Shaohua Xu1, Saket M Nigam1,2, Lennart Brodin1. 1. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. 2. Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.
Abstract
BACKGROUND: Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)β peptide production. OBJECTIVE: To examine the role of SNX3 in Aβ production and APP processing. METHODS: The effect of increased expression of SNX3 was studied in HEK293T cells. Aβ peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels. RESULTS: We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aβ and soluble N-terminal APP fragments (sAPPβ). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP. CONCLUSION: These results provide evidence that SNX3 regulates Aβ production by influencing the internalization of APP.
BACKGROUND: Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)β peptide production. OBJECTIVE: To examine the role of SNX3 in Aβ production and APP processing. METHODS: The effect of increased expression of SNX3 was studied in HEK293T cells. Aβ peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels. RESULTS: We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aβ and soluble N-terminal APP fragments (sAPPβ). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP. CONCLUSION: These results provide evidence that SNX3 regulates Aβ production by influencing the internalization of APP.