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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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Related in: MedlinePlus

Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Materials and Method. Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; *P<0.05, **P<0.001 compared with control). (C) Microglial phagocytosis of Aβ1–42 peptide after pre-treatment with PD98059 or SB203580 for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 and LPS. Supernatants and cell lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer (*P<0.05, **P<0.001). (D) Phosphorylation of p38 MAPK and inhibition of this effect by CD45RB antibody. Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38/total p38; n = 3 for each condition presented; *P<0.05 compared with LPS or LPS/IgG). (E) Microglial activation is evidenced by mean TNF-α and IL-6 release±1 SD (n = 3 for each condition presented; *P<0.05; **P<0.001 compared with LPS or LPS/IgG; P<0.05 compared LPS/SB with LPS/CD45RB Aβ/SB). For A–E, one-way ANOVA followed by post hoc Bonferroni testing was utilized. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
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pone-0002135-g002: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Materials and Method. Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; *P<0.05, **P<0.001 compared with control). (C) Microglial phagocytosis of Aβ1–42 peptide after pre-treatment with PD98059 or SB203580 for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 and LPS. Supernatants and cell lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer (*P<0.05, **P<0.001). (D) Phosphorylation of p38 MAPK and inhibition of this effect by CD45RB antibody. Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38/total p38; n = 3 for each condition presented; *P<0.05 compared with LPS or LPS/IgG). (E) Microglial activation is evidenced by mean TNF-α and IL-6 release±1 SD (n = 3 for each condition presented; *P<0.05; **P<0.001 compared with LPS or LPS/IgG; P<0.05 compared LPS/SB with LPS/CD45RB Aβ/SB). For A–E, one-way ANOVA followed by post hoc Bonferroni testing was utilized. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.

Mentions: It has been reported that the MAPK pathway is central to the biological activities of LPS [28]. This was evidenced by a rapid and transient increase in phosphorylation of both p38 and p44/42 in LPS-stimulated microglial cells. We treated microglial cells with SB203580 (SB, 5 µM; an inhibitor of p38 MAPK) or PD98059 (PD, 5 µM; a selective inhibitor of p44/42) for 1 h prior to treatment with LPS (100 ng/mL) for 30 minutes, and found both inhibitors markedly suppressed the activation of LPS-induced p38 or p44/42 MAPKs (Fig. 2A, B). To investigate whether p38 MAPK and/or p44/42 MAPK are involved in microglial phagocytosis of Aβ1–42 peptide, microglial cells were further pre-treated with either of these inhibitors for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 (500 nM) in complete medium for 2 h in the absence (control) or presence of LPS (100 ng/mL). As shown in Fig. 2C (top and bottom panels), both of SB203580 and PD98059 significantly increased microglial phagocytosis of Aβ1–42 peptide, with SB230580 showing a more potent effect, a phenomenon which was reversible by LPS stimulation. Together these data indicate p38 MAPK and/or p44/42 MAPK is involved in negative regulation of microglial phagocytosis of Aβ1–42 peptide.


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)

Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Materials and Method. Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; *P<0.05, **P<0.001 compared with control). (C) Microglial phagocytosis of Aβ1–42 peptide after pre-treatment with PD98059 or SB203580 for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 and LPS. Supernatants and cell lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer (*P<0.05, **P<0.001). (D) Phosphorylation of p38 MAPK and inhibition of this effect by CD45RB antibody. Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38/total p38; n = 3 for each condition presented; *P<0.05 compared with LPS or LPS/IgG). (E) Microglial activation is evidenced by mean TNF-α and IL-6 release±1 SD (n = 3 for each condition presented; *P<0.05; **P<0.001 compared with LPS or LPS/IgG; P<0.05 compared LPS/SB with LPS/CD45RB Aβ/SB). For A–E, one-way ANOVA followed by post hoc Bonferroni testing was utilized. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
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Related In: Results  -  Collection

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pone-0002135-g002: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Materials and Method. Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; *P<0.05, **P<0.001 compared with control). (C) Microglial phagocytosis of Aβ1–42 peptide after pre-treatment with PD98059 or SB203580 for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 and LPS. Supernatants and cell lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer (*P<0.05, **P<0.001). (D) Phosphorylation of p38 MAPK and inhibition of this effect by CD45RB antibody. Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38/total p38; n = 3 for each condition presented; *P<0.05 compared with LPS or LPS/IgG). (E) Microglial activation is evidenced by mean TNF-α and IL-6 release±1 SD (n = 3 for each condition presented; *P<0.05; **P<0.001 compared with LPS or LPS/IgG; P<0.05 compared LPS/SB with LPS/CD45RB Aβ/SB). For A–E, one-way ANOVA followed by post hoc Bonferroni testing was utilized. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
Mentions: It has been reported that the MAPK pathway is central to the biological activities of LPS [28]. This was evidenced by a rapid and transient increase in phosphorylation of both p38 and p44/42 in LPS-stimulated microglial cells. We treated microglial cells with SB203580 (SB, 5 µM; an inhibitor of p38 MAPK) or PD98059 (PD, 5 µM; a selective inhibitor of p44/42) for 1 h prior to treatment with LPS (100 ng/mL) for 30 minutes, and found both inhibitors markedly suppressed the activation of LPS-induced p38 or p44/42 MAPKs (Fig. 2A, B). To investigate whether p38 MAPK and/or p44/42 MAPK are involved in microglial phagocytosis of Aβ1–42 peptide, microglial cells were further pre-treated with either of these inhibitors for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 (500 nM) in complete medium for 2 h in the absence (control) or presence of LPS (100 ng/mL). As shown in Fig. 2C (top and bottom panels), both of SB203580 and PD98059 significantly increased microglial phagocytosis of Aβ1–42 peptide, with SB230580 showing a more potent effect, a phenomenon which was reversible by LPS stimulation. Together these data indicate p38 MAPK and/or p44/42 MAPK is involved in negative regulation of microglial phagocytosis of Aβ1–42 peptide.

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