Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Abstract

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.

Ethanol Neurotoxicity in the Developing Cerebellum: Underlying Mechanisms and Implications

“…Downstream, important effectors for active Rac1 and Cdc42 are the p21-activated kinases (PAKs), and for RhoA is Rho-kinase/ROCK. Previous studies from our laboratory and others confirm that ethanol alters the activity of Rho-GTPases and its effectors in neuronal cells [53] and non-neuronal cells [124,125,126], and regulates microtubules and F-actin cytoskeleton dynamics [127,128,129]. Using primary CGN culture from ethanol exposed seven-day-old pups, our laboratory found that moderate ethanol exposure (blood alcohol concentration 40 mM) inhibits the activation of RacI and stunts the formation of neurites, while RhoA activation at high dose of ethanol (blood alcohol 80 mM) promotes apoptosis [53].…”

Protein Traffic Is an Intracellular Target in Alcohol Toxicity

“…In addition to RhoGTPases RhoA, Rac1 and Cdc42, these proteins include clathrin, adaptor AP-2, sorting nexin SNX9, GTPases Rab5, Rab11, or endosomal marker EEA1. In addition, ethanol exposure also affects the cholesterol cellular levels related with clathrin-independent endocytosis [17]. A toxic effect of alcohol on endocytosis can affect some important neuronal activities that depend on the endocytic process, including synaptic vesicle recycling, regulation of the number of signaling receptors, trafficking of postsynaptic receptors, polarized axon elongation, growth cone navigation, neuronal migration and hippocampal plasticity.…”

Chronic exposure to alcohol alters network activity and morphology of cultured hippocampal neurons

“…For example, neuronal death has been reported in the cerebellum (Pickering et al, 2010), but not in the hippocampus (e.g. Marín et al, 2010).…”