Fig. 1: stb cKO neurons display impaired axonal transport of presynaptic cargos, reduced density of synapses and spines, and altered structures of AZs.

a–d, Reduced density of Bassoon (p < 0.001) (a) and synaptophysin (p < 0.001) (b) along axons and PSD95 (p < 0.001) along dendritic profiles (c) in stb cKO hippocampal neurons. Neurons cultured from E18 control and cKO mouse embryos were co-immunostained at DIV15 with the antibodies indicated. e, f, Reduced axonal delivery of Bassoon-labeled presynaptic cargos in DRG neurons isolated from adult cKO mice at P50 (anterograde transport, p = 0.001; flux rate, p = 0.001). DRG neurons at DIV0 were transfected with GFP-Bassoon and time-lapse imaged at DIV3-4 to measure the motility and axonal flux rate of Bassoon organelles (f). In kymographs (e), vertical lines represent stationary organelles; oblique lines or curves to the right indicate anterograde transport from the soma towards distal terminals (also see Videos S1 and 2). g Selective reduction of anterograde (p < 0.001), but not retrograde, transport of Bassoon-labeled presynaptic cargos in cKO hippocampal neurons. Neurons were transfected with GFP-Bassoon at DIV3 and time-lapse imaged at DIV7. h–k, Representative electron micrographs (h, i) and quantitative analyses (j, k) showing reduced density of AZs (red arrows) in 100-μm² micrograph images (h, j) and reduced total and docked synaptic vesicles per AZ (i, j) from the hippocampus stratum radiatum in cKO mice at 2.5-months of age. l, m, Golgi silver impregnation and quantitative analysis of spine density and maturation in CA1 pyramidal neurons of cKO mice. The red arrow (l) indicates distal basal dendrites (>60 μm from soma) where the spine density was counted. Spine length was defined as the distance from the tip of the spine head to the interface with the dendritic stalk. Filopodia (>2 μm) and long-thin spines (>1 μm in length and <0.2 μm in head diameter) were counted as immature spines, while thin, stubby, mushroom and branched spines were counted as mature spines. Note that both the spine density and percentage of mature spines (mushroom) were reduced (p < 0.001, p < 0.01, respectively) in stb cKO mice (m). Data were collected from the total number of axons or dendrites indicated within bars (d) and in parentheses (m), or the number of vesicles (v) in the total number of neurons (n) as indicated in parentheses (f, g) from three independent experiments, and presented as mean ± sem. Electron micrograph data were collected from the total number of electron micrographs (j, left panel) and total number of AZs indicated below the bars (j, k). The difference between genotypes was analyzed by an unpaired two-tailed Student’s t test. Scale bars: 50 μm (l left), 10 μm (a–c, e, l right), 1 μm (h), and 200 nm (i).