The extent of endogenous NLG1 loss from synapses observed using biochemical methods is greater than that observed by immunostaining (Figure 1). This disparity may be attributed to the broad specificity of the pan-NLG antibody used for immunolabeling and to the fact that it targets the C-terminal domain of NLGs, an epitope that is further regulated by the γ-secretase complex (Figures S3E and S3F). It will be important

to TSA HDAC address how other NLG isoforms are regulated by MMPs and whether NLG1-CTFs participate in intracellular signaling. Multiple soluble NLG1-NTF species can be detected in the brain (Figure 2D), suggesting that NLG1 contains more than one cleavage site. This observation provides a plausible explanation for why single amino acid point mutations in the juxtamembrane region of NLG1 fail to prevent MMP-dependent cleavage, which instead

requires substitution of a 24 amino acid segment (Figures 4A and 4B). Moreover, basal levels of NLG1-NTFs can be detected in brain extracts from MMP9 KO mice (Figure 7B), indicating the existence of MMP9-independent mechanisms cleaving NLG1. In addition, our data indicate that NLG1-NTFs are most abundant during the first postnatal weeks (Figures 2G and 2H), suggesting that NLG1 cleavage see more may have different functions during development. It will be important to define in detail the activity-independent and MMP9-independent mechanisms of NLG1 cleavage and their specific role in synapse development and plasticity. Nevertheless, our results indicate that MMP9 is required

for activity-dependent cleavage of NLG1 in multiple cellular contexts in vivo (Figure 7) and in vitro (Figures 3 and 4), consistent with the known involvement of MMP9 in multiple forms of synaptic plasticity (Bozdagi et al., 2007; Nagy et al., 2006; Szklarczyk et al., 2002; Wang et al., 2008; Wilczynski et al., 2008). Focal uncaging of glutamate triggers MMP9-dependent cleavage of postsynaptic NLG1, indicating Adenosine that this mechanism is regulated locally (Figures 4C and 4J). Interestingly, we detected a small heterosynaptic decrease of GFP-NLG1 from neighboring dendritic spines after synaptic stimulation (Figures 4C and 4J), suggesting that MMP9 activation may spread to adjacent dendritic regions. It remains unclear how NMDAR/CaMK signaling couples to MMP9 activity; however, considering the presence of MMP9 in spino-dendritic tubulovesicular structures (Wilczynski et al., 2008), one possibility is that CaMK activation triggers exocytosis of MMP9-containing vesicles in nearby dendritic regions or spines (Kennedy and Ehlers, 2011). It will be interesting to address if persistent cleavage of NLG1 can induce dendrite-wide effects and how the surface pools of NLGs are redistributed and replenished following periods of increased MMP-9 activity.