Fig 2: 8-month-old CAMKII-Cre:Cbsfl/fl mice demonstrated similar cognitive deficits to 18-month-old wild type counterparts after undergoing exploratory laparotomy. (A) Experimental timeline of surgery and behavioral tests. (B) Representative movement tracks in the open-field test. (C) Time spent in the central zone in the open-field test (ns: no significant difference, mean ± SD, unpaired t-test). PND: n=8, CBS-CKO: n=7. (D) Locomotive ability was evaluated by the average speed in the open field test (ns: no significant difference, mean ± SD, unpaired t-test). (E) and (F) show the statistical results of spatial learning (ns: no significant difference, mean ± SEM, two-way ANOVA) during four consecutive days and spatial memory (ns: no significant difference, mean ± SD, unpaired t-test) on the test day of the Barnes maze tests. (G) Contextual memory was also evaluated by the ratio of freezing time in the contextual fear conditioning test (ns: no significant difference, mean ± SD, unpaired t-test).

Fig 3: Effects of MR on redox status and CBS/H2S pathway in the brain of male APP/PS1 AD model mice(A) The GSSG/GSH ratio in the cortex (n = 8); (B) The levels of GSH in the cortex (n = 8); (C) The levels of GSSG in the cortex (n = 8); (D) The levels of MDA in the cortex (n = 8); (E) The levels of H2S in the cortex (n = 8); (F) mRNA levels of CBS in the cortex (n = 4); (G) Representative western blots of CBS protein levels in cortex (n = 3); (H) Quantification of the western blots of CBS protein levels in cortex (n = 6); (I) Representative images of immunofluorescence staining of CBS in the cortex (n = 3 slices per group); (J) Mean fluorescence intensity (AU) of CBS protein levels in cortex (n = 9 slices from 3 mice per group). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, compared with the WT + SD group, #p < 0.05, ##p < 0.01 compared with the AD + SD group. Significant differences between mean values were determined by two-way ANOVA with Tukey multiple comparisons test.

Fig 4: Generation of in vivo knockdown models of CBS and CGL, regulators of transsulfuration pathway involved in Hcy catabolism. (A) Simplified diagram showing the enzymes and metabolites of transsulfuration pathway (B) Representative western blot of 2 dpf embryos lysates showing that compared with SC MO injected embryos, CBS protein level is downregulated in CBS MO injected embryos. Expression of CGL, the other enzyme of the same pathway, remained unaltered. As a loading control β-actin was used. Corresponding bar plot showing fold change in protein expression (normalized to β-actin) as determined by densitometric analysis of the protein band. (C) Bar plot revealing no statistically significant difference in the viability of scrambled control and Hyperhomocysteinemic CBS MO embryos. (D) Bright field images exhibiting no apparent gross morphological defect in CBS morphants compared with scrambled Mo injected embryos of 2 dpf. Scale bar, 0.25 mm. (E) Representative western blot of embryo lysates at 2 dpf showing reduced protein level of CGL in the embryos injected with CGL MO. Expression of CBS, the upstream protein of the same pathway, remained unaltered. β-actin was used as a loading control. Corresponding bar diagram showing densitometric analysis of the fold change in protein expression (normalized to β-actin). (F) In comparison to SC MO injected embryos, viability of CGL MO injected embryos were not altered significantly. (G) Representative bright field images showing absence of any gross morphological defect in CGL MO injected embryos compared with SC MO injected ones at 2 dpf. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant (p > 0.05).

Fig 5: l-cysteine-induced PERK oligomerization is mediated by CBS and 3-MST. (A) Cells were treated with 2 mM l-cysteine for the indicated time, and PERK levels were analyzed in the whole-cell lysate by western blotting under nonreducing (A) or reducing (B) conditions. β-Tubulin was used as a loading control (bottom panel). (C) SH-SY5Y cells transfected with CBS, 3-MST, and CBS–3-MST (double knockout; dk) siRNA were treated with 2 mM l-cysteine for 6 h, and PERK levels were analyzed in the whole-cell lysates by western blotting. β-actin was used as a loading control (bottom panel). (D) SH-SY5Y cells transfected with CBS, 3-MST, and CBS–3-MST (dk) siRNA were treated with 2 mM l-cysteine for 6 h, and PERK levels were analyzed in the whole-cell lysate by western blotting under nonreducing conditions. The densities of CBS and 3-MST bands were measured, and the ratio to β-actin was calculated and expressed as the fold-change of the band levels relative to scrambled siRNA samples (A). The densities of PERK oligomer bands were measured, and the ratio to β-Tubulin was calculated and expressed as the fold-change of the band levels relative to 0 min (A) or scrambled siRNA treated with vehicle (D) samples. Values indicate means ± S.D. Dunnett's test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. All data are representative of at least three independent experiments.