Fig 1: Ebp1(+/-) mice display SZ-like behaviors. (A–C) Representative heat maps illustrating the time spent in different locations of the three chambers from the social preference test. (upper). (A) In the first 10 min, the test mouse was placed in the center and allowed to explore each chamber as habituation liberally. * p < 0.05. (B) In the second 10 min, an age- and gender-matched WT B6 mouse (mouse 1) that had never been exposed to the test mouse, was placed in the wired cage of left chamber. The other side remained empty. The test mouse was placed in the center of the cage and allowed to explore the chamber for 10 min liberally. * p < 0.05. (C) In the last experiment, a new stranger mouse (mouse 2) was introduced in the wired cage of right chamber. The test mouse was allowed to explore the chamber for 10 min liberally. * p < 0.05. E, empty; M1, mouse 1; M2, mouse 2. Ebp1(+/+), n = 12; Ebp1(+/+), n = 12. (D) Representative scheme and heat maps illustrating the time spent in different locations of novel object recognition test (upper). The test mouse was placed in the middle of objects and allowed to explore the cage and measured explore time with two objects for 10 min (bottom, left). Next day, one of the objects was replaced with a novel object and measured explore time with object1 and novel object for 10 min. ** p < 0.01. O1, object 1; O2, object 2; N, novel object. Ebp1(+/+), n = 12; Ebp1(+/+), n = 12. (E) Self-grooming assessed as repetitive grooming was scored during 10 min manually. * p < 0.05. Ebp1(+/+), n = 12; Ebp1(+/+), n = 12. (F,G) Open field test; the area was separated into two zones and included the center (28.5 × 28.5 cm) and the periphery (around of center zone) areas. * p < 0.05. (H) The number of entries in the center zone was counted. Open field test was performed for 20 min. Ebp1(+/+), n = 12; Ebp1(+/+), n = 12. (I) Marble-burying test; WT (n = 12) and mutant mice (n = 12) were placed in the cage for 30 min and allowed to roam liberally. The over 2/3 buried marbles were counted. * p < 0.05. (J–L) Y-maze experimental data; (J) Visiting of arms in the order 1–2-3 is an example of an alternation (left, AAR), whereas the order of 1–2-1 was considered as the non-alternation (right, SAR). (K) Total entry number and (L) latency to exit were counted. AAR, alternation arm returns; SAR, same arm returns. Ebp1(+/+), n = 12; Ebp1(+/+), n = 12. (M) Passive avoidance test; WT (n = 12) and Ebp1 mutant mice (n = 12) were tested and the duration of the freezing time was checked (maximum trial duration = 600 s). Light was used as the conditioned stimulus. ** p < 0.01. All data are shown as the mean ± s.e.m.
Fig 2: The loss of function/missense mutation of Ebp1 gene decreases GAD67 expression and disturbs epigenetic control. (A) Schematic presentation of two of loss of function mutations, mutation to stop codon, from SZ patients is shown. (B) RNA and protein expression levels of Gad67, Hdac1 and Dnmt1 were measured in transfected Ebp1(-/-) MEF cells. GFP-mock, Ebp1 WT, or Ebp1 G183Ter constructs were used for transfections. The RNA expression was tested using qRT-PCR with specific primer sets. The protein level was measured using IHC with anti-GAD67, HDAC1. and DNMT1 antibodies. (C) Ebp1(-/-) MEF cells were co-transfected with pGL-Gad67 promoter along with control or Ebp1 WT, G183Ter mutation construct plasmids. Luciferase activity of GAD67 was measured using the luciferase assay. ** p < 0.001. (D) Luciferase activity of Hdac1 (left) and Dnmt1 (right) were measured in the transfected Ebp1(-/-) MEF cells which over-expressed Ebp1 WT or G183Ter mutation proteins. ** p < 0.001. (E) Schematic illustration shows molecular mechanisms of EBP1 in GAD67 epigenetic effects. Created with BioRender. ** p < 0.001. All data are shown as the mean ± s.e.m.
Fig 3: Ebp1(+/-) mice displayed impaired neural development in stratum oriens (SO) of CA3/2 region in hippocampus. (A) representative hippocampus structure is displayed. Created with BioRender. (B) WT and Ebp1 heterozygous mouse brain were isolated and subjected to IHC staining with anti-EBP1 and TUJ1 antibodies, early neuronal markers. CA3 region and cropped specific region as SO are shown. Scale bar: 200 µm. (C) Brain tissues were isolated from Ebp1 WT and heterozygous mice and subjected to IHC analysis. The hippocampus was stained with anti-EBP1 (green) and TAU1 (red, axon specific marker) antibodies. The right-hand panel shows higher magnification of the CA3 region of the hippocampus; corresponding SO region is indicated by a white box. Intensity of Tau1 positive signals were quantified and displayed as bar graphs (right). Scale bar: 200 µm. (D) Fresh mouse brain tissues were stained with Golgi-Cox stain. Golgi-stained CA1 and CA3 hippocampal neurons (upper right) and their dendritic processes (bottom left) are shown, respectively. Total dendritic length was measured using image J. Dendrite numbers in the SO region of pyramidal neurons were counted within every 10 µm. * p < 0.05, ** p < 0.01. Created with BioRender. (E) Brain tissues, isolated from Ebp1(+/+) and Ebp1(+/-) mice, were stained with anti-PSD95 (red, marker for post-synapse) and MAP2 (green, marker for dendrites) antibodies. High power images show PSD95 and MAP2 double-labeling in the CA3 and corresponding SO regions (left). The intensity of PSD95 was quantified and shown as a bar graphs (right). *** p < 0.001. Scale bar: 200 µm. All data are shown as the mean ± s.e.m.
Fig 4: Increased DNMT1 levels in Ebp1(+/-) mice alters GAD67 promoter methylation. (A) Quantitative qRT-PCR analysis of gene expressions (Ebp1 and Gad67) was conducted in the samples from hippocampus of Ebp1 WT and heterozygous mice. The relative fold changes were quantified and shown in the bar graphs. The expression levels were normalized using Gapdh. ** p < 0.01, *** p < 0.001. (B) Endogenous protein expression levels of GAD67 in hippocampus were measured using IHC with anti-EBP1 (red) and GAD67 (green) antibodies or anti-NEUN (red, neuronal cell marker) and GAD67 (green) antibodies EBP1 and GAD67 intensities were quantified and shown as bar graphs (right). Scale bar: 500 µm. (C) Scatterplots indicates the correlation analysis between Ebp1 and Hdac1 expression levels from temporal cortex (left) and frontal cortex (right). ** p < 0.01. (D) Genomic DNA (gDNA) was extracted from brain hippocampus of Ebp1(+/+) and Ebp1(+/-) using gDNA extraction kit (Sigma-Aldrich, Saint Louis, MO, USA). The gDNA was subjected to CG island-cut using enzymes Hpl1 (cut only non-methylation CG island) or Msp1 (cut CG island as positive control). The gDNA was assessed for methylation levels using the methylation-specific PCR with GAD67 promoter primer sets. ** p < 0.01. (E) MEF cells were subjected to ChIP assay with anti-mouse IgG as negative control, RNA polymerase II as positive control, and with EBP1 or DNMT1 antibodies. The ChIP assay was performed suing ChIP assay kit (cat. 17-259, Millipore, Temecula, CA, USA). The two purified DNAs were observed using qRT-PCR with specific primer set to amplify Gad67 promoter region. *** p < 0.001. All data are shown as the mean ± s.e.m.
Fig 5: EBP1 repress histone deacetylase 1 (HDAC1) transcription and enhances Gad67 expression. (A) RNA expression levels of GAD67 and HDAC1 were measured using RT-PCR with samples of mouse hippocampus from Ebp1(+/+) or Ebp1(+/−) mice (left); RT-PCR quantification data is shown as bar graphs (right). *** p < 0.0001. (B) Chromatin immunoprecipitation analyses coupled with sequencing (ChIP-seq) analysis was performed with MEF cells and using anti-EBP1 antibody. Overview peak of the EBP1 binding site in the promoter region of HDAC1 gene (upper) is shown. ChIP assay was performed to confirmed EBP1 binding site on HdDAC1 promoter region was analyzed using RT-PCR (bottom). *** p < 0.0001. (C) MEF cells were subjected to ChIP assay with anti-IgG or RNA polymerase II antibodies to measure the binding of RNA pol II to GAD67 promoter region (upper). Quantification of the binding of RNA pol II to GAD67 promoter region is shown (bottom). *** p < 0.0001. (D) Luciferase activities of Dnmt1 (left), HDAC1 (middle), and GAD67 (right) were measured using the luciferase assay (Promega). Promoter regions of those genes were cloned using the pGL-4.12 vector and transfected into EBP1 MEF cells shown as Ebp1(+/+), Ebp1(+/−) or Ebp1(-/-). *** p < 0.0001. (E) Identification of EBP1 binding motif and mutation in the predicted EBP1 binding site in the Hdac1 promoter are shown. Cells were co-transfected using pGL-Hdac1 promoter constructs (WT or mutant) along with the control or Ebp1 plasmids. *** p < 0.0001. All data are shown as the mean ± s.e.m.
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