Additional file 1 of Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior

Additional file 1: Fig. S1. Cortical neurons were infected at 3 DIV with AVV encoding TdTomato as control and a combination of sgRNAs (1–6 / 2–4 / 1–4 / 2–6) targeting Vps50, as indicated. 10 days later genomic DNA was extracted, and the T7 endonuclease I assay was performed. (A) T7 endonuclease I assay for sgRNA combinations showing successful editing of the VPS50 locus with all sgRNA combinations as shown by lower size bands in the treated samples. (B) Sequences of VPS50 locus edited with sgRNAs in VPS50 KO cultured neurons showing the changes in the DNA sequence after gene editing using locus-specific sequencing. Fig S2. Proximity ligation assay (PLA) in Control and VPS50 KO neurons. PLA was performed using specific antibodies to determine proximity between proteins of interest. Low magnification (20x) for each condition are shown. Microtube-associated protein 2 (MAP2) was used to stain all neurons. (A) PLA for Synapsin1 (Syn1)-Synaptophysin (Syn) (pre-presynaptic) or (B) Synaptophysin-PSD95 (pre-post synaptic). Reaction controls where PLA signal was observed only for the Synapsin1-Synaptosophysin pair, the two proteins known to be in proximity. Lower magnification of PLA reactions in Fig. 2 to show different combinations of PLA reactions to determine proximity between (C) VPS50/PSD95, (D) VPS50/Syn1, (E) VPS50/v-ATPaseV1, (F) VPS50/v-ATPaseV1 in VPS50 KO neurons, (G) v-ATPaseV1/Syn, and (H) v-ATPaseV1/Syn in VPS50 KO neurons. (I) PSD95 and Synaptophysin protein expression in VPS50 KO and control neurons. N-cadherin was used as loading control. Scale bars, 25 μm. Fig. S3. VPS50 KO neurons show deficits in synaptic transmission. (A) Representative traces of AMPA-mediated EPSCs followed for 30 min for control and VPS50 KO neurons. VPS50 KO neurons show a robust reduction in both Amplitude (B) and Frequency (C) of AMPA EPSCs. (D-E) Cortical neurons were co-transduced with GCaMP7 to measure calcium events by changes in fluorescence over time. (D) Representative traces and quantification (E) of changes of fluorescence over time in control and VPS50 KO neurons. A significant reduction is observed in VPS50 KO neurons compared to control. Control n = 1214; VPS50 mKO n = 1175 cells from 3 independent experiments; Kolmogorov–Smirnov test was used to test for significance. For electrophysiology recordings at least 18 neurons from 3 independent experiments were analyzed. Unpaired t-test was used for statistical analysis; *p < 0.05, ***p < 0.001. Error bars represent ± SEM. Fig. S4. Anatomical changes of Control and VPS50 mKO brains. (A-B) Representative images of full brains from Control and VPS50 mKO animals. (C-D) Quantification of the total length (C) and weight (D) of brains extracted from Control or VPS50 mKO animals. (E–F) Representative bright field images of brain slices from Control (E) and VPS50 mKO (F) brains.