cytometry. Kruskal-Wallis analysis showed a dose-dependent increase in Aβ42 phagocytosis by MaR1. Pairwise comparison was analyzed by Mann-Whitney test. * indicates p < 0.05 and ** indicates p < 0.01 compared to vehicle. The data were normalized to the average of each individual experiment and presented as median ± non-outlier range (n = 5). MaR1 = maresin 1.
Fig. 7
Fig. 7
a–e. Actions of MaR1 on microglial phenotype. Human CHME3 microglia were treated with 0 – 1000 µM MaR1 for 1 and 6 h, and flow-cytometry analysis showed a down-regulation of CD33 (a), which is involved in inhibition of phagocytosis. MaR1 also down-regulated the pro-inflammatory markers activated form of CD11b (b), major histocompatibility complex class II (MHC-II), and (c) CD86 (d and e). * indicates p < 0.05 and *** indicates p < 0.005 compared to vehicle. The data were normalized to the average of each individual experiment and presented as median ± non-outlier. K-W = Kruskal-Wallis analysis of variance, MaR1 = maresin 1.
Fig. 8
Fig. 8
a–c. Expression of SPM receptors in differentiated neuroblastoma cells and in microglial cells. (a) Micrograph and underneath an immunoblot of differentiated human SH-SY5Y neuroblastoma cells with antibodies against G protein-coupled receptor 32 (GPR32). (b and c) Micrographs and underneath immunoblots of human CHME3 microglia cells with antibodies against LXA4 receptor/formyl peptide receptor 2 (ALX/FPR2) (b) and GPR32 (c), respectively. (a) Scale bar = 2.5 µm, (b and c) scale bar = 5 µm. LXA4 = lipoxin A4, SPM = specialized pro-resolving mediator.

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