UNCOVERING MICROGLIA ADAPTATIONS PRIOR TO DEPLETION IN RESPONSE TO PLX5622 TREATMENT

Colony‑stimulating factor 1 receptor (CSF1R), a cell‑surface receptor in myeloid cells, plays a crucial role in microglial survival, proliferation, and function. Pharmacological CSF1R inhibitors, such as Plexxikon 5622 (PLX5622), are widely used to deplete microglia in vivo, enabling researchers to uncover microglia roles in health and disease. However, less is known about the cellular and molecular events in microglia that occur before depletion. To investigate these early responses, we performed functional assays, transcriptomic analysis, and in vivo validation.
Initial experiments were conducted on BV‑2 cells and primary microglia exposed to 10 µM PLX5622 for 6 to 48 hours. The morphological changes observed between 24-48 hours prompted investigation into underlying mechanisms. RNA sequencing and Gene Ontology analyses revealed time‑dependent upregulation of lysosomal and mitochondrial pathways. These transcriptomic signatures aligned with functional alterations detected by live‑cell imaging, flow cytometry, targeted metabolomics, Seahorse assays, and electron microscopy. Together, the findings suggest that microglia undergo metabolic exhaustion rather than canonical cell death following PLX5622 treatment.
Additionally, immunofluorescence analysis of CD68, TOMM20, LGAL3, and AIF1 was performed on brain tissue from mice fed a PLX5622‑chow diet (1,200 ppm; Plexxikon) based on AIN‑76A chow. Mice received the diet ad libitum for 10, 24, 48, or 72 hours, while controls were fed an identical PLX5622‑free diet. These approaches refine our understanding of early PLX5622 effects on microglia before depletion and provide deeper insight into CSF1R inhibition and its regulatory dynamics, broadening the framework for its applications in neuroimmunology.