Part 6 of our 11-part Eibsee conference series. See also Parts 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, and complete PDF.
Of the three major APP processing steps, the richest and most varied biology appears to lie ahead for investigators who probe α cleavage. Long dismissed as “the other” cleavage that precluded Aβ formation, it languished in the shadows while the field trained its glare squarely on Aβ.
γ cleavage was the α male of the pack and, to be sure, tweaking it to shift the Aβ42 ratio remains a main focus in AD drug development. Even so, academic research on α-secretase is beginning to flourish. Below are summaries of five Eibsee talks on different angles of its biology. Paul Saftig, at Christian-Albrechts University of Kiel, Germany, introduced the topic broadly by noting that α cleavage, also called ectodomain shedding, is proving to be a critical step in the function of a wide range of a cell’s surface molecules. Scientists to date have identified about 40 different sheddases, or ADAMs. Of those, ADAM10 and 17 cleave APP but, to put APP in perspective, ADAM10 and 17 substrates also include a formidable list of type 1 transmembrane proteins that extends beyond Notch, the VEGF and transferrin receptors, the EGF and TNF families, and the large Ig superfamily of cell adhesion molecules (CAMs).
In his Eibsee talk, Saftig noted that deleting ADAM10 and 17 proved to be embryonically fatal and that mouse strains carrying subtler conditional knockouts are in the works. Meanwhile, Saftig and postdoc Karina Reiss have studied cell lines of the original knockouts to assess their ability to still perform shedding, and in this way get a sense of what ADAM10 and 17 do. The overarching finding so far from this ongoing work is that ADAM10/17 cleavage releases two protein stubs, each of which can have a different functional consequence on either side of the membrane.
Saftig focused on cell adhesion molecules (CAMs), a large group of proteins that originally became known for their role in allowing cells to stick together in forming tissues. They also participate in wound healing, inflammation, and tumor metastasis when the balance of cell adhesion versus detachment gets out of kilter. Saftig cited three examples to illustrate principles of ADAM10/17 function. First, consider N-cadherin. It is expressed in fibroblasts but also brain, where it colocalizes with β-catenin and plays a role in synaptic function. The scientists found that ADAM10 releases N-cadherin’s ectodomain, regulating cell adhesion, while internally, this same cleavage alters β-catenin signaling and expression of its target genes (Reiss et al., 2005). Second, ADAM10 turned out to shed E-cadherin in skin cells, and to influence their behavior in this way. Specifically, ADAM10 appears to promote wound healing as a physiological function in skin but, on the flip side, can promote eczema when proinflammatory cytokines induce its expression and excessive E-cadherin shedding then loosens cell-cell contacts in the skin (Maretzky et al., 2005). Third, the cell adhesion protein L1 is known to be important, in development and adult life, for axonal outgrowth and neuronal migration, but it is also present in neuroblastoma. L1 turned out to be constitutively “shedded” by ADAM10, and particular conditions induced additional shedding by ADAM17. The ectodomain locally loosened adhesion and stimulated migration, neurite outgrowth, and synaptic plasticity. L1’s remaining membrane stub became a substrate for none other than γ-secretase (Maretzky et al., 2005).
With APP, ADAM10 shedding also precedes γ-secretase action, but since ADAM10 splits the Aβ sequence in two, subsequent γ-secretase degradation does not release this peptide. The ectodomain of APP, too, is quite potent. Called APPsα, its neuroprotective and growth-promoting properties were worked out a decade ago. This raised the prospect of up-regulating APP shedding as an alternative therapeutic strategy that would kill two birds with one stone by dampening Aβ production while exploiting APPsα’s benefits. Alas, scientists did not know which enzyme did the job in adult human brain. In 1999, Falk Fahrenholz and colleagues at the University of Mainz, Germany, pinpointed ADAM10, at least in human cell lines (Lammich et al., 1999). Later research by Fahrenholz and Rolf Postina, using Fred Van Leuven’s APPV717I transgenic mice, revived interest in this cleavage when it proved the principle that tuning up ADAM10 expression even modestly could prevent amyloid deposition and a learning deficit in the Morris water maze (Postina et al., 2004).
The Fahrenholz group is now exploring ways of cranking up ADAM10 with an eye toward future therapeutics. The Alzforum has previously described ongoing research on the neuropeptide PACAP (see commentary and related news link at Kojro et al., 2006). At the Eibsee, Fahrenholz noted that his group is now attempting to deliver PACAP nasally to mice. A microarray study measuring the effect of chronic ADAM10 up-regulation in the APP-transgenic mice on gene expression found that mRNAs for inflammatory proteins were down, that typical AD-related mRNAs such as BACE and PS were unchanged, and that mRNAs encoding retinoid acid (RA) receptors were more abundant. Fahrenholz pursued this last clue, and this metabolite of vitamin A has since become a focus in the lab. The promoter of the ADAM10 gene contains binding sites for the retinoic acid receptor (Prinzen et al., 2005). Follow-up studies in neuroblastoma cells showed that RA selectively activates expression of ADAM10 but not ADAM17, together with the expression of ADAM10 substrates APP and APLP2. This common up-regulation of enzyme and substrates by retinoic acid might provide enhanced specificity of ADAM10 for APP and APLP2 cleavage (Endres et al., 2005).
RA is not a stranger to AD research. Dietary, metabolic, genetic, and epidemiological studies have implicated it before (see ARF conference report; Puchades et al., 2003; Rinaldi et al., 2003). Adult rats fed a vitamin A-deficient diet develop AD-like pathology (Corcoran et al., 2004), and a hippocampal LTP deficit induced by vitamin A deficiency was reported to be reversed by RA application (Misner et al., 2001). Taken together, the data suggested a working hypothesis whereby vitamin A, through a metabolite, might activate RA-responsive elements and in this way activate ADAM10 to boost the α-secretase pathway of APP processing. To test this concept, the Fahrenholz group is currently keeping mice on a vitamin A-deficient diet and reconstituting it with RA.—Gabrielle Strobel.