Proteasome serves as pivotal regulator in Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis

Abstract

Proteasome primarily degrades the unneeded or damaged proteins by proteolysis. Disruption of the brain barrier and its resulting meningoencephalitis caused by Angiostrongylus cantonensis are important pathological events in non-permissive hosts. In this study, the results showed upregulated proteasome during A. cantonensis infection. Occludin degradation and matrix metalloproteinase-9 (MMP-9) activity were significantly increased in infected mice than in uninfected mice. Moreover, confocal immunoflourescence microscopy showed that occludin was co-localized with MMP-9. The infected-mice were treated with proteasomal activity inhibitor MG132 by 1.5 and 3.0 mg/kg/day, which resulted in significantly reduced protein levels of phosphorylated IκBα (P<0.05) compared with the untreated control. The phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) showed similar result. In addition, MMP-9 activity and occludin degradation were reduced because of MG132 treatment. These results suggested that the proteasome in A. cantonensis infection degraded phosphorylated IκBα, modulated phosphorylated NF-κB, and then regulated the activation of MMP-9 and occludin degradation. Proteasome alterations were presented in eosinophilic meningitis of BALB/c mice and may contribute to the pathophysiology of eosinophilic meningitis by increasing occludin degradation. This molecule would serve as pivotal regulator in A. cantonensis-induced eosinophilic meningoencephalitis.

Fig 1. Time-course studies for proteasome.

The protein levels of proteasome from the (a) brain and (c) cerebrospinal fluid (CSF) of mice infected with Angiostrongylus cantonensis. β-actin was used as a loading control. (b, d) Quantification and normalization of the proteasome to β-actin was performed with a computer-assisted imaging densitometer system. The data are presented as the mean±SD of three independent experiments in duplicate. * indicates a statistically significant difference.

Fig 2. Time-course studies for occludin.

(a) The protein levels of occludin from the brain of mice infected with Angiostrongylus cantonensis. β-actin was used as a loading control. (b) Quantification and normalization of occludin to β-actin was performed with a computer-assisted imaging densitometer system. ^# indicates a statistically significant increase. * indicates a statistically significant decrease. (c) The protein levels of occludin from the CSF of mice infected with A. cantonensis. (d) The relative density was quantified using a computer-assisted imaging densitometer system. * indicates a statistically significant increase.

Fig 3. Localization of occludin and matrix metalloproteinase (MMP)-9 in the choroid plexus of the mouse brain.

(a) The epithelium of the choroid plexus were stained with 4’,6-diamino-2-phenylindole (DAPI) for nuclei (blue); (b) DyLight 488-stained for occludin (green); (c) Rhodamine red-stained for MMP-9 (red); (d) The merged image shows colocalization of Dylight 488-labeled peptide H4 with Rhodamine B-labeled lipid in the epithelial cells of choroid plexus (yellow, d). Scale bar = 20 μm.

Fig 4. Interaction between occludin and MMP-9 in mice infected with Angiostrongylus cantonensis.

Occludin interacted with MMP-9 in brain homogenates. Control, uninfected mice. Infection, mice infected with A. cantonensis on day 20 post-infection. The arrowheads indicate MMP-9 bands. H, immunoglobulin heavy chain. L, immunoglobulin light chain. Immunoprecipitates (IP): occludin. Immunoblotting (IB): MMP-9.

Fig 5. Influence of treatment on IκB-α and p-IκB-α in the brain.

(a) Protein bands of IκB-α and p-IκB-α from the brain of mice infected with A. cantonensis and treated with MG132. β-actin was used as a loading control. (b) Quantification of phosphorylation ratios [intensity of phosphorylated IκB-α (p-IκB-α) divided by the intensity of total IκB-α (t-IκB-α)] following normalization for β-actin. The data are presented as the mean± SD of three independent experiments in duplicate. ^# indicates a statistically significant increase.* indicates a statistically a significant decrease.

Fig 6. Influence of treatment on NF-κB in the brain.

(a) Protein bands of NF-κB p65 and p-p65 from the brain of mice infected with A. cantonensis and treated with MG132. β-actin was used as a loading control. (b) Quantification of phosphorylation ratios [intensity of phosphorylated p65 (p-p65) divided by the intensity of total p65 (t-p65)] following normalization for β-actin. The data are presented as the mean±SD of three independent experiments in duplicate. ^# indicates a statistically significant increase. * indicates a statistically significant decrease.

Fig 7. Influence of treatment on MMP-9.

The activities of MMP-9 from the (a) brain and (c) CSF of mice infected with A. cantonensis and treated with MG132. (b, d) The relative intensity was quantified using a computer-assisted imaging densitometer system. Bars represent mean ± S.D. (n = 5 per group). ^# indicates a statistically significant increase.* indicates a statistically significant decrease.

Fig 8. Influence of treatment on occludin.

(a) The protein levels of occludin from the brain of mice infected with Angiostrongylus cantonensis and MG132 treatment. β-actin was used as a loading control. (b) Quantification and normalization of occludin to β-actin were performed with a computer-assisted imaging densitometer system. ^# indicates a statistically significant decrease. * indicates a statistically significant increase. (c) The protein levels of occludin from the CSF of mice infected with A. cantonensis and MG132 treatment. (d) The relative density was quantified using a computer-assisted imaging densitometer system. ^# indicates a statistically significant increase. * indicates a statistically significant decrease. Bars represent mean ± S.D. (n = 5 per group).

Fig 9. Influence of MG132 on BBB permeability.

Mouse BBB permeability was detected by performing Evans Blue extravasation during Angiostrongylus cantonensis infection. The Evans blue units were significantly decreased (P < 0.05) in the MG132 treatment group compared with those of the A. cantonensis-infected mice. ^# indicates a statistically significant increase. * indicates a statistically significant decrease.