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Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy.

Ito T, Yoshida K, Negishi T, Miyajima M, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Int. J. Mol. Med. (2014)

Bottom Line: In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control.These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia.Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan.

ABSTRACT
Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (-/-) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1-/- mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1-/- mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

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Plexin-A1 is expressed in Iba-1-positive microglia in mouse hippocampus. (A) Plexin-A1-positive immunoreactivity shows a similar distribution pattern with the positive immunostaining for microglial marker Iba-1. (B) Overlaid image of Iba-1 and Plexin-A1 immunofluorescence in WT hippocampus is shown at a higher magnification of the selected area in (A). Bars, 100 μm. A1−/−, Plexin-A1−/− mouse hippocampus; DAPI, DAPI nuclear staining.
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f3-ijmm-33-05-1122: Plexin-A1 is expressed in Iba-1-positive microglia in mouse hippocampus. (A) Plexin-A1-positive immunoreactivity shows a similar distribution pattern with the positive immunostaining for microglial marker Iba-1. (B) Overlaid image of Iba-1 and Plexin-A1 immunofluorescence in WT hippocampus is shown at a higher magnification of the selected area in (A). Bars, 100 μm. A1−/−, Plexin-A1−/− mouse hippocampus; DAPI, DAPI nuclear staining.

Mentions: Primary microglia were isolated and purified from postnatal mouse brain tissue. For all microglial cultures, purity as determined by lectin cytochemistry analysis was >95%. RNA was prepared from hippocampi and primary microglia and analyzed by RT-PCR. As a result, gene transcripts for Plexin-A1 and Neuropilin-1 were detected in the hippocampus and in primary microglia (Fig. 1A). RT-PCR also detected the expression of Sema3A mRNA in the mouse hippocampus (Fig. 1A). To confirm the expression of Plexin-A1 protein in mouse microglia, western blotting was performed using protein extracts from WT and PlexinA1−/− primary microglia (Fig. 1B). The analysis detected Plexin-A1 protein in WT, but not in Plexin-A1−/− microglia (Fig. 1B). Western blotting also detected Sema3A protein, which in mouse hippocampus is a ligand of Plexin-A1 (Fig. 1B). Expression of Plexin-A1 was detected in primary microglia by double labeling with lectin staining for microglia identification and by Plexin-A1 immunocytochemistry (Fig. 2A and B). In total, 98.6±2.3% (mean ± SEM) of the primary microglia exhibited positive staining for Plexin-A1. To examine the localization of Plexin-A1 in mouse brain, immunohistochemical analyses were performed on hippocampi of WT and Plexin-A1−/− mice. The antibodies against Plexin-A1 detected Plexin-A1 in the microglia in WT (Fig. 3A and B), but not Plexin-A1−/− mice (Fig. 3A).


Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy.

Ito T, Yoshida K, Negishi T, Miyajima M, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Int. J. Mol. Med. (2014)

Plexin-A1 is expressed in Iba-1-positive microglia in mouse hippocampus. (A) Plexin-A1-positive immunoreactivity shows a similar distribution pattern with the positive immunostaining for microglial marker Iba-1. (B) Overlaid image of Iba-1 and Plexin-A1 immunofluorescence in WT hippocampus is shown at a higher magnification of the selected area in (A). Bars, 100 μm. A1−/−, Plexin-A1−/− mouse hippocampus; DAPI, DAPI nuclear staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020473&req=5

f3-ijmm-33-05-1122: Plexin-A1 is expressed in Iba-1-positive microglia in mouse hippocampus. (A) Plexin-A1-positive immunoreactivity shows a similar distribution pattern with the positive immunostaining for microglial marker Iba-1. (B) Overlaid image of Iba-1 and Plexin-A1 immunofluorescence in WT hippocampus is shown at a higher magnification of the selected area in (A). Bars, 100 μm. A1−/−, Plexin-A1−/− mouse hippocampus; DAPI, DAPI nuclear staining.
Mentions: Primary microglia were isolated and purified from postnatal mouse brain tissue. For all microglial cultures, purity as determined by lectin cytochemistry analysis was >95%. RNA was prepared from hippocampi and primary microglia and analyzed by RT-PCR. As a result, gene transcripts for Plexin-A1 and Neuropilin-1 were detected in the hippocampus and in primary microglia (Fig. 1A). RT-PCR also detected the expression of Sema3A mRNA in the mouse hippocampus (Fig. 1A). To confirm the expression of Plexin-A1 protein in mouse microglia, western blotting was performed using protein extracts from WT and PlexinA1−/− primary microglia (Fig. 1B). The analysis detected Plexin-A1 protein in WT, but not in Plexin-A1−/− microglia (Fig. 1B). Western blotting also detected Sema3A protein, which in mouse hippocampus is a ligand of Plexin-A1 (Fig. 1B). Expression of Plexin-A1 was detected in primary microglia by double labeling with lectin staining for microglia identification and by Plexin-A1 immunocytochemistry (Fig. 2A and B). In total, 98.6±2.3% (mean ± SEM) of the primary microglia exhibited positive staining for Plexin-A1. To examine the localization of Plexin-A1 in mouse brain, immunohistochemical analyses were performed on hippocampi of WT and Plexin-A1−/− mice. The antibodies against Plexin-A1 detected Plexin-A1 in the microglia in WT (Fig. 3A and B), but not Plexin-A1−/− mice (Fig. 3A).

Bottom Line: In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control.These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia.Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan.

ABSTRACT
Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (-/-) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1-/- mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1-/- mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

Show MeSH
Related in: MedlinePlus