Abstract
BackgroundAmyloid-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.MethodsAβ1-42 (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.ResultsAβ42-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.ConclusionsURMC-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0646-z) contains supplementary material, which is available to authorized users.
Highlights
Amyloid-β (Aβ)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer’s disease (AD)
We tested whether URMC-099 could affect the MAPK cascades in Aβ42stimulated microglia (Fig. 1)
While a 30-min exposure to Aβ42 showed a rapid induction of both p38 and p46/p54-Jun amino-terminal kinase (JNK) phosphorylation, URMC-099 treatment resulted in significant reduction in p38 and JNK
Summary
Amyloid-β (Aβ)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer’s disease (AD). 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. For treatment of AD, Aβ immunization reduces the Aβ42 load with immunomodulation, including microglial phagocytosis and changed lysosomal and scavenger markers [20, 21] Such immunizations can result in the development of meningoencephalitis (~6 %) [22], spongiosis, and neuronal loss [23]. Since removal of plaques at later stages of disease marked by neurofibrillary tangle formation was not proven to be beneficial, alternative treatment strategies are likely required These might improve clinical outcomes if administered years before clinical signs and symptoms emerge. Such a therapeutic approach could speed clearance of Aβ and modify inflammatory activities before disease develops
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