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

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.

Keywords: Alzheimer’s disease; Amyloid-β; Endolysosomal pathway; Microglia; Mixed-lineage kinase 3; Phagocytosis.

Fig. 1

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,d p < 0.05, ^aa,cc,dd p < 0.01, ^a vs control, ^cvs URMC-099, ^dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test

Fig. 2

URMC-099 inhibits Aβ-activated microglial pro-inflammatory cytokines. a A conventional RT2-qPCR was performed to measure IL-1β, IL-6, or TNF-α expression using primer sets (Table 1) and synthesized cDNA with total RNA isolated from murine microglia (n = 3 per group). b Quantification of IL-1β, IL-6, or TNF-α protein secretion from murine microglia (n = 3 per group). Data are presented as mean ± SEM, ^a,c,d p < 0.05, ^aa,cc,dd p < 0.01, ^aaa p < 0.001, ^avs control, ^cvs URMC-099, ^dvs Aβ + URMC-099, one-way ANOVA, Newman–Keuls post hoc test

Fig. 3

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,b p < 0.05, ^avs control, ^bvs Aβ, one-way ANOVA, Newman–Keuls post hoc test

Fig. 4

URMC-099 facilitates microglial Aβ-uptake. a Primary mouse microglia were incubated with soluble Aβ42 for 30 min, followed by immunofluorescence with anti-Aβ Ab (green) and DAPI (blue) for nuclear staining (blue). Merged captured images were shown. Scale bar, 100 μm. b Dense intensity of Aβ42 fluorescence was measured using ImageJ. Bars represent mean ± SEM. **p < 0.01 by Student’s t test

Fig. 5

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,cc p < 0.01, ^ccc p < 0.001, ^avs control, ^cvs Aβ, one-way ANOVA, Newman–Keuls post hoc test

Fig. 6

URMC-099 facilitates subcellular co-localization of Aβ42 with Rab7 and Lamp1 in murine microglia. Confocal microscopy shows cellular localization of Rab7 late endosomal component (a, red) or Lamp1 lysosomal compartment (b, red) and Aβ42 (green). Merged images of Rab7 and Lamp1 are shown. Aβ42 co-localization with Rab7 (c) or Lamp1 (d) was quantified by ImageJ with a co-localization plugin. Scale bar = 50 μm. Bars represent mean ± SEM. *p < 0.05 by Student’s t test

Fig. 7

URMC-099 facilitates Aβ42 degradation in murine microglia. a Microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and harvested for immunoblot using a 10 % SDS-polyacrylamide Tris-Tricine gel and 6E10 antibody. 1-mer, monomeric Aβ42. 2-mer, dimeric Aβ42. Asterisk indicates high molecular weight (HMW) Aβ42. b Band luminescent intensities for monomeric, dimeric, and HMW Aβ42 were quantified by ImageJ software. The amounts of Aβ42 in culture media (c) and microglial cell lysates (d) were measured by human Aβ42-specific ELISA. Bars represent mean ± SEM. (n = 3 per group). *p < 0.05 vs Aβ, as determined by Student’s t test