NLG1 predominantly partitions to and regulates glutamatergic synapses (Chubykin et al., 2007; Graf et al., 2004). selleck screening library The loss of NLGs from PSD95-positive sites prompted us to test whether depolarization depletes NLG1 from the postsynaptic density (PSD). Due to the lack of NLG1-specific antibodies suitable for immunocytochemistry, we performed biochemical fractionation of DIV21 cortical cultures after KCl stimulation (Ehlers, 2003) and measured NLG1 levels using an antibody targeted against the extracellular N-terminal domain. Immunoblot analysis of isolated fractions revealed enrichment of NLG1 in the PSD (Figure S1

available online). KCl depolarization resulted in a significant loss (47.6% ± 1.8%) of NLG1 from total extracts (Figures 1C and 1D). This reduction in NLG1 was observed in both synaptic plasma membrane (SPM) and PSD fractions (30.5% ± 1.5% decrease in SPM; 23.9% ± 7.3% in PSDI; 45.1% ± 5.4% in PSDIII) and was particularly pronounced in Triton-insoluble PSDII fractions (72.9% ± 4.9% reduction), in which NLG1 is most highly enriched (Figure S1). We next tested whether KCl incubation increases NLG1 internalization and lysosomal degradation using surface biotinylation (Ehlers, 2000). To inhibit lysosomal proteolysis, cells were preincubated with leupeptin for 1 hr. In basal conditions, 5.3% ±

1.2% of surface NLG1 was internalized Gamma-secretase inhibitor over 2 hr (Figures 1E and 1F). This low internalization rate was unaltered by KCl (5.7% ± 0.8% of surface NLG1 internalized), indicating that KCl-induced NLG1 loss is not due to increased internalization. By contrast, the GluA1 receptor exhibited a marked increase in old internalization upon KCl stimulation (Figure 1F, right panel), similar to previous reports (Ehlers, 2000). We further

addressed whether KCl-induced loss of total NLG1 was sensitive to proteasome or lysosome inhibition. Incubation with KCl for 2 hr resulted in a 48.1% ± 2.6% reduction of total NLG1 levels, which was unaffected by proteasome inhibition (MG132, 50 μM), blockade of lysosomal degradation (leupeptin, 200 μM), or both together (MG132, 51.5% ± 5.7%; leupeptin, 46.3% ± 6.7%; both, 47.4% ± 4.2% of control; Figures 1G and 1H). However, incubation with the broad-spectrum MMP inhibitor GM6001 (10 μM) abolished KCl-induced loss of NLG1 (102.9 ± 1.1% of control; Figures 1G and 1H), whereas incubation with GM6001, MG132, or leupeptin alone did not significantly alter NLG1 levels under basal conditions (MG132, 103.6% ± 4.4%; leupeptin, 102.1% ± 0.8%; GM6001, 109.8% ± 5.1% of control; Figures S1B and S1C). To test whether NLG1 is cleaved at the plasma membrane, we developed an assay based on surface biotinylation (Figure 2A). Briefly, DIV21 neuronal cultures were covalently labeled with cell impermeable biotin (Sulfo-LC-Biotin-NHS, 1 mg/ml) to exclusively label surface proteins.