Fig 2: Neuronal apoptosis and spatial learning ability in AppTg/Cebpd −/− mice. a Spatial learning ability of wild-type (WT; C57BL/6), AppTg, and AppTg/Cebpd −/− mice. Time required by WT (n = 5), AppTg (n = 5), and AppTg/Cebpd −/− (n = 3) mice to reach the hidden platform (latency to hidden) was analyzed in the Morris water maze. b The apoptosis of neurons was attenuated in AppTg/Cebpd −/− mice. The brain tissues were subjected to immunofluorescence staining with the antibody recognized NeuN or cleaved caspase 3 (cCap3; active caspase 3). Scale bar = 100 μm. c Primary Cebpd −/− neurons have no effect to reverse IL-1β-induced cell death. The MTT assay was conducted as indicated. (d) Attenuated Cebpd expression in primary astrocytes does not contribute to neuronal death. The conditioned medium came from either IL-1β-pretreated primary WT or Cebpd −/− astrocytes. Primary cortical neurons in WT and Cebpd −/− mice were seeded and grown on coverslips with the mixture medium (9:1) of neuronal maintenance medium and above conditioned medium for 72 h. The experimental neurons were performed with immunofluorescence staining by anti-MAP2 antibodies for morphological examination and anti-cleaved caspase 3 (cCap3) antibodies for cell death detection. Scale bar = 100 μm. Data represent two independent experiments with three mice/group in B or three independent experiments in c and d. Data are expressed as mean ± SEM by a Student’s t test. (*P < 0.05; **P < 0.01; ***P < 0.001; NS not significant)

Fig 3: A Schematic representation of the targeting vector and the targeted allele of the FBXO7 gene. The 3.2-kb region of the mouse FBXO7 gene, including exons 3–4, was followed by an FRT-flanked PGK-neo expression cassette in the opposite transcriptional orientation. In the targeting construct, a 5.0-kb 5′ fragment and a 3.0-kb 3′ fragment were used as the long and short homologous arms, respectively. B Southern blot analysis of genomic DNA from ES cells that had undergone homologous recombination. Genomic DNA was digested with EcoRV and hybridized with the 5′ probe or 3′ probe. Upper panel: Bands detected by the 5′ probe. Lower panel: Bands detected by the 3′ probe. C Immunoblot of anti-FBXO7 antibody (Millipore #ABN1038) and actin (Millipore #MAB1501). Lanes 1–3: whole-brain tissues of 3-week-old FBXO7flox/flox mice; lanes 4–6: whole-brain tissues of 3-week-old FBXO7flox/flox: Nestin-Cre mice. The genotypes of Nestin-Cre mice were determined by PCR using two primers: 5′-TTT GCC TGC ATT ACC GGT CGA TGC AAC-3′ and 5′-TGC CCC TGT TTC ACT ATC CAG GTT ACG GA-3′; these permitted the detection of the 1000-bp Nestin-Cre-targeted allele (lower panel). D 3-week-old FBXO7flox/flox mice (left) and 3-week-old FBXO7flox/flox: Nestin-Cre mice (right). Scale bars: 1 cm. E Body weight of mice 2 or 3 weeks of age (2-week-old FBXO7flox/flox mice and FBXO7flox/flox: Nestin-Cre mice, n = 10; 3-week-old FBXO7flox/flox mice and FBXO7flox/flox: Nestin-Cre mice, n = 10). Data are presented as means ± SE (error bars); **p < 0.01 (significance was evaluated using Student’s t-test). F Kaplan–Meier analysis of survival of FBXO7flox/flox mice (n = 20) and FBXO7flox/flox: Nestin-Cre mice (n = 20). G Footprint test in FBXO7flox/flox mice and FBXO7flox/flox: Nestin-Cre mice. Red footsteps indicate forepaws; black footsteps indicate hindpaws. Each stride length was recorded. Data are means ± SE (error bars); **p < 0.01 (significance was evaluated using Student’s t-test). H Runway test of 3-week-old FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). The runway test was performed using a narrow, horizontally fixed beam. FBXO7flox/flox: Nestin-Cre mice could hardly move on the beam, and their hindpaws frequently slipped. I The number of hindlimb slips of mice crossing the 2-cm pole was recorded. Data are presented as means ± SE (3-week-old FBXO7flox/flox mice and FBXO7flox/flox: Nestin-Cre mice, n = 10); data are means ± SE (error bars); **p < 0.01 (significance was evaluated using Student’s t-test). J Immunofluorescence labeling of Tom20 (red; Abcam #ab78547 Anti-Tom20) or TH (green; Merck Millipore #MAB318 Anti-Tyrosine Hydroxylase Antibody) in the SN area of FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Scale bars: 2 μm. K For conventional electron microscopy, mice were fixed by cardiac perfusion with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.2). Brain slices were embedded in epoxy resin, and ultrathin sections (70-nm thickness) were prepared and imaged on an HT7700 electron microscope (Hitachi, Japan). Electron micrographs of the cerebral cortex (a, b, c, d) and dopaminergic neurons in the SN (e, f, g, h); 3-week-old FBXO7flox/flox mice (n = 3) (a, b, e, f) and 3-week-old FBXO7flox/flox: Nestin-Cre mice (n = 3) (c, d, g, h). The right of each image (a, c, e, g) shows enlarged images. Scale bars: a, c, e, g, 2 µm; b, d, f, h: 500 nm. L Quantitation of mitochondrial area (dopaminergic and cerebral cortical cells from 3-week-old mice, n = 20). The mean mitochondrial area in dopaminergic neurons was smaller in 3-week-old FBXO7flox/flox: Nestin-Cre mice than in 3-week-old FBXO7flox/flox mice. Significance was evaluated using Student’s t-test. *p < 0.01. M Histological analyses of the SN area in 3-week-old FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Paraffin sections were immunostained for TH (Merck Millipore #MAB318 Anti-Tyrosine Hydroxylase Antibody) and are indicated by a square. Scale bars: 200 µm. N For stereological quantification, the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) were selected. Every other 40-μm section of serial coronal brain slices for each genotype was stained for DAB. Quantification was performed with a design-based stereology system (Stereo-Investigator version 2020; MBF Bioscience, Williston, VT, USA). The sampling parameters were defined according to the software guide to achieve a coefficient of error ranging from 0.05 to 0.09 as determined using the Gundersen test. Data are means ± SE (3-week-old FBXO7flox/flox mice, n = 3; 3-week-old FBXO7flox/flox: Nestin-Cre mice, n = 3); **p < 0.01 (Student’s t-test). N.S.: Not significant. O Histological analyses of p62 (PROGEN #GP62-C Anti-p62/SQSTM1) in 3-week-old FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). The area in the small rectangle is enlarged in the inset image at the lower left. Scale bars: 10 µm. P Immunoblot for p62 (PROGEN #GP62-C Anti-p62/SQSTM1) and GAPDH (Proteintech #10494-1-AP). Lanes 1–3: 3-week-old FBXO7flox/flox mice; lanes 4–6: whole-brain tissues of 3-week-old FBXO7flox/flox: Nestin-Cre mice. Data are means ± SE (3-week-old FBXO7flox/flox mice, n = 3; 3-week-old FBXO7flox/flox: Nestin-Cre mice, n = 3); *p < 0.05 (Student’s t-test). Q Immunofluorescence labeling of p62 (red; PROGEN #GP62-C Anti-p62/SQSTM1) or Synuclein (green; Merck Millipore #AB5038P Anti-Synuclein Alpha Antibody) in the SN area of FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Scale bars: 2 μm. R Immunofluorescence labeling of p62 (red; PROGEN #GP62-C Anti-p62/SQSTM1), MAP2 (gray; Genetex #GTX11267 Anti-MAP2), or TH (green; Merck Millipore #MAB318 Anti-Tyrosine Hydroxylase Antibody) in the SN area of FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Arrows indicate interneurons. Scale bars: 5 μm. S Immunofluorescence labeling of p62 (red; PROGEN #GP62-C Anti-p62/SQSTM1) or CD31 (green; R&D #AF3628 Anti-CD31) in the SN area of FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Scale bars: 10 μm. T Immunofluorescence labeling of p62 (red; PROGEN #GP62-C Anti-p62/SQSTM1) or iba1 (green; Wako #NCNP24 iba1 Monoclonal Antibody) in the SN area of FBXO7flox/flox mice (upper panel) and FBXO7flox/flox: Nestin-Cre mice (lower panel). Scale bars: 10 μm

Fig 4: Iba-1 and MAP2 expression in the inflamed brain of LPS-induced mice. (A) Representative immunofluorescent staining in the hippocampus for Iba-1 (red) and MAP2 (green). Scale bar = 400 μm. (B) Quantification of Iba-1 and MAP2 cell numbers in the hippocampus for each group. (C) Representative immunofluorescent staining in the cortex for Iba-1 (red) and MAP2 (green). Scale bar = 200 µm. (D) Quantification of Iba-1 and MAP2 cell numbers in the cortex for each group. (E) Representative images of LFB staining in the corpus callosum for four groups. (F) Bar graph of the mean thickness of the corpus callosum. Key: *, #, and † denote significant differences from the Sham, LPS, and LPS+LIPUS 0.5 groups, respectively; *, p < 0.05; ***, ###, †††, p < 0.001 (n = 5).