Inhibition of IkappaB kinase-beta protects dopamine neurons against lipopolysaccharide-induced neurotoxicity

Abstract

Parkinson's disease (PD) is a progressive neurological disorder characterized by a selective loss of dopamine (DA) neurons in the substantia nigra (SN). Although current therapy can control symptoms of this disorder, there is no effective therapy available to halt its progression. Recently, neuroinflammation has been recognized as an important contributor to the pathogenesis of PD, and nuclear factor-kappaB (NF-kappaB) plays a key role in regulating neuroinflammation. Hence, the modulation of NF-kappaB pathway may have therapeutic potential for PD. Activation of NF-kappaB depends on the phosphorylation of its inhibitor, IkappaB, by the specific IkappaB kinase (IKK) subunit IKK-beta. Compound A (7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1, 4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride), a potent and selective inhibitor of IKK-beta, has recently been reported to provide cardioprotection through specific suppression of NF-kappaB signaling. The present study, for the first time, elucidates neuroprotective effects of compound A. Daily subcutaneous injection of compound A (1 mg/kg) for 7 days inhibited the activation of microglia induced by nigral stereotaxic injection of lipopolysaccharide (LPS) and significantly attenuated LPS-induced loss of DA neurons in the SN. In vitro mechanistic studies revealed that neuroprotective effects of compound A were mediated by 1) suppressing the activity of microglial NADPH oxidase and decreasing the production of reactive oxygen species, and 2) inhibiting NF-kappaB-mediated gene transcription of various proinflammatory mediators in microglia via IKK-beta suppression. These findings indicate that compound A afforded potent neuroprotection against LPS-induced neurodegeneration through selective inhibition of NF-kappaB activation and may be of potential benefit in the treatment of PD.

Fig. 1.

Compound A protected DA neurons against LPS-induced neurotoxicity. Rat primary mesencephalic neuron-glia cultures seeded in 24-well culture plates at 5 × 10⁵/well were pretreated with various concentrations of compound A for 30 min before the addition of 10 ng/ml LPS. A and B, 7 days later, the LPS-induced DA neurotoxicity was quantified by the [³H]DA uptake assay (A) and TH-positive neuron counting using immunocytochemical analysis (B). C, representative images of immunostaining from three experiments are shown. Scale bar, 200 μm. For the time course study, the DA neurotoxicity was measured by [³H]DA uptake assay at 1, 3, 5 and 7 days after LPS treatment. Results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 2.

Microglia were indispensable to compound A-mediated neuroprotection. Four types of cultures prepared as described in Materials and Methods were pretreated with compound A (0.1 μM) for 30 min followed by the administration of MPP⁺ (0.5 μM). Seven days after MPP⁺ treatment, DA neurotoxicity was quantified by [³H]DA uptake assay. Results are expressed as a percentage of vehicle control cultures and are the mean ± S.E. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with MPP⁺-treated cultures.

Fig. 3.

Compound A inhibited the production of proinflammatory factors by LPS-activated microglia. Neuron-glia cultures were pretreated with compound A (0.025–0.1 μM) for 30 min and then stimulated with 10 ng/ml LPS. A, after 3 h (TNFα) or 1 day (NO and IL-1β), an aliquot of the supernatant was collected for enzyme-linked immunosorbent assay analysis of TNFα and IL-1β and Griess reaction analysis of nitrite levels. For mRNA assessment, total RNA was harvested 1 h (TNFα) or 3 h (iNOS and IL-1β) after LPS treatment. B, the gene expressions were determined by real-time reverse transcription–PCR. C, Western blot analysis revealed iNOS protein expression 1 day after LPS treatment. Results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 4.

Compound A inhibited NF-κB signaling pathway by Western blot analysis. Primary microglia-enriched cultures were pretreated with compound A (0.1 μM) for 30 min and then stimulated with LPS for 15 min. The levels of phosphorylated p65 and IKK-β compared with total p65 and IKK-β (A) and phosphorylated IκBα and total IκBα relative to β-actin (B) were investigated by Western blot analysis. Quantified results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 5.

Compound A suppressed MAPK phosphorylation in activated microglia by Western blot analysis. Primary microglia-enriched cultures were pretreated with compound A (0.1 μM) for 30 min and then stimulated with LPS for 15 min. After the treatment, the whole cell protein was harvested. A, the levels of phosphorylated MAPKs (ERK1/2, p38, and JNK) compared with total MAPKs were investigated by Western blot analysis. B, autoradiographs were analyzed by densitometry. Quantified results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 6.

Compound A attenuated LPS-induced production of ROS through the inhibition of NADPH oxidase. Primary microglia-enriched cultures were pretreated with compound A (0.1 μM) for 30 min followed by LPS treatment. A and B, the release of superoxide was determined immediately by measuring the SOD-inhibitable reduction of WST-1 (A), and the levels of intracellular ROS were detected with DCFH-DA (B). After LPS treatment for 15 min, subcellular fractions were isolated for Western blot analysis for p47^PHOX levels in membrane and cytosolic fractions of microglia. Lanes C, cytosolic extract; M, membrane extract. C, β-actin and gp91^PHOX were used as internal cytosolic and membrane controls, respectively. Results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments performed in triplicate. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.

Fig. 7.

Compound A attenuated DA neuronal loss in the SN induced by LPS in vivo. A, the treatment schedule of LPS-intoxicated rats with compound A is shown. Rats were pretreated with compound A (1 mg/kg/day s.c.) before a single intranigral injection of LPS (5 μg) into the SN pars compacta on the right side of the rat brain. Seven days after LPS injection, brains were harvested and sectioned. B and C, the sections were immunostained with an anti-TH antibody (B) and the number of TH-positive neurons in the SN was counted (C). Scale bar, 200 μm. Results are expressed as a percentage of the TH-positive number of the vehicle control group and are the mean ± S.E. from six rats. #, p < 0.05 compared with control groups; *, p < 0.05 compared with LPS-treated groups.

Fig. 8.

Compound A inhibited LPS-induced microglial activation. Rats were treated with compound A (1 mg/kg/day s.c.) before the injection of LPS (5 μg). A and B, 7 days later, the brains were harvested, sectioned, and immunostained with OX-42 antibody. The images are representative of six rats in each group. Scale bar, 200 μm; inset, 100 μm. B, the quantification of the OX-42 staining. Densitometry analysis of nigral OX-42-positive microglia from six evenly spaced brain sections from each rat was performed with Image J software. Quantified results are expressed as a percentage of the control and are the mean ± S.E. from six rats. To further confirm the inhibitory effects of compound A on LPS-induced microglia activation, primary midbrain neuron-glia cultures were pretreated with compound A (0.1 μM) for 30 min followed by LPS (10 ng/ml) treatment. C, 7 days later the levels of Iba-1 were determined by Western blot analysis. Quantified results are expressed as a percentage of the vehicle control cultures and are the mean ± S.E. from three independent experiments. #, p < 0.05 compared with control cultures; *, p < 0.05 compared with LPS-treated cultures.