1Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
2Department of Anatomy and Neuroscience, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
3Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia.
Abstract
Epilepsy is a common neurological disease worldwide, and one of its causes is genetic abnormalities. Here, we identified a point mutation in KIF4A, a member of kinesin superfamily molecular motors, in patients with neurological disorders such as epilepsy, developmental delay, and intellectual disability. KIF4 is involved in the poly (ADP-ribose) polymerase (PARP) signaling pathway, and the mutation (R728Q) strengthened its affinity with PARP1 through elongation of the KIF4 coiled–coil domain. Behavioral tests showed that KIF4-mutant mice exhibited mild developmental delay with lower seizure threshold. Further experiments revealed that the KIF4 mutation caused aberrant morphology in dendrites and spines of hippocampal pyramidal neurons through PARP1-TrkB-KCC2 pathway. Furthermore, supplementing NAD, which activates PARP1, could modulate the TrkB-KCC2 pathway and rescue the seizure susceptibility phenotype of the mutant mice. Therefore, these findings indicate that KIF4 is engaged in a fundamental mechanism regulating seizure susceptibility and could be a potential target for epilepsy treatment.
The genomic background of patients and the Kif4-mutant mice exhibit a high embryonic mortality rate and developmental delay. (A) The pedigree chart of a Kif4 mutation-carrying family. Note that III-1, IV-1, and IV-2 (Kif4aWT/Mut) exhibited no symptoms, whereas IV-3 (Kif4aMut/Y) showed global developmental delay, severe intellectual disability, and intractable seizure. (B) Illustration of the mouse KIF4 protein with its point mutation identified from the patients. LCR, low complexity region; CC, coiled coil; CXC, tesmin/TSO1-like CXC domain. (C) Coiled–coil prediction of the mouse KIF4 peptide from amino acid 524–732. Note that the Mut-KIF4 has a higher coil probability, as indicated with the asterisks. (D) Mouse mating strategy and the mortality rate. The embryonic mortality rate of Kif4 mutation-carrying mice (Mut, 26%) is significantly higher than that of wild-type mice (WT, 7%) Data are presented as mean ± SEM (n = 12 mating mouse pairs). ***P < 0.001 (Student’s t tests). (E) The composition of the offspring genotypes from the Kif4-mutant background exhibited a lower Kif4Mut/Y pup population (n = 6 mating mouse pairs). (F) The photograph of Kif4WT/Y and Kif4Mut/Y mice at E16.5 and the statistical analysis results of embryo size. Data are presented as mean ± SEM (n = 8 mouse pairs). ****P < 0.0001 (Student’s t tests). (G) The postnatal weight comparison between Kif4WT/Y and Kif4Mut/Y mice from P3 to P14. Data are presented as mean ± SEM (n = 12 mouse pairs). *P < 0.05, **P < 0.01 (two-way ANOVA). (H) The assessment of Kif4WT/Y and Kif4Mut/Y mouse developmental milestones. Note that Surface Righting, Auditory, Open field, and Air Righting exhibited statistical significance. Data are presented as mean ± SEM (n = 12 mouse pairs). *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA). (I) HE staining of sagittal mouse brain sections from the indicated genotypes at E16.5 and the corresponding statistical analysis of the hippocampal cell density. The red stars in the left panels indicate the hippocampus regions that are magnified in the right panels. Scale bars, 1000 μm (left panel) and 100 μm (right panel). Data are presented as mean ± SEM (n = 12 hippocampi from 6 mouse pairs). ****P < 0.0001 (Student’s t tests).The in vivo PARP1 activity regulates epileptic behavior (A-D) and graphical model summarizing the molecular mechanism of the KIF4 mutation-induced epilepsy (E). (A–D) The results of the PTZ kindling test. The chart representing the seizure stage classification (A) of the individual scores from mice with indicated genotypes and treatments, before and after the PTZ injection. Statistical analysis results of the cumulative number of maximum (B) and all (C) seizure stage, and the ratio of the occurrence of stage 4 (D). Data are presented as mean ± SEM (n = 10 mouse pairs). ****P < 0.0001 (two-way ANOVA). (E) Schematic model of the involvement of KIF4 in epileptogenesis. (a) The WT-KIF4 moderately binds to PARP1, and the cell exhibits a baseline level of PAR in the nucleus. (a′) Mut-KIF4 excessively binds to PARP1, and the cell exhibits a dramatically inhibited PARP1 activity. (b and b′) The suppressed PARP1 activity can alter the TrkB-KCC2 expression in the mutant group. Meanwhile, the aberrant protein expression triggers a higher intracellular chloride concentration. (c–d′) Subsequently, the hyper-branched dendrites and immature dendritic spines are dominantly observed in the mutant neuron.
