Investigation of the influence of sex and prenatal steroids on synapse formation in human cerebral organoids.

Several prominent neurodevelopmental disorders have differing prevalence between males and females (McCarthy 2016). For example, there appears to be a male bias in autism spectrum condition (ASC), with an estimated 3:1 ratio of males to females with ASC (Loomes, Hull, and Mandy 2017). It is still unclear why this difference exists, although there is increasing evidence that prenatal steroids including androgens and estrogens are associated with increased likelihood of autism (Baron-Cohen et al. 2020), with increased testosterone levels associated with higher ASC likelihood or traits (Baron-Cohen et al. 2011; Bos et al. 2016). Prenatal steroids have key roles in the development and sexual differentiation of the brain. Levels of testosterone in serum are elevated in males between 18 and 24 weeks of gestation and in the first months after birth, coinciding with key stages of neurodevelopment (Hines, Constantinescu, and Spencer 2015). Fetal steroids may contribute to latent sex differences in rodents, such that the end result is the same but the developmental mechanisms differ in males and females (Sellers et al. 2020). Human neural stem cell treatment with androgens increases their proliferation and affects expression of ~200 genes, including genes associated with or involved in ASC (Quartier et al. 2018). Kelava et al. show that steroids, specifically androgens, affect early corticogenesis in human cerebral organoids (2020). However, it is not known how steroids affect synapse development, which is affected in ASC. Several highly penetrant mutations associated with ASC have been linked to the regulation of synaptic function, including mutations in the SHANK3 and NEUREXIN (NRXN1) genes (Zoghbi and Bear 2012). Emerging evidence shows steroids can also act as neurotransmitters and influence neuronal activity in mammals (Rudolph et al. 2016). However, little is known about steroid action in the developing human cortex. There are now established protocols for reproducibly growing cerebral organoids (Lancaster et al. 2013; Velasco et al. 2019), which can be used as a model of early human brain development (Camp et al. 2015) to study such questions. Given also the proposed role of steroids in ASC likelihood, an investigation of the effect of prenatal steroids on cortical and synapse development is worthwhile. An intriguing link between the ASC-linked gene SHANK3 and steroids has been reported, whereby sex hormones regulate SHANK expression (Berkel et al. 2018). Existing published and unpublished data from the Srivastava lab suggests that male human induced pluripotent stem cell-derived (hiPSC) forebrain neurons can respond to a range of fetal steroids, for example 17B-estradiol, with effects on neurite outgrowth, synaptic protein expression, and synaptic gene expression, such as synaptic proteins interacting with NRXN1 (Shum et al. 2015). Lab data from male and female hiPSC lines with mutations in SHANK3 and NRXN1 indicates potential male-female differences in neuronal network activity (SHANK3 hiPSC lines) or gene expression differences (NRXN1 hiPSC lines).