Aβ degradation—the inside story

Two decades have passed since the discovery of the first proteases that degrade the amyloid β-protein (Aβ) (Roher et al., 1994; Turner et al., 2004), the primary constituent of the amyloid plaques that characterize Alzheimer disease (AD) (Selkoe, 2000). While significant progress has been made, this is an appropriate juncture to reflect on what has been accomplished and ask which research directions are most likely to bear fruit going forward. Herein, I argue that a renewed focus on intracellular Aβ-degrading proteases (AβDPs) is a highly promising direction for future studies, one that is not only likely to advance our understanding of the fundamental molecular pathogenesis of AD, but also to critically inform the development of effective therapies for use clinically. To date, most studies of AβDPs have focused predominantly on proteases that act extracellularly (LaFerla et al., 2007; Saido and Leissring, 2012; Leissring and Turner, 2013). This is not surprising— Aβ is, after all, a secreted peptide, and amyloid plaques form extracellularly. However, there is a growing body of evidence implicating intracellular pools of Aβ in the pathogenesis of AD (Saido and Leissring, 2012; Leissring and Turner, 2013). Generally speaking, it has been challenging to study specific pools of Aβ in conventional animal models of AD, since most models rely upon overexpression of the β-amyloid precursor protein (APP), which necessarily increases the levels of all pools of Aβ simultaneously. The study of AβDPs, by contrast, offers a unique window into the pathogenic role of Aβ, in no small part because individual AβDPs have unique subcellular localizations and pH profiles, which can be exploited to selectively target different pools of Aβ (e.g., extracellular, lysosomal, etc.) (Leissring and Turner, 2013). This can be readily achieved by overexpression, genetic deletion or pharmacological manipulation of appropriate AβDPs, either alone or in tandem with APP overexpression. There is a surprisingly long list of reasons to focus particular attention on intracellular AβDPs. First and foremost is the fact that the production of Aβ occurs intracellularly. Aβ is produced from APP by the successive action of two proteases, known as β-secretase—or β-site APP cleaving protease 1 (BACE1)—and γsecretase, an intramembraneous complex of four proteins, with presenilin-1 or -2 comprising the active site (De Strooper et al., 2010). Of note, βand γ-secretase are both aspartyl proteases and, as such, require an acidic environment to effect their proteolytic activity (De Strooper et al., 2010). As a consequence, although there is some evidence for limited production of Aβ at the cell surface (Chyung et al., 2005), the vast majority of Aβ is produced intracellularly, within acidified compartments. From a therapeutic perspective, it is logical to study AβDPs that are located closest to the sites of Aβ production, as they are best positioned to efficiently regulate Aβ levels, including Aβ in the extracellular space. Second, somewhat counter-intuitively, the fraction of Aβ amenable to degradation (i.e., non-aggregated) is primarily located intracellularly, not extracellularly as is widely assumed. In the human brain, the extracellular space comprises ∼5% of the total volume (Wyckoff and Young, 1956). By contrast, intracellular compartments contiguous with the extracellular space (e.g., ER, Golgi, endosomes, lysosomes, etc.) make up an estimated 17% of total cell volume (Alberts et al., 2008). Significantly, this figure ignores the many other intracellular compartments where Aβ has been detected (e.g., mitochondria, cytosol). The reason this point is not more widely appreciated may stem from the fact that extracellular/intracellular volume ratio is dramatically reversed in cultured cells—where, not coincidentally, most known AβDPs were discovered and many studies of Aβ metabolism were performed. Third, the fraction of Aβ present in the extracellular space is to a large degree bound to carrier proteins, notably apolipoproteins E and J (ApoE, ApoJ) (Bu, 2009). When bound to ApoE or other proteins, Aβ is protected from clearance by AβDPs. Moreover, a principal function of these same molecules is, in fact, to transport Aβ to intracellular sites for degradation (Bu, 2009; Fuentealba et al., 2010). Fourth, intracellular Aβ is far more prone to aggregation than extracellular Aβ, because Aβ aggregation is dramatically accelerated under acidic conditions (Su and Chang, 2001). Not only is newly synthesized Aβ produced within acidic compartments, but as mentioned, extracellular Aβ is also transported to lysosomes by ApoE and other Aβ-binding proteins (Bu, 2009). This point is key, because aggregated Aβ is far less amenable to clearance by proteolytic degradation or other means. In addition to the preceding, largely theoretical, arguments for focusing on intracellular Aβ degradation, there is a compelling body of empirical evidence that is supportive, as well. In animal studies, overexpression or genetic deletion of extracellular AβDPs have had the expected effects on cerebral Aβ levels and amyloid