Gag‐like proteins: Novel mediators of prenatal alcohol exposure in neural development

Background We previously showed that ethanol did not kill fetal neural stem cells (NSCs), but that their numbers nevertheless are decreased due to aberrant maturation and loss of self‐renewal. To identify mechanisms that mediate this loss of NSCs, we focused on a family of Gag‐like proteins (GLPs), derived from retroviral gene remnants within mammalian genomes. GLPs are important for fetal development, though their role in brain development is virtually unexplored. Moreover, GLPs may be transferred between cells in extracellular vesicles (EVs) and thereby transfer environmental adaptations between cells. We hypothesized that GLPs may mediate some effects of ethanol in NSCs. Methods Sex‐segregated male and female fetal murine cortical NSCs, cultured ex vivo as nonadherent neurospheres, were exposed to a dose range of ethanol and to mitogen‐withdrawal‐induced differentiation. We used siRNAs to assess the effects of NSC‐expressed GLP knockdown on growth, survival, and maturation and in silico GLP knockout, in an in vivo single‐cell RNA‐sequencing dataset, to identify GLP‐mediated developmental pathways that were also ethanol‐sensitive. Results PEG10 isoform‐1, isoform‐2, and PNMA2 were identified as dominant GLP species in both NSCs and their EVs. Ethanol‐exposed NSCs exhibited significantly elevated PEG10 isoform‐2 and PNMA2 protein during differentiation. Both PEG10 and PNMA2 were mediated apoptosis resistance and additionally, PEG10 promoted neuronal and astrocyte lineage maturation. Neither GLP influenced metabolism nor cell cycle in NSCs. Virtual PEG10 and PNMA2 knockout identified gene transcription regulation and ubiquitin‐ligation processes as candidate mediators of GLP‐linked prenatal alcohol effects. Conclusions Collectively, GLPs present in NSCs and their EVs may confer apoptosis resistance within the NSC niche and contribute to the abnormal maturation induced by ethanol. Retroviral Gag‐like proteins (GLPs) PEG10 and PNMA2 are expressed in fetal neural stem cells (NSCs) and their secreted extracellular vesicles. Ethanol‐exposed NSCs exhibit elevated PEG10 and PNMA2 during differentiation, and both GLPs mediate apoptosis resistance. Additionally, PEG10 promoted neuronal/astrocyte lineage maturation. Virtual GLP knockout identified gene transcription regulation and ubiquitin‐ligation processes as candidate mediators of GLP‐linked developmental ethanol effects. GLPs present in and secreted by NSCs may confer apoptosis resistance, but contribute to abn