FAD Synthase (FADS) localization and function in neuronal cell models: a possible involvement of FADS in the pathogenesis of neurodegenerative diseases.

Abstract

BACKGROUND AND AIMS: FAD synthase (FADS, EC 2.7.7.2), coded in humans by FLAD1 gene, is the last enzyme in the pathway converting riboflavin into the redox cofactor FAD, essential for the activity of hundreds of flavoenzymes. In non-neuronal cells FADS performs a citosolic, mitochondrial and nuclear localization (Giancaspero TA et al., 2013). Interestingly, a case report (Lin J et al., 2009) showed that in a ALS patient with an IgA gammopathy, the neuronal surface antigen was represented by FADS. Subcellular localization of FADS in human neuronal cells is still unknown. Also, a significant reduction of FADS mRNA levels was observed in the blood of ALS patients. Thus, here we aimed at studying the subcellular localization and the effect of altered expression levels of FADS on cellular bioenergetics in experimental models, i.e. neuronal cell models. METHODS: Confocal and sub-fractionation studies were performed on human neuroblastoma (SK-N-SH) and mouse motor neuron (NSC-34) cell lines. After overexpression and silencing, FADS levels were assessed by RT-PCR and WB, while Riboflavin, FMN and FAD content was measured by HPLC. The effects of altered FADS levels on neuronal bioenergetics were evaluated by measuring oxygen consumption rate and levels of ATP, ROS and glutathione reductase. RESULTS: Confocal data collected in neuronal cells showed a partial co-localization of FADS with a lipid raft marker and a high degree of co-localization with a vesicle marker in both the neuronal cell lines. Also, a clear toxic effect on bioenergetics was shown in FADS overexpressing SK-N-SH cells, as indicated by the specific reduction in complex I functionality. CONCLUSIONS: These data straightforwardly demonstrate the localization of FADS in membrane domains of two neuronal cell models, suggesting a novel role for FADS in neuronal physiology and, possibly, in neurotransmission. The relationships with results collected in other models will be discussed