SAT014 Involvement Of Cytochrome P450 1B1 In Synthesis Of Neurosteroid Pregnenolone In Human Brain Cells

Disclosure: Y. Lin: None. G. Cheung: None. Z. Zhang: None. V. Papadopoulos: None. Neurosteroids, steroids synthesized in the nervous system, have important functions in modulating brain function, neuroprotection, and reducing neuroinflammation. The neurosteroid pregnenolone, the precursor to all other neurosteroids, is one of the most abundant neurosteroids in the brain. Still, the enzyme known to make it, CYP11A1, has been difficult to detect in the human brain. We recently reported that unlike in peripheral steroidogenic organs, pregnenolone is not synthesized by CYP11A1 in human brain cells but rather by another mitochondrial cytochrome P450 enzyme (J Biol Chem. 2022 Jul;298(7):102110). Therefore, we aimed to identify the enzyme responsible for producing pregnenolone in the current study. Upon bioinformatic analyses of publicly available datasets for mitochondrial CYP450s expressed in the human brain, we identified three potential candidates: CYP27A1, CYP1A1, and CYP1B1. Given that CYP27A1 is highly expressed in the brain and is involved in cholesterol metabolism, we first tested the effects of CYP27A1 inhibition via drug treatment and siRNA knockdown on pregnenolone synthesis in steroidogenic MGM-1 human glioma cells. Neither treatment with CYP27A1 inhibitors anastrozole or dexmedetomidine nor CYP27A1 siRNA knockdown resulted in a significant reduction in pregnenolone formation by MGM-1 cells, suggesting that CYP27A1 likely is not involved in pregnenolone synthesis in human brain cells. On the other hand, MGM-1 cells treated with the CYP1A1/CYP1B1 inhibitors α-napthoflavone and 2,4,3’,5’-tetramethoxystilbene showed a significant reduction in pregnenolone production in the presence of the pregnenolone precursor 22R-hydroxycholesterol. To investigate whether this effect was due to the inhibition of CYP1A1 or CYP1B1, we knocked down CYP1A1 or CYP1B1 in MGM-1 cells using siRNA. While CYP1A1 knockdown did not result in any significant changes to pregnenolone synthesis, CYP1B1 knockdown significantly decreased secreted pregnenolone formation by MGM-1 cells. Together, these results suggest that CYP1B1 is likely involved in the alternative pathway for pregnenolone synthesis in human brain cells. Presentation: Saturday, June 17, 2023