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Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

View Article: PubMed Central - PubMed

ABSTRACT

Background: Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer’s disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations.

Methods: We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11–12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1β, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aβ40 and Aβ42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities.

Results: Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1β and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 μm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aβ40 and Aβ42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells.

Conclusions: Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0845-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

No effect of MW150 on Aβ in APP/KI mice. a Aβ and microglia volume were measured using immunofluorescent staining with 6E10 for Aβ and IBA1 for microglia. A z-stack of images were taken using confocal microscopy then were analyzed using the surface tool in Imaris software. Representative confocal images and 3D surface reconstructions with Imaris software are shown. b Aβ volume occupied by the surface reconstruction was reduced in KI + MW150; however, the decrease was not significant. The data represents average of 3–4 independent z-stacks from each mouse (n = 11 KI + veh; n = 14 KI + MW150). c PBS- and FA-soluble Aβ40 or Aβ42 levels were measured by MSD ELISA. No significant effect of MW150 treatment was found in the Aβ ELISA. (n = 11 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 1: Table S1 and Additional file 3: Table S3
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Fig5: No effect of MW150 on Aβ in APP/KI mice. a Aβ and microglia volume were measured using immunofluorescent staining with 6E10 for Aβ and IBA1 for microglia. A z-stack of images were taken using confocal microscopy then were analyzed using the surface tool in Imaris software. Representative confocal images and 3D surface reconstructions with Imaris software are shown. b Aβ volume occupied by the surface reconstruction was reduced in KI + MW150; however, the decrease was not significant. The data represents average of 3–4 independent z-stacks from each mouse (n = 11 KI + veh; n = 14 KI + MW150). c PBS- and FA-soluble Aβ40 or Aβ42 levels were measured by MSD ELISA. No significant effect of MW150 treatment was found in the Aβ ELISA. (n = 11 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 1: Table S1 and Additional file 3: Table S3

Mentions: We previously reported that MW150 had no effect on Aβ plaque burden in the APP/PS1 KI mice [14], as assessed by IHC staining. To test whether a potential effect of MW150 on Aβ might be revealed with quantitative and more detailed assays, we performed immunofluorescent staining and confocal analysis, as well as quantitative Aβ ELISAs. Aβ plaques were stained with the 6E10 antibody, and microglia were stained with IBA1 (Fig. 5a). 3D reconstructions were generated from the confocal z-stacks using Imaris software, and Aβ plaque volume was calculated. A slight reduction was found in the total volume occupied by Aβ plaques in the KI + MW150 compared to the KI + veh-treated mice, but the difference did not reach significance (Fig. 5b). Measurement of Aβ40 and Aβ42 levels in PBS soluble and formic acid (FA) soluble fractions of APP/PS1 KI mice cortex by quantitative Aβ ELISA showed that MW150 had no effect on Aβ levels (Fig. 5c).Fig. 5


Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes
No effect of MW150 on Aβ in APP/KI mice. a Aβ and microglia volume were measured using immunofluorescent staining with 6E10 for Aβ and IBA1 for microglia. A z-stack of images were taken using confocal microscopy then were analyzed using the surface tool in Imaris software. Representative confocal images and 3D surface reconstructions with Imaris software are shown. b Aβ volume occupied by the surface reconstruction was reduced in KI + MW150; however, the decrease was not significant. The data represents average of 3–4 independent z-stacks from each mouse (n = 11 KI + veh; n = 14 KI + MW150). c PBS- and FA-soluble Aβ40 or Aβ42 levels were measured by MSD ELISA. No significant effect of MW150 treatment was found in the Aβ ELISA. (n = 11 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 1: Table S1 and Additional file 3: Table S3
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Related In: Results  -  Collection

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Fig5: No effect of MW150 on Aβ in APP/KI mice. a Aβ and microglia volume were measured using immunofluorescent staining with 6E10 for Aβ and IBA1 for microglia. A z-stack of images were taken using confocal microscopy then were analyzed using the surface tool in Imaris software. Representative confocal images and 3D surface reconstructions with Imaris software are shown. b Aβ volume occupied by the surface reconstruction was reduced in KI + MW150; however, the decrease was not significant. The data represents average of 3–4 independent z-stacks from each mouse (n = 11 KI + veh; n = 14 KI + MW150). c PBS- and FA-soluble Aβ40 or Aβ42 levels were measured by MSD ELISA. No significant effect of MW150 treatment was found in the Aβ ELISA. (n = 11 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 1: Table S1 and Additional file 3: Table S3
Mentions: We previously reported that MW150 had no effect on Aβ plaque burden in the APP/PS1 KI mice [14], as assessed by IHC staining. To test whether a potential effect of MW150 on Aβ might be revealed with quantitative and more detailed assays, we performed immunofluorescent staining and confocal analysis, as well as quantitative Aβ ELISAs. Aβ plaques were stained with the 6E10 antibody, and microglia were stained with IBA1 (Fig. 5a). 3D reconstructions were generated from the confocal z-stacks using Imaris software, and Aβ plaque volume was calculated. A slight reduction was found in the total volume occupied by Aβ plaques in the KI + MW150 compared to the KI + veh-treated mice, but the difference did not reach significance (Fig. 5b). Measurement of Aβ40 and Aβ42 levels in PBS soluble and formic acid (FA) soluble fractions of APP/PS1 KI mice cortex by quantitative Aβ ELISA showed that MW150 had no effect on Aβ levels (Fig. 5c).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer’s disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations.

Methods: We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11–12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1β, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aβ40 and Aβ42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities.

Results: Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1β and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 μm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aβ40 and Aβ42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells.

Conclusions: Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0845-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus