TSPO REGULATES HIPPOCAMPAL NEURONAL DEVELOPMENT THROUGH ESTRADIOL/BDNF/ NGN3 PATHWAY IN A SEX DEPENDENT MANNER

Abstract

TSPO is involved in the rate-limiting step of de novo neuroactive steroids (NAS) production. Although studies have proved the effects of NAS on neuritogenesis in the hippocampus, none of them have yet investigated the potential role of TSPO in promoting neuronal development, as well as whether TSPO could have sex-dimorphic activities on brain cells. Primary hippocampal neuronal cultures were established using mouse embryos at 17 days of gestation and cultured separately based on sex. TSPO mRNA and protein levels were evaluated on different days in vitro (DIVs). To establish the role of TSPO in neuronal development, neurons were treated with the selective TSPO ligands, PK11195, XBD-173, or PIGA1138, or cotreated with the inhibitors aminoglutethimide, letrozole, or k252a, thus morphological analysis was performed. Neuronal TSPO was silenced by nucleoporation and the expression of neuronal markers and of the estradiol-regulated expression of BDNF and Neurogenin3 (Ngn3) were investigated. Sex dimorphisms in neuronal TSPO expression were observed only at the DIV1. Treatment with TSPO ligands increased axon length and branching with major effects in female neurons. Co-treatment with TSPO ligands and inhibitors abolished the effects of TSPO ligands on neuronal development, suggesting the role of estradiol (E2) as an inter-player for TSPOmediated neuritogenesis. Increased levels of E2, BDNF, and Ngn3 also confirmed the involvement of this cellular pathway. Notably, TSPO silencing affected the expression of neuronal markers related to proper neuronal functions and development only in female neurons, with particular effects on Ngn3 expression. Results show that the regulation of neuronal TSPO activity may occur in a sex-dependent manner. TSPO-mediated regulation of endogenous levels of E2 and of the pro-neural factors BDNF and Ngn3 paves the way for using TSPO as a possible therapeutic target to promote neuronal development and synapse formation.