Abstract:
Prenatal ethanol exposure induces aberrations in brain development leading to CNS dysfunctions. Astrocytes contribute to neuronal development and functions through the release of proteins and peptides. Stimulation of astrocytes with the cholinergic agonist carbachol induces hippocampal neuron neuritogenesis, which was inhibited when astrocytes were co-incubated with carbachol and ethanol. The effect of carbachol-treated astrocytes on neuritogenesis is mediated by soluble factors released by astrocytes. The goals for this research were to investigate the astrocyte secretome with the purpose of identifying astrocyte-released proteins involved in neuronal development, and to characterize proteins whose release by astrocytes is modulated by carbachol and/or ethanol.
A shotgun proteomic method with Gene Ontology analysis identified 133 secreted proteins. Many of the identified extracellular proteins were involved in neuronal development, underscoring the role of astrocyte secretome in neuronal – astrocyte communication, especially during brain development. Spectral counting quantitation identified carbachol treatment increased release of 15 proteins and decreased the release of 17 proteins. The second aim of this research was to examine the effect of carbachol on the secretion of laminin, fibronectin and PAI-1, and their potential roles in hippocampal neuronal neuritogenesis stimulated by carbachol-treated astrocytes. Exposure of astrocytes to carbachol increased the expression and release of fibronectin and laminin in these cells. This effect was mediated in part by an increase in laminin and fibronectin mRNA levels, and in part by increased production and release of PAI-1, an inhibitor of extracellular matrix proteolytic degradation. Carbachol-induced production and release of these three proteins was due to the activation of MS muscarinic receptors. The inhibition of fibronectin activity strongly lessened the effect of carbachol on neurite elongation, while inhibition of laminin activity only reduced the minor neurite elongation. PAI-1 not only increased fibronectin and laminin extracellular levels when incubated with astrocytes, but it also exhibited direct neuritogenic effects to the neurons. Ethanol treatment reduced the release of fibronectin and PAI-1 from carbachol-treated astrocytes. Thus the inhibition of carbachol-induced release of fibronectin and PAI-1 by ethanol may contribute to CNS abnormalities associated with prenatal ethanol exposures, by inhibiting the release of neuritogenic factors by astrocytes.