title: The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-β degradation.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.|Department of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, 350122, People's Republic of China.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Embury
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.
authors.forename: Yaman
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Whitmire
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.
authors.forename: Bhagyalaxmi
authors.initials: B
authors.lastname: Dyavarshetty
authors.affiliation: Department of Neurology, Center for Neural Development & Disease, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, 14642, NY, USA.
authors.forename: Harris A
authors.initials: HA
authors.lastname: Gelbard
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.|Department of Internal Medicine, University of Nebraska Medical Center, Omaha, 68198-5880, NE, USA.
authors.forename: Howard E
authors.initials: HE
authors.lastname: Gendelman
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA. tkiyota@unmc.edu.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kiyota
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000187:drug effects*; D018613:Microscopy, Confocal; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D051116:Receptors, Scavenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018709:Statistics, Nonparametric; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D016229:Amyloid beta-Peptides; D016207:Cytokines; D052118:Lysosomal-Associated Membrane Protein 1; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011758:Pyrroles; D051116:Receptors, Scavenger; C583037:URMC-099; C075222:amyloid beta-protein (1-42); D000090802:rab7 GTP-Binding Proteins; D020929:Mitogen-Activated Protein Kinase Kinases; D020691:rab GTP-Binding Proteins
keywords: Alzheimer’s disease; Amyloid-β; Endolysosomal pathway; Microglia; Mixed-lineage kinase 3; Phagocytosis