A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis

Abstract

Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. It was found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from β2-adrenergic receptor (β2-AR)-deficient mice, suggesting that novel pathways other than β2-AR are involved. To this end, It was found that submicromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (-) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism.

Keywords: DSP-4; extra-synaptic; neurodegeneration; neurotransmitter; volume transmission.

Figure 1. NE depletion produces a greater loss of nigral dopaminergic neurons in LPS-injected mice

(A) Schematic drawing of the treatment regimen for LPS and DSP-4. Six months after an initial single systemic injection of LPS, mice received 4 repeated injections of DSP-4 at two-week intervals. Two-week after the last injection of DSP-4, mice were sacrificed for immunohistological staining of nigral sections. (B) Quantitative measurement of TH-ir cells of the SN by stereological analysis. Counts were expressed as the total TH-ir cell numbers per SN and were of Mean ± SEM from 3 animals for each group. LPS injection alone caused 30% dopaminergic neuron loss (4691 ± 152 TH-ir neurons; n=3) compared with saline (7012 ± 88 TH-ir neurons; n=3). And depletion of NE by DSP-4 enhanced the neuronal loss into 50% (3439 ± 94 TH-ir neurons; n=3) (one-way ANOVA; F _{(3, 8)} = 153.5, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test). **p< 0.01 compared with saline control group; ^##p < 0.01 compared with LPS- treated group. (C) Immunohistological staining showed that depletion of brain NE with DSP-4 produced a greater loss of both the number of cell bodies and neuritis of dopaminergic neurons (TH-ir cells) in LPS-treated mice. Scale bar, 200 μm. (D) The quantitative measurements of Neu-Nir neuronal loss were performed by auto-counting using ImageJ software on the pictures of fluorescence staining. The results are expressed as the percentage of time-matched saline controls and are Mean ± SEM from 3 mice. (E) Representative pictures of double immunofluroscence staining showed that the loss of Neu-Nir neurons (red) were comparable to the decrease of THir neurons (green) in LPS or LPS + DSP-4 groups compare to saline controls.

Figure 2. Sub-micromolar concentrations of NE protects cultured dopaminergic neurons from inflammation-driven neurodegeneration

Rat mesencephalic neuron-glia cultures were pretreated with vehicle control or indicated concentrations of NE for 30 minutes before the addition of LPS (15 ng/ml). Seven days later, inflammation-elicited damage of dopaminergic neurons were assessed by quantifying functional changes with [³H]DA uptake assay and loss of TH-immunoreactive neurons. (A) NE pretreatment restored [³H]DA uptake capacity in a dose-dependent manner (53.6 ± 1.4, 65.8 ± 1.6, 77.2 ± 1.8, 88.5 ± 0.8 % of control at 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M NE, respectively; n = 5) compared with LPS alone group (47.4 ± 1.7% of control; n = 12) (one-way ANOVA; F _{(6, 42)} = 276.5, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test). (B) Cell count showed pretreatment of NE dose-dependently mitigated LPS-induced TH-ir neurons number decrease (125 ± 5, 154 ± 6, 194 ± 3, 215 ± 4 per well at 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M NE, respectively; n = 5) compared with LPS alone group (101 ± 3 per well; n = 5) (one-way ANOVA; F _{(6, 28)} = 79.08, p<0.0001; post hoc analysis by Tukey's multiple comparisons test). (C) Immunostaining showed clear reduced neurite numbers and shortened processes of dopaminergic neurons in LPS-treated group. Results in (A) and (B) are means ± SEM from 5 independent experiments in triplicate.*p< 0.05, **p< 0.01 compare with LPS-treated cultures. Scale bar, 50 μm.

Figure 3. Microglia are essential for sub-micromolar NE-mediated dopaminergic neuroprotection

Mesencephalic neuron-glia (which contains mainly neurons, astroglia and about 10% microglia) and microglia-depleted neuron-glia cultures (microglia-depleted N-G, which contains neuron, astroglia and less than 0.01% of microglia) were pretreated with either vehicle or NE (10⁻⁷ M) for 30 min prior to the addition of MPP⁺ (0.25 μM). Seven days later, [³H]DA uptake assays were performed for the measurement of dopaminergic neuron function. (A) In neuron-glia culture, NE pretreatment restored MPP⁺-induced [³H]DA uptake decrease by 20% (57.9 ± 2.4 % of control; n = 3) compared with MPP⁺ alone group (38.5 ± 1.7 % of control; n = 3) (one-way ANOVA, F _{(3, 8)} = 271.4, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test). (B) In microglia- depleted neuron-glia cultures, NE pretreatment had no protective effect (one-way ANOVA, F _{(3, 8)} = 269.3, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test, p > 0.05). Results were expressed as a percentage of the vehicle treated control cultures and were the means ± SEM from three independent experiments in triplicate. **p< 0.01 compare with the MPP⁺-treated group.

Figure 4. Sub-micromolar NE attenuates LPS-induced microglial activation and release of pro-inflammatory factors

Rat primary mesencephalic neuron-glia cultures were pretreated with NE for 30 minutes before LPS stimulation. (A) Microglia morphology was assessed using an antibody against Iba-1 at 24 hours after LPS treatment. (B) The number of Iba-1-immunoreactive cells of each well was counted under the microscope. NE pretreatment reduced the number of Iba-1-positive cell in NE/LPS group (251.7 ± 10.3 % of control; n = 5) compared to LPS alone group (332.2 ± 7.6 % of control; n = 5) (one-way ANOVA, F _{(3, 16)} = 221.5, p <0.0001; post hoc analysis by Tukey's multiple comparisons test). Data was shown as the percentage of control and expressed as the means ± SEM from 5 independent experiments in triplicate. (C) The concentrations of TNF-αin the supernatant of neuron-glia culture were determined after 3h of LPS treatment. The result showed that NE pretreatment reduced TNF-α production in a dose-dependent manner (650.0 ± 25.1, 585.4 ± 21.0, 454.3 ± 13.2, 320.8 ± 48.1 pg/ml at 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M NE, respectively versus LPS alone 790.0 ± 22.1 pg/ml; n = 5) (one-way ANOVA, F _{(6, 43)} = 318.1, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test). (D) The concentrations of NO in the supernatant of neuron-glia culture were determined after 24h of LPS treatment. The result showed that NE pretreatment reduced NO in a dose-dependent manner (3.25 ± 0.16, 2.51 ± 0.12, 1.93 ± 0.11, 1.44 ± 0.06 μM at 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M, respectively versus LPS alone group 4.93 ± 0.20 μM; n = 5) (one-way ANOVA, F_{(6, 43)} = 234.7, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test).(E) The production of superoxide was measured by SOD-inhibitable reduction of WST-1in rat primary mix-glia culture prepared from postnatal day-1 pups. The result showed that NE pretreatment inhibited superoxide production in a dose-dependent manner (79.4 ± 7.1, 61.4 ± 8.2, 46.7 ± 6.0, 26.4 ± 10.8 % of LPS-treated group at 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M, respectively versus LPS alone group) (one-way ANOVA, F _{(6, 27)} = 23.98, p < 0.0001; post hoc analysis by Tukey's multiple comparisons test). Results were expressed as the means ± SEM from 5 independent experiments in triplicate. *p< 0.05, **p< 0.01 compare with the LPS-treated cultures.

Figure 5. β2-AR is partially involved in the anti-inflammatory and neuroprotective effects of sub-micromolar NE

Mixed-glia and mesencephalic neuron-glia cultures prepared from β2-adrenergic receptor (β2-AR)-deficient mice or Balb/C mice (wild type, WT) were pretreated with NE (10⁻⁷ M) for 30 minutes prior to LPS challenge. (A) Three hours later, the levels of TNF-α in the mixed-glia supernatant were determined using commercial ELISA kit. The inhibitory effect of NE was reduced in the β2-AR^−/− culture (NE/LPS 1219.4 ± 52.64 versus LPS 1482.44 ± 67.03 pg/ml; n = 3) compared to WT (NE/LPS 911.09 ± 34.66 versus LPS 1450.98 ± 44.39 pg/ml; n = 3), but still had 20% significant change (two-way ANOVA, F _{(3, 16)} = 8.577, p = 0.0013; post hoc analysis by Tukey's multiple comparison test). Results were expressed as means ± SEM from 3 independent experiments in triplicate. (B) The effects of NE on [³H]DA uptake capacity were measured after 7 days following LPS treatment in neuron-glia cultures and the results were expressed as a percentage of the control cultures. The result showed that the protective effect in the β2-AR^−/− culture was 15% (NE/LPS 62.0 ± 0.8 % versus LPS 47.0 ± 1.3 % of control; n = 3) whereas it was 38% in the WT culture (NE/LPS 80.8 ± 8.9 % versus LPS 42.6 ± 1.3 % of control; n = 3) (two-way ANOVA, F _{(3, 16)} = 7.712, p = 0.0021; post hoc analysis by Tukey's multiple comparison test). Results were expressed as the means ± SEM from 3 independent experiments in triplicate. *p< 0.05, **p< 0.01 compared with the corresponding LPS alone cultures, ^#p < 0.05 compared to WT.

Figure 6. Sub-micromolar NE inhibits NOX2-generated superoxide in a β2-AR-independent manner

(A-B) Mixed-glia cultures prepared from rat postnatal day-1 pup, or COS7-PHOX cells transfected with all subunits of NOX2 were pre-treated with NE, including (−)- or (+)-isomers, for 15 minutes before LPS or phorbolmyristate acetate (PMA) stimulation, respectively. The production of extracellular superoxide was detected by SOD-inhibitable reduction of WST-1. (A) In LPS-treated mixed-glia cultures, (+)-NE and (−)-NE were equi-potent in inhibiting LPS-induced superoxide production ((−)-NE 49.8 ± 7.2 versus (+)-NE 43.7 ± 4.0 % of LPS alone; n = 5) (one-way ANOVA, F _{(5, 24)} = 22.03, p< 0.0001;post hoc analysis by Tukey's multiple comparison test). Results were expressed as the percentage of LPS alone-treated group and were the means ± SEM from 5 independent experiments in triplicate. **p< 0.01 compare with the LPS-treated cultures. (B) In phorbolmyristate acetate (PMA)-treatedCOS7-NOX2 cells, (+)-NE and (−)-NE also showed the comparable potency in inhibiting superoxide production ((−)-NE 43.5 ± 3.3 versus (+)-NE 37.4 ± 5.2 % of LPS alone; n = 5) (one-way ANOVA, F _{(5, 24)} = 55.07, p< 0.0001; post hoc analysis by Tukey's multiple comparison test). Results were expressed as the percentage of PMA alone-treated group and were the means ± SEM from 5 independent experiments in triplicate. **p< 0.01 compare with the PMA-treated cultures. (C) Mixed-glia cultures prepared from β2-AR^−/− and WT mice were pretreated with NE for 15 minutes before the addition of LPS. The effects of NE on superoxide production were determined. The similar superoxide inhibition was found in wild type (43.6 ± 1.9 % of LPS alone; n = 5) and β2-AR^−/−mice (46.6 ± 2.4 % of LPS alone; n = 5)(two-way ANOVA, F _{(3, 32)} = 0.6678, p = 0.5780; post hoc analysis by Tukey's multiple comparison test). Results were expressed as the percentage of LPS alone-treated group and were the means ± SEM from 5 independent experiments in triplicate. **p< 0.01 compare with the LPS-treated cultures. (D) Mixed-glia cultures prepared from rat postnatal day-1 pup were incubated with the non-selective α-AR blocker phentolamine or β-AR blocker propranolol alone or in combination for 15 minutes before the pretreatment of NE. Fifteen minutes after NE addition, the production of LPS-induced extracellular superoxide was measured. A similar experiment was performed with a protein kinase A (PKA) inhibitor H-89. Blockade of adrenergic receptors or inhibition of PKA didn't affect the inhibitory effect of NE on LPS-induced superoxide production (one-way ANOVA, F _{(6, 14)} = 8.652, p = 0.0005;post hoc analysis by Tukey's multiple comparison test). Results were expressed as the percentage of LPS alone-treated group and were the means ± SEM from 3 independent experiments in triplicate. **p< 0.01 compare with the LPS-treated cultures. (E) NE isomers failed to inhibit xanthine/xanthine oxidase-generated superoxide.(−)- or (+)-NE, xanthine oxidase (10 mU), and WST-1 (250 μM) were mixed in potassium phosphate buffer (PBS, 50 mM, pH 7.6). Xanthine (50 μM, final concentration) was added to initiate the reaction (final volume, 1 ml). Absorbance at 450 nm was continuously monitored. Results are expressed as the means of O.D. value ± SEM from 5 independent experiments in triplicate. Either (−)-NE or (+)-NE failed to scavenge the produced free radical compared to positive control SOD.

Figure 7. Sub-micromolar NE prevents LPS-induced membrane translocation of the cytosolic subunit p47^phox

HAPI microglia cells were pretreated with NE for 30 minutes and followed by LPS stimulation. Fifteen minutes later, cells were harvested, and the fractions of membrane and cytosol were isolated for Western blot analysis of p47^phoxlevels. GAPDH and gp91^phoxwere used for internal cytosolic and membrane controls, respectively. The band density including cytosolic and membrane fractions were quantified. (A) The results showed that NE pretreatment inhibited the LPS-induced p47^phox increase in cell membrane (NE/LPS 108.8 ± 4.1 % of control vs. LPS alone 147.7 ± 10.7 % of control; n = 3) (one-way ANOVA, F _{(3, 8)} = 10.70, p = 0.0036; post hoc analysis by Tukey's multiple comparison test). (B) The band showed the decreased level of p47^phox in cytosolic. The quantified data described 20% decrease of p47^phox after LPS stimulation, but not NE pre-treated group (NE/LPS 92.3 ± 5.6 % of control vs. LPS 71.3 ± 4.2 % of control; n = 3)(one-way ANOVA, F _{(3, 8)} = 10.43, p = 0.0039; post hoc analysis by Tukey's multiple comparison test). The results were expressed as a percentage of the control and were mean ± SEM from three independent experiments performed in triplicate.**p< 0.01 compared with control group, ^#p< 0.05 compared with LPS alone group.

Figure 8. NOX2 mediates β2-AR-independent anti-inflammatory/neuroprotective effects of sub-micromolar NE

(A-B) β2-AR genetic ablated neuron-glia cultures were pre-treated with 10⁻¹³ M DPI for 30 minutes. Cultures were then treated with NE for 30 minutes prior to LPS insult. (A) Levels of TNF-α in the supernatant were measured 3 hours after LPS treatment. In β2-AR^−/− culture, the inhibitory effects of DPI (495.14 ± 31.68 pg/ml; n = 5) on LPS-induced TNF-α production were comparable to those of NE (471.07 ± 29.28 pg/ml; n = 5) and no further reduction was observed when DPI and NE were combined (one-way ANOVA, F _{(4, 20)} = 87.62, p< 0.0001; post hoc analysis by Tukey's multiple comparisons test). The results were expressed as means ± SEM from 5 independent experiments in triplicate. (B) The protective effects of NE on dopaminergic neurons were determined by [³H]DA uptake assay after 7 days of LPS insult and the results were expressed as percentage of controls (means ± SEM) from 5 independent experiments in triplicate. The neuroprotective effect of DPI (70.40 ± 2.43 % of control; n = 5) on LPS-induced neurotoxicity were comparable to those of NE (77.22 ± 1.91 % of control; n = 5) (one-way ANOVA, F _{(4, 20)} = 97.76, p< 0.0001) and no additive protection was observed when DPI and NE were combined (post hoc analysis by Tukey's multiple comparisons test, p> 0.05). (C-D) Neuron-glia cultures prepared from gp91^−/− (CYBB) and C57BL/6 mice were pre-treated with a β2-AR antagonist (ICI-118,551) for 30 minutes followed by 30 minutes NE treatment. Then the cultures were stimulated by LPS of 20 ng/ml for C57BL/6 and 50 ng/ml for gp91^−/−. TNF-α production in the supernatant were detected at 3 hours after LPS addition. The results were expressed as means ± SEM from 3 independent experiments in triplicate. With β2-AR blockade in C57BL/6 culture, NE still reduced TNF-α production (966.7 ± 40.9 pg/ml) compared to LPS alone (1151.9 ± 55.6 pg/ml) (one-way ANOVA, F _{(6, 14)} = 283.0, p< 0.0001, post hoc analysis by Tukey's multiple comparison test), while NE failed to reduce TNF-α production in CYBB culture after β2-AR blockade (one-way ANOVA, F _{(6, 14)} = 80.12, post hoc analysis by Tukey's multiple comparison test, p> 0.05). (E-F) The DA uptake assay was performed at 7 days after treatment. The results were expressed as percentage of control alone group (means ± SEM) from 3 independent experiments in triplicate. In C57BL/6 neuron-glia culture, NE pretreatment restored DA uptake (73.54 ± 2.68% of control) compared to LPS alone (51.56 ± 2.93% of control) even with β2-AR blockade (one-way ANOVA, F _{(6, 14)} = 86.31, p< 0.0001; post hoc analysis by Tukey's multiple comparison test); Whereas in CYBB neuron-glia culture, NE failed to restore DA uptake with β2-AR blockade (one-way ANOVA, F _{(6, 14)} = 59.18, p<0.0001; post hoc analysis by Tukey's multiple comparison test, p> 0.05).*p< 0.05, **p< 0.01, compare with the LPS alone-treated cells.