Fig 2: PAL treatment diminishes 8-OHdG generation in neuronal mitochondria via improved BER. (a) PAL treatment markedly increased the expression of OGG1 and MTH1 but suppressed the expression of MUTYH in APP/PS1 mouse brains. n = 8. (b) Mitochondrial 8-OHdG generation was inhibited in APP/PS1 mouse brains after PAL treatment. Three sections/brain, n = 6. (c) PAL treatment dramatically suppressed mitochondrial 8-OHdG generation in NeuN-positive neurons from APP/PS1 mouse brains. Three sections/brain, n = 6. **p < .01 (Student's-t-test).

Fig 3: PAL treatment reduces senile plaque burden in APP/PS1 mice. (a) Immunofluorescent labelling of Aβ showing the Aβ plaque in the cortex and hippocampus of APP/PS1 mice. Three sections/brain, n = 6. (b-c) Quantification of Aβ fluorescence revealed reduced number of Aβ plaque in the cortex and reduced areas of Aβ plaque in both the cortex and hippocampus of APP/PS1 mice after PAL treatment. (d) PAL treatment decreased Aβ oligomer protein levels. n = 8. (e-f) ELISA results showed a slight but significant reduction in Aβ40 in the hippocampus and a considerable reduction in Aβ42 in the cortex and hippocampus of APP/PS1 mice after PAL treatment. n = 8. (g) The ratios of soluble Aβ42/40 were decreased in both the cortex and hippocampus after PAL treatment. n = 8. (h-i) The insoluble Aβ40 was decreased in the hippocampus and the insoluble Aβ42 were reduced in both the cortex and hippocampus of APP/PS1 mice after PAL treatment. n = 8. (j) The ratio of insoluble Aβ42/40 was decreased in hippocampus after PAL treatment. n = 8. *p < .05, **p < .01 (Student's-t-test).

Fig 4: Rer1 deficiency leads to the degradation of nicastrin and presenilin-1 in lysosomes.(A) Cycloheximide (CHX) chase experiments for nicastrin (NCT) and presenilin-1 C-terminal fragment (PS1 CTF) in WT or Rer1-deficient (KO) HAP1 cells. HAP1 cells were treated with 10 μg/ml CHX, and protein levels of NCT, PS1 CTF, and α-actin were measured at the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (B) Quantitative analysis of NCT and PS1 CTF during CHX chase experiments. Graphs show relative levels of NCT and PS1 CTF normalized to α-actin. Values are the mean ± standard error of the mean (SEM) of three independent experiments. Two-way analysis of variance (ANOVA) was performed to determine the significance of the differences. *P < 0.05, **P < 0.01 (ANOVA). (C) Effects of ERAD inhibitor on the stability of NCT and PS1 CTF in WT or Rer1 KO HAP1 cells. WT and Rer1 KO HAP1 cells were cultured for 16 h in the presence (+) or absence (-) of 2.5 μM eeyarestatin I (EerI). Cell lysates were immunoblotted with the indicated antibodies. (D, E) Quantitative analysis of the effects of ERAD on PS1 CTF (D) and NCT (E) in WT and Rer1 KO cells. Graphs show fold changes for γ-secretase components in cells treated with EerI (+) relative to those in vehicle (DMSO)-treated cells (-). Values are the mean ± SEM of three independent experiments. *P < 0.05, ****P < 0.0001 (Student t-test). n.s.: not significant. Note that the total amount of NCT and PS1 CTF are reduced in Rer1 KO HAP1 cells. (F-I) Lysosomal degradation of NCT and PS1 CTF in Rer1 KO HAP1 cells. (F) Rer1 KO HAP1 cells were cultured for 16 h in the presence (+) or absence (-) of 200 nM bafilomycin A1 (BafA1). Cell lysates were immunoblotted with the indicated antibodies. (G) Quantitative analysis of the effects of BafA1 on NCT and PS1 CTF. Graphs show fold changes of NCT and PS1 CTF in cells treated with BafA1 (+) relative to vehicle (DMSO) (-) treated cells. Values are the mean ± SEM of three independent experiments as shown in panel F. *P < 0.05 (Student t-test). (H) Immunoblot analysis of Rer1 KO cells expressing GFP-Rer1 with the indicated antibodies. Cells were treated as shown in Fig 5F (I) Quantification of immunoblot experiments as shown in panel H. (J) Accumulation of NCT in lysosomes in BafA1-treated Rer1 KO cells. WT and Rer1 KO cells were cultured for 12 h in presence (+) or absence (-) of BafA1 and immunostained using anti-NCT (red) and anti-Lamp1 (green) antibodies. Nuclei were visualized with DAPI (blue). Immunostained cells were observed using confocal laser-scanning microscopy. Scale bar, 20 μm. (K) Quantitative analysis of colocalization between NCT- and Lamp1-positive area. The percentage of NCT-positive area on Lamp1-positive area was calculated using cellSens software (Olympus). The graph shows the average colocalization percentage per cell. Error bars represent the SEM (n = 58 and n = 46 of WT and Rer1 KO HAP1 cells, respectively). ***P < 0.001 (Student t-test). (L) γ-Secretase complex formation in WT and Rer1 KO HAP1 cells. WT and Rer1 KO HAP1 cells were treated with or without 200 nM BafA1 for 24 h. Cell lysates were subjected to Blue-Native (BN-PAGE) and SDS-PAGE analysis and were immunoblotted with the indicated antibodies. (M) Underglycosylation of NCT in Rer1 KO HAP1 cells. WT or Rer1 KO HAP1 cells were immunoblotted with the indicated antibodies.

Fig 5: Sequencing traces of the PSEN1 locus and gRNA design(A) All human fibroblast samples were sequenced for the presence of the PSEN1 M146L (A > C) mutation in exon 5 of the PSEN1 gene. The results from one PSEN1 M146L mutated and one non-mutated subject are shown. (B) This mutation creates a 5'-NGG-3' PAM site on the reverse DNA strand that allows for generation of the 20-bp-long allele-specific M146L gRNA.