Substituting 60 aa of the NLG1 stalk domain (aa 636–695) with the polylinker GAAAAA resulted in a mutant (NLG1-ΔSDfull) that is resistant to APMA (Figures 4A and 4B). Within this 60 residue stretch, deletion of aa 672–695 (NLG1-ΔSD3) and replacement PLX3397 mw with the polylinker GAAAAA likewise abolished APMA-induced cleavage, whereas mutation of more membrane-distal sequences did not (aa 636–660, NLG1-ΔSD1; aa 654–677, NLG1-ΔSD2). Notably, we attempted to further resolve the precise cleavage site, but shorter deletions or single site mutants were all cleaved upon APMA treatment, potentially due to the presence of multiple MMP target sequences within this domain. Importantly, the ΔSD3 mutation

does not alter NLG1 localization, as GFP-NLG1-ΔSD3 exhibited a similar distribution pattern and synaptic enrichment as wild-type GFP-NLG1 when expressed in DIV21 hippocampal neurons (Figure S4A). Moreover, GFP-NLG1-ΔSD3 and GFP-NLG1 induced quantitatively similar spine formation when expressed in mouse cortical neurons in vivo from E15.5 to P17 and P18, indicating that GFP-NLG1-ΔSD3 retains the synaptogenic properties of wild-type NLG1 (Figures S4D and S4E). To address if NLG1-ΔSD3 is resistant to activity-dependent cleavage in neurons, we tested the effect of KCl depolarization in neurons expressing GFP-NLG1 and GFP-NLG1-ΔSD3. Following

2 hr of KCl incubation, synaptic GFP-NLG1 fluorescence decreased to 55.9% ± 5.4% of initial value, whereas GFP-NLG1-ΔSD3 exhibited no change upon KCl treatment (101.9% ± 6.9% of initial fluorescence Bafilomycin A1 cell line level; Figures S4A and S4B). To address if NLG1 cleavage occurs locally in response to increased synaptic activity, we released glutamate at single dendritic spines by two-photon laser-induced photolysis of (4-methoxy-7-nitroindolinyl)-glutamate (MNI-glutamate), while imaging dendrites mafosfamide of neurons expressing GFP-NLG1-WT or GFP-NLG1-ΔSD3 (Figures 4C–4L). Analysis was performed in CA1 pyramidal neurons in organotypic hippocampal slices at a time corresponding to P14 with tdTomato (tdT) coexpression used as a cell fill. Stimulation near (∼1 μm) the distal

head of a dendritic spine (Spine 1) with 80 4-ms laser pulses at 2 Hz induced rapid loss of spine GFP-NLG1 within 1 min, whereas no change in fluorescence was detected in the neighboring dendritic shaft (ΔGFP/tdT: Spine 1: 0.61 ± 0.04, dendrite: 1.01 ± 0.02; Figures 4C, 4F, and 4J). We observed a smaller, partial loss of NLG1-GFP in neighboring spines (Spine 2, ΔGFP/tdT: 0.79 ± 0.15), possibly due to the diffusion of intracellular signals. Incubation with the MMP2/MMP9 inhibitor II (0.3 μM) or GM6001 (10 μM) abrogated glutamate-induced GFP-NLG1 loss (MMP2/MMP9i: ΔGFP/tdT: Spine 1, 0.89 ± 0.10; Spine 2, 0.97 ± 0.12; dendrite, 0.98 ± 0.11; Figures 4D, 4G, and 4K; GM6001: ΔGFP/tdT: Spine 1, 0.91 ± 0.04; dendrite, 0.96 ± 0.03; Figures S4F–S4H).