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Ethanol Exposure Transiently Elevates but Persistently Inhibits Tyrosine Kinase Activity and Impairs the Growth of the Nascent Apical Dendrite

  • Dandan Wang
  • Joshua Enck
  • Brian W. Howell
  • Eric C. OlsonEmail author
Article
  • 72 Downloads

Abstract

Dendritogenesis can be impaired by exposure to alcohol, and aspects of this impairment share phenotypic similarities to dendritic defects observed after blockade of the Reelin-Dab1 tyrosine kinase signaling pathway. In this study, we find that 10 min of alcohol exposure (400 mg/dL ethanol) by itself causes an unexpected increase in tyrosine phosphorylation of many proteins including Src and Dab1 that are essential downstream effectors of Reelin signaling. This increase in phosphotyrosine is dose-dependent and blockable by selective inhibitors of Src Family Kinases (SFKs). However, the response is transient, and phosphotyrosine levels return to baseline after 30 min of continuous ethanol exposure, both in vitro and in vivo. During this latter period, Src is inactivated and Reelin application cannot stimulate Dab1 phosphorylation. This suggests that ethanol initially activates but then silences the Reelin-Dab1 signaling pathway by brief activation and then sustained inactivation of SFKs. Time-lapse analyses of dendritic growth dynamics show an overall decrease in growth and branching compared to controls after ethanol-exposure that is similar to that observed with Reelin-deficiency. However, unlike Reelin-signaling disruptions, the dendritic filopodial speeds are decreased after ethanol exposure, and this decrease is associated with sustained dephosphorylation and activation of cofilin, an F-actin severing protein. These findings suggest that persistent Src inactivation coupled to cofilin activation may contribute to the dendritic disruptions observed with fetal alcohol exposure.

Keywords

Fetal alcohol syndrome disorder Cortical development Src kinases Dab1 Dendritogenesis Cofilin 

Abbreviations

FASD

fetal alcohol spectrum disorder

SFKs

Src family kinases

EtOH

ethanol

PAO

phenylarsine oxide

MβCDs

methyl-β-cyclodextrins

RM

Reelin-conditioned medium

Notes

Acknowledgements

We thank members of the Olson lab and Krysten O’Hara for assistance and valuable input on the project.

Author Contribution

EO and DW designed the study with significant input from BH. DW and JE performed the study. EO and DW wrote the manuscript with comments and edits from BH, JE, and members of the Developmental Exposure to Alcohol Research Center (DEARC).

Funding Information

This work was supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) via the Developmental Exposure to Alcohol Research Center (DEARC) P50AA017823-10.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1473_Fig8_ESM.png (360 kb)
Supplementary Figure 1

a and b, Exposure to increasing concentrations (0-0.75%) of EtOH did not reduce cell viability of embryonic cortical neurons as determined by Calcein AM / Propidium Iodide (PI) assay. Live cells internalize the Calcein dye but are not stained with propidium iodide. Cortical cultures were treated with different concentration of EtOH (0.25%, 0.5% and 0.75%) for different time (1h, 4h or 16h) followed with 30 min incubation of 3 μM Calcein AM and 5 μM PI. Wells containing cells treated with 0.2% Triton X-100 (15min) was used as control. Fluorescence signal was detected at 485nm and 528nm. Data was normalized to untreated cells. c, Membrane cholesterol manipulation did not significantly alter EtOH-induced tyrosine phosphorylation. Cortical cultures were treated with 10 min of 0.5% EtOH after cholesterol supplementation (500μM, 30 min) or cholesterol depletion using methyl-β-cyclodextrin (MβCD, 500μM, 30 min). PY99 expression was determined by western blotting. GAPDH was used as loading control. d, Quantification of blots from figure c. One-way ANOVA followed by Bonferroni post hoc test was used between different groups. NS, p > 0.05, # p < 0.001. (PNG 360 kb)

12035_2019_1473_MOESM1_ESM.tif (2.9 mb)
High Resolution Image (TIF 2930 kb)

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Neuroscience and PhysiologySUNY Upstate Medical UniversitySyracuseUSA
  2. 2.Developmental Exposure to Alcohol Research Center (DEARC)Binghamton UniversityBinghamtonUSA

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