Prenatal alcohol exposure alters p35, CDK5 and GSK3β in the medial frontal cortex and hippocampus of adolescent mice

Abstract

Fetal alcohol spectrum disorders (FASDs) are the number one cause of preventable mental retardation. An estimated 2-5% of children are diagnosed as having a FASD. While it is known that children prenatally exposed to alcohol experience cognitive deficits and a higher incidence of psychiatric illness later in life, the pathways underlying these abnormalities remain uncertain. GSK3β and CDK5 are protein kinases that are converging points for a vast number of signaling cascades, including those controlling cellular processes critical to learning and memory. We investigated whether levels of GSK3β and CDK5 are affected by moderate prenatal alcohol exposure (PAE), specifically in the hippocampus and medial frontal cortex of the adolescent mouse. In the present work we utilized immunoblotting techniques to demonstrate that moderate PAE increased hippocampal p35 and β-catenin, and decreased total levels of GSK3β, while increasing GSK3β Ser9 and Tyr216 phosphorylation. Interestingly, different alterations were seen in the medial frontal cortex where p35 and CDK5 were decreased and increased total GSK3β was accompanied by reduced Tyr216 of the enzyme. These results suggest that kinase dysregulation during adolescence might be an important contributing factor to the effects of PAE on hippocampal and medial frontal cortical functioning; and by extension, that global modulation of these kinases may produce differing effects depending on brain region.

Prenatal Alcohol Exposure Leads to Enhanced Serine 9 Phosphorylation of Glycogen Synthase Kinase‐3β (GSK‐3β) in the Hippocampal Dentate Gyrus of Adult Mouse

“…Although we did not directly measure GSK‐3 β catalytic activity in this study, it is likely substantially suppressed because GSK‐3 β is a constitutively active kinase predominantly regulated via inactivation through phosphorylation at Ser9 (Doble and Woodgett, ). These findings corroborate prior observations of elevated p(Ser9)GSK‐3 β in whole hippocampus of adolescent PAE mice exposed to an identical gestational drinking paradigm, but without EE (Goggin et al., ). Taken together, these studies demonstrate that exposure to even moderate levels of alcohol during gestation leads to long‐term elevation of p(Ser9)GSK‐3 β through young adulthood.…”

“…Tissue was homogenized in 100 μ l of buffer containing the following components: 20 mM Tris‐HCl, pH 7.4, 1 mM EDTA, 320 mM sucrose, 20 mM β ‐glycerophosphate, 20 mM sodium pyrophosphate, 10 mM sodium fluoride, 200 μ M sodium orthovanadate, and protease inhibitor cocktail (1:1,000; Sigma‐Aldrich). The homogenates were centrifuged twice at 1,000× g for 6 minutes at 4°C, and the supernatants were combined as a “postnuclear lysate.” Protein concentrations were determined using the Bradford protein assay (Bio‐Rad, Hercules, CA) and subjected to analysis by routine Western immunoblotting as previously described (Diaz et al., ; Goggin et al., ). Briefly, samples were diluted in NuPAGE LDS Sample Buffer (#NP0007; InVitrogen, Grand Island, NY), heated at 70°C for 5 minutes, and loaded (10 or 20 μ g protein/well) into 4 to 12% Bis‐Tris precast gels (#NP0336; InVitrogen).…”

“…The blots were scanned using 2‐channel infrared direct detection (Odyssey Imaging System; LI‐COR Biosciences) and quantified using Image Studio (version 3.1; LI‐COR Biosciences). Coomassie staining was performed as a within‐lane loading control as previously described (Goggin et al., ). Following immunodetection, membranes were stained with Coomassie Brilliant Blue R‐250 (#161‐0400; Bio‐Rad) and quantified using Quantity One 1‐D analysis software (Bio‐Rad).…”

CB1R regulates CDK5 signaling and epigenetically controls Rac1 expression contributing to neurobehavioral abnormalities in mice postnatally exposed to ethanol

“…In contrast, conditional ablation of CDK5 in the HP exhibited loss of the cAMP pathway and impaired CREB phosphorylation [44]. Interestingly, ethanol exposure throughout the gestational period was shown to reduce p35 and CDK5 levels in the adolescent medial frontal cortical tissues [45]. These observations together suggest that postnatal ethanol-activated CB1R facilitates the generation of the CDK5 activator, p25 (CDK5 activation), and impairs the downstream signaling leading to neurodegeneration in neonatal mice.…”

Early ethanol exposure and vinpocetine treatment alter learning‐ and memory‐related proteins in the rat hippocampus and prefrontal cortex

“…Rodents exposed to alcohol prenatally or during the early postnatal period have been shown to exhibit deficits in learning and memory (Filgueiras et al, 2010), depression-like behavior and hyperactivity (Nunes et al, 2011;Brocardo et al, 2012;Brys et al, 2014), and a greater vulnerability to developing addiction-like behaviors (Barbier et al, 2008). Brainderived neurotrophic factor (BDNF) and its signaling pathways, comprising, among others, mitogen-activated protein kinase phosphatase (MKP-1), extracellular signalregulated kinase 1/2 (ERK1/2), and glycogen synthase kinase 3 b (GSK3b) have all been implicated in developmental and learning and memory processes (Lee and Son, 2009;Schmitt et al, 2009;Peng et al, 2010;Goggin et al, 2014). Several studies have indicated changes in neurotrophins and their signaling pathways after early ethanol exposure (Barbier et al, 2008;DuPont et al, 2014;Goggin et al, 2014).…”