Tracy Young, from Dennis Selkoe’s lab at Harvard Medical School, also addressed the role of APP in development. Some of Young’s data was presented at a subsequent slide session. Young uses in utero electroporation of short hairpin RNAs to knock down APP to create mouse mosaics, with some cells expressing APP while others do not. This may be a crucially important point, because some of the phenotypes observed are not seen in APP knockout mice.
Young traced the APP-deficient cells by virtue of a green fluorescent protein (GFP) construct that is coexpressed with the shRNA. She showed how precursor cells that lack APP fail to migrate past the cortical plate. This does not seem to be a problem of motility, since the cells appear to migrate laterally. In fact, when Young focused on embryonic day-16 brains, 3 days after electroporation, she was able to see that GFP cells migrate out of the intermediate zone, before getting trapped at the cortical plate (ARF related conference story. Interestingly, the trapped cells begin to express the neuronal marker MAP2, suggesting that they begin to differentiate into neurons despite having their journey cut short.
By what mechanism does APP contribute to neuronal migration? Young has started to perform rescue experiments with various APP constructs to tease out factors that retard precursors at the cortical plate. She found that the migration defect does not appear when she electroporates in human APP constructs that are not recognized by the shRNA and are therefore expressed. Both the 751 and the 695 amino acid isoforms of APP rescue migration, as do APLP1, APLP2, and even APP with the Swedish mutation, Young reported. However, she found that the complete holoprotein is required; the extracellular or intracellular domains alone failed to rescue. Young also reported that rescue with full-length APP depended on an intact NPTY motif, found in the cytoplasmic tail. This motif binds a variety of proteins, including phosphotyrosine-binding proteins such as Dab1. In fact, Young reported that electroporation of Dab1 shRNAs lead to a similar phenotype as APP shRNA, with cells getting trapped at the cortical plate. Interestingly, Dab1 expression could partially rescue APP knockdown but not the other way around. Young suggested that this means that Dab1 works downstream of APP. It is worth noting that Dab1 has been shown to increase cell surface expression and processing of APP (see Parisiadou and Efthimiopoulos, 2006).
Both Hongmei Li from Tom Sudhof’s lab at the University of Texas Southwest Medical Center, Dallas, and Muller also addressed the role of various parts of the APP molecule. Li noted that although plenty of molecules have been discovered to bind to the cytoplasmic tail of APP, 80 percent of APP is in the extracellular space. Which is more important, she asked, the head or the tail of APP? To address this, Li attempted to rescue APP/APLP2 double KO mice.
Despite having what appears to be normal brain morphology and normal expression of synaptic protein, about 80 percent of these animals die early. Li tried to keep them alive with a flag-tagged construct that expresses only sAPPβ. Knocked in to the APP KO mice, this construct did not extend survival past postnatal day 21. It did bind to the molecular chaperone GRP78, otherwise known as BiP, however. Li said that presently it is unclear whether this binding is physiologically relevant or is merely related to misfolding of the flagged-APP protein. It is also unclear if the sAPPβ gets retained in the endoplasmic reticulum or is released from the cells.
For her part, Muller has had better success with rescue experiments using the extracytoplasmic end of APP. In San Diego, she summarized findings published last summer that sAPPα is sufficient to rescue APP KO mice (see Ring et al., 2007 and commentary).
Muller introduced a stop codon into the APP gene such that knock-in mice only expressed secreted sAPPα, the extracellular part of APP that is normally cleaved off by α-secretase. Muller found that this knock-in was sufficient to rescue the prominent phenotypes of APP-deficient mice, including their small body weight, weak grip, learning deficit in the Morris water maze, and loss of hippocampal synaptic plasticity. When crossed with APP/APLP2 double knockouts, the knock-in also survived longer. Muller noted that sAPPα levels are lowered in Alzheimer disease, which may partially explain learning and memory deficits in patients.
Overall, one important theme that emerged from the symposium was the role of the various parts of the APP molecule, said Guenette. In both C. elegans and in mice, the extracellular piece of APP, E1 or E2 in the case of worms or sAPPα in rodents, can rescue APP deficiency. In worms this seems particularly noteworthy, since these animals only have one APP homolog, APL-1, while in mice, APLP1 and APLP2 may partially compensate for APP loss. The ability of sAPPα to rescue at least some of the lethality seen in APP/APLP2 knockouts indicates the importance of this part of the molecule in mammals, as well. But the fact that the full-length molecule seems necessary to rescue developmental defects in mice suggests that different parts of the APP molecule may have different targets.—Tom Fagan.
This is Part 1 of a two-part story. See Part 2.