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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 induces gene expression of anti-inflammatory cytokines in Aβ42-stimulated microglia. A conventional RT2-qPCR was performed to measure IL-4 and IL-13 expression using primer sets (Table 1) and synthesized cDNA with total RNA isolated from murine microglia (n = 3 per group). Data are presented as mean ± SEM, a,bp < 0.05, avs control, bvs Aβ, one-way ANOVA, Newman–Keuls post hoc test
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Fig3: URMC-099 induces gene expression of anti-inflammatory cytokines in Aβ42-stimulated microglia. A conventional RT2-qPCR was performed to measure IL-4 and IL-13 expression using primer sets (Table 1) and synthesized cDNA with total RNA isolated from murine microglia (n = 3 per group). Data are presented as mean ± SEM, a,bp < 0.05, avs control, bvs Aβ, one-way ANOVA, Newman–Keuls post hoc test

Mentions: To determine the neuroinflammatory phenotype in URMC-099-treated microglia, total RNA was isolated and RT2-qPCR was performed for genes specific to IL-4 and IL-13. Data were shown as fold change compared to control (Fig. 3). While 30-min Aβ42 stimulation did not alter gene expression of IL-4 and IL-13, URMC-099 significantly induced this expression pattern (increases of 111.1 and 345.7 % in IL-4 and IL-13, respectively, Fig. 3), further supporting the anti-inflammatory effects of URMC-099 on Aβ-stimulated microglia.Fig. 3


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 induces gene expression of anti-inflammatory cytokines in Aβ42-stimulated microglia. A conventional RT2-qPCR was performed to measure IL-4 and IL-13 expression using primer sets (Table 1) and synthesized cDNA with total RNA isolated from murine microglia (n = 3 per group). Data are presented as mean ± SEM, a,bp < 0.05, avs control, bvs Aβ, one-way ANOVA, Newman–Keuls post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940949&req=5

Fig3: URMC-099 induces gene expression of anti-inflammatory cytokines in Aβ42-stimulated microglia. A conventional RT2-qPCR was performed to measure IL-4 and IL-13 expression using primer sets (Table 1) and synthesized cDNA with total RNA isolated from murine microglia (n = 3 per group). Data are presented as mean ± SEM, a,bp < 0.05, avs control, bvs Aβ, one-way ANOVA, Newman–Keuls post hoc test
Mentions: To determine the neuroinflammatory phenotype in URMC-099-treated microglia, total RNA was isolated and RT2-qPCR was performed for genes specific to IL-4 and IL-13. Data were shown as fold change compared to control (Fig. 3). While 30-min Aβ42 stimulation did not alter gene expression of IL-4 and IL-13, URMC-099 significantly induced this expression pattern (increases of 111.1 and 345.7 % in IL-4 and IL-13, respectively, Fig. 3), further supporting the anti-inflammatory effects of URMC-099 on Aβ-stimulated microglia.Fig. 3

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