It has been proposed that the dynamic balance

It has been proposed that the dynamic balance between longer, more toxic Aβ peptides, in particular the 42-amino-acid Aβ42, and shorter Aβ peptides is a more significant determinant of disease rho kinase inhibitor and progression than total Aβ production (Findeis, 2007; Kuperstein et al., 2010). This suggests that modulating rather than inhibiting processing may be an effective strategy while avoiding adverse effects due to altered proteolysis of other substrates of γ-secretase. After initial β-secretase cleavage, the remaining membrane-bound fragment of APP is subjected to endopeptidase and then stepwise carboxypeptidase cleavage by γ-secretase, generating progressively shorter Aβ peptides (Takami et al., 2009). By augmenting the carboxypeptidase efficiency of γ-secretase with γ-secretase modulators (GSMs), it is possible to shift the production of Aβ peptides away from longer more toxic species toward shorter forms, without affecting total Aβ production or γ-secretase targeting of other substrates. While the results of larger clinical trials are yet to be reported, GSMs have demonstrated target engagement in patients (Soares et al., 2016; Toyn et al., 2016; Yu et al., 2014) and remain a promising avenue for development. APP has a complex life cycle: in addition to its processing by β- and γ-secretase, it undergoes proteolytic turnover in a number of different cellular compartments by numerous different proteases (Small and Gandy, 2006). This complexity suggests that it may be possible to alter amyloidogenic APP processing in a secretase-independent manner to shift Aβ peptide production toward shorter forms, at the expense of production of longer, toxic peptides. The aim of this study was to ask whether it was possible to identify secretase-independent, small molecule modulators of Aβ processing that would shift the production of Aβ fragments in human cortical neurons away from Aβ42 to shorter, non-toxic forms. To do so, we performed a small-molecule phenotypic screen in TS21 cortical neurons, which we have previously shown to produce highly elevated levels of Aβ peptides (Shi et al., 2012b). Using this approach, we identified a family of macrocyclic lactone anthelminthic compounds, the avermectins, which reproduce the effects of GSMs, without acting directly on the γ-secretase complex or causing accumulation of γ-secretase substrates. These data demonstrate that phenotypic screening in human stem cell models of AD provides a potentially powerful strategy for identifying disease-modifying pathways and compounds, independent of known approaches to modulating APP processing.
Results
Discussion We report here the identification, by phenotypic screening in a human stem cell model of AD, of γ-secretase-independent modulators of APP processing. A class of anthelmintic macrocyclic lactones, the avermectins, increases the relative production of short forms of Aβ and reduces the relative production of longer Aβ fragments in human cortical neurons. This effect is independent of the known molecular targets of avermectins in the mammalian nervous system and, moreover, despite an effect phenotypically similar to that of existing GSMs, is not due to a direct interaction with the core γ-secretase complex. The avermectins phenocopy γ-secretase modulation in healthy control neurons and multiple different models of genetic forms of AD, including TS21, APP duplication, and PSEN1 and APP mutations. In addition to the discovery of avermectins as modifiers of Aβ peptide production, this study demonstrates the utility of human stem cell models for small-molecule phenotypic screens of disease-relevant biology in neurodegeneration. Future investigation will focus on elucidating the mechanisms and molecular targets behind this phenomenon. The effect of the avermectins on Aβ production mimics that of GSMs, a diverse class of compounds that, either through action on γ-secretase or APP, alters APP cleavage in favor of shorter Aβ peptides. With a phenotypic effect opposite to that of the PSEN1 mutations leading to early-onset fAD, which drives an accumulation of longer Aβ peptides due to impaired carboxypeptidase cleavage efficiency of γ-secretase (Chavez-Gutierrez et al., 2012), the promise of GSMs lies in their ability to reduce the accumulation of toxic longer forms of Aβ while preserving γ-secretase cleavage of its other substrates. Of note, the very first GSMs were NSAIDs, whose effect on increasing Aβ38 while reducing Aβ42 production was first discovered thorough a phenotypic screen in transfected cells and ascribed to a mechanism independent of their previously known molecular targets (Weggen et al., 2001). Target identification of the early GSMs has led to more potent and selective derivatives, a number of which are currently in human trials for AD (Hall and Patel, 2014). We have demonstrated that the avermectins elicit changes in Aβ production that are comparable with those in existing GSMs, and similarly avoid accumulation of γ-secretase substrates, but act through a γ-secretase-independent pathway. It is hoped that target identification in future studies may lead to derivatives with higher potency, in addition to more favorable physicochemical and pharmacokinetic properties than the existing avermectins, which are highly lipophilic (Prichard et al., 2012) and poorly CNS penetrant (Schinkel et al., 1994).