Animal and clinical studies support the biological vulnerability to chronic central nervous system (CNS) diseases in FASD populations is driven by dysfunctional CNS-immune interactions, which include signaling and transcriptional regulatory factors often engaged following toll-like 4 (TLR4) immune receptor activation, and may underlie cognitive deficits precipitated by typical stressors; a secondary insult or “second hit”. Frequently, these problems do not manifest until adolescence or early adulthood. One potential mechanism driving CNS immune dysfunction as a consequence of prenatal alcohol exposure (PAE) (“first hit”) may involve circular RNAs (circRNAs), which are brain-enriched non-coding RNAs capable of acting as diverse regulators of gene expression and immune function via both transcriptional and post-transcriptional mechanisms. We recently published that a subset of circRNAs is dysregulated in the prenatal brain as a result of PAE. Preliminary data show dysregulated circRNAs from PAE in rodent blood and spinal cord, and sex differences were present between elevated proinflammatory immune factors in the rodent female amygdala when stimulated by a “second minor hit”, an acute stressor, with PAE. These immune factors overlap with the intracellular TLR4 activation pathway thus narrowing the breadth of immune factors that may be dysregulated by PAE following a secondary challenge. Leveraging clinical data from adolescent children with an FASD (Component 3; current Phase III P50) who are evaluated for cognitive regulation, the goals of Component 5 is to evaluate and correlate the impact of PAE on the expression of a subset of circRNAs, and associated mRNAs and proteins from (1) adolescent mouse blood and brain regions that support cognitive function involving working memory following minor challenges, and (2) saliva and blood of adolescent children with an FASD undergoing specific cognitive tasks. The overarching hypothesis of Component 5 is that a subset of circRNAs is dysregulated by PAE, creating brain immune sensitization that: 1) is necessary and sufficient for cognitive deficits precipitated by minor challenges in adolescent mice, and (2) predicts cognitive deficits and stress perception in human adolescents with FASD. Sex differences are integrated into the three Aims proposed to test this hypothesis. Component 5 of Phase III will complement other research components by evaluating middle adolescent cognitive function of mouse offspring with PAE using age-appropriate cognitive tests, which may predict and complement cognitive deficits that develop in young adulthood, as assessed by other components of Phase III. CircRNA, mRNA and protein analyses from Component-3 adolescent mice can be correlated with measured neuroimmune factors from brains of behaviorally validated young adult mice from other components. Newly identified circRNAs that regulate associated mRNA/protein immune factors will be correlated with Component 3’s clinical study o...