**1. Introduction**

The accumulation of amyloid-beta (Aβ) peptides in the brain is a key pathological hallmark of Alzheimer's disease (AD). These peptides vary between 37–43 amino acids in length and exist in a range of conformations and assembly states, from monomers to oligomers, protofibrils, and finally insoluble fibrils and plaques [1]. Aβ<sup>40</sup> and the more hydrophobic and aggregation-prone Aβ<sup>42</sup> are the most common isoforms, with a higher ratio of Aβ42: Aβ<sup>40</sup> being associated with increased neurotoxicity, and accelerated disease pathology and cognitive decline [2]. In the brain, these peptides are constitutively produced in neurons and astrocytes following enzymatic cleavage of the transmembrane amyloid precursor protein (APP), and subsequently rapidly cleared [3–5]. Studies in late-onset AD patients have shown that the production rate of Aβ remains unaltered; rather, impaired cellular clearance mechanisms are responsible for their accumulation in the brain [6]. Excess levels of soluble oligomeric Aβ have been demonstrated to impair long-term potentiation, drive synaptic and receptor dysfunction, propagate tau pathology, neuroinflammation, and oxidative stress, and correlate with disease severity [7–9].

Both intra- and extracellular Aβ accumulation have been implicated in neurotoxicity, with the former reported to precede the latter [9–12]. This raises the question of how

**Citation:** Chai, A.B.; Hartz, A.M.S.; Gao, X.; Yang, A.; Callaghan, R.; Gelissen, I.C. New Evidence for P-gp-Mediated Export of Amyloid-β Peptides in Molecular, Blood-Brain Barrier and Neuronal Models. *Int. J. Mol. Sci.* **2021**, *22*, 246. https://doi .org/10.3390/ijms22010246

Received: 2 December 2020 Accepted: 25 December 2020 Published: 29 December 2020

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intraneuronally-generated peptides are able to exit neurons and enter the extracellular space. The hydrophobic, aggregation-prone and highly membrane-associated nature of the Aβ peptide suggests that its constitutive release from cells relies on active transport [13,14]. P-glycoprotein (P-gp), also known as ATP-binding cassette (ABC) transporter B-family subtype 1 (ABCB1) or multi-drug resistance protein 1 (MDR1), is an ATP-dependent exporter protein with broad substrate specificity, that is ubiquitously expressed on cells with barrier or excretory functions [15]. Several studies have provided evidence that P-gp at the blood-brain barrier (BBB) is responsible for Aβ export out of the brain [16]. Consequently, we hypothesised that such a mechanism could also occur in the neuron. However, there remains overall skepticism about the capacity of P-gp to carry these peptides due to their considerably larger size than most known P-gp substrates [14,17].

The aim of the present study was two-fold: firstly, we sought to provide unequivocal evidence that P-gp is able to transport Aβ peptides using in vitro and ex vivo model systems that have been validated previously [18]. Secondly, we investigated the role of neuronal P-gp, in a cell culture system utilised extensively in AD research, to ascertain whether P-gp plays a role at the site of peptide generation and peptide-mediated damage.
