Fig. 2: The human autism-linked STB missense variant is a loss-of-function mutant. | Molecular Psychiatry

Fig. 2: The human autism-linked STB missense variant is a loss-of-function mutant.

From: Defects in syntabulin-mediated synaptic cargo transport associate with autism-like synaptic dysfunction and social behavioral traits

Fig. 2

a, b, Schematic diagram of human STB (a) and the human autism-associated de novo variant R178Q (b) that changes arginine at 178 into glutamine within the KIF5-binding domain (KBD). c, GST pull-down assay showing impaired binding capacity of STB-R178Q to the KIF5 motor heavy chain (KHC) (p = 0.004). GFP-tagged STB or mutant STB-R178Q (140-230), or GST alone was incubated with lysates of HEK cells expressing GFP-KHC (814-930), the cargo-binding domain of KHC. The bound protein complexes were blotted with an anti-GFP antibody. Coomassie blue staining shows loaded GST-STB or GST-STB-R178Q, or GST. Pull-down of GFP-KHC (814-930) was quantified from three repeats, calibrated and normalized to the band in the STB pull-down group. d, f, Representative kymographs (d) and quantitative analyses (e, f) showing impaired axonal delivery of Bassoon-labeled presynaptic cargos in control DRG neurons expressing mutant STB-R178Q. DRG neurons were isolated from adult control mice at P40 and transfected with GFP-Bassoon together with STB or STB-R178Q at DIV0. Note that expressing STB-R178Q selectively reduced anterograde (p < 0.001) but not retrograde (p = 0.280) transport of Bassoon organelles in axons (f), thus reducing flux rate into distal axons (p = 0.006) (e). g, i, Representative kymographs (g) and quantitative analyses (h, i) showing failed rescue of impaired Bassoon transport in cKO DRG neurons by expressing STB-R178Q. DRG neurons were isolated from adult cKO mice at P40 and co-transfected with GFP-Bassoon and control vector, STB, or STB-R178Q at DIV0. Note that expressing STB-R178Q failed to rescue axonal flux rate (One-way ANOVA, Ctrl vector vs. STB, p < 0.001; Ctrl vector vs. STB-R178Q, p = 0.678) (h) and anterograde transport of presynaptic cargos (One-way ANOVA, Ctrl vector vs. STB, p < 0.001; Ctrl vector vs. STB-R178Q, p = 0.826) (i) (also see Videos S3 and 4). j–l, Representative kymographs (j) and quantitative analyses (k, l) showing failed rescue of defective Bassoon transport in cKO cortical neurons by expressing STB-R178Q. Embryonic cortical neurons were co-transfected with GFP-Bassoon and control vector, STB, or STB-R178Q at DIV4 and imaged at DIV6-7. Note that expressing STB-R178Q failed to rescue axonal flux rate (One-way ANOVA, Ctrl vector vs. STB, p = 0.007; Ctrl vector vs. STB-R178Q, p = 0.526) (k) and anterograde transport of presynaptic cargos (One-way ANOVA, Ctrl vector vs. STB, p < 0.0001; Ctrl vector vs. STB-R178Q, p = 0.998) (l). Axonal Bassoon flux rate (100 μm/3 min) (e, h, k) and relative Bassoon motility (f, i, l) of anterograde (antero), retrograde (retro), or stationary (stat) were examined by time-lapse imaging at 2 s intervals for a total of 3 min. m, n, Representative images (m) and quantitative analysis (n) showing failed rescue of reduced Bassoon density in axons of cKO cortical neurons by expressing STB-R178Q at DIV14. Note that expressing STB effectively reverses the phenotype (One-way ANOVA, Ctrl vector vs. STB, p < 0.001; Ctrl vector vs. STB-R178Q, p = 0.212). Data were collected from the total number of axons indicated within bars from three experiments and presented as mean ± sem and analyzed with an unpaired t-test with Welch’s correction (e, f), one-way ANOVA with Tukey’s multiple comparisons test (h, i, k, l) or Dunnett’s multiple comparisons test (n), or Student’s t test (c). **p < 0.01; ***p < 0.001, ****p < 0.0001, ns, not significant. Scale bars: 10 μm.

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