Advancing therapeutic strategies for CSF1R-ALSP.

Figure 1: Proposed CSF1R-mediated signaling in microglial survival and proliferation. Upon stimulation by its ligands CSF1 or IL-34, CSF1R undergoes rapid dimerization and autophosphorylation at key tyrosine residues, leading to the activation of PI3K/Akt, JNK, and ERK1/2 pathways that regulate microglial survival and proliferation. CSF1R also interacts with the TREM2/DAP12 complex, further supporting microglial survival. Note: Figure adapted from Hu et al., 2021, licensed under CC BY 4.0 [4].

Disruption of CSF1R signaling, as observed in loss-of-function mutations, leads to microglial depletion and subsequent neurodegeneration. This highlights the critical role of CSF1R in maintaining microglial homeostasis and underscores its therapeutic relevance not only in rare diseases like CSF1R-ALSP but also in more common neurodegenerative disorders. Consequently, CSF1R has emerged as a promising target for drug development aimed at modulating microglial function and mitigating neurodegeneration.

Exploring CSF1R as a therapeutic target

Recent clinical trials, such as the Phase 2 IGNITE trial involving iluzanebart—a human monoclonal IgG1 antibody agonist of human TREM2 (hTREM2) designed to selectively activate TREM2 [2]—have highlighted both the potential and challenges in this approach. Despite encouraging preclinical results [1], the trial did not meet its efficacy endpoints.

A June 2025 update from Vigil Neuroscience reported that iluzanebart was safe and well-tolerated at both 20 and 40 mg/kg doses with expected pharmacokinetics. However, it failed to improve key biomarkers or clinical metrics, leading to the discontinuation of the long-term extension study. Following Sanofi’s acquisition of Vigil, development of iluzanebart was halted in favor of alternative TREM2-targeting approaches, including the small-molecule agonist VG-3927. These results emphasize the ongoing need for robust, scalable tools capable of elucidating microglial signaling with precision, particularly in pathways as complex and dynamic as CSF1R.

AlphaLISA™ SureFire® Ultra™ provides a validated and effective approach for measuring intracellular signaling events with high sensitivity and throughput. This no-wash, homogeneous immunoassay platform delivers robust performance in complex cellular models, making it an ideal tool for advancing CSF1R-targeted drug discovery and assessing pathway modulation in disease-relevant systems.

Results: Measuring CSF1R activation with AlphaLISA SureFire® Ultra

In a study using THP-1 cells stimulated with macrophage colony-stimulating factor (M-CSF, 100 ng/mL), a key ligand that activates CSF1R and supports macrophage survival and function, robust phosphorylation of CSF1R was observed at multiple tyrosine residues following treatment (Figure 2). Phosphorylation peaked at 5 minutes post-treatment, with fold changes of 18.6-fold at Tyr561, 14-fold at Tyr699, 3-fold at Tyr708, and 11.7-fold at Tyr723. Concurrently, total CSF1R levels decreased over time, consistent with receptor internalization or degradation following activation.

Phosphorylation of downstream effectors ERK1/2 at Thr202 and Tyr204 (5-fold) and AKT1/2/3 at Ser473 (6-fold) at 5 minutes further confirmed activation of key signaling pathways involved in microglial and macrophage function.

Overall, the assays exhibited high sensitivity, reproducibility, and dynamic range, highlighting the AlphaLISA SureFire® Ultra platform as a reliable and precise tool for measuring CSF1R signaling and evaluating pathway modulation in complex cellular models.