Nedd4-1 regulates human sodium-dependent vitamin C transporter-2 functional expression in neuronal and epithelial cells

The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is cur-rently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we deter-mined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways. & COPY; 2023 Elsevier Inc. All rights reserved.