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. 2018 Jun 6;38(23):5289–5301. doi: 10.1523/JNEUROSCI.3305-17.2018

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Copyright © 2018 the authors 0270-6474/18/385289-13$15.00/0

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Figure 4. — Knockdown of USP8 leads to a loss of synaptic puncta and regulates SHANK1 puncta size and intensity. A, Rat hippocampal dissociated neurons were transfected at DIV14 with a scrambled shRNA or one of two shRNAs that target rat USP8 (USP8_A, USP8_B) for 72 h. At DIV17, neurons were fixed and stained for endogenous synaptic proteins with antibodies to SHANK1, SHANK2, PSD-95, GKAP, and HOMER1A. B, Collapsed z-stack images were thresholded and a mask was developed to identify synaptic protein puncta by a single blinded experimenter in a 20 × 5 μm region of interest. Knockdown of USP8 causes a reduction in SHANK1 synaptic puncta intensity and size, while SHANK2, PSD-95, GKAP, and HOMER1A puncta are unaffected. The number of synaptic protein puncta per 20 μm are reduced for all, with the exception of HOMER1A not reaching significance. n = 3–5 independent cultures, ∼10 neurons quantified for each condition per culture. Puncta intensity SHANK1: one-way ANOVA: F = 12.37, p < 0.0001; post hoc Student's t test: scrambled vs USP8_A, p < 0.0001; scrambled vs USP8_B p = 0.0014; USP8_A vs USP8_B, p = 0.5416; puncta intensity SHANK2: one-way ANOVA: F = 0.5540, p = 0.5772; puncta intensity PSD-95: one-way ANOVA: F = 2.583, p = 0.0837; puncta intensity GKAP: one-way ANOVA: F = 0.4559, p = 0.6356; puncta intensity HOMER1A: one-way ANOVA: F = 1.899, p = 0.1566; puncta size SHANK1: one-way ANOVA: F = 4.553, p = 0.0147; post hoc Student's t test: scrambled vs USP8_A, p = 0.0371; scrambled vs USP8_B, p = 0.0304; USP8_A vs USP8_B, p = 0.9995; puncta size SHANK2: one-way ANOVA: F = 0.5615, p = 0.5728; puncta size PSD-95: one-way ANOVA: F = 1.804, p = 0.1732; puncta size GKAP: one-way ANOVA: F = 0.7936, p = 0.4559; puncta size HOMER1A: one-way ANOVA: F = 1.222, p = 0.3002; puncta number SHANK1: one-way ANOVA: F = 9.892, p = 0.0002; post hoc Student's t test: scrambled vs USP8_A, p = 0.0002; scrambled vs USP8_B, p = 0.0054; USP8_A vs USP8_B, p = 0.4991; puncta number SHANK2: one-way ANOVA: F = 6.297, p = 0.0030; post hoc Student's t test: scrambled vs USP8_A, p = 0.0031; scrambled vs USP8_B, p = 0.0245; USP8_A vs USP8_B, p = 0.7621; puncta number PSD-95: one-way ANOVA: F = 13.88, p < 0.0001; post hoc Student's t test: scrambled vs USP8_A, p = 0.0006; scrambled vs USP8_B, p < 0.0001; USP8_A vs USP8_B, p = 0.5508; puncta number GKAP: one-way ANOVA: F = 5.567, p = 0.0054; post hoc Student's t test: scrambled vs USP8_A, p = 0.0066; scrambled vs USP8_B, p = 0.0302; USP8_A vs USP8_B, p = 0.8321; puncta number HOMER1A: one-way ANOVA: F = 1.621, p = 0.2040. B, Representative images. Scale bar, 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001.