Fig 1: De novo LOF mutations reported in patients with bipolar disorder. (A) Plot of RVIS percentile and pLI of the genes in which de novo LOF mutations were previously found (13). Arrows show the genes coding calcium binding proteins (MACF1, EHD1 and UNC13B). (B) Structures of MACF1 isoforms. An arrowhead indicates the location of the de novo mutation on a minor exon. Data are retrieved from the UCSC database. (C) RT-PCR of the minor isoform of MACF1. The minor exon of MACF1 in which the de novo mutation was found was amplified using cDNA derived from human cerebral cortex and thalamus. RT, Reverse-transcription. (D) Schematic diagram of the domain structure of EHD1 and the location of the de novo mutation of EHD1. The mutation, 1-bp deletion, was found in the last exon, and a truncated protein lacking most of the EH-domain is predicted to be expressed. aa, amino acids.
Fig 2: Functional analyses of the de novo LOF mutation in EHD1 in PC12 cells. (A) Diagram of the EHD1 proteins fused with mCherry. (B) Western blot for EHD1 in PC12 cells transfected with the EHD1 expression vectors. (C) Representative fluorescence microscope images of PC12 cells stimulated with ß-NGF. mCherry-EHD1-(red) and Venus-(green) expression vectors were co-transfected into PC12 cells. The images of Venus were utilized for the analysis of the neurite length. Scale bars, 200 µm. (D) Neurite length. mCherry-EHD1-WT-expressing PC12 cells showed significantly longer neurites. n > 81 cells. Data shown are the mean ± standard error of the mean (SEM). Tukey’s multiple test was used for multiple comparisons. *P < 0.05. Effect size, ?2 = 0.027. (E) Schematic diagram of quantification of endocytosis. The uptake of Alexa Fluor 488-conjugated transferrin by starved PC12 cells was measured using FACS. (F) The mean of Alexa Fluor 488 fluorescent intensity measured by FACS. The fluorescent intensity was the average of Alexa Fluor 488 of mCherry positive cells. Transferrin uptake was significantly lower in mCherry-EHD1-Mut-expressing PC12 cells compared with mCherry-expressing cells. The trial was triplicated (n = 3). Data shown are the mean ± SEM. Tukey’s multiple test was used for multiple comparisons. *P < 0.05. Effect size, ?2 = 0.64.
Fig 3: Higher activity in light-phase in a wheel running test of female mice. (A) Ehd1 mutant female mice showed significantly higher rate of activity in light-phase per total activity. The test was performed for 18 weeks. Data shown are the mean ± standard error of the mean (SEM). The Kruskal–Wallis test was used for comparisons. n = 15 for each group. The post-hoc test was performed by Dunn’s multiple comparison test. WT versus Ehd1 mutant; P = 0.0025. WT versus Macf1 mutant; P = 0.40. Effect size, WT versus Ehd1 mutant; r = 0.59, WT versus Macf1 mutant; r = 0.23. (B) Representative actogram showing daily activity of each female mouse. The Ehd1 mutant mouse exhibited high activities in light-phase. Each row means the data of one day. The X-axis indicates local time. Y-axis of each row shows the wheel running activity per 10 min.
Fig 4: HDR-mediated genome editing to establish Ehd1 and Macf1 mutant mouse lines. (A) Sequences of Ehd1 mutant mice. 1-bp deletion of G modeling the de novo mutation of the patient was introduced in the exon 5 of Ehd1. Sequence of ssODN utilized that of the strand in which PAM sequence exists. PAM, proto-spacer adjacent motif. (B) Western blot for EHD1 in the hippocampus of Ehd1 mutant mice. The truncated protein was caused by the mutant allele of the heterozygous Ehd1 mutant mice. (C) sequences of Macf1 mutant mice. 1 bp insertion of C was introduced in the exon 1 of the minor isoform of Macf1 to model the de novo mutation. (D) Splicing isoforms of Macf1 in mice. The knock-in mutation on a minor exon of Macf1 is indicated by an arrowhead.
Supplier Page from Abcam for Anti-EHD1 antibody [EPR4955]