Long-Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin-Mediated Intracellular Signaling and in Particular Protein Kinase C

Background: Revealing the molecular changes in chronic ethanol‐impaired neuronal differentiation may be of great importance for understanding ethanol‐related pathology in embryonic development but also in the adult brain. In this study, both acute and long‐term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain‐derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal‐regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. Methods: The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT‐PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK‐cascade, protein kinase C (PKC) isoforms and Raf‐Kinase‐Inhibitor‐Protein (RKIP). Results: Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF‐mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf‐1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down‐regulated. Conclusions: Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC‐dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf‐1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long‐term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.