lucifer-yellow and Neuroblastoma

Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF-1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF-1 functionally bound to Gria2 promoter. Silencing of NRF-1 with small interference RNA prevented the depolarization-stimulated up-regulation of Gria2 and COX, and over-expression of NRF-1 rescued neurons from tetrodotoxin-induced down-regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF-1 is a critical agent in this process.

Topics: Animals; Animals, Newborn; Cells, Cultured; Chromatin Immunoprecipitation; Cyclooxygenase 2; Electron Transport Complex IV; Electrophoretic Mobility Shift Assay; Energy Metabolism; Isoquinolines; Mice; Mutagenesis; Neuroblastoma; Neurons; Nuclear Respiratory Factor 1; Potassium Chloride; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Receptors, AMPA; RNA, Small Interfering; Transfection; Visual Cortex

Alpha-synuclein has been implicated in the pathology of certain neurodegenerative diseases, including Parkinson disease (PD) and dementia with Lewy bodies (LBs). Overexpression of human alpha-synuclein in neuronal cells reduces cell viability, but the precise cellular and molecular mechanisms remain poorly understood. Gap junctional intercellular communication (GJIC) is thought to be essential for maintaining cellular homeostasis and growth control. In the present study, the effect of alpha-synuclein overexpression on GJIC in human dopaminergic neuroblastoma SH-SY5Y cells was investigated. Cells overexpressing wild-type alpha-synuclein were more vulnerable to hydrogen peroxide and 6-hydroxydopamine. GJIC was decreased in cells overexpressing alpha-synuclein. In addition, alpha-synuclein binds directly to connexin-32 (Cx32). As such, the post-translational modification of Cx32 was enhanced in cells overexpressing alpha-synuclein. These findings suggest that alpha-synuclein can modulate GJIC in a dopaminergic neuronal cell line through specific binding to Cx32.

Topics: Adrenergic Agents; alpha-Synuclein; Cell Communication; Cell Line, Tumor; Cell Survival; Connexins; Dopamine; Gap Junction beta-1 Protein; Gap Junctions; Humans; Hydrogen Peroxide; Immunoprecipitation; Isoquinolines; Mutation; Neuroblastoma; Oxidopamine; Transfection