Tara A Lindsley1, Samit N Shah, Elizabeth A Ruggiero. 1. Center for Neuropharmacology & Neuroscience, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA. lindslt@mail.amc.edu
Abstract
BACKGROUND: The effects of ethanol on development of postmitotic neurons include altered neurite outgrowth and differentiation, which may contribute to neuropathology associated with fetal alcohol spectrum disorders. We previously reported that ethanol exposure alters axon growth dynamics in dissociated cultures of rat hippocampal pyramidal neurons. Given the important regulatory role of small Rho guanosine triphosphatases (GTPases) in cytoskeletal reorganization associated with axon growth, and reports that ethanol alters whole cell Rho GTPase activity in other cell types, this study explored the hypothesis that ethanol alters Rho GTPase activity specifically in axonal growth cones. METHODS: Fetal rat hippocampal pyramidal neurons were maintained in dissociated cultures for 1 day in control medium or medium containing 11 to 43 mM ethanol. Some cultures were also treated with brain-derived neurotrophic factor (BDNF), an activator of Rac1 and Cdc42 GTPases that promotes axon extension. Levels of active Rho GTPases in growth cones were measured using in situ binding assays for GTP-bound Rac1, Cdc42, and RhoA. Axon length, growth cone area, and growth cone surface expression of tyrosine kinase B (TrkB), the receptor for BDNF, were assessed by digital morphometry and immunocytochemistry. RESULTS: Although ethanol increased the surface area of growth cones, the levels of active Rho GTPases in axonal growth cones were not affected in the absence of exogenous BDNF. In contrast, ethanol exposure inhibited BDNF-induced Rac1/Cdc42 activation in a dose-dependent manner and increased RhoA activation at the highest concentration tested. Similar TrkB expression was observed on the surface of axonal growth cones of control and ethanol-treated neurons. CONCLUSIONS: These results reveal an inhibitory effect of ethanol on growth cone signaling via small Rho GTPases during early stages of hippocampal development in vitro, and suggest a mechanism whereby ethanol may disrupt neurotrophic factor regulation of axon growth and guidance.
BACKGROUND: The effects of ethanol on development of postmitotic neurons include altered neurite outgrowth and differentiation, which may contribute to neuropathology associated with fetal alcohol spectrum disorders. We previously reported that ethanol exposure alters axon growth dynamics in dissociated cultures of rat hippocampal pyramidal neurons. Given the important regulatory role of small Rho guanosine triphosphatases (GTPases) in cytoskeletal reorganization associated with axon growth, and reports that ethanol alters whole cell Rho GTPase activity in other cell types, this study explored the hypothesis that ethanol alters Rho GTPase activity specifically in axonal growth cones. METHODS: Fetal rat hippocampal pyramidal neurons were maintained in dissociated cultures for 1 day in control medium or medium containing 11 to 43 mM ethanol. Some cultures were also treated with brain-derived neurotrophic factor (BDNF), an activator of Rac1 and Cdc42 GTPases that promotes axon extension. Levels of active Rho GTPases in growth cones were measured using in situ binding assays for GTP-bound Rac1, Cdc42, and RhoA. Axon length, growth cone area, and growth cone surface expression of tyrosine kinase B (TrkB), the receptor for BDNF, were assessed by digital morphometry and immunocytochemistry. RESULTS: Although ethanol increased the surface area of growth cones, the levels of active Rho GTPases in axonal growth cones were not affected in the absence of exogenous BDNF. In contrast, ethanol exposure inhibited BDNF-induced Rac1/Cdc42 activation in a dose-dependent manner and increased RhoA activation at the highest concentration tested. Similar TrkB expression was observed on the surface of axonal growth cones of control and ethanol-treated neurons. CONCLUSIONS: These results reveal an inhibitory effect of ethanol on growth cone signaling via small Rho GTPases during early stages of hippocampal development in vitro, and suggest a mechanism whereby ethanol may disrupt neurotrophic factor regulation of axon growth and guidance.
Authors: Yong Qian; Jia Luo; Stephen S Leonard; Gabriel K Harris; Lyndell Millecchia; Daniel C Flynn; Xianglin Shi Journal: J Biol Chem Date: 2003-02-21 Impact factor: 5.157
Authors: Jorge Santos Da Silva; Miguel Medina; Cecilia Zuliani; Alessia Di Nardo; Walter Witke; Carlos G Dotti Journal: J Cell Biol Date: 2003-09-29 Impact factor: 10.539
Authors: Shufeng Li; Bradley W Tuft; Linjing Xu; Marc A Polacco; Joseph C Clarke; C Allan Guymon; Marlan R Hansen Journal: Biomaterials Date: 2015-03-12 Impact factor: 12.479
Authors: Ellen Merrick Schill; Jonathan I Lake; Olga A Tusheva; Nandor Nagy; Saya K Bery; Lynne Foster; Marina Avetisyan; Stephen L Johnson; William F Stenson; Allan M Goldstein; Robert O Heuckeroth Journal: Dev Biol Date: 2015-11-14 Impact factor: 3.582