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The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-β degradation.

Dong W, Embury CM, Lu Y, Whitmire SM, Dyavarshetty B, Gelbard HA, Gendelman HE, Kiyota T - J Neuroinflammation (2016)

Bottom Line: Co-localization of Aβ and endolysosomal markers associated with enhanced Aβ42 degradation was observed.URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aβ degradation.Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.

ABSTRACT

Background: Amyloid-β (Aβ)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer's disease (AD). Mixed-lineage kinases (MLKs) regulate upstream MAPK signaling that include p38 MAPK and c-Jun amino-terminal kinase (JNK). However, whether MLK-MAPK pathways affect Aβ-mediated neuroinflammation is unknown. To this end, we investigated if URMC-099, a brain-penetrant small-molecule MLK type 3 inhibitor, can modulate Aβ trafficking and processing required for generating AD-associated microglial inflammatory responses.

Methods: Aβ1-42 (Aβ42) and/or URMC-099-treated murine microglia were investigated for phosphorylated mitogen-activated protein kinase kinase (MKK)3, MKK4 (p-MKK3, p-MKK4), p38 (p-p38), and JNK (p-JNK). These pathways were studied in tandem with the expression of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Gene expression of the anti-inflammatory cytokines, IL-4 and IL-13, was evaluated by real-time quantitative polymerase chain reaction. Aβ uptake and expression of scavenger receptors were measured. Protein trafficking was assessed by measures of endolysosomal markers using confocal microscopy.

Results: Aβ42-mediated microglial activation pathways were shown by phosphorylation of MKK3, MKK4, p38, and JNK and by expression of IL-1β, IL-6, and TNF-α. URMC-099 modulated microglial inflammatory responses with induction of IL-4 and IL-13. Phagocytosis of Aβ42 was facilitated by URMC-099 with up-regulation of scavenger receptors. Co-localization of Aβ and endolysosomal markers associated with enhanced Aβ42 degradation was observed.

Conclusions: URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aβ degradation. These data demonstrate a new immunomodulatory role for URMC-099 to inhibit MLK and to induce microglial anti-inflammatory responses. Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

No MeSH data available.


Related in: MedlinePlus

URMC-099 inhibits p38 and JNK MAPK signaling cascades in Aβ-stimulated microglia. Immunoblots of p-MKK3 and total MKK3 (a), p-MKK4 (asterisk; non-specific bands) and total MKK4 (c), p-p38 and total p38 (e), p-JNK p54 (top), p46 (bottom), and total JNK (g). Quantification of p-MKK3 (b), p-MKK4 (d) p-p38 (f), p-JNK p54 (h), and p46 (i) levels. Data are presented as mean ± SEM, a,c,dp < 0.05, aa,cc,ddp < 0.01, a vs control, cvs URMC-099, dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test
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Fig1: URMC-099 inhibits p38 and JNK MAPK signaling cascades in Aβ-stimulated microglia. Immunoblots of p-MKK3 and total MKK3 (a), p-MKK4 (asterisk; non-specific bands) and total MKK4 (c), p-p38 and total p38 (e), p-JNK p54 (top), p46 (bottom), and total JNK (g). Quantification of p-MKK3 (b), p-MKK4 (d) p-p38 (f), p-JNK p54 (h), and p46 (i) levels. Data are presented as mean ± SEM, a,c,dp < 0.05, aa,cc,ddp < 0.01, a vs control, cvs URMC-099, dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test

Mentions: Aβ activates p38 and JNK MAPK cascades to produce pro-inflammatory mediators in microglia [36–38], and has previously been shown to activate MLK3 in cortical neurons [39], but not microglia. Thus, we tested whether URMC-099 could affect the MAPK cascades in Aβ42-stimulated microglia (Fig. 1). As MKK3/6 and MKK4/7 regulate p38 and JNK phosphorylation downstream MLK3 [40], we examined the phosphorylation patterns of MKK3 and MKK4 (Fig. 1a, c). A 30-min Aβ42 treatment increased MKK3 phosphorylation 32.1 % as compared to untreated controls (Fig. 1b). Moreover, trends in MKK4 phosphorylation were also seen with an 8.0 % increase when compared to untreated controls (Fig. 1d). However, URMC-099 treatment significantly reduced both MKK3 and MKK4 phosphorylation in the Aβ-stimulated microglia with decreases of 26.9 and 20.5 % in MKK3 and MKK4 phosphorylation, respectively (Fig. 1b, d). The phosphorylation of p38 and p46/p54-JNK downstream MKK3 and MKK4 was also assayed (Fig. 1e, g). While a 30-min exposure to Aβ42 showed a rapid induction of both p38 and p46/p54-JNK phosphorylation (increases of 84.8, 18.7, and 25.1 % in p38, p46-JNK, and p54-JNK, respectively, as compared to untreated controls, Fig. 1f, h, i), URMC-099 treatment resulted in significant reduction in p38 and JNK phosphorylation in the Aβ-stimulated microglia. Here, decreases were seen at 21.5, 17.3, and 23.7 % in p38, p46-JNK, and p54-JNK, respectively (Fig. 1f, h, i). To assess the effects of URMC-099 on Aβ42-mediated microglial cytotoxicity, we measured cell viability using the MTT assay. Aβ42-treatment reduced cell viability (Additional file 2: Figure S2) as observed previously [31]. Although URMC-099 treatment did not ameliorate Aβ42-mediated cytotoxicity, trends in protection of cell viability were observed with 9.4 and 16.0 % increase in 3- and 48-h incubation, respectively, when compared to Aβ42 treatment (Additional file 2: Figure S2). These results support the notion that URMC-099 inhibits MLK3-MKK3/4-mediated activation of p38 and JNK MAPK cascades in Aβ-activated microglia.Fig. 1


The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-β degradation.

Dong W, Embury CM, Lu Y, Whitmire SM, Dyavarshetty B, Gelbard HA, Gendelman HE, Kiyota T - J Neuroinflammation (2016)

URMC-099 inhibits p38 and JNK MAPK signaling cascades in Aβ-stimulated microglia. Immunoblots of p-MKK3 and total MKK3 (a), p-MKK4 (asterisk; non-specific bands) and total MKK4 (c), p-p38 and total p38 (e), p-JNK p54 (top), p46 (bottom), and total JNK (g). Quantification of p-MKK3 (b), p-MKK4 (d) p-p38 (f), p-JNK p54 (h), and p46 (i) levels. Data are presented as mean ± SEM, a,c,dp < 0.05, aa,cc,ddp < 0.01, a vs control, cvs URMC-099, dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940949&req=5

Fig1: URMC-099 inhibits p38 and JNK MAPK signaling cascades in Aβ-stimulated microglia. Immunoblots of p-MKK3 and total MKK3 (a), p-MKK4 (asterisk; non-specific bands) and total MKK4 (c), p-p38 and total p38 (e), p-JNK p54 (top), p46 (bottom), and total JNK (g). Quantification of p-MKK3 (b), p-MKK4 (d) p-p38 (f), p-JNK p54 (h), and p46 (i) levels. Data are presented as mean ± SEM, a,c,dp < 0.05, aa,cc,ddp < 0.01, a vs control, cvs URMC-099, dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test
Mentions: Aβ activates p38 and JNK MAPK cascades to produce pro-inflammatory mediators in microglia [36–38], and has previously been shown to activate MLK3 in cortical neurons [39], but not microglia. Thus, we tested whether URMC-099 could affect the MAPK cascades in Aβ42-stimulated microglia (Fig. 1). As MKK3/6 and MKK4/7 regulate p38 and JNK phosphorylation downstream MLK3 [40], we examined the phosphorylation patterns of MKK3 and MKK4 (Fig. 1a, c). A 30-min Aβ42 treatment increased MKK3 phosphorylation 32.1 % as compared to untreated controls (Fig. 1b). Moreover, trends in MKK4 phosphorylation were also seen with an 8.0 % increase when compared to untreated controls (Fig. 1d). However, URMC-099 treatment significantly reduced both MKK3 and MKK4 phosphorylation in the Aβ-stimulated microglia with decreases of 26.9 and 20.5 % in MKK3 and MKK4 phosphorylation, respectively (Fig. 1b, d). The phosphorylation of p38 and p46/p54-JNK downstream MKK3 and MKK4 was also assayed (Fig. 1e, g). While a 30-min exposure to Aβ42 showed a rapid induction of both p38 and p46/p54-JNK phosphorylation (increases of 84.8, 18.7, and 25.1 % in p38, p46-JNK, and p54-JNK, respectively, as compared to untreated controls, Fig. 1f, h, i), URMC-099 treatment resulted in significant reduction in p38 and JNK phosphorylation in the Aβ-stimulated microglia. Here, decreases were seen at 21.5, 17.3, and 23.7 % in p38, p46-JNK, and p54-JNK, respectively (Fig. 1f, h, i). To assess the effects of URMC-099 on Aβ42-mediated microglial cytotoxicity, we measured cell viability using the MTT assay. Aβ42-treatment reduced cell viability (Additional file 2: Figure S2) as observed previously [31]. Although URMC-099 treatment did not ameliorate Aβ42-mediated cytotoxicity, trends in protection of cell viability were observed with 9.4 and 16.0 % increase in 3- and 48-h incubation, respectively, when compared to Aβ42 treatment (Additional file 2: Figure S2). These results support the notion that URMC-099 inhibits MLK3-MKK3/4-mediated activation of p38 and JNK MAPK cascades in Aβ-activated microglia.Fig. 1

Bottom Line: Co-localization of Aβ and endolysosomal markers associated with enhanced Aβ42 degradation was observed.URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aβ degradation.Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.

ABSTRACT

Background: Amyloid-β (Aβ)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer's disease (AD). Mixed-lineage kinases (MLKs) regulate upstream MAPK signaling that include p38 MAPK and c-Jun amino-terminal kinase (JNK). However, whether MLK-MAPK pathways affect Aβ-mediated neuroinflammation is unknown. To this end, we investigated if URMC-099, a brain-penetrant small-molecule MLK type 3 inhibitor, can modulate Aβ trafficking and processing required for generating AD-associated microglial inflammatory responses.

Methods: Aβ1-42 (Aβ42) and/or URMC-099-treated murine microglia were investigated for phosphorylated mitogen-activated protein kinase kinase (MKK)3, MKK4 (p-MKK3, p-MKK4), p38 (p-p38), and JNK (p-JNK). These pathways were studied in tandem with the expression of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Gene expression of the anti-inflammatory cytokines, IL-4 and IL-13, was evaluated by real-time quantitative polymerase chain reaction. Aβ uptake and expression of scavenger receptors were measured. Protein trafficking was assessed by measures of endolysosomal markers using confocal microscopy.

Results: Aβ42-mediated microglial activation pathways were shown by phosphorylation of MKK3, MKK4, p38, and JNK and by expression of IL-1β, IL-6, and TNF-α. URMC-099 modulated microglial inflammatory responses with induction of IL-4 and IL-13. Phagocytosis of Aβ42 was facilitated by URMC-099 with up-regulation of scavenger receptors. Co-localization of Aβ and endolysosomal markers associated with enhanced Aβ42 degradation was observed.

Conclusions: URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aβ degradation. These data demonstrate a new immunomodulatory role for URMC-099 to inhibit MLK and to induce microglial anti-inflammatory responses. Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

No MeSH data available.


Related in: MedlinePlus