To examine surface levels of APP, cells were rinsed two times with ice cold 1 PBS. 2004; Young-Pearse et al., 2007). Aged mice of APP single knockouts show impairment in spatial learning (Mller et al., 1994; Phinney et al., 1999; Ring et al., 2007) and long-term potentiation (Seabrook et Targapremir-210 al., 1999; Ring et al., 2007; Tyan et al., 2012). Furthermore, a reduced number of dendritic spines (Lee et al., 2010; Tyan et al., 2012; Weyer et al., 2014) and a reduced overall dendritic length in the CA1 region has been reported (Seabrook et al., 1999). APP/APLP2 double knockout (dko) mice die shortly after birth and display profound neuronal defects in the central and peripheral nervous system. Analysis of the neuromuscular junction (NMJ) revealed incomplete apposition of the pre- and postsynaptic structures (Wang et al., 2005), a reduced number of docked presynaptic vesicles and an impaired synaptic transmission (Wang et al., 2005). Mice that express only sAPP in an APP/APLP2 dko background show Targapremir-210 less pronounced, but also severe defects in the peripheral as well as in the central nervous system, including motor and learning deficits (Weyer et al., 2011). This argues that sAPP, although representing the major secreted species of APP, only partially rescues APP function. Notably, APP family members are expressed pre- and postsynaptically (Kim et al., Targapremir-210 1995; Lyckman et al., 1998; Back et al., 2007; Hoe et al., 2009; Wang et al., 2009; Wilhelm et al., 2014), a prerequisite for synaptic adhesion molecules (Siddiqui and Craig, 2011; Baumk?tter et al., 2012). A recent publication showed Targapremir-210 APP to be predominantly located at the surface of synaptosomes (Wilhelm et al., 2014). Further, tissue specific deletion of APP in either presynaptic motor neurons or postsynaptic muscle cells in APLP2?/? mice demonstrated similar NMJ defects as observed in APP/APLP2 dko mice (Wang et al., 2009). In conclusion neither sAPP nor expression of APP only at the pre- or postsynaptic site is sufficient for proper formation of the NMJ. In line with these analyses, co-culture assays of a non-neuronal cell line seeded on primary neurons (Biederer and Scheiffele, 2007) revealed that expression of APP in non-neuronal cells promotes presynaptic differentiation of contacting axons (Wang et al., 2009; Baumk?tter et al., 2014), similar to Neuroligin-1 (NLG-1; Scheiffele et al., 2000; Wang et al., 2009). Synapse promoting activity of APP in the hemisynaptic assay depends on expression of APP containing the E1 domain on both sides, similarly to what was shown for cell adhesion properties of APP (Soba et al., 2005; Wang et al., 2009; Dahms et al., 2010). Recent publications suggest that the synaptogenic activity of synaptic adhesion molecules (SAM) is regulated by ectodomain shedding (Suzuki et al., 2012; Pettem et al., 2013). Since APP is heavily processed by secretases, we investigated the influence of proteolytic processing on trans-interaction properties of APP and its effect on APP synaptogenic function. Results Generation of secretion deficient APP mutants We have previously shown using a Schneider (S2) cell based aggregation assay (Tsiotra et al., 1996; Klueg and Muskavitch, 1999; Islam et al., 2004) that APP possesses adhesion properties and can induce cellular aggregation (Soba et al., 2005). To investigate the consequences of -secretase processing on APP-mediated cell adhesion, we designed different putative secretion deficient APP mutants: N-terminally myc-tagged APP carrying either an amino acid substitution (F615P) previously shown to lower -secretase cleavage (Sisodia, 1992), small deletions removing the -secretase and -secretase cleavage site (APPF616, Rabbit Polyclonal to RFX2 APPS622), and deletion of A10C24 including amino acid substitutions with aspartates to increase electrostatic repulsion of -secretase (APP-D8; Figure ?Figure1A1A). Open in a.