Sulfanegen stimulates 3-mercaptopyruvate sulfurtransferase activity and ameliorates Alzheimer's disease pathology and oxidative stress in vivo

Abstract

Increased oxidative stress and inflammation are implicated in the pathogenesis of Alzheimer's disease. Treatment with hydrogen sulfide (H₂S) and H₂S donors such as sodium hydrosulfide (NaSH) can reduce oxidative stress in preclinical studies, however clinical benefits of such treatments are rather ambiguous. This is partly due to poor stability and bioavailability of the H₂S donors, requiring impractically large doses that are associated with dose-limiting toxicity. Herein, we identified a bioavailable 3-mercaptopyruvate prodrug, sulfanegen, which is able to pose as a sacrificial redox substrate for 3-mercaptopyruvate sulfurtransferase (3MST), one of the H₂S biosynthetic enzymes in the brain. Sulfanegen is able to mitigate toxicity emanating from oxidative insults and the Aβ_1-42 peptide by releasing H₂S through the 3MST pathway. When administered to symptomatic transgenic mouse model of AD (APP/PS1; 7 and 12 months) and mice that were intracerebroventricularly administered with the Aβ_1-42 peptide, sulfanegen was able to reverse oxidative and neuroinflammatory consequences of AD pathology by restoring 3MST function. Quantitative neuropathological analyses confirmed significant disease modifying effect of the compound on amyloid plaque burden and brain inflammatory markers. More importantly, sulfanegen treatment attenuated progressive neurodegeneration in these mice, as evident from the restoration of TH+ neurons in the locus coeruleus. This study demonstrates a previously unknown concept that supplementation of 3MST function in the brain may be a viable approach for the management of AD. Finally, brought into the spotlight is the potential of sulfanegen as a promising AD therapeutic for future drug development efforts.

Keywords: 3MST; Alzheimer's disease; Hydrogen sulfide; Neurodegeneration; Neuroinflammation; Sulfanegen.

Graphical abstract

Fig. 1

Sulfanegen displays antioxidant property due to release of H₂S via 3MST pathway and mitigates peroxide and Aβ_1-42cytotoxicity. (A) Radical scavenging property of sulfanegen was analyzed by a well-known DPPH assay as described in the methods section. (B) Cellular ROS generation was measured using DCFDA where cells were treated with H₂O₂ in the presence (top right) or absence of sulfanegen (bottom right) and images were taken after 8 h treatment. The left panel of images shows cells without H₂O₂ or sulfanegen exposure (top) and sulfanegen only (bottom controls). (C) Under similar conditions as B, fluorescence intensity was measured using a multimode plate reader. (D) Data represents dose dependent release of H₂S from sulfanegen. (E) H₂S release from sulfanegen was measured in the presence or absence of 3MST or CBS or CSE inhibitors as described in the methods section. (F) Protection of H₂O₂ cytotoxicity in the cells treated with sulfanegen in the presence or absence of 3MST inhibitor, I3MT3. (G) Cytotoxicity in the cells treated with Aβ_1-42 +/− sulfanegen in the presence or absence of I3MT3 after 24 h treatment. Statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2

Sulfanegen administration shows improved cognitive behavior and inflammatory pathology in the brain tissues of mice injected intracerebroventricularly with oligomeric Aβ_1-42solution. (A) Time course of the i.c.v. Aβ-model, created using BioRender.com. (B) Cognitive assessment was conducted using T-maze test on day 10 after Aβ_1-42 injection. A significant reduction in the alternation behavior observed in Aβ_1-42 only mice was improved in sulfanegen treated mice. Similarly, significant improvement in number of repetitive arm entries was observed in the compound treated group (C). There was no innate spatial bias displayed by these mice as evident from the ration of left and right arm entries (D). Neuroinflammatory markers such as TNF-α (E) and IL-6 (F) were elevated in Aβ_1-42-only group and sulfanegen treatment rescued against this inflammatory phenotype in this mouse model. Statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparison test. *p < 0.05, * *p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3

Sulfanegen administration improves cognitive behavior and reduced Aβ plaque burden in APP/PS1 mice. (A–C) Cognitive assessment of 10 and 15 mo old APP/PS1 cohorts was conducted using the T-maze test. Percent alternation (A) and repetitive arm entries (B) showed significant improvement in the sulfanegen treated mice compared to transgenic vehicle controls. No spatial bias (C) was observed as deemed by ration of arm entries. (D–E) Representative images of immunohistochemical detection of amyloid plaques using 4G8 antibody in cortex and hippocampal regions. Quantitation of the plaque-covered area in cortex (F) and hippocampus (G) regions of the 15 mo old cohort displayed significant reduction in compound treated group. (H–I) ELISA-based quantitation of Aβ levels showed significant reduction in the insoluble Aβ levels in the cortex (H) and hippocampus (I) of 15 mo old cohort. Statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparison test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4

Administration of sulfanegen in APP/PS1 mice reverses oxidative stress and reduces inflammation. (A–D) Overall oxidative stress was assessed by measurement of hallmark markers such as redox ratio (GSH:GSSG ratio, A), lipid peroxidation by thiobarbituric acid reactive substance, malondialdehyde (B), protein carbonyls using western blots with anti-DNPH antibody (C) in the cortex. Quantitation of dot blot in C is displayed in D. Assessment of inflammatory markers such as TNF-α (E) and IL-6 (F) was conducted in 10 and 15 mo old cohorts. Significant reduction in the levels of oxidative and inflammatory markers was observed in sulfanegen treated groups. Statistical significance was assessed by a one-way ANOVA with Dunnett's multiple comparison test. *p < 0.05, **p < 0.01, ** *p < 0.001.

Fig. 5

Sulfanegen treatment reduces reactive astrogliosis and microglial reactivity in symptomatic 10- and 15-mo APP/PS1 mice. (A–C) Representative images of reactive astrocytes visualized by GFAP antibody in cortex and dentate gyrus of 15 mo NTG (A) and APP/PS1 mice treated with vehicle (B) or sulfanegen (C). Scale bar, 100 μm. (D–E) Quantification of GFAP staining in S1BF (E) and hippocampus (E) of the 15 mo old cohort. (F–H) Representative images of microglia visualized using CD68 antibody in cortex and hippocampal regions of 15 mo NTG (F) and vehicle (G) or sulfanegen (H) treated APP/PS1 mice. Scale bar, 100 μm. (I–J) Quantification of CD68 staining in S1BF (I) and hippocampus (J) of the 15 mo old cohort. Data are presented as mean ± S.E.M. For comparisons between NTG, saline-, and sulfanegen-treated APP/PS1 groups, a one-way ANOVA with Tukey's post-hoc test was used for data analysis. **p < 0.01, ****p < 0.001.

Fig. 6

Sulfanegen treatment after the onset of AD-like neuropathology halts the progressive degeneration of the NAergic cortical afferent axons in symptomatic 15 mo APP/PS1 mice. (A–C) Representative images of TH+ axonal projections in S1BF of the 15 mo old cohort; (A) NTG, (B) APP/PS1 vehicle and (C) APP/PS1 sulfanegen. Scale bar, 20 μm. (D) Quantification of TH+ axonal density in 15 mo old cohort. Density estimation (μm/μm³) was determined using the Spherical probe on Stereo Investigator Software. (E–G) Representative images of TH+ neurons of the LC in both the 15 mo NTG (E) and vehicle (F) or sulfanegen (G) treated APP/PS1 mice. Scale bar, 100 μm. (H) Quantification of TH+ neurons in the LC in 15 mo old cohort. Estimated population was determined by the Optical Fractionator probe on Stereo Investigator software. (I–K) Higher magnification images of the TH+ neurons in the LC of 15 mo old cohort comprising NTG (I) and vehicle (J) or sulfanegen (K) treated APP/PS1 groups. Scale bar, 50 μm. (L) Quantification of the TH+ neuronal volume in 15 mo old cohort. Data are presented as mean ± S.E.M. A one-way ANOVA with Tukey's post-hoc test was used for statistical comparisons (b). *p < 0.05, **p < 0.01, ***p < 0.005.

Fig. 7

Reduced 3MST function in the APP/PS1 mice is restored by sulfanegen treatment. (A) ELISA-based quantitation of 3 MST expression in the cortex region of 15 mo old cohort showed no changes in the levels of 3MST protein. (B) Quantitation of 3MST substrate, 3-mercaptopyruvate (3-MP) showed significantly reduced levels in the APP/PS1 mice, which were increased after sulfanegen treatment. (C) Endogenous activity of 3MST enzyme was significantly reduced in both 10 mo and 15 mo transgenic cohorts. Sulfanegen treatment restored 3MST function to NTG control levels. (D) 3MST enzymatic reaction showing substitution of 3-MP by sulfanegen in the enzymatic reaction, restoring its function. Statistical significance was assessed by a one-way ANOVA with Tukey's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.