BaP inhibits human GnRH neuron migration by altering the RhoA pathway

Benzo[a]pyrene (BaP) is a widespread pollutant that can act as an endocrine-disrupting chemical, negatively affecting various physiological functions, including reproduction. The central network of reproduction is controlled by gonadotropin-releasing hormone (GnRH) neurons, which originate in the olfactory placode and migrate to the hypothalamus during fetal development. Using human fetal GnRH neuroblasts (FNCB4), we recently demonstrated that BaP (10µM, 24h) interferes with the migratory ability and, therefore, maturation of GnRH neurons. In this study, we used RNA-sequencing (RNA-seq) to clarify the mechanisms by which BaP affects FNCB4 migration. The differential expression analysis identified 585 significant differentially expressed genes (DEGs; FDR<0.05) in BaP-treated (10µM, 24h) compared to untreated cells, including 272 up-regulated and 313 down-regulated genes. According to functional analysis by Gene Ontology (GO) enrichment, BaP specifically altered 86 genes related to cell adhesion, cell migration and extracellular matrix organization processes. Similarly, Reactome enrichment analysis indicated that BaP exposure significantly changed genes involved in cell motility pathways, such as syndecan interactions, extracellular matrix proteoglycans, integrin and non-integrin cell surface interactions. Among these, we found a down-regulation of syndecan-2, syndecan-4 and CD44 which are strictly related to the RhoA pathway, an important signaling implicated in cell adhesion, cytoskeletal remodeling and migration, especially in neurons. To better understand the BaP mechanism of action and confirm the implication of RhoA, we analyzed its subcellular localization in FNCB4. Accordingly, immunofluorescence analysis showed that BaP exposure inhibited RhoA membrane translocation and, therefore, its activation, thus compromising the downstream signaling. In conclusion, our findings suggest the alteration of the RhoA pathway as a possible mechanism through which BaP affects GnRH neuron development. This work was supported by #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DR. 1553 11.10.2022).