Abstract

Environmental estrogenic compounds are increasingly recognized as neurotoxicants, yet their effects on microglia intracellular homeostasis in vivo remain poorly understood. Using zebrafish larvae as a live imaging model, we identify the synthetic estrogen mestranol as a potent inducer of a reversible lysosomal storage-like state in microglia. Mestranol exposure causes pronounced microglia hypertrophy and reduces neutral red staining, without affecting microglia number or neuronal apoptosis. Functional analyses show that mestranol-treated microglia retain phagocytic capacity toward apoptotic neurons and bacterial particles but fail to efficiently digest internalized cargo, leading to intracellular accumulation. Although acidic vesicles and protease‑associated compartments expand and disperse throughout the cytoplasm, lysosomal degradative efficiency is markedly impaired, suggesting that cargo digestion is compromised despite the formation of acidic phagolysosomal compartments. Transcriptomic profiling of flow‑sorted macrophage/microglia populations reveals coordinated down‑regulation of lysosomal-phagosomal and immune gene networks, including MIT/TFE family transcription factors (TFEB/TFEC) and immune master regulators such as SPI1, IRFs, BATF, MAFB, and RUNX3. Overexpression of TFEC partially rescues both estradiol and mestranol-induced microglia hypertrophy and neutral red loss, although the rescue is only partial, indicating a more complex mechanism involving additional TFEC-independent pathways. Notably, microglia lysosomal dysfunction and hypertrophy are reversible upon drug withdrawal. Together, our findings demonstrate that mestranol induces an acquired and regulatable lysosomal stress state in microglia, characterized by immune transcriptional suppression and impaired intracellular digestion.