Fig 2: Characterization of the growth and behavior of AIF and AIF3 splicing mice. a-b Survival curves of male (a, AIFfl/Y/CamKIIa-iCre+, n = 82) and female (b, AIFfl/fl/CamKIIa-iCre+, n = 25) AIF3 splicing mice and their littermate AIF controls (AIFfl/Y/CamKIIa-iCre- and AIFfl/fl/CamKIIa-iCre-). Data were shown as mean and analyzed for statistical significance by two-way ANOVA. *** p < 0.001. c-d Measurement of the body weight of male (c, n = 20) and female (d, n = 20) AIF3 splicing mice and their littermate AIF controls. Data are shown as mean ± S.E.M. and analyzed for statistical significance by two-way ANOVA. *** p < 0.001. e-h Open field tests of AIF3 (n = 13) and their littermate controls (n = 10) at P60–75. Central zone travel distance (e), percentage of time in the Central zone (f), total travel distance (g) and walk speed (h) were analyzed. Data are shown as mean ± S.E.M. and analyzed for statistical significance by Student t test. ** p < 0.01; *** p < 0.001, n = 10–13

Fig 3: Effects of AIF and AIF3 expression on mitochondrial functions in vitro and in vivo. a OCR in AIFfl/Y/AIFfl/fl cortical neurons 3 days after GFP or Cre-GFP lentivirus infection was measured after exposure to 1 µM oligomycin, 3 µM FCCP, and 1 µM rotenone. Data are shown as mean ± S.E.M. n = 3. ***p < 0.001 by two-way ANOVA. b OCR in Hq cortical neurons 3 days after AIF or AIF3 lentiviral infection was measured by Seahorse Bioscience XF24 Extracellular Flux Analyzer after exposure to 1 µM oligomycin, 3 µM FCCP, and 1 µM rotenone. Data are shown as mean ± S.E.M. n = 3. ***p < 0.001 by two-way ANOVA. c Intracellular ATP levels were measured in Hq cortical neurons 3 days after AIF or AIF3 lentiviral infection using a luminescence ATP assay kit. Data are shown as mean ± S.E.M. n = 3. *p < 0.05, ***p < 0.001. d Mitochondrial structure of neurons in the cortex, hippocampus and cerebellum of AIF3 splicing mice (AIFfl/Y/CamKIIa-iCre+) and their littermate control AIF mice (AIFfl/Y/CamKIIa-iCre-) by electron microscopy. e Quantification of abnormal mitochondria (mito) in the cortex, hippocampus (Hip) and cerebellum of AIF3 splicing mice and their littermate control AIF mice. Data are shown as mean ± S.E.M. n = 5. ****p < 0.0001 by one-way ANOVA. f AIF3 splicing reduced mitochondrial biogenesis in brain, but not heart or liver of AIF3 splicing mice. The ratio of mitochondrial DNA relative to nuclear genomic DNA was determined by quantitative real-time PCR using primers for cytochrome b (mitochondrial) and ribosomal protein S2 (RPS2, nuclear). The data observed in AIF3 splicing mice were normalized to the littermate control AIF mice. Data are shown as mean ± S.E.M. n = 3. ***p < 0.001, **p < 0.01, *p < 0.05 by one-way ANOVA

Fig 4: Characterization of neuron loss in the cortex of AIF3 splicing mice at P60. a Representative images of TBR1+ neurons in AIF3 splicing mice (AIFfl/Y/CamKIIa-iCre+) and littermate control AIF mice (AIFfl/Y/CamKIIa-iCre-) at P60. Scale bar, 200 µm. b Representative images of CUX1+ neurons in AIF3 splicing mice (AIFfl/Y/CamKIIa-iCre+) and littermate control AIF mice (AIFfl/Y/CamKIIa-iCre-) at P60. Scale bar, 200 µm. c Representative images of NeuN+ neurons in AIF3 splicing mice (AIFfl/Y/CamKIIa-iCre+) and littermate control AIF mice (AIFfl/Y/CamKIIa-iCre-) at P60. Scale bar, 200 µm. Representative images are from at least three independent experiments

Fig 5: Characterization of AIF3 neurotoxicity effects in cortical neurons and its biochemical properties in vitro. a Expression of AIF and AIF3 in Hq cortical neurons 3 days after lentiviral transduction. b AIF3 expression in Hq cortical neurons caused neuronal death 4 days after lentiviral transduction as indicated by propidium iodide staining. Scale bar, 20 µm. c Quantification of AIF3-induced cytotoxicity in Hq cortical neurons 4 days after lentiviral transduction. Data were shown as mean ± S.E.M. n = 4. ***p < 0.001 by one-way ANOVA. d Expression of AIF3 in AIF fl/fl cortical neurons 3 days after GFP or Cre-GFP lentiviral infection (1.0 × 109 T.U./ml). e AIF3 expression in AIFfl/Y/AIFfl/fl cortical neurons caused neuronal death 5 days after GFP or CRE-GFP lentiviral infection as indicated by propidium iodide staining. Scale bar, 20 µm. f Quantification of AIF3-induced cytotoxicity in cortical neurons 5 days after GFP or CRE-GFP lentiviral infection. Data are shown as mean ± S.E.M. n = 4. ***p < 0.001 by Student t test. g AIF (62 kDa) and AIF3 protein purification. BSA was used as purity and quantity control. h The NADH oxidase activity of purified AIF (3 µM) and AIF3 (3 µM) was determined by monitoring changes in absorbance at OD340 nm. Data were shown as mean ± S.E.M. n = 3. ***p < 0.001 by two-way ANOVA. i FAD binding of purified AIF (1 µM) and AIF3 (1 µM) was determined by spectrophotometric wavelength scanning. j DNA retardation assay of purified AIF (3 µM) and AIF3 (3 µM) by incubating with 1 kb plus DNA ladder (200 ng) for 30 min. BSA was used as a negative control