Norepinephrine promotes microglia to uptake and degrade amyloid beta peptide through upregulation of mouse formyl peptide receptor 2 and induction of insulin-degrading enzyme

Abstract

Locus ceruleus (LC) is the main subcortical site of norepinephrine synthesis. In Alzheimer's disease (AD) patients and rodent models, degeneration of LC neurons and reduced levels of norepinephrine in LC projection areas are significantly correlated with the increase in amyloid plaques, neurofibrillary tangles, and severity of dementia. Activated microglia play a pivotal role in the progression of AD by either clearing amyloid beta peptide (Abeta) deposits through uptake of Abeta or releasing cytotoxic substances and proinflammatory cytokines. Here, we investigated the effect of norepinephrine on Abeta uptake and clearance by murine microglia and explored the underlying mechanisms. We found that murine microglia cell line N9 and primary microglia expressed beta(2) adrenergic receptor (AR) but not beta(1) and beta(3)AR. Norepinephrine and isoproterenol upregulated the expression of Abeta receptor mFPR2, a mouse homolog of human formyl peptide receptor FPR2, through activation of beta(2)AR in microglia. Norepinephrine also induced mFPR2 expression in mouse brain. Activation of beta(2)AR in microglia promoted Abeta(42) uptake through upregulation of mFPR2 and enhanced spontaneous cell migration but had no effect on cell migration in response to mFPR2 agonists. Furthermore, activation of beta(2)AR on microglia induced the expression of insulin-degrading enzyme and increased the degradation of Abeta(42). Mechanistic studies showed that isoproterenol induced mFPR2 expression through ERK1/2-NF-kappaB and p38-NF-kappaB signaling pathways. These findings suggest that noradrenergic innervation from LC is needed to maintain adequate Abeta uptake and clearance by microglia, and norepinephrine is a link between neuron and microglia to orchestrate the host response to Abeta in AD.

Figure 1.

Norepinephrine and isoproterenol induce mFPR2 gene expression in microglia and brain. N9 cells (A, C) or murine primary microglia (B, D) were incubated with different concentrations of NE (A, B) for 6 h or ISO (C, D) for 9 h, and then total RNA was extracted and examined for mFPR2 mRNA level by real-time PCR. Results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus cells cultured with control medium.

Figure 2.

Norepinephrine and isoproterenol upregulate mFPR2 expression through activation of β₂AR. A, The expression of β adrenergic receptors in N9 cells and mouse primary microglia was examined by RT-PCR. B, C, N9 cells pretreated with 10 μm propranolol (Pro) for 1 h were incubated with or without 1 μm NE for 6 h (B) or 10 μm ISO for 9 h (C), and total RNA was extracted and examined for mFPR2 mRNA level by real-time PCR. Results represent the mean ± SD of three independent experiments. *p < 0.05 versus cells cultured with control medium. ^#p < 0.05, ^##p < 0.01, compared with cells treated with NE or ISO alone.

Figure 3.

Isoproterenol treatment of microglia increases calcium mobilization but not cell migration in response to mFPR2 agonists. A–C, N9 cells were treated with or without 40 μm ISO for 24 h and then loaded with Fura-3-AM and stimulated with different concentrations of W peptide and measured florescence (RFU) (A, B) or examined for mFPR1 and mFPR2 expression by RT-PCR (C). Data shown in A and B are representative of three independent experiments with similar results. Data in C represent the mean ± SD of three independent experiments. **p < 0.01, compared with cells cultured in medium alone. D, E, N9 cells were cultured in the presence or absence of 500 ng/ml LPS or 10 μm ISO at 37°C for 24 h and then examined for migration in response to W peptide (D), and MMK-1 (E). The results are expressed as the mean ± SD of migrated cells in three high-power fields in triplicate samples, **p < 0.01, cell migration in response to W peptide or MMK-1 compared to medium control; ^##p < 0.01, comparison of spontaneous cell migration between N9 cells treated with ISO and LPS. F, Visualization of N9 cell migration in response to W peptide.

Figure 4.

Isoproterenol enhances Aβ₄₂ uptake by microglia. A, E, Primary microglia (A), N9 cells (E) transduced with control lentivirus (Mock), or mFPR2 shRNA-c lentivirus (shRNA-c) were treated with or without 10 μm ISO for 24 h followed by incubation with 10 μm Aβ₄₂ for 30 min and then examined for Aβ uptake with antibody against Aβ (red). Nuclei were stained with Hoechst (blue). Scale bars, 20 μm. Dara are expressed as mean ± SD (n = 2, with duplicate samples in each experiment); **p < 0.01, compared with Aβ uptake by primary microglia or mock transduced N9 cells without ISO treatment; ^##p < 0.01, compared with Aβ uptake by mock transduced N9 cells pretreated with ISO. B–D, mFPR2/293 cells were transduced with control lentiviruses (Mock) or mFPR2 shRNA lentiviruses, mFPR2 mRNA level was examined by RT-PCR (B), and cell migration in response to mFPR2 agonists (C: MMK-1; D: Aβ₄₂) was examined by chemotaxis assay. Data are expressed as mean ± SD (n = 3); *p < 0.05, **p < 0.01, chemotaxis index of mFPR2 shRNA lentivirus transduced cells versus control lentivirus transduced cells (Mock).

Figure 5.

Activation of β₂AR on microglia cells increases the protein level of IDE and enhances Aβ degradation. A, N9 cells were treated with 10 μm ISO for 12 and 24 h and cell lysates were subjected to immunoblot analysis with antibodies against IDE, neprilysin (NEP), or β-actin. B, Primary microglia were treated with 10 μm ISO or 1 μm NE for 12 and 24 h and then examined for IDE expression by immunoblot analysis. Data are the mean ± SD (n = 3); *p < 0.05, **p < 0.01, compared with cells cultured in medium alone. C–F, N9 cells (C, D) or primary microglia (E, F) pretreated with control medium, 10 μm ISO, or 10 μm ISO combined with 100 μg/ml insulin for 24 h were incubated with 2 μm Aβ₄₂ for an additional 12 h and then examined for Aβ in cell lysate and supernatant with Western blot. Data are the mean ± SD (n = 3); **p < 0.01, compared with Aβ in cells treated with Aβ₄₂ alone; ^##p < 0.01, compared with Aβ in cells treated with ISO followed by Aβ₄₂.

Figure 6.

Isoproterenol induces mFPR2 mRNA expression in microglia through activation of MAP kinases. N9 cells were treated with 10 μm ISO for different periods of time (A) or treated with or without different concentrations of PD98059 (PD) (C) for 1 h followed by 10 μm ISO for 30 min and then examined for p38 or ERK1/2 phosphorylation by Western blot. N9 cells pretreated with different concentrations of SB203580 (SB) (B) or PD (D) for 1 h were stimulated with or without 10 μm ISO for 9 h and then examined for mFPR2 mRNA level by RT-PCR. All data are mean ± SD (n = 3); **p < 0.01, compared with cells without stimulation.

Figure 7.

Isoproterenol induces mFPR2 mRNA expression through ERK/p38-NF-κB pathway. A, N9 cells pretreated with or without different concentrations of BAY117082 (BAY) for 1 h were stimulated with 10 μm ISO for 9 h and then examined for mFPR2 expression by RT-PCR. B, C, N9 cells (B) or primary microglia (C) were stimulated with 10 μm ISO for 30 min and then examined for IκBα phosphorylation by Western blot. D, N9 cells pretreated with or without 50 μm PD98059 (PD), 50 μm SB203580 (SB), or 20 μm BAY for 1 h were stimulated with 10 μm ISO for 30 min and then examined for IκBα level by Western blot. All data are mean ± SD (n = 3); *p < 0.05, **p < 0.01, compared with cells cultured in medium alone; ^##p < 0.01, compared with cells treated with ISO alone.