Abstract
The ATP-binding cassette transporter, P-glycoprotein (P-gp), has been demonstrated to facilitate the clearance of amyloid-beta (Aβ) peptides, exporting the neurotoxic entity out of neurons and out of the brain via the blood–brain barrier. However, its expression and function diminish with age and in Alzheimer’s disease. P-gp is known to undergo ubiquitination, a post-translational modification that results in internalisation and/or degradation of the protein. NEDD4-1 is a ubiquitin E3 ligase that has previously been shown to ubiquitinate P-gp and reduce its cell surface expression. However, whether this effect translates into altered P-gp activity remains to be determined. siRNA was used to knockdown the expression of Nedd4 in CHO-APP cells. Western blot analysis confirmed that absence of Nedd4 was associated with increased P-gp protein expression. This was accompanied by increased transport activity, as shown by export of the P-gp substrate calcein-AM, as well as enhanced secretion of Aβ peptides, as shown by ELISA. These results implicate Nedd4 in the regulation of P-gp, and highlight a potential approach for restoring or augmenting P-gp expression and function to facilitate Aβ clearance from the brain.
Highlights
Amyloid-β (Aβ) peptides are a normal cellular product generated by the routine enzymatic cleavage of amyloid precursor protein (APP)
It is estimated that 25–60% of Aβ peptides are cleared from the brain via active transport across the blood–brain barrier (BBB) [6–8], and multiple lines of evidence have highlighted the involvement of the 170 kDa ATP-binding cassette (ABC)
Utilising an in vitro approach, we examined the effects of the knockdown of Nedd4 on P-gp protein expression, transport function, and Aβ peptide transport capacity in Chinese hamster ovary cells stably overexpressing APP (CHO-APP)
Summary
Amyloid-β (Aβ) peptides are a normal cellular product generated by the routine enzymatic cleavage of amyloid precursor protein (APP). By engendering complexity and diversification of protein expression and activity beyond what is encoded by the genome, these modifications determine the ultimate biological function of the protein Ubiquitination is one such post-translational modification that P-gp has been demonstrated to undergo [22,23]. Akkaya et al showed that NEDD4-1 directly tags eight lysine residues on the cytosolic surface of P-gp with ubiquitin, resulting in reduced plasma membrane expression of the transporter [32]. In this investigation, we sought to expand upon the present understanding of the NEDD4-1/P-gp relationship, and hypothesise that downregulating NEDD4-1 could enhance P-gp expression and activity in the brain. Our data suggest that Nedd is implicated in the post-translational regulation of P-gp, and that enhancing P-gp function by modulating Nedd could facilitate Aβ clearance from the brain
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