1-Mesityl-3-(3-Sulfonatopropyl) Imidazolium Protects Against Oxidative Stress and Delays Proteotoxicity in C. elegans

Abstract

Due to the increase in life expectancy worldwide, age-related disorders such as neurodegenerative diseases (NDs) have become more prevalent. Conventional treatments comprise drugs that only attenuate some of the symptoms, but fail to arrest or delay neuronal proteotoxicity that characterizes these diseases. Due to their diverse biological activities, imidazole rings are intensively explored as powerful scaffolds for the development of new bioactive molecules. By using C. elegans, our work aims to explore novel biological roles for these compounds. To this end, we have tested the in vivo anti-proteotoxic effects of imidazolium salts. Since NDs have been largely linked to impaired antioxidant defense mechanisms, we focused on 1-Mesityl-3-(3-sulfonatopropyl) imidazolium (MSI), one of the imidazolium salts that we identified as capable of improving iron-induced oxidative stress resistance in wild-type animals. By combining mutant and gene expression analysis we have determined that this protective effect depends on the activation of the Heat Shock Transcription Factor (HSF-1), whereas it is independent of other canonical cytoprotective molecules such as abnormal Dauer Formation-16 (DAF-16/FOXO) and Skinhead-1 (SKN-1/Nrf2). To delve deeper into the biological roles of MSI, we analyzed the impact of this compound on previously established C. elegans models of protein aggregation. We found that MSI ameliorates β-amyloid-induced paralysis in worms expressing the pathological protein involved in Alzheimer's Disease. Moreover, this compound also delays age-related locomotion decline in other proteotoxic C. elegans models, suggesting a broad protective effect. Taken together, our results point to MSI as a promising anti-proteotoxic compound and provide proof of concept of the potential of imidazole derivatives in the development of novel therapies to retard age-related proteotoxic diseases.

Keywords: Caenorhabditis elegans; imidazolium salts; neurodegenerative disease; oxidative stress; proteotoxicity.

FIGURE 1

Effect of imidazolium salts on oxidative stress resistance in wild-type C. elegans. (A). Chemical structures of imidazolium salts with their corresponding oral bioavailability radar below. The pink area represents the optimal range for each property. Compounds 1: 1-(3-sulfonatopropyl) imidazolium, 2: 1-Methyl-3-(3-sulfonatopropyl) imidazolium, 3: 1-Mesityl-3-(3-sulfonatopropyl) imidazolium, 4: 1-(2,6-Diisopropylphenyl)-3-(3-sulfonatopropyl) imidazolium and 5:1,3-bis(2,6-diisopropyl-4-sodiumsulfonatophenyl) imidazolium. (B). Oxidative stress resistance of wild-type worms in the absence (-) or presence of 10 μM of each imidazolium salt (60–80 animals per condition per experiment, n = 3–8). Oxidative stress was induced by 10 mM FeSO4 for 1 h. Survival was scored immediately after this treatment. Data are normalized to the resistance of wild-type animals in the absence of compound assayed the same day. Data in the bar graph are represented as mean ± SEM. Statistical significance was evaluated by One-way ANOVA followed by Holm–Sidak’s test for multiple comparisons against the control group (-) (**p < 0.01). (C). Pharyngeal pumping rates (pumps per min) of wild-type worms in the absence (-) or presence of 10 μM of SI and MSI (30 animals per condition). Data is represented as a box plot with a line at the median. Statistical analysis of the data was performed with a Kruskal–Wallis one-way ANOVA on ranks. No statistical differences (ns) compared to worms without compound treatment (-). (D). Developmental rate of wild-type worms grown in the absence (-) or presence of 10 and 50 μM of SI (left) and MSI (right). A color code was used to represent each of the following animal stages: L1–L3: early larval stages, L4: last larval stage, > L4: adult stage. Animal classification was evaluated at the indicated time points (24, 48, and 72 h). Data are represented in a stacked bar chart as mean ± SEM. No statistical differences (ns) were found compared to worms without compound treatment (-) (One-way ANOVA for MSI and One-way ANOVA on ranks for SI, n = 4 independent experiments).

FIGURE 2

Molecular pathways involved in the mechanism of stress resistance of MSI. (A). Schematic diagram showing classical transcription factors involved in the DAF-2/IIS signaling pathway. Activation of the insulin/IGF-1 receptor ortholog, DAF-2 receptor, leads to phosphorylation and cytoplasmatic sequestration of the transcription factors (TFs) DAF-16/FOXO, HSF-1/HSF, and SKN-1/Nrf2. In contrast, downregulation of the DAF-2/IIS signaling permits the nuclearization of these TFs and the triggering of cytoprotective mechanisms. (B). Oxidative stress resistance (10 mM FeSO4, 1 h) of wild-type, GR1307 daf-16(mgDf50), PS3551 hsf-1(sy441), and QV225 skn-1(zj15) mutant animals exposed to MSI. Data are shown as mean ± SEM from at least 4 independent experiments (n = 60–80 animals per experiment per condition). Statistical differences were evaluated by comparison of means (Student’s t-test for daf-16 animals) or medians (Mann-Whitney Rank Sum Test for wild-type and hsf-1 worms). *represents p-value compared with the same strain without MSI and # represents p-value for wild-type and hsf-1 mutant comparison upon MSI exposure (ns, not significant, *p < 0.05, **p < 0.01, ##p < 0.01). (C). Representative fluorescence micrographs depicting the expression of the stress-responsive gene hsp-16.2 in the absence and presence of 50 μM MSI. (D). Quantification of hsp-16.2 expression by measuring fluorescence intensity in the pharynx region. Data are shown in a scatter-dot plot graph, with a horizontal line indicating the median of each dataset, from five independent experiments and 25–40 animals per experiment per condition. Statistical differences were evaluated by Mann-Whitney Rank Sum Test (***p < 0.001).

FIGURE 3

Evaluation of MSI impact on proteotoxic-associated phenotypes in C. elegans models of NDs. (A). Influence of MSI on the paralysis of animals expressing human β-amyloid_3–42 in muscles throughout its lifetime. Data show a representative Kaplan-Meier curve in the absence and presence of 50 μM MSI. Statistical differences were evaluated by a log-rank test (***p < 0.001). (B). Evaluation of paralysis after a heat shock (35°C for 30 min). Data in the bar graph represent the mean ± SEM from 4 independent experiments with 50–80 animals per condition per experiment. Statistical significance was evaluated by One-way ANOVA followed by Holm–Sidak’s test for multiple comparisons against the control group (0 μM MSI) (*p < 0.05, **p < 0.01). (C,D). Swimming behavior evaluated in C. elegans strains expressing α-synuclein (C) and poly-Q repeats (D) in their body wall muscles. Data were classified into three speed categories based on thrashing rates of young adult animals: normal includes those animals moving at a speed between 25% and 75% of the average thrashing rate at day 0, slow accounts for those animals moving at a speed lower than 25% of average thrashing rate of day 0 adults, and fast comprises those animals moving at a higher speed than 75% of control. Numbers inside bars show the number of animals falling in each category.

FIGURE 4

MSI effects on protein aggregation in C. elegans models of ND. (A). Quantification of α-synuclein expression levels upon MSI treatment. Left: Representative fluorescence images depicting α-syn::yfp in 7-day adult worms grown in the absence or presence of 50 μM MSI. Right: Relative fluorescence intensity in the head area of the worm at different ages represented in a scatter-dot plot (line at the median). Each dot symbolizes one worm (n = 30–60). Statistical analysis was performed by comparing differences between median values for 0 and 50 μM MSI at each indicated stage (Mann-Whitney Rank Sum Test). Statistical symbols represent **p < 0.01 and ns not significant. (B) Quantification of poly-Q aggregation upon MSI treatment. Top: Representative fluorescence images depicting Q-40::YFP in 1-day adult worms grown in the absence or presence of 50 μM MSI. Bottom: The number of aggregates per animal and their relative mean size at days 0, 1, and 2 of adulthood were quantified using ImageJ FIJI and represented in their corresponding scatter-dot plot (line at the median, n = 60–80). Statistical analysis was performed by comparing differences between the median for 0 vs. 50 μM MSI (Mann-Whitney Rank Sum Test, ns not significant).

FIGURE 5

Schematic diagram depicting the cytoprotective activities of the imidazolium salt MSI, supporting its role as an antiproteotoxic agent. Cellular protein quality control systems, such as autophagy, proteosome and antioxidants, act to prevent or degrade misfolded proteins. When the capacity of these protective mechanisms is overwhelmed, abnormal proteins can accumulate into toxic protein aggregations that generate cellular damage. MSI reduces oxidative stress injury and pathological phenotypes associated with proteotoxicity accumulation.

SCHEME 1

Synthesis of sulfonated-imidazolium salts.