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A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation
Published on: September 19, 2010
The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-β degradation
Weiguo Dong1,2, Christine M Embury1, Yaman Lu1
1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.
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.
Insights
URMC-099, an MLK inhibitor, reduces Alzheimer's disease-associated neuroinflammation by enhancing microglial phagocytosis and promoting amyloid-beta degradation. This suggests URMC-099 as a potential novel therapy for Alzheimer's disease.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglial inflammatory responses are activated by Amyloid-beta (Aβ) via mitogen-activated protein kinase (MAPK) pathways in Alzheimer's disease (AD).
- Mixed-lineage kinases (MLKs) are upstream regulators of MAPK signaling, including p38 MAPK and c-Jun amino-terminal kinase (JNK).
- The role of MLK-MAPK pathways in Aβ-mediated neuroinflammation remains unclear.
Purpose of the Study:
- To investigate if URMC-099, a brain-penetrant MLK type 3 inhibitor, can modulate Aβ trafficking and processing.
- To determine the effect of URMC-099 on Aβ-associated microglial inflammatory responses.
Main Methods:
- Murine microglia were treated with Aβ1-42 (Aβ42) and/or URMC-099.
- Phosphorylation of MKK3, MKK4, p38, and JNK, and expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-4, IL-13) were measured.
- Aβ uptake, scavenger receptor expression, and endolysosomal trafficking were assessed.
Main Results:
- Aβ42 treatment activated microglial inflammatory pathways, evidenced by increased MKK3/4, p38, JNK phosphorylation, and pro-inflammatory cytokine expression.
- URMC-099 modulated these responses, inducing anti-inflammatory cytokines IL-4 and IL-13.
- URMC-099 enhanced Aβ42 phagocytosis via scavenger receptor upregulation and promoted Aβ degradation through improved phagolysosomal trafficking.
Conclusions:
- URMC-099 effectively reduces microglial inflammatory responses in the context of Aβ exposure.
- The compound facilitates phagolysosomal trafficking and enhances Aβ degradation.
- URMC-099 demonstrates potential as a novel immunomodulatory therapeutic agent for Alzheimer's disease by inhibiting MLK and promoting anti-inflammatory microglial activity.
