18 kDa TSPO-mediated neurosteroidogenesis controls cholesterol homeostasis in tuning microglia response to inflammatory stimulus

In neurodegenerative diseases, microglia commonly accumulate toxic levels of cholesterol, compromising their ability to resolve the neuroinflammatory response. Here, we explored the role of neurosteroidogenesis, a complex cholesterol-metabolizing pathway that produces anti-inflammatory neurosteroids, in regulating cholesterol dynamics in IL-1β-activated human microglia. Our investigation focused on the Translocator protein (TSPO, 18 kDa), which plays a pivotal role in initiating neurosteroidogenesis, representing its rate-limiting step. Microglia in which TSPO was knocked down (TSPO KD) exhibited a marked reduction in neurosteroid biosynthesis suggesting an impaired cholesterol metabolism. Concurrently, these cells showed upregulated expression of the SREBP/HMGCR/Fdft-1/CEH axis, indicating compensatory activation of the systems aimed at increasing cholesterol availability, and downregulated cholesterol membrane efflux. As a result of these dynamics, excessive cholesterol significantly accumulated, suggesting inadequate cholesterol clearance. This dysregulation was further exacerbated in IL-1β-treated TSPO KD microglia. The impairment of neurosteroidogenic biosynthetic capacity, combined with pronounced downregulation of SREBP/HMGCR/Fdft-1/CEH axis and cholesterol membrane efflux, resulted in a severe cholesterol overload, highlighting a complete disruption of cholesterol trafficking mechanism cross-talks. Noteworthy, these cells exhibited hallmarks of neurodegenerative diseases-associated microglia (MGnD), including heightened inflammatory reactivity. On the other hand, the stimulation of TSPO-mediated neurosteroidogenesis, known to promote a reparative microglial phenotype, significantly reduced cholesterol accumulation. Neurosteroids such as allopregnanolone and estradiol emerged as key mediators in enhancing cholesterol clearance. In conclusion, these findings underscore that native neurosteroidogenesis is a crucial autocrine regulator of cholesterol trafficking in activated human microglia. They further highlight the therapeutic potential of targeting TSPO-mediated neurosteroidogenesis for the treatment of neuroinflammatory-based diseases by restoring cholesterol homeostasis and promoting reparative functions in MGnD.