Rare variants in KIF1A detected in a family and KIF1A are conserved across species. (A) Rare mutation in KIF1A detected in a family. (B) Electroencephalogram (EEG) of the proband (III2) showing generalized sharp waves. (C) The analysis of sequences of affected girl (III2), affected mother (II3), and unaffected father (II4). (D) The amino acids coded by variation are conserved across species as showing in the boxed. The last column shows identity with the human protein. The gray boxes highlight the amino acids mentioned in the manuscript.

Schematic representation of KIF1A protein and locations of mutations in human KIF1A associated with various neuronal disorders. These mutations are distributed in various locations and domains of the KIF1A subunit protein peptide (Niwa et al., 2016; Iqbal et al., 2017; Riviere et al., 2011; Chiba et al., 2019).

Effect of mutant KIF1A on the intrinsic excitability of primary cultured neurons. (A) Recording paradigm of passive excitability in the excitatory cultured neurons. (B) Examples of the AP responses to superimposed current steps recorded from primary cultured GFP-positive hippocampal neurons transfected with the mutant or wild-type KIF1A plasmid. (C) Resting membrane potential of the examined neurons from two groups (n = 8 neurons in each group, Student's t test). (D) Injected currents used to induce the first spikes (n = 8 neurons in each group, Student's t test). (E) Number of APs induced by the injected currents in the primary cultured GFP-positive hippocampal neurons transfected with the mutant or wild-type KIF1A plasmid (n = 8 neurons in each group, two-way ANOVA).

Effect of mutant KIF1A on the miniature excitatory post-synaptic currents (mEPSCs) and miniature inhibitory post-synaptic currents (mIPSCs) of primary cultured neurons. (A) Representative trace of mEPSCs from cultured hippocampal neurons transfected with the mutant or wild-type KIF1A plasmid at a holding potential of −70 mV. (B, C) Bar graph analysis of the mEPSC frequency and amplitude (n = 8 neurons in each group, ***p < 0.001, Student's t test). (D) Representative trace of mIPSCs from cultured hippocampal neurons transfected with the mutant or wild-type KIF1A plasmids at a holding potential of −70 mV. (E, F) Bar graph analysis of the mIPSC frequency and amplitude [n = 10 in the mutant group and 8 in the wild type (WT) group, Student's t test].

Effect of mutant KIF1A on neuronal development. (A) Representative image of cultured primary hippocampal neurons obtained through a Sholl analysis. The radius interval between circles was 10 μm per step and ranged from 10 to 210 μm from the center of the neuronal soma. (B) Sholl analysis of neurons expressing the mutant (n = 15 neurons) or wild-type protein (n = 13 neurons) (two-way ANOVA). (C, D) Number of primary and secondary neurites of neurons expressing the mutant (n = 15 neurons) or wild-type protein (n = 13 neurons) (Student's t test).

Effect of mutant KIF1A on excitatory synapses. (A) Representative image of dendritic spines at DIV16 from neurons transfected with the mutant or wild-type KIF1A plasmid. (B) Total spines/10 μm of neurons expressing the mutant (n = 26 neurons) or wild-type protein (n = 23 neurons). (***p < 0.001, Student's t test). (C) Representative image of vGLUT at DIV16 from neurons transfected with the mutant or wild-type KIF1A plasmid. (D) Total vGLUT/10 μm of neurons expressing the mutant (n = 24 neurons) or wild-type protein (n = 21 neurons). (**p < 0.01, Student's t test). (E) Representative image of excitatory synapses (vGLUT-positive PSD-95 clusters) at DIV16 from neurons transfected with the mutant or wild-type KIF1A plasmid. (F) Total excitatory synapses (vGLUT-positive PSD-95 clusters)/10 μm of neurons expressing the mutant (n = 19 neurons) or wild-type protein (n = 18 neurons). (*p < 0.05, Student's t test).

Overexpression of mutant Kif1aa causes abnormal behavior and epileptiform discharges in transgenic zebrafish larvae. (A) Graphic representation of the experimental protocol used for the zebrafish studies. (B) Representative images of zebrafish larvae microinjected with A433D-kif1aa (n = 64) or wild type (WT)-kif1aa (n = 61) selected for local field recordings. The distribution of the labeled protein is shown by green fluorescence. (C) Seizure behaviors of zebrafish larvae. The sample locomotion tracking plots are shown on the top (S0, baseline activity; S1, small increase in swim activity; and S2, large increase in movement. The bar plot shows the percentage of tested larvae recorded during locomotion tracking (*p < 0.05, χ² test). (D) Representative trace of spontaneous epileptiform activity recorded from zebrafish larvae. The top trace represents a typical epileptiform pattern observed in gap-free recordings. The bottom trace shows a high-resolution magnification of the selected epileptiform events, and the frequency spectrum corresponding to the selected local field potentials (LFPs) is shown on the right. Color scale: blue (low magnitude) to red (high magnitude). (E) Percentage of larvae with abnormal epileptiform activity after overexpression of A433D (n = 64) or WT Kif1aa (n = 61). (**p < 0.01, χ² test).