Craniofacial Morphogenesis in Prenatal Alcohol Exposure

Univ Of North Carolina Chapel Hill, Chapel Hill NC

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Abstract

ABSTRACT Prenatal alcohol exposure (PAE) remains a leading cause of permanent neurodevelopmental disability. Diagnosis is often initiated by a distinctive craniofacial appearance that originates, in part, from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the Neural Crest. In the prior award period, we demonstrated that alcohol causes nucleolar stress in neural crest through its suppression of Ribosome Biogenesis (RBG), and this suppression is causative in their MDM2/p53-mediated apoptosis. We further showed that this nucleolar stress originates from alcohol’s activation of AMPK, which suppresses TORC1 and the RPS6K-mediated stimulation of RBG. How alcohol activates AMPK is unknown. Studies in this competing renewal will identify the underlying mechanism through which AMPK initiates this nucleolar stress in neural crest. Specifically, we test the novel hypothesis that alcohol causes nucleolar stress because it suppresses the metabolic reprogramming (Warburg effect) that is essential to sustain their high rates of RBG and proliferation. Aim 1 shows that neural crest rely on glycogen to fuel their Warburg effect, and that alcohol reduces their glycolysis, in part, because it suppresses glycogenolysis. Aim 2 shows that this reduction in glycolysis activates AMPK, which then suppresses this glycolytic reprogramming via the Hippo-mediated repression of YAP/TAZ, which upregulates glycolytic gene expression during reprogramming. Aim 3 shows that alcohol’s parallel suppression of Oxidative Phosphorylation (OxP), plus the neural crest’s exclusive reliance on glucose, prevents OxP from rescuing the cells from apoptosis. Studies utilize the established mouse neural crest primary cell line, O9-1, which recapitulates cranial neural crest fates and development. The involvement of these signaling pathways is tested functionally using molecular approaches including small molecule inhibitors and transfection with gain- and loss-of-function effectors. Their metabolic activity is directly quantified using real-time cellular respirometry (Seahorse, Oroboros O2k) to dissect their fuel choice and OxP activity. RBG consumes ~80% of total ATP generation in dividing cells, and the neural crest cell doubling time is just 19- 22hr. Our pilot data show they have an exceptional metabolic rate that is necessary to meet their high nucleotide and ATP demands. Thus, like cancer cells, neural crest relies on the Warburg effect to support these processes, and alcohol’s disruption of this metabolism initiates their nucleolar stress and p53-mediated apoptosis. This mechanism explains why the cranial neural crest is so vulnerable to alcohol. Moreover, because we reported elsewhere that nucleolar stress also drives the apoptosis of primary neural stem cells, this may represent a broader and novel mechanism that underlies alcohol’s developmental toxicity.