Moreover, overexpression of ADAM10 in mouse primary neurons increased the production of sNLG1 ( Figure S2B). Lastly, to demonstrate the physiological significance of ADAM10 in NLG1 processing in vivo, we incubated the brain microsome from postnatal day (P) 18 neuron-specific conditional ADAM10 knockout

mice (Adam10flox/flox; Selleckchem Screening Library CamKII-Cre) ( Figure 3I). Notably, the levels of NLG1-FL were increased in the microsome fractions from brains of Adam10 conditional knockout mice, whereas sNLG1 production was significantly decreased ( Figures 3J and 3K). Taken together, we concluded that the major physiological sheddase of NLG1 in brain is ADAM10. We next examined the proteolytic processing of NLG1 at the ectodomain in more detail. According to the molecular Smad inhibitor weight of the CTF, the stalk region of NLG1 is predicted as the candidate cleavage site for shedding. To obtain further precise information on the location and characteristics of the cleavage site, we

analyzed mutant forms of NLG1 overexpressed in COS-1 cells (Figure 4A). Recombinant NLG1-FL was sequentially cleaved similarly to the endogenous NLG1-FL in primary neurons, whereas NLG1-ICD was not observed (Figures 4B and 4C). To identify the extracellular cleavage site of NLG1, we have systematically generated a series of alanine substitutions around the juxtamembrane stalk region, at sites consistent with the molecular weight of sNLG1 and NLG1-CTF: K674QDD/AAAA, P678KQQ/AAAA, P682SPF/AAAA, S686VDQ/AAAA, R690DYS/AAAA, and T694E/AA (Figure 4A). Among these mutants, generation of sNLG1 was significantly decreased in the PKQQ/AAAA mutant, whereas the level of

mutant FL protein was increased (Figures 4D and 4E). The observation that overexpressed PKQQ/AAAA mutant NLG1 accumulated at dendritic spines in rat primary neurons similarly to wild-type (WT) NLG1 and showed spinogenic effects supported the view that introduction of PKQQ/AAAA mutation did not affect the trafficking of NLG1 (see Figure 8C). In contrast, alanine substitutions at regions proximal to the membrane caused an augmentation of NLG1 shedding. Intriguingly, the levels of sNLG1 were significantly increased in the PSPF/AAAA and SVDQ/AAAA mutants, whereas the corresponding FL proteins were these decreased, suggesting that the region from Pro682 to Gln689 negatively regulates the NLG1 shedding. These data suggest that the region between Pro678 to Gln681 is critical for the sNLG1 production. We then overexpressed NLG1-ΔE, a recombinant polypeptide corresponding to NLG-CTF starting from residue Val682 fused with a signal peptide (Figure 4A). The levels of NLG1-ΔE were increased by DAPT treatment, indicating that NLG1-ΔE was processed by γ-secretase (Figure 4F). However, NLG1-ICD was again undetectable in lysates of transfected cells.