Cadmium-induced neuroinflammatory cytokines impairs BBB permeability by altering tight junction morphological localization

There is increasing evidence that environmental pollutants like polycyclic aromatic hydrocarbons (PAHs) and heavy metals may act as endocrine disruptors, negatively affecting various physiological functions, including reproduction. In particular, both pollutants may impact the neuroendocrine circuits of the reproductive axis, with the hypothalamic gonadotropin-releasing hormone (GnRH) system being the crucial target. Our previous research using human fetal GnRH neuroblasts (FNCB4) demonstrated that treating cells with benzo(a) pyrene (BaP, 10μM, 24h), a representative PAH, interferes with their migratory properties and therefore maturation of GnRH neurons [1]. Here, we extended our studies with the main purpose of clarifying the mechanisms through which BaP affects FNCB4 migration. In the gene expression profile analysis, performed with the RNA-seq technique, we identified 2,324 differentially expressed genes (DEGs) in BaP-treated compared to untreated cells, including 1,128 up-regulated and 1,196 down-regulated. Reactome enrichment analysis indicated that BaP exposure induced significant changes in genes involved in cell motility pathways, such as Rho GTPase signaling, semaphorin interactions, ECM proteoglycans, integrin and non-integrin cell surface interactions. Interestingly, we found 31 DEGs that are related to RhoA/ROCK pathway, an important signaling implicated in cell adhesion, cytoskeletal remodeling and migration, especially in neurons. To better investigate the BaP mechanism of action and confirm the implication of RhoA/ROCK pathway, we analyzed the subcellular localization of the small GTPase RhoA in FNCB4. Immunofluorescence analysis showed that BaP exposure inhibited RhoA membrane translocation and, therefore, its activation, thus compromising the downstream signaling. Interestingly, exposing cells to cadmium (Cd, 10μM, 24h), a widespread pollutant belonging to the heavy metal category, also affected FNCB4 cell migration without interfering with RhoA membrane translocation. In conclusion, our findings suggest different molecular mechanisms of action for the two pollutants and identified the alteration of the RhoA/ROCK 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).