Novel Agonist TREM2 Monoclonal Antibodies Activate Syk-Associated Cell Signaling Pathways

Abstract

Triggering receptor expressed on myeloid cells 2 (TREM2), a protein expressed on the surface of microglia, is genetically linked to, and a therapeutic target for Alzheimer’s disease (AD). As a myeloid cell receptor, TREM2 binds a variety of ligands, including AD-related proteins apolipoprotein E (ApoE) and β-amyloid. Upon stimulation, TREM2 activates various cell signaling pathways that drive microglial functions capable of ameliorating or accelerating AD progression and pathology (Ennerfelt et al., 2022; Gratuze et al., 2018; Leyns et al., 2017; Price et al., 2020; Takahashi et al., 2007; Takahashi et al., 2005; Wang et al., 2015; Wang et al., 2016; Wang et al., 2020; Zhao et al., 2018). Characterization of TREM2 signaling pathways mediating these functions, however, is incomplete. Further investigation of these signaling cascades is required to understand the function of TREM2 in AD beyond its genetic component. The goal of this research was to generate novel agonist monoclonal antibodies to activate cells in vitro in a TREM2-dependent manner, and to examine the phosphorylation state of potential downstream targets. We screened a library of rabbit host monoclonal antibodies that were raised against the ectodomain of human or mouse TREM2 by western blot to identify clones that were target specific. We then identified several clones that could activate cell signaling pathways downstream of TREM2 in vitro. These pathways include the activation of spleen tyrosine kinase (Syk), which is suggested to be downstream of TREM2/DAP12 microglial activation (Ennerfelt et al., 2022; Gratuze et al., 2018; Jay et al., 2017; McQuade et al., 2020; Schlepckow et al., 2020; Wang et al., 2020; Zhao et al., 2018). Syk activation was characterized using phospho-specific anti-Syk monoclonal antibodies. Having established tools to activate TREM2, we screened other downstream proteins related to Syk signaling that may also be engaged by TREM2 activation. Based on our findings, we suggest that Syk phosphorylation is indeed a reliable readout for TREM2-dependent microglia activation. We further suggest that the antibodies validated herein can be leveraged to further characterize signaling events and cellular responses (phagocytosis, inflammation, proliferation, etc.) downstream of TREM2. Full characterization of TREM2 signaling cascades can be applied to AD therapeutic research, targeting the benefits of upregulating or downregulating TREM2-dependent microglial activation to attenuate AD pathology.