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CD45RB is a novel molecular therapeutic target to inhibit Abeta peptide-induced microglial MAPK activation.

Zhu Y, Hou H, Nikolic WV, Ehrhart J, Rrapo E, Bickford P, Giunta B, Tan J - PLoS ONE (2008)

Bottom Line: Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-alpha and IL-6 release through p44/42 and/or p38 pathways.Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Abeta lowering, and inflammation reducing, properties that are particularly targeted at microglial cells.Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Rashid Laboratory Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, Florida, United States of America.

ABSTRACT

Background: Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer's disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed beta-amyloid (Abeta) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.

Methodology and results: In this study, CD45RB modulation of Abeta peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with "aged" FITC-Abeta(1-42) and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Abeta(1-42) peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-alpha and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Abeta(1-42) peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Abeta(1-42) peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-alpha and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Abeta(1-42) peptide, inhibits co-localization of microglial MHC class II and Abeta peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.

Conclusion: In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Abeta phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Abeta lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

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Cross-linking of CD45RB markedly inhibits phen and Aβ peptide-induced microglial activation.(A) Microglial cells were treated as indicated for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD, picograms per milligram of total protein) in cultured media (top and bottom panels). Data are representative of three independent experiments (**P<0.001). (B–D) Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated p38 MAPK at the indicated time points. Phosphorylation of p38 MAPK is indicated in (B) and inhibition of this effect by CD45RB Ab is indicated in Fig C or by SB in Fig. D. Histograms below the immunoblots represent the mean band density ratio±1 SD (p38/total p38 MAPK; n = 3 for each condition presented). (E) Microglial cells were pre-treated with SB230580 or CD45RB antibody for 1 h, then co-treated with phen and/or aged Aβ1–42 for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD) in cultured media (top and bottom panels; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant differences (*P<0.05, ** P<0.001). Note: SB = SB203580, Ab = antibody, pp = phosphorylation).
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pone-0002135-g004: Cross-linking of CD45RB markedly inhibits phen and Aβ peptide-induced microglial activation.(A) Microglial cells were treated as indicated for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD, picograms per milligram of total protein) in cultured media (top and bottom panels). Data are representative of three independent experiments (**P<0.001). (B–D) Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated p38 MAPK at the indicated time points. Phosphorylation of p38 MAPK is indicated in (B) and inhibition of this effect by CD45RB Ab is indicated in Fig C or by SB in Fig. D. Histograms below the immunoblots represent the mean band density ratio±1 SD (p38/total p38 MAPK; n = 3 for each condition presented). (E) Microglial cells were pre-treated with SB230580 or CD45RB antibody for 1 h, then co-treated with phen and/or aged Aβ1–42 for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD) in cultured media (top and bottom panels; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant differences (*P<0.05, ** P<0.001). Note: SB = SB203580, Ab = antibody, pp = phosphorylation).

Mentions: It has been reported that a tyrosine phosphorylation cascade plays an important role in Aβ-induced microglial activation [30], [31]. Our previous studies demonstrated phen (a specific tyrosine phosphatase inhibitor) synergistically enhanced Aβ-stimulated microglial activation [27]. CD45, a protein-tyrosine phosphatase that is constitutively expressed on microglia [25], is markedly increased on microglia from AD frontal cortices [24], [32]. To investigate the role of CD45RB in microglial activation, we treated primary cultured microglial cells with monoclonal CD45RB antibody before stimulation with phen and Aβ peptide. Microglial activation, as evidenced by TNF-α and IL-6 release after co-treatment of microglia with phen and Aβ peptide, was significantly inhibited by cross-linking CD45RB (Fig. 4A, top and bottom panels). Our previous studies have confirmed this result in vivo [11].


CD45RB is a novel molecular therapeutic target to inhibit Abeta peptide-induced microglial MAPK activation.

Zhu Y, Hou H, Nikolic WV, Ehrhart J, Rrapo E, Bickford P, Giunta B, Tan J - PLoS ONE (2008)

Cross-linking of CD45RB markedly inhibits phen and Aβ peptide-induced microglial activation.(A) Microglial cells were treated as indicated for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD, picograms per milligram of total protein) in cultured media (top and bottom panels). Data are representative of three independent experiments (**P<0.001). (B–D) Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated p38 MAPK at the indicated time points. Phosphorylation of p38 MAPK is indicated in (B) and inhibition of this effect by CD45RB Ab is indicated in Fig C or by SB in Fig. D. Histograms below the immunoblots represent the mean band density ratio±1 SD (p38/total p38 MAPK; n = 3 for each condition presented). (E) Microglial cells were pre-treated with SB230580 or CD45RB antibody for 1 h, then co-treated with phen and/or aged Aβ1–42 for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD) in cultured media (top and bottom panels; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant differences (*P<0.05, ** P<0.001). Note: SB = SB203580, Ab = antibody, pp = phosphorylation).
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pone-0002135-g004: Cross-linking of CD45RB markedly inhibits phen and Aβ peptide-induced microglial activation.(A) Microglial cells were treated as indicated for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD, picograms per milligram of total protein) in cultured media (top and bottom panels). Data are representative of three independent experiments (**P<0.001). (B–D) Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated p38 MAPK at the indicated time points. Phosphorylation of p38 MAPK is indicated in (B) and inhibition of this effect by CD45RB Ab is indicated in Fig C or by SB in Fig. D. Histograms below the immunoblots represent the mean band density ratio±1 SD (p38/total p38 MAPK; n = 3 for each condition presented). (E) Microglial cells were pre-treated with SB230580 or CD45RB antibody for 1 h, then co-treated with phen and/or aged Aβ1–42 for 16 h. Microglial activation was determined by TNF-α and IL-6 production (mean±1 SD) in cultured media (top and bottom panels; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant differences (*P<0.05, ** P<0.001). Note: SB = SB203580, Ab = antibody, pp = phosphorylation).
Mentions: It has been reported that a tyrosine phosphorylation cascade plays an important role in Aβ-induced microglial activation [30], [31]. Our previous studies demonstrated phen (a specific tyrosine phosphatase inhibitor) synergistically enhanced Aβ-stimulated microglial activation [27]. CD45, a protein-tyrosine phosphatase that is constitutively expressed on microglia [25], is markedly increased on microglia from AD frontal cortices [24], [32]. To investigate the role of CD45RB in microglial activation, we treated primary cultured microglial cells with monoclonal CD45RB antibody before stimulation with phen and Aβ peptide. Microglial activation, as evidenced by TNF-α and IL-6 release after co-treatment of microglia with phen and Aβ peptide, was significantly inhibited by cross-linking CD45RB (Fig. 4A, top and bottom panels). Our previous studies have confirmed this result in vivo [11].

Bottom Line: Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-alpha and IL-6 release through p44/42 and/or p38 pathways.Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Abeta lowering, and inflammation reducing, properties that are particularly targeted at microglial cells.Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Rashid Laboratory Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, Florida, United States of America.

ABSTRACT

Background: Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer's disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed beta-amyloid (Abeta) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.

Methodology and results: In this study, CD45RB modulation of Abeta peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with "aged" FITC-Abeta(1-42) and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Abeta(1-42) peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-alpha and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Abeta(1-42) peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Abeta(1-42) peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-alpha and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Abeta(1-42) peptide, inhibits co-localization of microglial MHC class II and Abeta peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.

Conclusion: In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Abeta phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Abeta lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

Show MeSH
Related in: MedlinePlus