Department of Pharmacology and Pharmaceutical Sciences, Alfred E. Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, USA.
Department of Pharmacology and Pharmaceutical Sciences, Alfred E. Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, USA.
J Biol Chem. 2023 Aug;299(8):105035. doi: 10.1016/j.jbc.2023.105035. Epub 2023 Jul 11.
Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.
神经甾体是神经系统合成的甾体，可以通过基因组和非基因组途径发挥神经调节和神经保护作用。神经甾体和主要的甾体前体孕烯醇酮在各种疾病中具有治疗潜力，如精神和疼痛障碍，并且可能在髓鞘形成、神经炎症、神经递质传递和神经可塑性中发挥重要作用。尽管孕烯醇酮是由外周甾体生成器官中的 CYP11A1 合成的，但我们最近的研究表明，在人类神经胶质细胞中，孕烯醇酮必须由另一种线粒体细胞色素 P450（CYP450）酶而不是 CYP11A1 合成。因此，我们试图确定人类大脑中负责孕烯醇酮生成的 CYP450。在筛选具有线粒体定位的人脑中表达的 CYP450 后，我们确定了三种酶候选物：CYP27A1、CYP1A1 和 CYP1B1。我们发现，通过抑制剂和 siRNA 敲低抑制 CYP27A1 不会对人神经胶质细胞中的孕烯醇酮合成产生负面影响。同时，用 CYP1A1/CYP1B1 抑制剂处理人神经胶质细胞会显著降低 22(R)-羟基胆固醇存在时的孕烯醇酮生成。我们在人神经胶质细胞中进行了 CYP1A1 或 CYP1B1 的 siRNA 敲低，发现只有 CYP1B1 敲低显著降低了孕烯醇酮的生成。此外，线粒体靶向 CYP1B1 的过表达在基础条件下以及在羟胆固醇和低密度脂蛋白存在下显著增加了孕烯醇酮的生成。通过抑制剂或 siRNA 敲低抑制 CYP1A1 和/或 CYP1B1 不会显著减少人肾上腺皮质细胞中的孕烯醇酮合成，这表明 CYP1B1 不是外周的主要孕烯醇酮生成酶。这些数据表明，线粒体 CYP1B1 参与了人神经胶质细胞中的孕烯醇酮合成。
