Abstract
Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.
Highlights
The CDC estimates that 0.2–1.5 per 1000 live births are children with fetal alcohol syndrome disorder (FASD), a syndrome characterized by disrupted fetal brain development and postnatal intellectual disability (ID) [1, 2]
We found that phosphotyrosine levels in lysates of primary cortical cultures derived from Swiss Webster (SW) embryos was transient and largely absent after 30 min of continuous ethanol exposure, a timeline that was paralleled by the in vivo response
We provide evidence that maternal exposure to ethanol leads to rapid activation of Src family kinases and an elevation of tyrosine phosphorylation in multiple fetal cortical regions and cell types
Summary
The CDC estimates that 0.2–1.5 per 1000 live births are children with FASD, a syndrome characterized by disrupted fetal brain development and postnatal intellectual disability (ID) [1, 2]. Disrupted connectivity including altered dendritic structure, axonal pathfinding, and white matter tracts are common findings in FAS and are thought to be major contributors to ID [3,4,5,6]. This study extends our recent finding that ethanol exposure leads to SFK activation, inappropriate tyrosine phosphorylation in cultured primary neurons [7]. One consequence of the disruption is that the Reelin signaling pathway can no longer be activated in the presence of ethanol [7]. The Reelin-Dab signaling is absolutely required for proper cortical development [8], and genetic disruptions of this pathway are associated with disorganized cortical architecture and severe intellectual disability in humans [9]. Complete absence of Reelin [10], the receptors [12], the essential adaptor protein Dab1 [13], or the Src family kinases (SFKs) Src and Fyn [14] leads to similar histological
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