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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.


No effect of MW150 on IBA1+ microglia volume. a Representative 3D surface reconstructions of IBA1+ cells generated from confocal microscopic imaging using Imaris software. b Mean volume ± standard deviation (SD) of rendered IBA1 cells is shown for the APP/PS1 KI + veh and APP/PS1 KI + MW150 groups. Data represent mean of 3–4 independent z-stacks from each mouse. (n = 11 KI + veh; n = 14 KI + MW150)
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Fig3: No effect of MW150 on IBA1+ microglia volume. a Representative 3D surface reconstructions of IBA1+ cells generated from confocal microscopic imaging using Imaris software. b Mean volume ± standard deviation (SD) of rendered IBA1 cells is shown for the APP/PS1 KI + veh and APP/PS1 KI + MW150 groups. Data represent mean of 3–4 independent z-stacks from each mouse. (n = 11 KI + veh; n = 14 KI + MW150)

Mentions: IBA1 is a commonly used pan-marker of microglia and macrophages. Change in morphology (i.e., hypertrophy) of IBA1-positive cells is used as a marker of a reactive microglia response. To determine if MW150 had an effect on the reactive microglia response, we performed immunofluorescent staining for IBA1, followed by confocal microscopy and three-dimensional (3D) reconstruction using Imaris software. For each animal, 3–4 confocal z-stacks were collected. A 3D surface rendering was made of the IBA1 staining, and the total volume of the 3D z-stack that was occupied by the rendered IBA1+ staining was determined, for approximately 40–70 microglia per animal. The volume of microglia staining included all IBA1+ staining captured in the z-stack including processes not associated with cell bodies. As shown in Fig. 3, there was no difference in the volume of microglia in the KI mice treated with MW150 or vehicle.Fig. 3


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 IBA1+ microglia volume. a Representative 3D surface reconstructions of IBA1+ cells generated from confocal microscopic imaging using Imaris software. b Mean volume ± standard deviation (SD) of rendered IBA1 cells is shown for the APP/PS1 KI + veh and APP/PS1 KI + MW150 groups. Data represent mean of 3–4 independent z-stacks from each mouse. (n = 11 KI + veh; n = 14 KI + MW150)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382362&req=5

Fig3: No effect of MW150 on IBA1+ microglia volume. a Representative 3D surface reconstructions of IBA1+ cells generated from confocal microscopic imaging using Imaris software. b Mean volume ± standard deviation (SD) of rendered IBA1 cells is shown for the APP/PS1 KI + veh and APP/PS1 KI + MW150 groups. Data represent mean of 3–4 independent z-stacks from each mouse. (n = 11 KI + veh; n = 14 KI + MW150)
Mentions: IBA1 is a commonly used pan-marker of microglia and macrophages. Change in morphology (i.e., hypertrophy) of IBA1-positive cells is used as a marker of a reactive microglia response. To determine if MW150 had an effect on the reactive microglia response, we performed immunofluorescent staining for IBA1, followed by confocal microscopy and three-dimensional (3D) reconstruction using Imaris software. For each animal, 3–4 confocal z-stacks were collected. A 3D surface rendering was made of the IBA1 staining, and the total volume of the 3D z-stack that was occupied by the rendered IBA1+ staining was determined, for approximately 40–70 microglia per animal. The volume of microglia staining included all IBA1+ staining captured in the z-stack including processes not associated with cell bodies. As shown in Fig. 3, there was no difference in the volume of microglia in the KI mice treated with MW150 or vehicle.Fig. 3

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.