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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 GFAP immunostaining. a Representative images of GFAP immunohistochemical (IHC) staining in cortex of WT or APP/PS1 KI mice treated with vehicle (veh) or MW150. b Digital quantification of GFAP in the cortex was done using the Aperio ScanScope with the entire cortex used as the region of interest. Quantification using the positive pixel algorithm showed a significant increase in GFAP staining in the KI + veh compared to WT + veh (p < 0.0001). No significant difference was found between the KI + veh compared to the KI + MW150. (F2,41) = 34.66; p < 0.0001). (n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 2: Table S2
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Fig2: No effect of MW150 on GFAP immunostaining. a Representative images of GFAP immunohistochemical (IHC) staining in cortex of WT or APP/PS1 KI mice treated with vehicle (veh) or MW150. b Digital quantification of GFAP in the cortex was done using the Aperio ScanScope with the entire cortex used as the region of interest. Quantification using the positive pixel algorithm showed a significant increase in GFAP staining in the KI + veh compared to WT + veh (p < 0.0001). No significant difference was found between the KI + veh compared to the KI + MW150. (F2,41) = 34.66; p < 0.0001). (n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 2: Table S2

Mentions: To investigate whether astrocyte activation might be modulated by MW150 treatment, we measured a marker of reactive astrocytes, GFAP. The APP/PS1 KI mice showed higher levels of GFAP immunostaining than WT mice (Fig. 2a). KI mice treated with MW150 showed a trend of reduced GFAP staining in the cortex compared to KI + veh-treated mice, but this difference did not reach significance (Fig. 2b).Fig. 2


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 GFAP immunostaining. a Representative images of GFAP immunohistochemical (IHC) staining in cortex of WT or APP/PS1 KI mice treated with vehicle (veh) or MW150. b Digital quantification of GFAP in the cortex was done using the Aperio ScanScope with the entire cortex used as the region of interest. Quantification using the positive pixel algorithm showed a significant increase in GFAP staining in the KI + veh compared to WT + veh (p < 0.0001). No significant difference was found between the KI + veh compared to the KI + MW150. (F2,41) = 34.66; p < 0.0001). (n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 2: Table S2
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Fig2: No effect of MW150 on GFAP immunostaining. a Representative images of GFAP immunohistochemical (IHC) staining in cortex of WT or APP/PS1 KI mice treated with vehicle (veh) or MW150. b Digital quantification of GFAP in the cortex was done using the Aperio ScanScope with the entire cortex used as the region of interest. Quantification using the positive pixel algorithm showed a significant increase in GFAP staining in the KI + veh compared to WT + veh (p < 0.0001). No significant difference was found between the KI + veh compared to the KI + MW150. (F2,41) = 34.66; p < 0.0001). (n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file 2: Table S2
Mentions: To investigate whether astrocyte activation might be modulated by MW150 treatment, we measured a marker of reactive astrocytes, GFAP. The APP/PS1 KI mice showed higher levels of GFAP immunostaining than WT mice (Fig. 2a). KI mice treated with MW150 showed a trend of reduced GFAP staining in the cortex compared to KI + veh-treated mice, but this difference did not reach significance (Fig. 2b).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Brain p38&alpha; 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 p38&alpha;MAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer&rsquo;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&nbsp;mg/kg/day) was administered daily to 11&ndash;12-month-old KI mice for 14&nbsp;days, and levels of proinflammatory cytokines IL-1&beta;, TNF&alpha;, 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 A&beta;40 and A&beta;42 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-1&beta; and TNF&alpha; but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15&nbsp;&mu;m radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and A&beta;40 and A&beta;42 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.