The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-[beta] degradation

Background Amyloid-[beta] (A[beta])-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 A[beta]-mediated neuroinflammation is unknown. To this end, we investigated if URMC-099, a brain-penetrant small-molecule MLK type 3 inhibitor, can modulate A[beta] trafficking and processing required for generating AD-associated microglial inflammatory responses. Methods A[beta]1-42 (A[beta]42) 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)-1[beta], IL-6, and tumor necrosis factor (TNF)-[alpha]. Gene expression of the anti-inflammatory cytokines, IL-4 and IL-13, was evaluated by real-time quantitative polymerase chain reaction. A[beta] uptake and expression of scavenger receptors were measured. Protein trafficking was assessed by measures of endolysosomal markers using confocal microscopy. Results A[beta]42-mediated microglial activation pathways were shown by phosphorylation of MKK3, MKK4, p38, and JNK and by expression of IL-1[beta], IL-6, and TNF-[alpha]. URMC-099 modulated microglial inflammatory responses with induction of IL-4 and IL-13. Phagocytosis of A[beta]42 was facilitated by URMC-099 with up-regulation of scavenger receptors. Co-localization of A[beta] and endolysosomal markers associated with enhanced A[beta]42 degradation was observed. Conclusions URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated A[beta] 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: Mixed-lineage kinase 3, Alzheimer's disease, Amyloid-[beta], Microglia, Phagocytosis, Endolysosomal pathway