UC Irvine

The ubiquitin pathway regulates the functional expression of several transporters in many cellular systems. However, currently, nothing is known about the role of ubiquitination E3 ligase, neural precursor cell expressed developmentally down-regulated gene 4 (Nedd4-1) in regulating human vitamin C transporter-2 (hSVCT2, the product of the SLC23A2 gene) in neuronal cells. Nedd4-1 has been identified as a putative interactor with hSVCT2 and in the brain, hSVCT2 is predominantly expressed and mediates the cellular uptake of ascorbic acid (AA). Our aim in this investigation was to address this issue using in vitro (SH-SY5Y and HEK-293 cells), and in vivo (mouse) models, together with an array of physiological, cellular and molecular biological approaches. The results showed that the expression of Nedd4-1 in neuronal samples (human and mouse hippocampi and SH-SY5Y cells) is markedly higher than that of Nedd4-2. Interestingly, Nedd4-1 expression was shown to be significantly higher in the hippocampi of patients with Alzheimer’s disease (AD) and a J20 mouse model of AD. In addition, Nedd4-1-GFP and hSVCT2-DsRed co-localized in intracellular vesicles and at the cell membrane in HEK-293 cells. Further, the co-expression of Nedd4-1 with hSVCT2 showed a marked decrease in AA uptake. In contrast, Nedd4-1 siRNA knockdown significantly increased the AA uptake. Further, we have mutated a classical Nedd4 protein interacting motif (‘PPXY’) within the hSVCT2 polypeptide and observed significantly reduced AA uptake due to the intracellular retention of the hSVCT2 mutant compared to hSVCT2 wild-type. Collectively, our data demonstrate that Nedd4-1 dependent ubiquitination regulates hSVCT2 functional expression in neuronal cells. Supported by NIH grant DK 107474 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.