Fig 2: dCas9-Dnmt3a-mediated APP methylation enhanced neuroprotection in APP-KI mouse primary neurons. a Quantitative real-time PCR analysis of APP expression in WT and APP-KI mouse primary neurons treated with − 189 sgRNA and dCas9-Dnmt3a. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. b, c Western blot analysis of APP in WT and APP-KI mouse primary neurons treated with − 189 sgRNA and dCas9-Dnmt3a. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. d Bisulfite sequencing analysis of the promoter region of APP in APP-KI mouse primary neurons treated with − 189 sgRNA and dCas9-Dnmt3a. e Quantification of methylated amplicons. In 3 independent experiments, 10 to 30 sequencing data were measured in total. Data are expressed as mean ± SEM. **P < 0.01, two-sided Student’s t-test. f Immunostaining of Tuj1 (green), NeuN (red), and DAPI (blue) in APP-KI mouse primary neurons treated with − 189 sgRNA and dCas9-Dnmt3a. g The number of Tuj1-positive neurons in f. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. h Quantification of neurite length. In 3 independent experiments, over 100 neurites were measured. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. i Map2 (green), Aβ42 (red), and DAPI (blue) immunostaining in APP-KI mouse primary neurons treated with − 189 sgRNA and dCas9-Dnmt3a. j Percentage of Aβ42+/Map2+ cells from i. In 3 independent experiments, over 100 neurites were measured. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. k ELISA of the Aβ42/40 protein ratio in APP-KI mouse primary neurons transduced with − 189 sgRNA and dCas9-Dnmt3a. Data are expressed as mean ± SEM (n = 3). **P < 0.01, two-sided Student’s t-test. The images in b, f, i are representative of three or more similar experiments

Fig 3: Vestibular, retinal, and central exclusion. Error bars represent standard deviation, *P < 0.05. All scale bars represent 20 μm. (A) Vestibular thresholds were determined to be the lowest intensity at which a characteristic Peak 1 could be identified in both positive and negative directions. Tracings represent the average of 256 responses. (B) Vestibular thresholds at 90 dpi shows no detectable physiologic vestibular difference between WT and DTR animals (0 dpi = P30, n = 10 mice per group). (C) Utricle cell counts per 130 μm2 section show a statistically significant reduction in DTR animals that were not physiologically significant. Each point represents the average of a striolar and extrastriolar count per animal (n = 10 mice per group). (D) H&E stained retina shows characteristic architecture: extraocular muscles (EOM), choroid coat (CC), outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). (E) GCL cell counts were performed in an automated fashion using spot detection in the DAPI channel along 210 μm sections (40 μm original slice thickness). (F) No loss of RCG cells were observed at 2 and 10 dpi. Each point represents the average of 6 sections per eye (n = 8 eyes from 4 animals per group). (G) Striatal cells were counted in 420 μm2 sections (40 μm original slice thickness) using anti-NeuN with DAPI counter-stain. (H) Striatal nuclei (all cell types) counts were performed in an automated fashion using spot detection in the DAPI channel. (I) Striatal neuron counts were performed in an automated fashion using spot detection in the NeuN channel. (J) No loss of striatal nuclei or neurons were detected were observed at 2 and 10 dpi. Each point represents the average of 8 sections per hemisphere (n = 8 hemisphere from 4 animals per group).

Fig 4: Protein Levels of DMT1 in Neurons and Glia in Four Cerebral Regions. (A–L) After treatment with 2 mg/kg/day iron dextran for 6 days, the images of DMT1 (green) costained with NeuN, GFAP, or Iba1 (red) and DAPI (blue) were visualized in FC (A–C), HP (D–F), SN (G–I), and ST (J–L). Scale bar, 50 µm. (M) The fluorescence intensity of DMT1 normalized to control for neurons, astrocytes, and microglia is presented as mean ± SD, n = 6. (N) Changes in fluorescent intensity of GFAP and Iba1 induced by treatment with 2 mg/kg/day iron dextran for 6 days. Data are presented as mean ± SD, n = 6. *P < 0.05—statistically significant difference from control group.

Fig 5: Effects of Excess Iron on Neuronal Apoptosis and Behavior. (A–D) After treatment with 2 mg/kg/day iron dextran for 6 days, TUNEL assays were applied to FC (A), HP (B), SN (C), and ST (D). Neurons were also stained with NeuN (red) and nuclei were labeled with DAPI (blue). (E) Ratios of TUNEL+/DAPI+ cells presented as mean ± SD, n = 6. Scale bar, 50 µm. *P < 0.05, statistically significant difference from control group. (F) ROS levels in four cerebral regions. Data presented as mean ± SD, n = 6. *P<0.05, statistically significant difference from control group. (G–N) After treatment with 2 mg/kg/day iron dextran for 6 days, the mice behaviors were tested. (G) GAP scores of animals treated with 2 mg/kg/day iron dextran from 0 to 6 days. Data represent mean ± SEM, n = 6. *P < 0.05, statistically significant difference from 0 day. (H) Appearances of mice treated with 2 mg/kg/day iron dextran from Day 0 to Day 6. (I) Weight of mice in control group and in a group treated with 2 mg/kg/day iron dextran for 6 days. (J) Movement time from the pole top to the floor (T-LA time). (K) The dwelling time on rotating bar. (L) Time spent in target quadrant in Morris water maze test. (M and N) The total traveled distance (M) and time spent in the central area (N). Data are presented as mean ± SD, n = 6. *P < 0.05, statistically significant difference from control group.