Kif15 deficiency leads to impaired development of dendritic spines in PFC and hippocampus of mice.

A: Experimental design. WB: Western Blot; IF: immunofluorescence staining. B-D: Representative images of neuronal dendrites by Golgi staining in layer2/3 neurons of PFC at 3,6 and 11 week-old Kif15^+/+ and Kif15^-/- mice, Scale bar = 5μm. H-J: Representative images of neuronal dendrites by Golgi staining in CA1 of hippocampus at 3,6 and 11 week-old Kif15^+/+ and Kif15^-/- mice, Scale bar = 5μm. B, C, E and F: Decreased densities of total spine and mushroom-like spines in the PFC neurons from Kif15^-/- in 3, 6 week-old mice, compared with Kif15^+/+ mice, n = 15 dendrites of neurons from 3 mice in each group. H, I, K and L: Decreased densities of total spine and mushroom-like spines in the CA1 of hippocampus from Kif15^-/- in 3, 6 week-old mice, compared with Kif15^+/+ mice, n = 15 dendrites of neurons from 3 mice in each group. D and G: Similar total dendritic spine density between Kif15^+/+ and Kif15^-/- in 11 week-old mice expect for filopodia in PFC. J and M: Similar total dendritic spine density between Kif15^+/+ and Kif15^-/- in 11 week-old mice expect for mushroom like spine in hippocampus. The densities of different types of dendritic spines in PFC neurons from different weekly mice were quantified, n = 15 dendrites of neurons from 3 mice in each group. N: Diagram to show the whole-cell recording of pyramidal neurons in layer 2/3 of PFC from Kif15^+/+ and Kif15^-/- mice. (O-Q): Representative mEPSCs traces of neurons in PFC from 3, 6 and 11 week-old mice. R: Decreased mEPSCs frequency and similar amplitude of neurons in 3 week-old, n = 20 neurons from 6 Kif15^+/+ mice, n = 14 neurons from 5 Kif15^-/- mice. S: Decreased frequency but increased amplitude of mEPSCs in PFC neurons in 6 week-old Kif15^-/- mice, compared with Kif15^+/+ mice, n = 12 neurons from 5 Kif15^+/+ mice, n = 17 neurons from 5 Kif15^-/- mice. T: Similar frequency and decreased amplitude of mEPSCs in PFC Kif15^-/- mice at 11 week old, n = 23 neurons from 4 Kif15^+/+ mice, n = 23 neurons from 4 Kif15^-/- mice. All the data are presented as mean±SD, *P < 0.05, **P < 0.01,two-tailed student’s t test between two groups, Multiple t test performed in different morphology spine group.

Kif15 deficiency affects the expression of excitatory postsynaptic receptors in PFC and hippocampus.

(A and D): Western Blot analysis showed decreased protein levels of PSD95 and NMDA receptor GluN2A in the PFC of 3 week-old Kif15^-/- mice, n = 7 ~ 8 in Kif15^+/+ group, n = 6 in Kif15^-/- group. (B and E): Decreased expression of PSD95 in the PFC at 6 week-old Kif15^-/- group mice, n = 6 in Kif15^+/+ group, n = 8 in Kif15^-/- group. (C and F): Increased expression of PSD95 in the PFC of Kif15^-/- group compared with Kif15^+/+ group mice at 11 week-old, n = 7 in each group. (G and J): The expression of PSD95 in hippocampus of Kif15^-/- mice decreased at 3 weeks, n = 8 in Kif15^+/+group, n = 6 in Kif15^-/- group (H and K): The expression of PSD95 and GluN2A in hippocampus of Kif15^-/- mice was lower than that of wild type mice at 6 weeks, n = 6 in Kif15^+/+ group, n = 8 in Kif15^-/- group. (I and L): The expression of PSD95, GluA1 and GluN2A in the hippocampus of Kif15^-/- mice was significantly higher than that of Kif15 WT mice at 11 weeks, n = 7 in each group. All the data are presented as mean±SD, *P < 0.05, **P < 0.01, two-tailed student’s t test were used for comparison between two groups, expect for the GluA1 of J, which was used as a nonparametric test.

Kif15 deficiency affects the expression of Gephyrin and GABRB1 in PFC and hippocampus.

A-C: Western blot analysis of inhibitory postsynaptic proteins expression in 3, 6 and 11 week-old Kif15^+/+ and Kif15^-/- mice in PFC. G-I: Western blot analysis of inhibitory postsynaptic proteins expression in 3, 6 and 11 week-old Kif15^+/+ and Kif15^-/- mice in Hippocampus. D and J: Similar expression of gephyrin between two groups at 3 week-old in both PFC and hippocampus, n = 6 mice in each group. E and K: Increased gephyrin or GABRB1 expression in Kif15^-/- mice compared with Kif15^+/+ mice at 6 week-old in PFC and hippocampus, n = 6 mice in each group. F and L: High expression of gephyrin and GABRB1 in Kif15^-/- mice compared with Kif15^+/+ mice in PFC and hippocampus, n = 6 mice in each group. All the data are presented as mean±SD, *P < 0.05, **P < 0.01, two-tailed student’s t test were used for comparison between two groups, expect for the GABRB1 of E, which was used as a nonparametric test.

Kif15 deficient mice exhibit both depressive and manic behaviors in adulthood.

A-D: Kif15^-/- mice exhibited longer immobility times in Tail suspension test and the forced swimming test at 9, 12, 16, and 20 week-old, n = 7 or 8 in WT group, n = 8 or 9 in Kif15^-/- group; E: Representative tracking path for Kif15^-/- and Kif15 WT mice during 10 minutes open field test in 9, 12, 16 and 20 week-old. F: Kif15^-/- mice traveled further in the 10 minutes open field test at 16 and 20 week-old; n = 8 in WT group, n = 9 in Kif15^-/- group, G: Schematic of the mechanism by which Kif15^-/- mice exhibit depression and mania. The image was created with Microsoft Power Point, and some icons are free downloaded from OPENCLIPART (https://openclipart.org/). All the data are presented as mean±SD, *P < 0.05, **P < 0.01,two-tailed student’s t test between two groups.

The depressive behavior in Kif15^-/- zebrafish was rescued following Kif15 supplementation.

A: Sequencing results of Kif15^-/- zebrafish. B: Representative plots of spontaneous swimming trajectories of zebrafish from different groups on day 4 after fertilization. C: On day 4 after fertilization, Kif15 overexpression rescued the depressive behavior induced by kif15 knockout, with 8 zebrafish larvae in each group. All the data are presented as mean±SD, *P < 0.05, **P < 0.01,two-tailed student’s t test between two groups.

KIF15 interacts with PSD95 and affect its localization in neuronal cytoplasm neurons of the PFC.

A and C: Co-IP result of cortical protein homogenate from P1 Kif15^+/+ mouse. B: Result of co-IP assays from 293T cells which simultaneous overexpression of KIF15-GFP and PSD95-Flag. Asterisks indicate interaction bands in the Western blot assays, triangles represent negative bands with no interaction. D: Immunofluorescent images showing the distribution of PSD95 expression in the PFC neurons at 3, 6 and 11 week-old mouse (Right panel, higher magnification of dashed boxed area). E: Wildtype and Kif15 knockout DIV18 neurons were immunostained for PSD95 and presynaptic synaptophysin. The colocalization of PSD95 and synaptophysin in confocal images was considered as a synapse (solid white arrows). F: The synaptic density in KIF15 knockout neurons was significantly lower compared to the wildtype group, n = 15 neurons per condition. All the data are presented as mean±SD, ***P < 0.001,two-tailed student’s t test between two groups.

KIF15 mediates the trafficking of PSD95 along microtubules.

A and B: Overexpression or knockdown of Kif15 in 293T cells affected the expression and distribution of PSD95 on the cell membrane. n ≥ 20 neurons in each group, All the data are presented as mean±SD, *P < 0.05, **P < 0.01,two-tailed student’s t test between two groups. C: FRAP analysis of fluorescence recovery of PSD95-mCherry in 293T cells, The white dashed box in the upper panel is the magnified area of the image in the lower panel, and the white solid box is the bleached area. D: Fluorescence recovery intensity in the bleached region (white solid-line box) was measured at fixed time intervals.

Intrauterine supplementation of Kif15 in E15 Kif15^-/- mice rescued the distribution of PSD95 in Kif15^-/- PFC neurons.

A: Changes in PSD95 expression in the cortex and hippocampus of P1 mice were observed following cortical injection of adenovirus overexpressing Kif15 into the cortex of E15 Kif15^-/- embryos. The right panel is a magnified view of the white dashed box in the left panel. B: The abnormal distribution of PSD95 within neurons in the PFC region of P1-day Kif15^-/-mice was rescued by cortical injection of adenovirus overexpressing Kif15 into E15 Kif15^-/- embryos. Bar = 20 μm.