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. 2009 Nov 15;57(15):1600-9.
doi: 10.1002/glia.20873.

beta2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK-dependent/protein kinase A-independent pathway

Li Qian  1 , Xiaoming Hu, Dan Zhang, Amanda Snyder, Hung-Ming Wu, Yachen Li, Belinda Wilson, Ru-Band Lu, Jau-Shyong Hong, Patrick M Flood
Affiliations

beta2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK-dependent/protein kinase A-independent pathway

Li Qian et al. Glia. .

Abstract

Activation of the beta2 adrenergic receptor (beta2AR) on immune cells has been reported to possess anti-inflammatory properties, however, the pro-inflammatory properties of beta2AR activation remain unclear. In this study, using rat primary mesencephalic neuron-glia cultures, we report that salmeterol, a long-acting beta2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the major superoxide-producing enzyme in microglia. A key role of PHOX in mediating salmeterol-induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene-iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47(phox) to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol-induced superoxide production, p47(phox) translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that beta2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK-dependent/PKA-independent pathway.

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Figures

FIGURE 1
FIGURE 1
Salmeterol produces a dose-dependent and selective toxicity on DA-producing neurons. Rat primary mesencephalic neuron-glia cultures were treated with indicated concentrations of salmeterol. After seven days, the salmeterol-induced DA neurotoxicity was quantified by the [3H]-DA uptake assay (A) and TH neuron count (B). The effect of salmeterol on overall neuron number was determined at 7 days post-treatment with immunocytochemical staining using Neu-N antibody (C). Representative light micrographs of the immunostained TH neurons or total neurons were shown in (D); Results in (A–C) were expressed as a percentage of the vehicle-treated control cultures and were the mean ± SE. from three independent experiments in triplicate. **P<0.01 when compared with the vehicle-treated cultures. Bar = 50 μm.
FIGURE 2
FIGURE 2
Salmeterol-induced neurotoxicity is through β2AR. ICI118,551was added to rat mesencephalic midbrain neuron-glia cultures for 30 min prior to the addition of salmeterol. DA neurotoxicity was quantified on day 7 using the [3H]-DA uptake assay. Data are percentage of control cultures, and are presented as the mean ± SE of 3 separate experiments. *P<0.05 compared with the salmeterol-alone treated cultures without ICI118,551, ##P<0.01 when compared with the vechicle-treated cultures.
FIGURE 3
FIGURE 3
Microglia mediate salmeterol-induced DA neurotoxicity. Mesencephalic midbrain neuron-glia cultures (open bars, NG), neuron-astrocyte (solid bars, NA) and neuron-enriched cultures (cross-hatched bars, N) were treated with either vehicle or 1 μM salmeterol. DA neurotoxicity was measured at 7 days post-treatment using the [3H]-DA uptake assay. Data are percentage of control cultures, presented as the mean ± SE of 3 separate experiments. *P<0.05 when compared with the salmeterol-treated NG cultures, ##P<0.01 when compared with the vechicle-treated cultures.
FIGURE 4
FIGURE 4
Salmeterol increases production of ROS from purified microglia. Microglia-enriched cultures were treated with either vehicle or indicated concentrations of salmeterol for 30 min. Production of superoxide was measured by the superoxide dismutase (SOD) inhibitable reduction of WST-1 (A). Production of intracellular ROS was measured 1 h post-stimulation using DCFDA (B). The results are the mean ± SE and the average of 3 separate experiments. *P<0.05 compared with the vehicle alone-treated cultures.
FIGURE 5
FIGURE 5
NADPH oxidase plays an important role in salmeterol-induced DA neurotoxicity. DA neurotoxicity was determined by the [3H]-DA uptake assay in neuron-glia cultures 7 days after pretreatment with DPI (0.01 μM) for 30 min and treatment with salmeterol (1 μM). The results are the mean ± SE, and values are the average of 3 separate experiments. *P<0.05 salmeterol + DPI group compared with salmeterol alone-treated cultures, ##P<0.01 salmeterol only group compared with the vechicle alone-treated cultures.
FIGURE 6
FIGURE 6
Salmeterol-induced neurotoxicity is PKA independent. Mesencephalic midbrain neuron-glia cultures were pretreated with Rp-cAMPS (25 and 50 μM) for 30 min prior to the addition of salmeterol (1 μM). Neurotoxicity was assessed by DA uptake 7 days after salmeterol addition (A). Superoxide production was measured 30 min after salmeterol addition in purified microglia cultures (B). The results are the mean ± SE, and values are the average of 3 separate experiments. #P<0.05, ##P<0.01 compared with the vehicle alone-treated cultures.
FIGURE 7
FIGURE 7
ERK activation is required for salmeterol-mediated neurotoxicity and superoxide production. Purified microglia were incubated with 1 μM salmeterol for 15 min. The cells lysates were subjected to western blot. The levels of phosphorylated MAPK relative to total MAPK were determined by western blot using specific antibodies against phosphorylated or total ERK, p38 or JNK respectively. Representative western blots are shown from 3 independent experiments (A). ImageJ software was used to quantitate the intensity of the phosphorylated MAPK and total MAPK bands in western blot, respectively, normalized to the vehicle-treated control (B). Effects of additional three long-acting β2AR agonists, including bambuterol, formoterol, and clenbuterol, on the phosphorylation levels of ERK were studied. The representative pictures and quantitative data are shown (C and D). Neuron-glia cultures were pretreated with U0126 for 30 min prior to the addition of salmeterol. Neurotoxicity was assessed by DA uptake 7 days after salmeterol addition (E). Superoxide production was measured 30 min after salmeterol stimulation in purified microglia cultures (F). Results in B, C and D are the means ± SE, and are the average of 3 separate experiments. ##P<0.01, #P<0.05 compared with the vehicle alone-treated cultures, *P<0.05, **P<0.01, compared with the salmeterol alone-treated cultures.
FIGURE 8
FIGURE 8
Salmeterol-induced cytosolic p47phox protein translocation is ERK dependent, but PKA independent. HAPI cells were pretreated with vehicle, U0126 (10 μM) or Rp-cAMPS (50 μM) for 30 min prior to the addition of salmeterol (1 μM). Thirty minutes after salmeterol addition, subcellular fractions were isolated for western blot analysis. Representative western blots are shown from 3 independent experiments (A). ImageJ software was used to quantitate the intensity of the bands in western blots, results given in B represents the percentage difference of the ratio of membrane p47phox protein compared with membrane gp91phox protein (left panel, solid bars), cytosolic p47phox protein compared with cytosolic GAPDH (right panel, cross-hatched bars), respectively, normalized to the vehicle-treated control (B).
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