Poster CC15 - Alzheimer’s pathogen Aβ re-wires CSF1R-induced MAPK pathway activation in MMC microglia

Introduction: Mitogen activated protein kinase (MAPK) signaling is a critical regulator of microglial phenotype, including phagocytic function, cytokine expression, and motility, among others. Importantly, both canonical (pErk1/2) and non-canonical (p-p38 and pJnk) MAPK signaling is directly activated by RTKs, including CSF1R and its ligands, which have been shown to be modified in human and mouse Alzheimer’s tissues. Interestingly, CSF1R is activated by two agonists, CSF1 and IL-34, which have been shown to activate the receptor in different ways that can lead to distinct kinase activities. However, little is known about how the AD-relevant CSF1R agonists CSF1 and IL-34 affect microglial MAPK signaling and whether their effects are dependent on disease state/Aβ exposure. In this study, we hypothesized that IL-34 and CSF-1 elicit distinct patterns of MAPK signaling activation in microglia, and MAPK activation would be dependent on whether the cells were exposed to Aβ.

Materials and

Methods: We applied agonists of CSF1R (i.e., IL-34, CSF1) to the mouse microglial cell (MMC) line, either pre-treated with 500nM of Aβ1-42 or vehicle (1% NH4OH) for 48 hours. Cells were serum-starved (1 hour), stimulated with each agonist for 5 minutes, and then lysed for Luminex quantification of a panel of 10 MAPK phosphoproteins. Principle component analysis (PCA) and cluster analysis were conducted to analyze the data.

Results, Conclusions, and Discussions: Cluster analysis revealed that in the absence of Aβ1-42, both CSF1R and IL-34 stimulated both canonical (pErk1/2) and non-canonical (p-p38, pJnk) MAPK signaling phospho-proteins (Figure 1, Yellow boxes, Wilcox test, p< 0.05 vs. control). While Aβ1-42 pre-treatment resulted in diminished activation in canonical MAPK signaling (i.e., pMek1, pErk1/2) following either CSF1 or IL-34 stimulation, CSF1 stimulation of non-canonical MAPK signaling through p-p38 and pJnk was preserved with Aβ1-42 pre-treatment (Figure 1, Blue boxes). PCA of the MAPK phospho-proteins demonstrated separation of the MMCs treated with Aβ1-42 from control (1%NH4OH) along PC2, illuminating a distinct pattern of MAPK activation in the presence of AD pathogen (Aβ) compared to control. Additionally, within Aβ1-42 treated group, MMCs treated with CSF1 were separated from PBS group along PC1. These findings indicate that CSF1-induced signaling is robust regardless of Aβ1-42 exposure. Furthermore, different agonists of the same receptor distinctly activate non-canonical MAPK pathway signaling (p-p38, Figure 1). Collectively, this finding supports the overall hypothesis that CSF-1R-induced signaling is dependent on Aβ and Aβ pre-treatments shift the canonical ERK signaling to non-canonical p38 signaling.
These findings show that different agonists (IL-34 and CSF1) of CSF1R can provoke distinct profiles of canonical and non-canonical MAPK pathway signaling in the presence of Aβ1-42. Understanding the differences in agonist-specific microglial signaling in healthy and diseased microglia will be essential to designing microglial-targeted therapeutics for AD treatment.

Acknowledgements (Optional): The authors would like to thank funding provided by the NSF CAREER 1944053 and NIA R01 NS114130 Awards.