Fig. 3

MEF2C enhancer mutation impairs MEF2C transcription and leads to mitochondrial dysfunction. (A) Demonstration of CRISPR-editing of rs304152-G mutation in HEK293T cells. Underlined sequence and blue sequence determine sgRNA and PAM, respectively. Scheme created with BioRender.com. (B) MEF2C mRNA level decreased in rs304152-G (MT) cells compared to rs304152-T (WT) cells. Statistics were calculated using Student’s t-test: *, P = 0.047. (C) Immunofluorescence staining of MEF2C (green) and outer mitochondria membrane marker, TOMM20 (red), in human cortical pyramidal neuron along with deconvolved and 3D reconstruction image made by Imaris 9 (Bitplane). Right panel shows line measurement analysis for MEF2C and TOMM20 colocalization signals. White dotted line indicates the colocalization analysis line. (D) Ultrafractionation of cellular compartments of mouse brain tissues. (E) Western blots of cell fractionation confirmed the presence of MEF2C in mitochondria in pure mitochondrial fractions. (F) qPCR results showed decrease of ND4 mRNA levels in MT cells. Statistics were calculated using Student’s t-test: **, P < 0.002. (G) Immunofluorescence staining of MEF2C and ND4 in WT and MT cells. Scale bars (white): 5 μm. Right: densitometry analysis showed decrease of MEF2C and ND4 levels in MT cells. Scatter plot represents positive correlation between MEF2C and ND4 levels. A total of 39 cells/group were counted (13 cells/well) from n = 3 wells/group (WT and MT). Statistics were calculated using LMM (***, P < 0.001). (H) Immunostaining of MitoTracker (red) and MitoSox (green) in WT and MT cells. The nuclei were counterstained with DAPI (blue). Right: quantification of MitoTracker and MitoSox levels. A total of 36 cells/group were counted (12 cells/well) from n = 3 wells/group (WT and MT). Statistics were calculated using LMM (*, P = 0.037; ***, P < 0.001). (I) Decrease of ATP level in MT cells compared to WT cells. The experiment was repeated three times. Statistics were calculated using Student’s t-test (**, P = 0.006). Error bars represent means ± SEM