Limits...
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 alters scavenger receptor expression in microglia. a Representative images of immunoblots for CD36 and CD47 in mouse microglia after a 30-min Aβ42 stimulation. b Quantification CD36 and CD47 expression. Bars represent mean ± SEM. aa,ccp < 0.01, cccp < 0.001, avs control, cvs Aβ, one-way ANOVA, Newman–Keuls post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: URMC-099 alters scavenger receptor expression in microglia. a Representative images of immunoblots for CD36 and CD47 in mouse microglia after a 30-min Aβ42 stimulation. b Quantification CD36 and CD47 expression. Bars represent mean ± SEM. aa,ccp < 0.01, cccp < 0.001, avs control, cvs Aβ, one-way ANOVA, Newman–Keuls post hoc test

Mentions: A previous study demonstrated that microglial phagocytic capacity is linked to scavenger receptor (SR) expression levels in microglia in AD mouse models [45]. To pursue the mechanism of how URMC-099 facilitates Aβ phagocytosis, we examined the expression of SRs. CD36 and CD47 expression was assessed by immunoblotting (Fig. 5a). CD36 expression was increased in all treated groups (increases of 44.4, 56.1, and 44.1 % in URMC-099 only, Aβ only, and Aβ plus URMC-099 to control, respectively, Fig. 5b), while CD47 expression was significantly increased with URMC-099 treatment (increases of 55.9 and 50.2 % in URMC-099 only and Aβ plus URMC-099 to control, respectively, Fig. 5b), demonstrating URMC-099-specific alteration in SR expression.Fig. 5


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 alters scavenger receptor expression in microglia. a Representative images of immunoblots for CD36 and CD47 in mouse microglia after a 30-min Aβ42 stimulation. b Quantification CD36 and CD47 expression. Bars represent mean ± SEM. aa,ccp < 0.01, cccp < 0.001, avs control, cvs 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

Fig5: URMC-099 alters scavenger receptor expression in microglia. a Representative images of immunoblots for CD36 and CD47 in mouse microglia after a 30-min Aβ42 stimulation. b Quantification CD36 and CD47 expression. Bars represent mean ± SEM. aa,ccp < 0.01, cccp < 0.001, avs control, cvs Aβ, one-way ANOVA, Newman–Keuls post hoc test
Mentions: A previous study demonstrated that microglial phagocytic capacity is linked to scavenger receptor (SR) expression levels in microglia in AD mouse models [45]. To pursue the mechanism of how URMC-099 facilitates Aβ phagocytosis, we examined the expression of SRs. CD36 and CD47 expression was assessed by immunoblotting (Fig. 5a). CD36 expression was increased in all treated groups (increases of 44.4, 56.1, and 44.1 % in URMC-099 only, Aβ only, and Aβ plus URMC-099 to control, respectively, Fig. 5b), while CD47 expression was significantly increased with URMC-099 treatment (increases of 55.9 and 50.2 % in URMC-099 only and Aβ plus URMC-099 to control, respectively, Fig. 5b), demonstrating URMC-099-specific alteration in SR expression.Fig. 5

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