1189 DEVELOPMENT AND DISEASE RESEARCH ARTICLE INTRODUCTION Amyloid precursor protein (APP) is a transmembrane protein that plays a key role in Alzheimer’s disease. This disease is characterized by intraneuronal tangles and extracellular plaques, and the amyloid beta-peptide (A), which is derived from APP upon cleavage by - and -secretases, is the major component of the plaques. Mutations in APP have been linked to familial Alzheimer’s disease, and the most widely accepted models of disease etiology propose that A aggregates or oligomers trigger a cascade of events causing damage to neuronal connections and cell death (Selkoe, 1999; Hardy and Selkoe, 2002; Tanzi and Bertram, 2005; Catalano et al., 2006). The functions of APP in normal physiology and development are not well understood. APP-deficient mice are viable and fertile, but have some abnormalities, including susceptibility to seizures (Steinbach et al., 1998) and a defect in corpus callosum formation (Magara et al., 1999), indicating roles in neural development and function. Triple knockout mice for APP and its two homologs, APLP1 (amyloid precursor like protein 1) and APLP2, exhibit a cortical defect reminiscent of human type 2 lissencephaly, suggesting a role in neuronal migration, and die perinatally, although the exact reasons for this remain mysterious (Herms et al., 2004). Since its initial identification, APP was thought likely to be a receptor, based on its transmembrane structure (Kang et al., 1987). By analogy to Notch (Mumm and Kopan, 2000), an APP signaling pathway has been proposed where -secretase cleavage yields the C-terminal CTF fragment, then -cleavage liberates the APP intracellular domain to participate in downstream pathways (Chan and Jan, 1998). Some evidence has accumulated to support this model of signaling (Cao and Sudhof, 2001; Kimberly et al., 2001; Kinoshita et al., 2002), although some aspects remain controversial (Cao and Sudhof, 2004; Hass and Yankner, 2005; Hebert et al., 2006). Several cytoplasmic binding partners for APP have been identified, delineating potential downstream pathways (Kerr and Small, 2005). However, the key signaling mechanisms remain unclear, especially as it is not clear which, if any, of the identified extracellular binding partners for APP might function as a physiological ligand. In addition to the potential receptor function of APP, its cleaved ectodomain, APPs, may function as a ligand. Numerous studies have shown that APPs can modulate cell behaviors including neurite outgrowth, synaptogenesis, neurogenesis and cell survival and proliferation (Mattson, 1997; Turner et al., 2003; Caille et al., 2004). Distinct domains of APPs have been implicated in these actions, suggesting the existence of more than one receptor. To date, however, no cell-surface receptor capable of mediating APPs- induced signaling has been identified. More than a decade of work has led to the identification of a number of extracellular partners that can interact with APP, directly or indirectly. Binding has been reported for extracellular matrix components, including heparan sulfate (Multhaup, 1994; Small et al., 1994), collagen (Beher et al., 1996) and fibulin 1 (Ohsawa et al., 2001); zinc and copper ions (Turner et al., 2003); and the lipoprotein receptors, scavenger receptor A (Santiago- Garcia et al., 2001) and LRP (Kounnas et al., 1995). More recently identified extracellular proteins that can interact with APP include F-spondin (also known as spondin 1) (Ho and Sudhof, 2004; Hoe et al., 2005), Drosophila FASII (Ashley et al., 2005), BRI2 (ITM2B) (Fotinopoulou et al., 2005; Matsuda et al., 2005), APLP1, APLP2 and APP itself (Soba et al., 2005), Notch family members (Fassa et al., 2005; Fischer et al., 2005; Oh et al., 2005; Chen et al., 2006), LRRTM3 (Majercak et al., 2006) and NgR (RTN4R) (Park et al., 2006). Some of these proteins can influence candidate downstream signaling pathways or APP processing. However, these interactions have generally not yet been characterized thoroughly with regard to whether they have affinity and specificity in the range of cognate receptor-ligand interactions, involve direct interaction with APP, and whether they can affect cell behavior. There is also generally little evidence regarding potential roles for these interactions in vertebrate neural development. Interaction of amyloid precursor protein with contactins and NgCAM in the retinotectal system Miriam Osterfield, Rikke Egelund, Lauren M. Young and John G. Flanagan* The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease, but its actions in normal development are not well understood. Here, a tagged APP ectodomain was used to identify extracellular binding partners in developing chick brain. Prominent binding sites were seen in the olfactory bulb and on retinal axons growing into the optic tectum. Co-precipitation from these tissues and tandem mass spectrometry led to the identification of two associated proteins: contactin 4 and NgCAM. In vitro binding studies revealed direct interactions among multiple members of the APP and contactin protein families. Levels of the APP processing fragment, CTF, were modulated by both contactin 4 and NgCAM. In the developing retinotectal system, APP, contactin 4 and NgCAM are expressed in the retina and tectum in suitable locations to interact. Functional assays revealed regulatory effects of both APP and contactin 4 on NgCAM-dependent growth of cultured retinal axons, demonstrating specific functional interactions among these proteins. These studies identify novel binding and functional interactions among proteins of the APP, contactin and L1CAM families, with general implications for mechanisms of APP action in neural development and disease. KEY WORDS: Amyloid precursor protein, Axon, Contactin, L1CAM, Retina Development 135, 1189-1199 (2008) doi:10.1242/dev.007401 Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA. *Author for correspondence (e-mail: flanagan@hms.harvard.edu) Accepted 9 January 2008 DEVELOPMENT