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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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Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Material and Methods. (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (*P<0.05, **P<0.001) and CD45RB Ab compared with isotype-control IgG, P<0.001. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
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pone-0002135-g003: Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Material and Methods. (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (*P<0.05, **P<0.001) and CD45RB Ab compared with isotype-control IgG, P<0.001. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.

Mentions: Our previous studies have shown either SB203580 or PD98059 inhibitor markedly attenuate CD40 signaling-stimulated activation of p38 or p44/42 MAPK in microglial cells [13], [29]. To determine the role of the p38 and/or p44/42 MAPK cascades in the CD45RB signaling-mediated microglial phagocytosis of Aβ peptide, microglial cells were pre-treated with SB203580 (5 µM) or PD98059 (5 µM) for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 (500 nM) for 2 h in the absence (control) or presence of CD45RB antibody or isotype control IgG. Cell culture supernatants were collected and cell lysates were prepared to measure Aβ by fluorometer (Fig. 3A). In parallel experiments, microglial cells were treated as above. Cell lysates were then prepared and assessed by Aβ ELISA (Fig. 3B for SB203580 and Fig. 3C for PD98059). These data collectively showed that cross-linking CD45RB boosts microglial phagocytosis of Aβ peptide, which was enhanced by inhibition of p38 or p44/42 activities.


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)

Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Material and Methods. (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (*P<0.05, **P<0.001) and CD45RB Ab compared with isotype-control IgG, P<0.001. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
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pone-0002135-g003: Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ1–42 peptide.Microglial treatment conditions are indicated and are further described in Material and Methods. (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (*P<0.05, **P<0.001) and CD45RB Ab compared with isotype-control IgG, P<0.001. Note: SB = SB203580, PD = PD98059, Ab = antibody, pp = phosphorylatioin.
Mentions: Our previous studies have shown either SB203580 or PD98059 inhibitor markedly attenuate CD40 signaling-stimulated activation of p38 or p44/42 MAPK in microglial cells [13], [29]. To determine the role of the p38 and/or p44/42 MAPK cascades in the CD45RB signaling-mediated microglial phagocytosis of Aβ peptide, microglial cells were pre-treated with SB203580 (5 µM) or PD98059 (5 µM) for 1 h, then co-treated with “aged” FITC-tagged Aβ1–42 (500 nM) for 2 h in the absence (control) or presence of CD45RB antibody or isotype control IgG. Cell culture supernatants were collected and cell lysates were prepared to measure Aβ by fluorometer (Fig. 3A). In parallel experiments, microglial cells were treated as above. Cell lysates were then prepared and assessed by Aβ ELISA (Fig. 3B for SB203580 and Fig. 3C for PD98059). These data collectively showed that cross-linking CD45RB boosts microglial phagocytosis of Aβ peptide, which was enhanced by inhibition of p38 or p44/42 activities.

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