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), Aß42 (red), and DAPI (blue) immunostaining in APP-KI mouse primary neurons treated with - 189 sgRNA and dCas9-Dnmt3a. j Percentage of Aß42+/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 Aß42/40 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: Gene expression changes in cultured cortical neurons after bisphenol-A (BPA) exposure. (A,B) Cell viability: WST-8 assay. Histograms represent the percentage, with respect to control cells, of viable cells after exposure to 0.01, 0.1, 1, 10, 100 or 1000 µM BPA for 24 h or 48 h. Statistical significance was determined by two-way ANOVA with Bonferroni correction. Data are shown as relative changes versus controls. NS, not significant; *P<0.05, **P<0.01. (C) BPA suppressed neurite outgrowth in cultured cortical neurons. Cultured cortical neurons were treated with 0.1% DMSO or BPA (100 µM) for 24 h. Neurites were assessed by immunostaining using anti-MAP2 and anti-TUJ1 antibodies. Scale bar: 10 µm. (D) BPA exposure changes the levels of mRNAs in the cortical neurons. Heatmap of RNA-sequencing transcriptome analysis for different genes in brains of control or BPA-exposed groups (fold change >2; P<0.05). (E) The significantly enriched GO_CC terms for upregulated genes by BPA. (F) Heatmap of differentially expressed genes associated with enriched GO_CC terms [axon initial segment (GO: 0043194): Kcnq3, Cck; neuronal cell body (GO:0030425): Kcnq3, Cck, Anxa3] and Rgs4.

Fig 4: LAMP2A protein levels and punctate localization in cortical neurons and astrocytes due to high glucose and PGRN treatment. (A) Total protein LAMP2A levels are unchanged among treatment groups in primary cortical neurons treated for 24 h. N = 6 samples. (B) Perinuclear LAMP2A punctate formation increased in neurons under HG (from 1.000 ± 0.137 AU to 1.463 ± 0.108 AU), an effect that was attenuated with PGRN (from 1.031 ± 0.083 AU to 1.246 ± 0.096 AU). N = 12-27 cells. (C) Representative immunofluorescence images of primary neurons after 72 h of treatment, with blue as DAPI, green as MAP2, and red as LAMP2A. Scale bar, 10 µm. (D) Perinuclear LAMP2A punctate formation appeared to show a trend toward an increase due to PGRN but was not statistically significant (F = 1.825, p = 0.151). N = 16-22 cells. (E) Representative immunofluorescence images of primary astrocytes after 72 h of treatment, with blue as DAPI, green as GFAP, and red as LAMP2A. Scale bar, 10 µm. **p < 0.01.

Fig 5: LC3B lipidation and punctate formation in cortical neurons due to high glucose and PGRN. (A) Western blot analysis of LC3 lipidation (i.e., LC3-II:I ratio) in primary cortical neurons after 72 h of treatment. The LC3-II:I ratio decreased from control to high glucose (1.000 ± 0.208 AU to 0.464 ± 0.092 AU), an effect that appeared unaffected when treated with PGRN alongside high glucose (0.474 ± 0.076 AU). No difference was observed between control and PGRN-treated samples (0.938 ± 0.309 AU). N = 9 samples. (B) Immunofluorescence analysis of LC3B puncta expression in neurons after 72 h of treatment. The amount of punctate expression trended toward an increase due to PGRN (from 1.000 ± 0.069 AU, to 1.183 ± 0.077 AU). This difference was significant under high-glucose conditions (from 0.916 ± 0.045 AU, to 1.190 ± 0.097 AU). N = 11-29 cells. (C) Representative immunofluorescence images of primary neurons after 72 h of treatment, with blue as DAPI, green as MAP2, and red as LC3B. Scale bar, 10 µm. (D) Immunofluorescence of LC3B puncta expression in astrocytes after 72 h of treatment increased significantly due to high glucose as well as PGRN treatment (from 1.000 ± 0.0374 AU to 1.512 ± 0.087 AU and 2.792 ± 0.099 AU, respectively). This decreased to control levels under HG + PGRN treatment, to 0.899 ± 0.049 AU. N = 12-29 cells. (E) Representative immunofluorescence images of primary astrocytes after 72 h of treatment, with blue as DAPI, green as GFAP, and red as LC3B. Scale bar, 10 µm. **p < 0.01; ***p < 0.001.