This interesting and technically innovative paper addresses the still enigmatic physiological function of APP. The study sheds new light on APP by comparing its “interactome” (i.e., the sum of its interactions with other proteins) with those of the APP paralogs, the APP-like proteins 1 and 2.

To achieve the goal of catching as many as possible interacting proteins in the act, a new way of cross-linking is employed. This method, termed "time-controlled transcardiac perfusion cross-linking," basically glues all protein complexes together while the animal still lives. This is advantageous as it reduces the risk of mapping artifactual interactions, which often occur after tissue is extracted, and it should better enable the identification of transient and low-affinity interactions. After immuno-affinity purification of the cross-linked protein complexes, the authors employ isobaric tagging and quantitative mass spec to map differences in the complexes formed by the three paralogous proteins.

This strategy poses a twofold advantage. First, non-specific interactions with...  Read more

This interesting and technically innovative paper addresses the still enigmatic physiological function of APP. The study sheds new light on APP by comparing its “interactome” (i.e., the sum of its interactions with other proteins) with those of the APP paralogs, the APP-like proteins 1 and 2.

To achieve the goal of catching as many as possible interacting proteins in the act, a new way of cross-linking is employed. This method, termed "time-controlled transcardiac perfusion cross-linking," basically glues all protein complexes together while the animal still lives. This is advantageous as it reduces the risk of mapping artifactual interactions, which often occur after tissue is extracted, and it should better enable the identification of transient and low-affinity interactions. After immuno-affinity purification of the cross-linked protein complexes, the authors employ isobaric tagging and quantitative mass spec to map differences in the complexes formed by the three paralogous proteins.

This strategy poses a twofold advantage. First, non-specific interactions with other proteins—a notorious problem for membrane proteins—are identified as they will likely be the same for a group of proteins with similar structural make-up. Second, any interactors identified that are specific for APP may yield clues to why APP but not its cousins can go awry in Alzheimer disease.

Indeed, the results obtained here are interesting. Firstly, it is always great to find interactions previously observed with other approaches, and a number of known APP binding proteins are identified. This includes cystatin C which has been proposed as a genetic marker for AD (see Alzgene). Secondly, a number of novel interacting proteins are identified. An intriguing case is LINGO-1, a neuronal transmembrane protein. The authors go on to demonstrate that siRNA-mediated reduction of LINGO-1 in APPsw-cells caused a reduction in Aβ production, while LINGO-1 overexpression caused increased β-secretase cleavage of APP. In summary, the combination of techniques used here carries a lot of promise for the elucidation of membrane-protein complexes, which should contribute to a better understanding of their role in disease processes and as drug targets.

View all comments by Gerard Drewes

Bai et al. use a novel approach for identifying APP family member-candidate interacting proteins. For identification of interacting proteins, the investigators used transcardiac perfusion cross-linking, followed by immunoprecipitation of the cross-linked complexes from mouse brain with APP family member-specific antibodies and LC/MS/MS analysis of recovered tryptic peptides. Validation of the putative interaction of APP with one of the identified proteins, LINGO-1, a transmembrane protein that binds p75, could only be demonstrated in immune complexes recovered from cross-linked mouse brain proteins, suggesting that this interaction is weak, transient, or mediated by an intermediate protein. However, manipulations of LINGO-1 protein levels in HEK293 APPSwe cells using siRNA or overexpression produced opposite effects on levels of APP proteolytic fragments, suggesting that this interaction is physiologically relevant for APP processing.

The data in this study confirm the previously identified interaction of APP family members with each other, as well as the interaction of APP...  Read more

Bai et al. use a novel approach for identifying APP family member-candidate interacting proteins. For identification of interacting proteins, the investigators used transcardiac perfusion cross-linking, followed by immunoprecipitation of the cross-linked complexes from mouse brain with APP family member-specific antibodies and LC/MS/MS analysis of recovered tryptic peptides. Validation of the putative interaction of APP with one of the identified proteins, LINGO-1, a transmembrane protein that binds p75, could only be demonstrated in immune complexes recovered from cross-linked mouse brain proteins, suggesting that this interaction is weak, transient, or mediated by an intermediate protein. However, manipulations of LINGO-1 protein levels in HEK293 APPSwe cells using siRNA or overexpression produced opposite effects on levels of APP proteolytic fragments, suggesting that this interaction is physiologically relevant for APP processing.

The data in this study confirm the previously identified interaction of APP family members with each other, as well as the interaction of APP with the membrane-associated proteins: calsyntenins (alcadeins) and PrP; the ER proteins BIP and calnexin, and the secreted proteins F-spondin and Cystatin C. One advantage of this approach is the identification of proteins that are located in the membrane or in the vicinity of membranes, including the plasma membrane. However, a striking absence of previously identified APP C-terminal binding proteins was observed using this experimental approach. In the case of the FE65 proteins, this cannot be attributed to lack of importance of this protein family for APP biology, since the FE65/FE65L1 double knockout mice display a neuronal positioning defect in the developing cortex that is remarkably similar to the APP triple knockout mice (Guénette et al., 2006; Herms et al., 2004).

Given that APLP2 and APLP1 were found in APP immunoprecipitates, it is unfortunate that no proteins common to all three APP family member immune complexes were identified. Such proteins would be good candidates for studies aimed at elucidating the molecular mechanisms underlying the phenotypes observed in APP/APLP1/APLP2 triple knockout mice. Nevertheless, it remains possible that disruptions of APP family member interactions with proteins identified by Bai et al. contribute to the type II lissencephaly-like phenotype observed in the APP family member knockout mice. In this respect, SPARC-L1, an extracellular protein known to localize to the surface of radial glial fibers in the outer layers of the developing cortex, is particularly interesting. That is because it was recently shown to block adhesion of migrating neurons to radial glia, thereby terminating radial glia-mediated neuronal migration (Gongidi et al., 2004). Continued adhesion of neurons to radial glia in mice deficient for the APP family members may lead to neuronal overmigration and heterotopia formation, similar to what is observed in the developing cortex of APP/APLP1/APLP2 triple knockout mice (Herms et al., 2004).

Finally, the identification of calsyntenins (alcadeins) as APP cross-linked proteins in the absence of X11L (X11β or Mint-2) is interesting, given that alcadein α1 (Alcα1) was identified as an interactor for X11L in a two-hybrid screen (Araki et al., 2003). Alcadein was initially proposed to form an APP-X11L-Alcα1 tripartite complex because all three proteins could be recovered from brain membrane fractions in immune complexes isolated with an APP C-terminal antibody.

However, the binding domain within X11L for alcadein was identified as the same domain that mediates binding to APP, the PTB domain of X11L; thus X11L cannot act as an adapter protein in the formation of this complex. The data provided by Bai et al. suggest that the tripartite complex is formed by the interaction of the N-terminal domains of APP and alcadein with binding of either alcadein or the APP C-terminus to X11L. Furthermore, this may allow for complex formation of secreted APP or alcadein with X11L-bound, membrane-associated alcadein or APP, respectively (Araki et al., 2004).

Unique APP and APLP-interacting proteins have been identified using different experimental strategies. Therefore, it seems reasonable to consider all candidate interactors when thinking of the APP/APLP interactome, and how it may shed light on our understanding of the physiological functions of APP and/or the APLPs through their protein-protein interactions.

View all comments by Suzanne Guenette