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

Dong W, Embury CM, Lu Y, Whitmire SM, Dyavarshetty B, Gelbard HA, Gendelman HE, Kiyota T - J Neuroinflammation (2016)

Bottom Line: 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.Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

URMC-099 facilitates Aβ42 degradation in murine microglia. a Microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and harvested for immunoblot using a 10 % SDS-polyacrylamide Tris-Tricine gel and 6E10 antibody. 1-mer, monomeric Aβ42. 2-mer, dimeric Aβ42. Asterisk indicates high molecular weight (HMW) Aβ42. b Band luminescent intensities for monomeric, dimeric, and HMW Aβ42 were quantified by ImageJ software. The amounts of Aβ42 in culture media (c) and microglial cell lysates (d) were measured by human Aβ42-specific ELISA. Bars represent mean ± SEM. (n = 3 per group). *p < 0.05 vs Aβ, as determined by Student’s t test
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Fig7: URMC-099 facilitates Aβ42 degradation in murine microglia. a Microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and harvested for immunoblot using a 10 % SDS-polyacrylamide Tris-Tricine gel and 6E10 antibody. 1-mer, monomeric Aβ42. 2-mer, dimeric Aβ42. Asterisk indicates high molecular weight (HMW) Aβ42. b Band luminescent intensities for monomeric, dimeric, and HMW Aβ42 were quantified by ImageJ software. The amounts of Aβ42 in culture media (c) and microglial cell lysates (d) were measured by human Aβ42-specific ELISA. Bars represent mean ± SEM. (n = 3 per group). *p < 0.05 vs Aβ, as determined by Student’s t test

Mentions: In response to Aβ binding to SRs, microglia start to engulf Aβ by phagocytosis, and then Aβ enters into the endolysosomal pathway. Thus, we investigated how URMC-099 affects the endolysosomal trafficking underlying Aβ phagocytosis. Microglia treated as described above were immunostained with antibodies to Rab7 (for late endosomes, Fig. 6a) and Lamp1 (for lysosomes, Fig. 6c) at 1-h post-incubation with Aβ. Confocal microscopy demonstrated that co-localization of Rab7 and Lamp1 with Aβ42 was increased in URMC-099-treated microglia, compared to untreated microglia (21.2 and 26.3 % increases in Rab7 and Lamp1, respectively, Fig. 6c, d). To investigate Aβ metabolism, microglia were exposed to Aβ42 for 30 min, washed and cultured in fresh media for additional 1 h, then harvested for immunoblotting (Fig. 7a). Co-treatment with URMC-099 significantly reduced immunoreactivity of monomeric, dimeric, and high molecular weight (HMW) Aβ42, as compared to Aβ42 treatment only (39.3, 30.6, and 42.3 % decreases in monomeric, dimeric, and HMW Aβ42, respectively, Fig. 7b). To validate these results, microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and then Aβ42 release and retention in microglia were quantified using ELISA. While URMC-099 had no effect on Aβ42 release (Fig. 7c), co-treatment with URMC-099 significantly reduced Aβ42 with a 50.2 % reduction (Fig. 7d). These data suggest that URMC-099 promotes microglial endolysosome-mediated degradation.Fig. 6


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

Dong W, Embury CM, Lu Y, Whitmire SM, Dyavarshetty B, Gelbard HA, Gendelman HE, Kiyota T - J Neuroinflammation (2016)

URMC-099 facilitates Aβ42 degradation in murine microglia. a Microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and harvested for immunoblot using a 10 % SDS-polyacrylamide Tris-Tricine gel and 6E10 antibody. 1-mer, monomeric Aβ42. 2-mer, dimeric Aβ42. Asterisk indicates high molecular weight (HMW) Aβ42. b Band luminescent intensities for monomeric, dimeric, and HMW Aβ42 were quantified by ImageJ software. The amounts of Aβ42 in culture media (c) and microglial cell lysates (d) were measured by human Aβ42-specific ELISA. Bars represent mean ± SEM. (n = 3 per group). *p < 0.05 vs Aβ, as determined by Student’s t test
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Fig7: URMC-099 facilitates Aβ42 degradation in murine microglia. a Microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and harvested for immunoblot using a 10 % SDS-polyacrylamide Tris-Tricine gel and 6E10 antibody. 1-mer, monomeric Aβ42. 2-mer, dimeric Aβ42. Asterisk indicates high molecular weight (HMW) Aβ42. b Band luminescent intensities for monomeric, dimeric, and HMW Aβ42 were quantified by ImageJ software. The amounts of Aβ42 in culture media (c) and microglial cell lysates (d) were measured by human Aβ42-specific ELISA. Bars represent mean ± SEM. (n = 3 per group). *p < 0.05 vs Aβ, as determined by Student’s t test
Mentions: In response to Aβ binding to SRs, microglia start to engulf Aβ by phagocytosis, and then Aβ enters into the endolysosomal pathway. Thus, we investigated how URMC-099 affects the endolysosomal trafficking underlying Aβ phagocytosis. Microglia treated as described above were immunostained with antibodies to Rab7 (for late endosomes, Fig. 6a) and Lamp1 (for lysosomes, Fig. 6c) at 1-h post-incubation with Aβ. Confocal microscopy demonstrated that co-localization of Rab7 and Lamp1 with Aβ42 was increased in URMC-099-treated microglia, compared to untreated microglia (21.2 and 26.3 % increases in Rab7 and Lamp1, respectively, Fig. 6c, d). To investigate Aβ metabolism, microglia were exposed to Aβ42 for 30 min, washed and cultured in fresh media for additional 1 h, then harvested for immunoblotting (Fig. 7a). Co-treatment with URMC-099 significantly reduced immunoreactivity of monomeric, dimeric, and high molecular weight (HMW) Aβ42, as compared to Aβ42 treatment only (39.3, 30.6, and 42.3 % decreases in monomeric, dimeric, and HMW Aβ42, respectively, Fig. 7b). To validate these results, microglia were exposed to Aβ42 for 30 min, followed by wash and culture in fresh media for additional 1 h, and then Aβ42 release and retention in microglia were quantified using ELISA. While URMC-099 had no effect on Aβ42 release (Fig. 7c), co-treatment with URMC-099 significantly reduced Aβ42 with a 50.2 % reduction (Fig. 7d). These data suggest that URMC-099 promotes microglial endolysosome-mediated degradation.Fig. 6

Bottom Line: 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.Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5930, USA.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus