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