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Deficiency of Kif15 impairing synaptic development leads to mood disorder in mice [1]

['Xiaomei He', 'Key Laboratory Of Neuroregeneration Of Jiangsu', 'Ministry Of Education', 'Co-Innovation Center Of Neuroregeneration', 'Medical School', 'Nantong University', 'Nantong City', 'Department Of Basic Medicine', 'Nantong Health Vocational College', 'Wei Zhang']

Date: 2025-09 

Abstract The harmony of neuronal excitation and inhibition is essential for precise neuronal circuitry in the developmental brain, and thus affects the human emotion. Abnormalities of synaptic morphology directly affect neuronal function and contribute to a variety of psychiatric disorders. Previous studies have shown that Kif15 (Kinesin-12), a microtubule-associated motor protein, affects neurite growth, navigation, and branching during neuronal development, revealing the potential of Kif15 to influence neuronal dendritic morphology. A GWAS study in a European population showed that there were variants in both exons and introns of the KIF15 gene on chromosome 3 in patients with depression. Therefore, we generated Kif15-/- mice using CRISPR/Cas9 technology. In this study, we found that Kif15-/- mice have exhibited significant impacts on dendritic morphology and function, which contributes to mood disorders. Compared with Kif15 wild-type mice, adolescent Kif15-/- mice showed a significant decrease in the excitatory postsynaptic scaffolding protein PSD95 and NMDA receptors, as well as a reduction in the total density of dendritic spines and the density of mushroom spines, and a decrease in the frequency of mEPSCs. Meanwhile, the inhibitory postsynaptic scaffold protein Gephyrin and GABRB1 significantly upregulated. However, the adult Kif15-/- mice simultaneously exhibited an obvious manic behavior and their PSD95 expression increased rapidly, even more than that of the Kif15 wild-type mice. Meanwhile, overexpression of Kif15 in kif15−/− zebrafish rescued their depressive behavior. In terms of molecular mechanism, we showed that KIF15 interacted with PSD95 protein using both endogenous and exogenous Co-IP assays. Furthermore, we found that PSD95 in Kif15-/- mice was distributed around neuronal nuclei, in contrast to PSD95 localized close to the cell membrane in Kif15 wild-type mice. In conclusion, our study has identified a microtubule-associated molecular motor, KIF15, that plays a novel role in bipolar disorder through its contributions to spine morphology and function.

Author summary Mood disorders are a common mental illness with a high incidence rate. The etiology of mood disorders is complex, involving both genetic and environmental factors, although the precise cause remains unknown. Previous GWAS analysis showed KIF15 gene is a candidate gene for depression, but the molecular mechanism is unknown. In our study, we found that KIF15 gene knockout mice exhibited mood disorder during the postnatal development. Further investigation showed an imbalance in the expression of excitatory and inhibitory postsynaptic proteins at different stages, which may lead to bipolar disorder, characterized by alternating episodes of depression and mania. KIF15, also named kinesin-12, is a microtubule-associated motor protein, and our study revealed that KIF15 involved in the PSD95 transportation and localization at postsynaptic membrane, and thus the deficiency of KIF15 gene disrupt the localization of PSD95, and hence impair the expression of AMPA receptors and NMDA receptors in synapses. Our data uncover a novel function of KIF15 in synapses and also offer valuable insights into the pathogenesis of mood disorders.

Citation: He X, Zhang W, Chen X, Dong Z, Wei C, Wu T, et al. (2025) Deficiency of Kif15 impairing synaptic development leads to mood disorder in mice. PLoS Genet 21(9): e1011839. https://doi.org/10.1371/journal.pgen.1011839 Editor: Brian D. Perkins, Cleveland Clinic Cole Eye Institute, UNITED STATES OF AMERICA Received: January 28, 2025; Accepted: August 13, 2025; Published: September 2, 2025 Copyright: © 2025 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (NO. 32070725 and NO.31171007 to ML; NO.32471038 to YL). The funder played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

1 Introduction Mood disorders, which can be roughly divided into depression and bipolar disorder [1,2], often involve multiple brain regions, including the prefrontal cortex and hippocampus. The prefrontal cortex (PFC) is a highly intricate brain region both in terms of its structure and function, encompassing working memory, emotion regulation, sensorimotor, cognition and decision-making, as well as other advanced features [3–6]. Hippocampus is one of the neurogenesis areas of the adult brain, which is mainly involved in learning and memory, and also has certain cognitive and emotional regulation functions [7,8]. Dysfunction of the prefrontal cortex or hippocampus is often linked to a range of psychiatric conditions, such as schizophrenia, depression, and bipolar disorder [9,10]. In the prefrontal cortex, the postsynaptic membrane of most glutamatergic excitatory synapses is composed of spines on dendrites [11]. In contrast, inhibitory synapses mainly occur in the shaft of dendrites, neuron bodies, or axon initial segments [12]. The number and morphology of dendritic spines undergo changes throughout mouse development and adulthood, and this plasticity of dendritic spines is closely associated with synaptic function. Previous studies have indicated alterations in both the density and morphology of dendritic spines in the prefrontal cortex of a mouse model exhibiting depressive symptoms [13,14]. However, the specific molecular mechanism underlying the relationship between dendritic spines and the mood disorders is worthy of further investigation. In the central nervous system, excitatory neurons modulate synaptic function by regulating postsynaptic scaffolding proteins such as PSD95 thereby altering neuronal excitability [15,16]. PSD95 is a member of the membrane-associated guanylate kinase (MAGUK) class of proteins in the synapse, which regulates the stabilization, recruitment and trafficking of postsynaptic glutamatergic receptors such as AMPA receptors and NMDA receptors to the postsynaptic membrane [17,18]. The postsynaptic scaffolding proteins at inhibitory synapses are mainly gephyrin, but the specific molecular mechanisms of how inhibitory postsynaptic scaffolding proteins and related receptor proteins are regulated have been less studied [19,20]. PSD95 deficiency causes an increase in the number of inhibitory GABAergic synapses and receptor expression, whereas overexpression of PSD95 causes a decrease in the number of inhibitory GABAergic synapses in the hippocampal neurons, thereby altering the ratio of excitatory to inhibitory synapses (E/I) [21,22]. A large number of studies have shown that both depression and bipolar disorder are associated with abnormal synaptic function [23,24]. Genome-wide association studies (GWASs) found that the variant risk genes of bipolar disorder were also mainly enriched in genes related to synaptic function [25]. Neurons are highly polarized cells, and intracellular material transport plays an important role in the maintenance of cell morphology and function. There are three main motor proteins involved in substance transport in neurons, including dynein, kinesin and myosin [26]. Kinesins transport a variety of proteins, mRNA or organelles along microtubules. A variety of kinesins have been reported to be involved in vesicular or postsynaptic protein trafficking [26,27]. Kif15 is a member of kinesin12 family, which is an N-terminal plus-end-directed mitotic motor protein involved in the establishment of bipolar spindle, cell proliferation, apoptosis, and differentiation. It plays a crucial role in neuronal development [28,29]. Our previous study indicated that Kif15 mediates axon growth, navigation, and branching through microtubule growth and sliding in neurons [28]. We previously reviewed a genome-wide association study (GWAS) [30] in major depressive patients of Europe, and identified the presence of SNP (Single nucleotide polymorphism) variants in the KIF15 gene on chromosome 3. The SNP variants of rs3804583, rs3804580 and rs7622843 are significantly above the threshold in control population. Therein, the rs3804583 was found to be located in the exon of the KIF15 gene, while rs3804580 and rs7622843 were found in the intron. (S1 Fig). Additionally, a SNP variant in the intron of KIF15 gene was also found in another GWAS [31] of depression patients from multi-ancestry. According to the NCBI, the SNP rs3804583, located in the exon, is mutated from C to A, G, or T at 44843155 (GRCh38) or 44884647 (GRCh37) on chromosome 3. These mutations occur at the same base site in the CDS region of the KIF15 gene. The mutation of this base led to a missense mutation of KIF15 protein, Leu1206Met or Leu1206Val. According to literature, the tail region of KIF15 consists mainly of amino acids 1149–1388 [32]. so this missense mutation primarily affects the tail function of KIF15. This region of kinesin is crucial for the binding of cargos [26,33]. Therefore, we propose that the mutation at this SNP ultimately disrupts the binding of KIF15 with its cargo. These findings suggest that the KIF15 gene may play a role in the onset or development of major depression. Therefore, we adopted the KIF15 knockout C57BL/6 mice to investigate whether Kif15 gene involving in the emotional disorder and the possible molecular mechanism.

3 Discussion In this study, the effects of Kif15 deficiency on the development of dendritic spines in the prefrontal cortex and hippocampus of mice were revealed. Meanwhile, the loss of Kif15 leads to an imbalance in the expression of excitatory and inhibitory synapses throughout the development of mice. Due to the deficiency of Kif15, the expression of excitatory postsynaptic scaffold protein PSD95 is decreased, while the expression of inhibitory postsynaptic scaffold protein gephyrin is increased during adolescence, and Kif15-/- mice mainly exhibit depressive behavior. After adulthood, the expressions of excitatory and inhibitory synaptic proteins in Kif15-/- mice increased, which may be one of the reasons why Kif15-/- mice develop bipolar disorders like behavior. In addition, overexpression of Kif15 in Kif15-/- zebrafish reversed the depression-like behavior, further confirming the important role of kif15 in depression-like behavior. It is evident that Kif15, as a microtubule plus-end-directed protein, plays an indispensable role in the developmental process for the transport on dendrites or the anchoring of postsynaptic membranes. Therefore, we propose that KIF15 plays a crucial role in the development of excitatory synapses in the prefrontal cortex of mice and influences the balance between excitatory and inhibitory synaptic expression. As a member of the kinesin superfamily, Kif15 is essential for neuronal development. It affects PSD95 localization in the neuronal cytoplasm through interaction with PSD95, which may contribute to the impaired dendritic spine density and morphology observed in Kif15-/- mice. In addition, NMDA receptors can directly bind to PSD95, but AMPA receptors do not [46,47], so the reduction of PSD95 caused by Kif15 deficiency may directly affect NMDA receptor expression or membrane anchoring. In addition, at 6 weeks, the whole-cell patch clamp results showed that the frequency of Kif15-/- mice decreased and the amplitude of mEPSCs increased. The amplitude of mEPSCs represents the change of the number or nature of glutamatergic receptors, but the protein expression of two excitatory postsynaptic receptors in the prefrontal cortex of Kif15-/- mice was not different from that of the wild group. Here it is more likely to affect the synaptic function. Surprisingly, at 11 weeks, there was no difference in the frequency of mEPSCs between the two groups, but the amplitude of mEPSCs was significantly lower than that of WT mice, reflecting the complexity of organism development and the importance of Kif15 in the development of mouse nervous system. It has been reported in the literature that reduced ratios of excitability and inhibition are exhibited in rodents with major depression or depressive behavior [41,48]. Our previous results showed that Kif15-/- mice already exhibit depressive tendencies during adolescence. In the present study, we examined both the excitatory postsynaptic scaffolding protein PSD95 and the inhibitory scaffolding protein gephyrin, and found that PSD95 was decreased in the Kif15-/- mice during adolescence, whereas the expression of gephyrin was increased, and we speculate that this imbalance between excitatory and inhibitory synaptic expression is one of the reasons for triggering depressive-like behavior in Kif15-/- mice, Moreover, the high expression of this inhibitory postsynaptic protein persisted into adulthood. Therefore, adult Kif15-/- mice not only change the expression of excitatory postsynaptic receptors, but also alter inhibitory postsynaptic receptors. It has been reported that patients with bipolar disorder have increased neuronal excitability and abnormal expression of γ-aminobutyric acid or its receptor. Therefore, we wondered whether Kif15-/- mice also have such emotional fluctuations, and the results of behavioral experiments actually supported our suggestion. Behaviorally, the depressive behavior of Kif15-/- mice also persisted from adolescence to adulthood. Although we detected increased expression of both excitatory and inhibitory proteins at 11 weeks, they did not show manic behavior until 16 weeks, and the mood swings of this depressive and manic behavior continued until 20 weeks. This not only reflects the consistency between molecular expression and behavior, but also suggests that the behavior is delayed. It has been reported in the literature [49] that patients with major depression often develop bipolar disorder with intermittent episodes of manic behavior, which may coincide with this biological mechanism we have revealed. Meanwhile, we will continue to study the mood swings of the mice after 20 weeks and even into old age. In a follow-up study, we further explored how Kif15 deficiency impacts dendritic spine morphology and subsequent emotional changes. Our preliminary results indicate that KIF15 interacts with the fourth class of cytoskeletal proteins, Septin7 and Septin3, the former are involved in maintaining cell morphology specifically [50]. Previous studies have shown that Septin7 interacts with PSD95 to influence dendritic spine morphology and synaptic distribution [51]. Additionally, Septin3 is a neuron-specific protein, suggesting that Kif15 may play a role in mediating synaptic development through interactions with the Septins family proteins. As a plus-end microtubule kinesin in neurons, Kif15 not only affects the growth of neuronal axons, but also mediates the expression and localization of postsynaptic protein in vivo, and then affects the mood disorders in mice. In addition, other kinesins are involved in PSD95 trafficking in neurons, including KIF5A, a member of the Kinesin-1 family, which mediates the transport of PSD95 on dendrites by binding to the C-terminal region of its tail. [27]. It has also been reported that KIF5B plays a crucial role in the localization of PSD95 on dendrites and in dendritic spine plasticity [52]. Beyond that, our previous studies have found that the main difference between KIF15 and KIF11 is that KIF15 can interact with actin and plays a role in maintaining the growth cone and branching of protrusions. However, the unique role of KIF15, distinct from KIF5A and KIF5B in transporting PSD95, was not thoroughly explored in this study, which represents a limitation of the research. In summary, our study revealed a novel role of Kif15 on the emotion, and also provided a new clinical target for the pathogenesis of bipolar disorder.

4 Materials and methods 4.1 Ethics statement All mice experiments were conducted in accordance with the requirements of National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by Nantong University. The studies were also reviewed and approved by institutional animal care and use committee(IACUC) of the Animal Center of Nantong University with approval number S20230408-012. 4.2 Details of Materials and methods are in supplementary information 4.2.1 Animals and genotype analysis. Kif15-/- mice were Kif15 systemic knockout mice made by CRISPR/Cas9 technology using C57BL/6 as background mice. In our breeding colony, two heterozygous females and one heterozygous male were mated to obtain homozygotes. All animals were housed in a constant environment where was maintain on a 12-hr light-dark cycle at a comfortable temperature. 4.2.2 Electrophysiology. In the present study, whole-cell patch clamp recordings were performed to record mEPSCs from Layer2/3 pyramidal cells in the mouse PFC. (see supplementary information). 4.2.3 Golgi staining. FD Rapid GolgiStain Kit was employed to examine the neuromorphopathological alterations. The mouse brain tissue was placed in a mixture of equal volumes of solution A and solution B for two weeks in the dark at room temperature. Tissue was placed in solution C for 4 days. Brain tissue were cut into sagittal slices (100 μm thick) and mounted on slides. Subsequent staining steps are performed according to the instructions. Dendritic spines on the basal dendrites of pyramidal neurons in layer 2/3 of the PFC were selected for statistics. Dendritic spine density was analyzed by Fiji software. 4.2.4 Western-blotting. As previously described [53], the brain tissues from the prefrontal cortex and hippocampus of mice were collected, and protein was cleaved, extracted, and then the samples were prepared by 6 × loading buffer for the next steps. The specific conditions for separating the proteins, transferred to PVDF membrane, blocking with milk and antibody incubation are described in the Supplementary material. Protein bands were visualized by chemiluminescence and quantified using Fiji software. 4.2.5 Immunofluorescence staining. After anesthesia, the mice were rapidly perfused with 0.9% saline solution and 4% paraformaldehyde, and the brain tissues were collected. Brain tissues were removed and fixed in 4% paraformaldehyde at 4°C for 16 hours, then dehydrated in 30% sucrose solution. Next, brain tissue were cut into sagittal slices at a thickness of 16 μm. The slices were blocked and incubated with primary and secondary antibody. Images were taken by Zeiss LSM 900 with Airyscan 2 confocal microscope. 4.2.6 Gene overexpression. Simultaneous overexpression of KIF15 and PSD95 in 293T cell lines, corresponding empty plasmids were used as controls. Lipo8000(Cat. C0533, Beyotime) was used as the transfection reagent, and the experimental procedure was carried out according to the instructions(see supplementary information). 4.2.7 Co-immunoprecipitation. Protein cleavage and extraction were performed as previously described. The primary antibody was added to the protein lysate and incubated overnight at 4 ° C by shaker, agarose beads were added on the second day and shaken at 4° C overnight, and on the third day the protein was separated from the beads by 95 ° C water bath, the supernatant protein for subsequent Western Blot. Exogenous co-IP directly added protein lysates to magnetic beads with tagged antibodies. 4.2.8 Behavioral assays. Tail suspension test and Forced swimming test were performed to detect depressive behavior. The manic behavior of mice was detected by open field test. Immobility time and movement trajectory of mice was recorded by EthoVision XT software system. 4.2.9 Microinjection and zebrafish behavior. Embryonic injections of cas9 protein, Kif15 sgRNA, and Rat Kif15 mRNA were performed at the one-cell stage, and the spontaneous swimming behavior of zebrafish larvae was assessed on day 4 post-fertilization. Detailed procedural steps are provided in the Supplementary Materials and Methods. 4.2.10 Statistical analysis. Data were analyzed by unpaired two tailed Student’s t test, Multiple t tests and expressed as mean±SD. p < 0.05 was considered statistically significant (see supplementary information).

Supporting information S1 Text. Materials and methods. https://doi.org/10.1371/journal.pgen.1011839.s001 (DOCX) S1 Fig. Manhattan plot for genome-wide association study (GWAS) in a European population with depression. A: The x-axis represents the chromosomal position, and the y-axis represents the significance on a –log10 scale. The rs3804583 marked in the figure is a variant SNP located in the exon of KIF15 in the population wit depression, rs3804580 and rs7622843 are two SNPs located in the intron variation of the KIF15 gene. The red line represents the genome-wide significance threshold of 5 × 10−8 and the blue line 10−5. B: The representative co-IP image of PSD95-Flag and KIF15-GFP or KIF15 mutant (C3636A or C3636G) plasmids co-expressed in 293T cells. C: Point mutations in the tail region of KIF15 significantly decreased its binding efficiency to PSD95, n = 3 (three independent experiments). All the data are presented as mean±SD, *P < 0.05, **P < 0.01, two-tailed student’s t test between two groups. https://doi.org/10.1371/journal.pgen.1011839.s002 (TIF) S2 Fig. Gene knockout strategy and genotype identification in KO mice. A: Kif15 gene knockdown strategy diagram; B: Results of mouse genotype identification, M, 2000 bp marker, lane 1、3、5: Kif15+/- mouse; lane 2: Kif15-/- mouse; lane 4: Kif15+/+ mouse; C: The knockout of Kif15 protein was confirmed in the PFC and hippocampus of Kif15-/- mice in P1. https://doi.org/10.1371/journal.pgen.1011839.s003 (TIF) S3 Fig. The distribution of PSD95 and KIF15 in the cortex and hippocampus of 3-week, 6-week, and 11-week Kif15+/+ and Kif15-/- mice was examined. https://doi.org/10.1371/journal.pgen.1011839.s004 (TIF) S4 Fig. Emotional changes in Kif15-/- mice from adolescence to adulthood. A: From 14W to 9M, the time or frequency of entering the open arm of Kif15-/- mice were significantly lower than those of Kif15+/+ mice. B: At 24 weeks of adulthood, the total distance traveled by Kif15-/- mice in the open field test was similar to that of Kif15+/+ mice. n = 7 ~ 9 mice 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. https://doi.org/10.1371/journal.pgen.1011839.s005 (TIF) S5 Fig. Kif15 deletion results in abnormal distribution of PSD95 in neuropil. A: Representative images of PSD95 and synaptophysin immunofluorescence staining in DIV18 neurons treated with KIF15 inhibitor (KIF15-IN-1), B: The KIF15-IN-1 significantly reduced the synaptic density of DIV18 neurons, n = 15 neurons per condition. C and D: The distribution of PSD95 in the dendrites of neurons in the PFC region of Kif15+/+ and Kif15-/- mice. The solid triangular arrows indicate PSD95 particles retained in the dendrites, and the hollow triangular arrows indicate PSD95 particles distributed in the postsynaptic membranes on both sides of the dendrites, n = 3 ~ 4 mice in each group. E: Representative images of immunofluorescence staining showing colocalization of PSD95 and KIF15 or IgG in the postsynaptic region of DIV18 neurons, The solid white arrowheads represent KIF15 fluorescent particles localized at the postsynaptic site. F: The Pearson coefficients for the postsynaptic localization of KIF15 and PSD95 were significantly higher than those of the IgG group, n = 10 neurons per condition. All the data are presented as mean±SD, *P < 0.05, **P < 0.01, ***P < 0.001 two-tailed student’s t test between two groups. https://doi.org/10.1371/journal.pgen.1011839.s006 (TIF) S6 Fig. Agarose gel electrophoresis validation of Kif15-/-zebrafish complement; M, 2000 bp marker. https://doi.org/10.1371/journal.pgen.1011839.s007 (TIF) S7 Fig. The knockdown efficiency of KIF15 in 293T cells was assessed. A: qRT-PCR showed the knockdown efficiency of KIF15 mRNA. B-C: Western Blot showed the knockdown efficiency of KIF15 protein, n = 3 (hree independent experiments), All the data are presented as mean±SD, *P < 0.05, ***P < 0.001 two-tailed student’s t test between two groups. https://doi.org/10.1371/journal.pgen.1011839.s008 (TIF)

Acknowledgments We thank Professor Yongjing Gao’s research group for providing the patch clamp platform and Professor Xiaobo Wu for providing technical support. At the same time, we thank Research Center of Clinical Medicine, Affiliated Hospital of Nantong University for providing a behavioral analysis platform.

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