, 2011). We also observed the metalloprotease-dependent production of soluble forms of endogenous NRXs in rat primary neurons (Figure 6A). Treatment with TAPI2 or GM6001 caused the accumulation of NRX-FL and inhibited the accumulation of NRX-CTF, which was detected upon DAPT treatment (Figure 6B). These data indicate that NRXs also are sequentially cleaved by metalloprotease and γ-secretase in primary neurons. To investigate

whether the binding of NRX regulates the production of sNLG1, we coincubated LY2109761 price primary neurons with the conditioned media of HEK293T cells expressing NRX1α or NRX1β, which contained soluble forms of the NRXs (Figure 6C). Accumulation of NRX1β immunoreactivity at endogenous NLG1 puncta was observed in rat primary neurons treated with the HEK293T conditioned media containing sNRX1β, suggesting that recombinant sNRX1β is capable of interacting with NLG1 at synapses (Figure S4). Intriguingly, release of sNLG1 from neurons was significantly increased by addition of the soluble NRX-containing media (Figures 6D and 6E). This result indicates that ligand binding at the cell surface regulates the shedding of NLG1. We also analyzed the activity-dependent NLG1 processing in vivo. Pilocarpine

treatment induces Ibrutinib glutamate-mediated synaptic activation, resulting in status epilepticus associated with synapse remodeling (Isokawa, 1998; Kurz et al., 2008). In agreement with the previous reports (Kamenetz et al., 2003), APP processing was promoted in the brains of 8-week-old epileptic mice (Figure 7A). Moreover, the level of sNLG1 was significantly increased, whereas that of the membrane-associated NLG1-FL was decreased, suggesting that NLG1 shedding was augmented in brains by pilocarpine-induced seizures (Figure 7B). Taken together, these data suggest that NLG1 processing is modulated by the excitatory activity in vivo as well as in vitro. To analyze the functional impact of NLG1 processing on its spinogenic

activity, we overexpressed NLG1 and its derivatives in dentate granule cells of the organotypic hippocampal slice culture obtained from P6 rat, in which local-circuit synaptic interactions are preserved. Overexpression of NLG1-FL significantly increased the spine density at the apical dendrites of granule cells. However, NLG2-FL failed to induce spines, suggesting Suplatast tosilate that NLG1 specifically increased the spine density at glutamatergic synapses as previously described (Figure 8A) (Scheiffele et al., 2000; Graf et al., 2004). Overexpression of NLG1ΔPDZ that lacks the C terminus failed to increase the spine number, suggesting that the spinogenic effect of NLG1 is dependent on the PDZ-binding motif in rat dentate granule cells. Reduction in the amount of transfected NLG1 cDNA led to loss of the spinogenic effect of NLG1 (see 0.1 μg HA-NLG1, Figures 8A and 8B), indicating that the protein level of NLG1 is critical to the de novo formation of the dendritic spine (Figure 8B).