Generally speaking, drug development research improves when a range of proteins becomes known that interacts with the target of choice in biologically important ways. A number of labs are now on the trail of proteins that control α-secretase cleavage of APP. While in its early days, this work is already showing that α-secretase may be subject to more intricate regulation than γ-secretase, noted Haass. Stefan Lichtenthaler, at Ludwig-Maximilians University, Munich, described some of those regulators, which he found in an expression cloning strategy devised to identify proteins that activate APP ectodomain shedding (Schoebel et al., 2006). The proteins fell into three groups. Some were intracellular signaling proteins, but their effect was small. A much stronger boost for APP shedding was measured with proteins that reduce endocytosis of APP from the cell membrane (where most α cleavage tends to occur), probably because endocytosis brings APP cheek to jowl with BACE lodged in endosomes. Intriguing proteins in this category include dynamin and a new sorting protein, Snx31. A third group of proteins that regulate APP shedding are intracellular adaptors that mediate the association of APP with other membrane proteins, famously LRP (see below). Broadly speaking, this work dovetails with a hypothesis about abnormalities in endocytosis, autophagy, and ensuing intraneuronal degradation blockages in early AD pathogenesis that Randy Nixon and Anne Cataldo have developed in recent years (see, e.g., Grbovic et al., 2003; Yu et al., 2005).

The endocytosis inhibitors Lichtenthaler found include some known and some novel ones. Known ones are endophilin A3, a member of the endophilin family of trafficking proteins, which activated α-secretase strongly and specifically. Another one is the GTPase dynamin. It mediates endocytosis by way of clathrin-coated pits and is necessary for synaptic vesicle recycling. In 2002, Paul Coleman’s group identified dynamin as a protein whose mRNA plummeted in microarray studies of single neurons from AD brain (see ARF related news story; Yao et al., 2003). Other scientists have since implicated dynamin in synaptic failure in AD (e.g., Kelly et al., 2005), or as a factor influencing APP processing (Chyung and Selkoe, 2003), and a dynamin mutant in Haass’ lab serves as a tool to hold back APP at the plasma membrane.

Dynamin draws added intrigue from a novel protein Lichtenthaler identified. Called Snx31, it belongs to a poorly studied group of sorting nexins (SNXs) that mediate endocytosis in various cell types. Snx31 is highly expressed in brain and strongly stimulates APP shedding, Lichtenthaler reported. Its mechanism is still unclear, but tinkering with its levels yielded phenotypes that mirrored those generated by dynamin manipulation. Possibly, Snx31 and dynamin might reduce APP endocytosis as binding partners, Lichtenthaler speculated. Snx31 appears more specific than merely being another protein in a generalized endocytosis machinery, however. Experiments in C. elegans, done with Ralf Baumeister, suggest that Snx31 is required for endocytosis of only a certain subset of cell surface proteins. Besides APP, an important one appears to be the insulin receptor, raising the possibility that Snx31-mediated endocytosis forms a trafficking nexus connecting APP α cleavage and insulin signaling.

Other APP shedding activators also interfere with endocytosis, though in a different way. APP’s cousin APLP1 came up in the expression screen; its effect just appeared in print (Neumann et al., 2006; see Dominic Walsh’s comment there). In brief, the scientists propose that APLP1 can form a tripartite complex with the transmembrane protein Low Density Lipoprotein Receptor Related Protein (LRP). The adaptor protein Fe65 holds this complex together just below the membrane. Prior work by Claus Pietrzik and others had shown essentially the same thing for APP (Kinoshita et al., 2001; Pietrzik et al., 2004). According to this model, APLP1 would draw LRP molecules away from APP, leaving more APP molecules in a state that is less likely to be endocytosed, and fewer in the LRP complex that gets readily internalized. Fe65 can “glue” together either APP and LRP, or APLP1 and LRP, by binding to a NPXY internalization motif that APP, APLP1, and LRP each carry in their cytoplasmic tails. (Each Fe65 molecule can bind this motif twice.) The rates by which these complexes form, then, may help determine the rate of endocytosis and of APP shedding, Lichtenthaler suggested.

Pietrzik, who is at the University of Mainz, Germany, expanded the LRP story. At 600 kD, LRP is a massive receptor protein, and boasts at least 30 ligands. It is intimately intertwined with AD, beginning with a genetic association and continuing through its role as a receptor for ApoE and α2 macroglobulin. LRP’s manifold interactions with molecules relevant to AD pathogenesis are only beginning to be exposed, but it is already clear that its importance extends beyond its role in lipoprotein metabolism to include strong influences on APP processing. Over the past few years, Pietrzik first developed the model of how LRP, Fe65, and APP might bind together to influence APP internalization and, by extension, APP shedding and Aβ secretion. (Pietrzik et al., 2002). While this model is controversial, Pietrzik pointed to supporting in-vivo data by scientists at Aventis, who bred Fe65-transgenic and APP-transgenic mice and found that Fe65 reduced Aβ levels in the double-transgenics (Santiard-Baron et al., 2005). This fits into Pietrzik’s model in that adding surplus Fe65 to the mix would lead to some Fe65 molecules to bind to LRP and other Fe65 molecules to bind to APP. Yet these dimeric complexes would preclude formation of the trimeric ones that are necessary for APP endocytosis. The result would be more APP shedding even though Fe65’s primary role is to mediate internalization, which ordinarily reduces shedding. This ties into the notion that endocytic processing is a major source of Aβ, an idea long held by Eddie Koo, Gunnar Gouras, and others.

Other work by this group is testing the idea that LRP might influence APP processing in yet another way, that is, by holding APP in early secretory compartments such as the ER, where γ-secretase will not process it. Ongoing work with an ER retention signal on LRP is suggesting that LRP and APP travel together through the secretory pathway, and that this journey may occur in the same tripartite setup with Fe65, Pietrzik said. Their relationship is even more intimate than that, as recent studies have shown that LRP itself first sheds its ectodomain and then undergoes γ-secretase cleavage, complete with LRP-CTF release. In this process, LRP competes with APP (Lleo et al., 2005; Zerbinatti and Bu, 2005). A take-home message from this evolving data is that any researcher studying APP processing would do well to include LRP in the experimental design, as this type 1 membrane protein influences APP at many levels, Pietrzik said.—Gabrielle Strobel.