25 ± 0.11 versus 1.49 ± 0.12, p = 0.1642; PSD-95-positive, 0.89 ± 0.07 versus 1.01 ± 0.08, p = 0.2809; double-positive, 0.86 ± 0.07 versus 0.93 ± 0.08, p = 0.4911, two-tailed t test; Kif1a−/−: synaptophysin-positive, 1.03 ± 0.09 versus 1.06 ±
0.10, p = 0.8125; PSD-95-positive, 0.74 ± GSK1349572 0.05 versus 0.75 ± 0.05, p = 0.8378; double-positive, 0.70 ± 0.05 versus 0.71 ± 0.06, p = 0.9163, two-tailed t test) ( Figures 5D–5F). These results suggest that KIF1A upregulation is required for BDNF-induced synaptogenesis. Finally, to confirm the role of KIF1A in synaptogenesis, we examined the effects of KIF1A overexpression on cultured hippocampal neurons. Neurons were cotransfected with KIF1A and synaptophysin-green fluorescent protein (syp-GFP), and the density of syp-GFP puncta along dendrites was compared with that of neurons transfected with syp-GFP alone (Figure 6A). Significant increases in the total number and density of syp-GFP puncta along dendrites were observed in cotransfected neurons (syp-GFP alone versus KIF1A+syp-GFP: total number, 55.6 ± 4.6 versus 73.3 ± 5.0, p < 0.05; density [per 10 μm], 0.40 ± 0.02 versus 0.54 ± 0.02, p < 0.0001, post hoc Dunnett's test) (Figures 6C and 6D). Furthermore, there was also a significant increase in the number of syp-GFP puncta colocalized
with PSD-95 in cotransfected neurons (syp-GFP alone versus KIF1A+syp-GFP: 37.1 ± 2.6 versus 47.2 ± DNA Synthesis inhibitor 3.6, p < 0.05, post hoc Dunnett's test) (Figures 6B and 6I). No significant differences TCL were detected in neurons cotransfected with KIF5B and syp-GFP (syp-GFP alone versus KIF5B+syp-GFP: total number, 55.6 ± 4.6 versus 57.2 ± 4.2, p > 0.05; density [per 10 μm], 0.40 ± 0.02 versus 0.42 ± 0.02, p > 0.05; number of colocalized puncta, 37.1 ± 2.6 versus 37.4 ± 2.9, p > 0.05, post hoc Dunnett’s test) (Figures
6C, 6D, and 6I). There were no differences in the number or total length of dendrites contacted by axons of transfected neurons among groups (syp-GFP alone, KIF1A+syp-GFP, and KIF5B+syp-GFP: number of dendrites, 18.3 ± 1.3, 19.1 ± 1.2, and 18.5 ± 1.3, respectively, p = 0.9053; total length [μm], 1,389 ± 111, 1,411 ± 118, and 1,394 ± 109, respectively, p = 0.9894, one-way ANOVA) (Figures 6E and 6F). Also no significant differences were observed in the number of neurons contacted by axons of transfected neurons or in the mean distance from transfected neurons to contacted neurons among groups (syp-GFP alone, KIF1A+syp-GFP, and KIF5B+syp-GFP: number of neurons, 6.95 ± 0.41, 7.10 ± 0.51, and 6.80 ± 0.48, respectively, p = 0.9034; mean distance [μm], 245 ± 20, 249 ± 20, and 238 ± 20, respectively, p = 0.9254, one-way ANOVA) (Figures 6G and 6H). Collectively, these results suggest that increased KIF1A levels promote synaptogenesis via the formation of presynaptic boutons.