This is distinct from the activation of the “conventional” γ-secretase substrates,

e.g., Notch, by γ-cleavage, although there also are several examples of negative regulation of the functions of γ-secretase substrates by cleavage, e.g., ephrin-B1 and DCC (Tomita et al., 2006; Parent et al., 2005). Taken together, our present results CX 5461 provide compelling evidence that proteolytic processing is a molecular mechanism regulating the NLG1 levels as well as its spinogenic function. Further functional analysis would be required to determine whether spines modulated by NLG1 shedding are functional. However, previous results showing that changes in spines by overexpression or knockdown of NLG1 correlated with synaptic transmission (Chih et al., 2006; Levinson et al., 2005; Chubykin et al., 2007) may support our view that the proteolytic cleavage by ADAM10 and γ-secretase downregulates the cell-surface levels of NLG1, which in turn negatively affects the synaptogenic function. Considering the recent

implication of aberrant levels of expression of NLGs or NRXs in ASD, it is tempting to speculate that alterations in the proteolytic processing of NLG1 may Galunisertib datasheet also be involved in the etiology of the neurodevelopmental abnormalities. All experimental procedures were performed in accordance with the guidelines for animal experiments of the University of Tokyo. Primary neuron culture, immunoblot analyses, and immunocytochemistry experiments were performed as previously described with some modifications (Tomita et al., 1998; Fukumoto et al., 1999). For in vitro Cre-mediated Adam10 ablation, primary cortical neurons were obtained from E16 pups of Adam10flox/flox mice, in which the first exon was floxed ( Yoda et al., 2011). For analysis of neuron-specific conditional Adam10 knockout mice, brains of P18 exon 2 floxed Adam10flox/flox mice ( Jorissen et al., 2010) crossed with CamKII-Cre mice (J.P. and P.S., unpublished data) were homogenized to obtain microsome fractions. Other animals

were obtained from Japan-SLC. See Supplemental Experimental Procedures for details. Male 8-week-old BALB/C mice were injected with scopolamine methylnitrate (Tokyo Chemical Industry) (1 mg/kg, intraperitoneally [i.p.]) to protect against peripheral autonomic Dipeptidyl peptidase effects caused by subsequent pilocarpine administration. Fifteen to thirty minutes later, mice were injected with pilocarpine-HCl (SIGMA) (330–380 mg/kg, i.p.) or saline (Otsuka), and then we scored the seizure intensity according to a previously described method (Patel et al., 1988). We defined status epilepticus (status epilepsy) as a continuous seizure lasting longer than 30 min. One hour after the injection of pilocarpine, mice were sacrificed to isolate the cerebrums. See Supplemental Experimental Procedures for details. Hippocampal slice cultures were prepared from P6 rats as previously described (Koyama et al.