In fact, the immunostaining of NLG1-ICD was relatively weak and predominantly detected in neuronal nuclei and somata, whereas NLG1-FL, as well as the other mutants, was localized to the somatodendritic compartments in transfected primary neurons (Figure S3). We further examined the significance of the PDZ-binding motif located at the C terminus of NLG1 and found that deletion of this selleck chemicals llc motif did not impact on the shedding as well as the γ-secretase-mediated cleavage (Figures 4A, 4B, 4C, and 4F). Collectively, these data support the notion that NLG1 is sequentially cleaved by ADAM10 and γ-secretase to release sNLG1 and a highly labile NLG1-ICD. It has been shown that some

γ-secretase substrates (e.g., APP, N-cadherin, and EphA4) undergo cleavage in an activity-dependent manner in neurons (Kamenetz et al., 2003; Marambaud et al., 2003; Reiss et al., 2005; Inoue et al., 2009). To investigate the effect of synaptic activity on NLG1

Palbociclib processing, we treated rat primary neuronal culture at day in vitro (DIV) 11 with a set of compounds. Fifteen minute treatments with glutamate or NMDA significantly increased the sNLG1 level in the conditioned media, which was abolished by addition of NMDA receptor antagonists (i.e., D-AP5 and MK-801) (Figures 5A and 5B). Intriguingly, pretreatment with MK-801 (Figures 5C and 5D), an open-channel blocker of NMDA receptor (Huettner and Bean, 1988), completely inhibited the NLG1 shedding induced by glutamate, suggesting that the physiological activation of functional NMDA receptors is sufficient for the generation of sNLG1 at the glutamatergic synapses. To examine whether the shedding regulates the cell surface level of NLG1, we performed a cell surface biotinylation experiment

in rat primary neurons (Figure 5F). Treatment with TAPI2 or GM6001 significantly increased the surface levels of NLG1 (Figures 5G and 5H). Moreover, secretion of biotinylated sNLG1 was detected in the conditioned media of labeled primary neurons (Figures 5I, 5J, and 5K). Notably, increased sNLG1 by NMDA treatment also was biotinylated, suggesting that the proteolytic processing of NLG1 occurs at the cell surface and regulates the levels of cell surface NLG1. Taken together with the results of synaptoneurosome incubation (Figure 1D), these data indicate that glutamatergic ADAMTS5 synaptic transmission through NMDA receptor activation modulates the levels of NLG1 at the synaptic membrane. It has been shown that several γ-secretase substrates are cleaved upon binding with cognate membrane-tethered or soluble ligands, e.g., Delta/Jagged for Notch (Mumm et al., 2000), Hyaluronan for CD44 (Sugahara et al., 2003), BDNF for p75 (Kenchappa et al., 2006), and VEGF-A for VEGF receptor (Swendeman et al., 2008). Recently, it was reported that NRXs undergo proteolytic processing, which is augmented by glutamate treatment (Bot et al.