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. 2024 Mar 1;17:1222935. doi: 10.3389/fnmol.2024.1222935

FIGURE 2.

FIGURE 2

Functional effect of SLITRK3 variants on inhibitory synapses. (A) Identification of SLITRK3-WT, SLITRK3-C566R, and SLITRK3-E606X expression in HEK-293 cells. Cell lysates from HEK-293 cells transfected with SLITRK3-WT-Myc, SLITRK3-C566R-Myc, or SLITRK3-E606X-Myc were blotted with anti-SLITRK3, anti-Myc and anti-tubulin antibodies. N = 3 independent repeats. (B–D) Human mutants in SLITRK3 are essential for GABAergic synaptic transmission. mIPSC recording showed that overexpression of human WT- SLITRK3 significantly increased mIPSC frequency, whereas overexpression of the SLITRK3-C566R mutant had no effect on the frequency of mIPSCs in cultured hippocampal neurons. Insets showed the mean ± SEM of mIPSC frequency and amplitude [Frequency (Hz): Control, 2.25 ± 0.50, n = 10; WT- SLITRK3, 4.67 ± 0.54, n = 9; SLITRK3-C566R, 2.67 ± 0.65, n = 11; SLITRK3-E606X, 2.08 ± 0.28, n = 10. One-way ANOVA test, **p = 0.0058; Mann-Whitney test, Control vs. SLITRK3-WT: **p = 0.004; Control vs. SLITRK3-C566R: p = 0.918; Control vs. SLITRK3-E606X: p = 0.579. Amplitude (pA): Control, 19.770 ± 1.91, n = 10; SLITRK3-WT, 15.44 ± 1.14, n = 9; SLITRK3-C566R, 19.65 ± 2.02, n = 11; SLITRK3-E606X, 21.30 ± 3.22, n = 10. One-way ANOVA test, p = 0.33; Mann-Whitney test, Control vs. SLITRK3-WT: p = 0.08; Control vs. SLITRK3-C566R: p = 0.76; Control vs. SLITRK3-E606X: p = 0.91. N = 5–6] Scale bar, 20 pA and 1 s. N = 6 independent repeats. (E–G) Identification of C566R and E606X in SLITRK3 that is important for GABAergic synapse density in hippocampal neurons. Representative images of dendrites and quantification analysis showed that overexpression of human SLITRK3-WT significantly increased vGAT and gephyrin puncta density in cultured hippocampal neurons, whereas overexpression of human SLITRK3-C566R and SLITRK3-E606X, did not change vGAT or gephyrin puncta density [vGAT: Control: 5.38 ± 0.39, n = 19, SLITRK3-WT: 8.36 ± 0.44, n = 17, SLITRK3-C566R: 5.17 ± 0.46, n = 16, SLITRK3-E606X: 5.22 ± 0.33. One-way ANOVA test, ****p < 0.0001, F = 12.22; Mann-Whitney test, Control vs. SLITRK3-WT: ****p < 0.0001; Control vs. SLITRK3-C566R: p = 0.62; Control vs. SLITRK3-E606X: p = 0.66. Gephyrin: Control: 5.07 ± 0.45, n = 19, SLITRK3-WT: 8.44 ± 0.80, n = 17, SLITRK3-C566R: 5.50 ± 0.58, n = 16, SLITRK3-E606X: 5.00 ± 0.40. One-way ANOVA test, ***p = 0.0001, F = 8.038; Mann-Whitney test, Control vs. SLITRK3-WT: **p = 0.002; Control vs. SLITRK3-C566R: p = 0.68; Control vs. SLITRK3-E606X: p = 0.90]. N = 4 independent repeats (SLITRK3 denoted as ST3 on figure).