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First published online 13 February 2008
doi: 10.1242/dev.007401

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Interaction of amyloid precursor protein with contactins and NgCAM in the retinotectal system

Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.

View larger version (73K): [in this window] [in a new window]   Fig. 1. APP-binding sites in embryonic chick brain. (A,B) Binding of AP-APPs versus AP control. Ventral view, E11.5-12.5 brains. Prominent binding in optic tectum (OT, arrow), with additional staining in olfactory bulbs (OB, arrowhead) and other regions. (C) AP-APPs staining of tectum at greater magnification, showing anterior-posterior striation characteristic of axons in the stratum opticum. Anterior-posterior is horizontal. Scale bar: 100 µm. (D-F) Time-course of AP-APPs binding to brains. Dorsal view. Anterior (A) and posterior (P) extremes of tecta. Arrows indicate posterior limit of staining. (G) Binding of AP-APPs to brain from embryo with single enucleated eye. Ventral view. Staining in contralateral tectum (arrow) is reduced to background levels. AP, alkaline phosphatase.

View larger version (80K): [in this window] [in a new window]   Fig. 2. Two different domains of APP exhibit distinct binding properties. AP in situs on E11.5-12.5 brains. Ventral views. Arrowheads indicate olfactory bulbs. Arrows indicate optic tecta. (A) Binding of AP-APP deletion series (numbered according to APP695). Complete AP-APPs is AP-APP(18-612). Binding pattern 1 refers to prominently striated staining specifically in the anterior tectum at E9.5. Binding pattern 2 refers to widespread staining of the brain, including throughout the tectum, and prominent olfactory bulb staining, at E11.5-12.5. Colored bars indicate regions of APP with strong binding in patterns 1 (green) and 2 (blue). (B,C) AP-APP(199-345) gave strong tectal staining. AP-APP(199-293) also bound to RGC axons in tecta, though less strongly. (D,E) PI-PLC or mock treatment of brains, then AP-APPs staining. PI-PLC treatment eliminated most olfactory bulb staining and some broader staining. (F,G) AP-APP(18-205) and AP-APLP1 exhibit widespread binding including in the tectum, with particular prominence in olfactory bulb. (H) Diagram of chick brain, ventral view.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Crosslinker-based affinity purification of APP-interacting proteins from tecta. AP probes were crosslinked to surface-biotinylated tecta, then immunoprecipitated from lysates to identify associated proteins. Crosslinker was cleaved before gel analysis. (A) Western blot using NeutrAvidin-HRP to detect surface-biotinylated co-precipitated proteins. Asterisks mark the two predominant bands. (B-G) 2D gels for large-scale co-precipitation. Western blots for biotinylated proteins (B,E) were compared with silver-stained gels (C,D,F,G). Arrows indicate the predominant biotinylated proteins in B and corresponding locations in the silver-stained gels. This region was excised from gels in C and D for tandem mass spectrometry. Arrowhead indicates an additional spot specific for the AP-APPs co-IP: mass spectrometry identified several proteins, but these lacked obvious signal peptide or transmembrane domains and were not characterized further.

View larger version (29K): [in this window] [in a new window]   Fig. 4. APP and APLP1 bind to specific contactin family members. (A) AP fusion proteins were tested for binding to contactin-Fc fusion proteins immobilized on Protein A beads. AP activity retained after washing beads is shown. APP bound prominently to contactins 3 and 4. APLP1 bound to contactins 3, 4 and 5. Some non-saturable binding indicating weak or non-specific binding was also seen with contactin 1-Fc (data not shown). (B-F) Saturation curves (insets) were generated by varying the concentration of AP fusion protein. Lines in corresponding Scatchard plots were generated by least squares fitting. KDs: contactin 4-APP, 17 nM; contactin 4-APLP1, 28 nM; contactin 3-APP, 22 nM; contactin 3-APLP1, 35 nM; and contactin 5-APLP1, 32 nM.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Characterization of interactions of APP with contactins and NgCAM. (A) AP-fused APP domains were tested for binding to Fc-fused contactin 3 or 4 domains [for concentrations, see Materials and methods; for domain selection see Rader et al. (Rader et al., 1996)]. Binding activity localizes to the fibronectin (FN) domains of contactins, and to the N-terminal domain of APP. (B) APP association with NgCAM. Western blots with antibodies against NgCAM after immunoprecipitating for AP tag. Asterisks mark major bands specifically co-immunoprecipitated with AP-APPs, consistent with published molecular weights of NgCAM. (C) Amino acids 199-345 of APP are sufficient for association with NgCAM. Non-cleavable crosslinker BS3 used here to examine net molecular weight of immunoprecipitated complexes. Resulting spread of signal might indicate the presence of additional molecules in the crosslinked NgCAM-APP complexes. (D) Model for interactions among proteins. N-terminal domain of APP (amino acids 18-205, `E1') binds directly to fibronectin domains of contactin 4, whereas amino acids 199-345 of APP interact, directly or indirectly, with NgCAM. Amino acids 199-345 of APP encompass the acidic domain (oval, `A') and the N-terminal portion of the central APP domain, termed E2, which includes the RERMS peptide previously implicated in APPs function (Reinhard et al., 2005). (E) Co-transfection of APP-HA and indicated constructs, followed by anti-HA western blot. Contactin 4-Fc or NgCAM-Fc constructs increased the CTF level compared with Fc control. (F) Co-migration with an artificial CTF polypeptide confirms the identity of the APP cleavage fragment observed after co-expression with contactin 4-AP.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Expression of APP, NgCAM, contactin 4 and contactin 3 in the developing visual system. RNA in situ hybridization in sagittal sections of E11.5 chick heads. (A-E) Retina. Each antisense probe detected RNA expression in the RGC layer (arrow). Pigmented epithelium (dark brown line) bounds the retina opposite the RGC layer. (F-J) Each antisense probe detected RNA expression in multiple layers of the tectum. Top, anterior extreme; bottom, posterior extreme; open arrowheads, stratum opticum as identified by GAP43 immunolabeling. (K-Z) Co-staining using RNA in situ hybridization (K-R) or immunohistochemistry (S-Z) for APP (green), DAPI nuclear stain (blue) and immunohistochemistry for GAP43 (red). Solid arrow, RGC layer; open arrow, retinal optic fiber layer; solid arrowhead, pigmented epithelium; open arrowheads, tectal stratum opticum as identified by GAP43 immunolabeling. Scale bars: 500 µm in A-J; 100 µm in K-V; 200 µm in W-Z.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Interactions among NgCAM, contactin 4 and APPs in RGC axon outgrowth. Retinal strips cultured on different substrates before axon imaging. (A-D) APPs and APP(18-205) both enhanced outgrowth on NgCAM. AP, n=12; other conditions, n=6. (E) APPs enhanced NgCAM-dependent, but not laminin-dependent, axon outgrowth. n=6. (F) Contactin 4-Fc inhibitied NgCAM-dependent, but not laminin-dependent, axon outgrowth. n=10. Protein coating was varied in different experiments; the high basal outgrowth here allowed for robust quantitation of the inhibitory effect of contactin 4-Fc. (G-K) Contactin 4 shRNA in replication-competent retrovirus inhibited axon outgrowth on NgCAM+AP-APPs, but not on laminin. Laminin, n=11 (shRNA), n=11 (control virus); NgCAM+APPs, n=21 (shRNA), n=19 (control virus). (L) Model of APPs, NgCAM and contactin 4 function based on the assays of RGC axon outgrowth shown here. Other cis and trans interactions might also occur. See text for details. *, P<0.025 by Student's t-test. Error bars, s.e.m. Scale bars: 300 µm.

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