Organoruthenium(II) Complexes Ameliorates Oxidative Stress and Impedes the Age Associated Deterioration in Caenorhabditis elegans through JNK-1/DAF-16 Signalling

New ruthenium(II) complexes were synthesised and characterized by various spectro analytical techniques. The structure of the complexes 3 and 4 has been confirmed by X-ray crystallography. The complexes were subjected to study their anti-oxidant profile and were exhibited significantly greater in vitro DPPH radical scavenging activity than vitamin C. We found that complexes 1–4 confered tolerance to oxidative stress and extend the mean lifespan of mev-1 mutant worms and wild-type Caenorhabditis elegans. Further, mechanistic study and reporter gene expression analysis revealed that Ru(ƞ6-p-cymene) complexes maintained the intracellular redox status and offers stress resistance through activating JNK-1/DAF-16 signaling axis and possibly by other antioxidant response pathway. Notably, complex 3 and 4 ameliorates the polyQ (a Huntington’s disease associated protein) mediated proteotoxicity and related behavioural deficits in Huntington’s disease models of C. elegans. From these observations, we hope that new Ru(ƞ6-p-cymene) complexes could be further considered as a potential drug to retard aging and age-related neurodegenerative diseases.


Results and Discussion
The reactions of 1:2 molar ratio of [(ƞ 6 -p-cymene)RuCl 2 ] 2 with various 3-methoxysalicylaldehyde-4(N) -substituted thiosemicarbazones (H 2 L 1 -H 2 L 4 ) in dichloromethane resulted in the formation of four new complexes, the analytical data of which confirmed the stoichiometry of the complexes (1-4) (Fig. 1). The structure of the complexes 3 and 4 were confirmed by X-ray crystallographic studies and attempts were made to grow single crystals of complex 1 and 2 suitable for crystallographic studies in various organic solvents were unsuccessful. The  (Table 1) and this band has been observed at 1574-1593 cm −1 in the complexes, indicating the coordination of the azomethine nitrogen atom to the ruthenium 52 . A sharp band that appeared at 783-818 cm −1 in the ligands corresponding to ν C=S vibration was appeared at 771-883 cm −1 in the complexes confirmed the binding of thione sulphur atom to the ruthenium 53 . Vibrations corresponding to phenolic OH (ν OH ) was found at 3439-3457 cm −1 in the ligands shifted slightly lower region at 3427-3454 cm −1 in complexes (1)(2)(3)(4), indicating the nonparticipation of the phenolic oxygen atom in coordination 54 . The electronic spectra of the complexes (1-4) ( Supplementary Fig. S1) have been recorded in dichloromethane, and they displayed two to three bands in the region around 210-342 nm. The bands appearing in the region 210-291 nm have been assigned to intra ligand transitions 55 , and the band at 342 nm have been assigned to ligand to metal charge transfer transitions (LMCT) 56 . The 1 H NMR spectral data of the ligands and the complexes were recorded in the DMSO and CDCl 3 . The 1 H-NMR spectral data of the complexes in CDCl 3 suggested a 1:1 molar ratio of the coordinated p-cymene and the Schiff base ligands (H 2 L 1 -H 2 L 4 ). The proton NMR spectra and the detailed description have been given in supporting information (Supplementary Figs S2-S13). The molar conductance for complex 1 and 2 was found to be 122 Ω −1 cm 2 mol −1 and 113 Ω −1 cm 2 mol −1 respectively found to be in good agreement with the reported 1:1 electrolytic behaviour 57 .
X-ray Crystallography. Complexes 3 and 4 crystallized in monoclinic space group P2 1 /n and P2 1 /c. The crystal structure of the complexes 3 and 4 are shown in Figs 2 and 3. Crystals of complexes 3 and 4 suitable for X-ray diffraction analysis were grown by slow diffusion of hexane into dichloromethane. Crystallographic data are listed in Table 1. Selected bond lengths and angles are listed for each compound in Supplementary Table S1. The molecular packing diagrams of the complexes were given in Supplementary Figs S14 and S15. In 55 . The other three sites are occupied by the chloride and the N,S donor Schiff base ligand. The ruthenium atom is situated 1.690 Å and 1.452 Å away from the centre of the ƞ 6 -p-cymene ring for complexes 3 and 4 respectively. The ruthenium-centroid distances are also in agreement with other structurally characterized cationic ƞ 6 -p-cymene complexes of ruthenium 58 . The Ru-S, Ru-Cl, and Ru-N distances are all in line with other structurally characterized ƞ 6 -arene ruthenium(II) complexes 58 . The variations in bond lengths and angles lead to a significant distortion from an ideal octahedral geometry for the complex 59 .
In addition, complex 3 contains three intermolecular hydrogen bonding through the hydrogen atom of the hydroxy group with the chloride atom (Cl2), hydrogen atom of the hydrazinic nitrogen (N2) group with the chloride atom (Cl2) and hydrogen atom of the terminal nitrogen (N3) group with the chloride atom (Cl2) with O(1)-H(1)···Cl (2), N(2)-H(2)···Cl (2) Table 2 showed that all complexes exhibited significantly greater in vitro radical scavenging activity, as it was able to quench DPPH radicals stronger than vitamin C (V C ). The radical scavenging activity of complexes 1-4 in the ranges of IC 50 DPPH = 0.211 ± 0.005, 0.274 ± 0.012, 0.192 ± 0.001 and 0.266 ± 0.013 μg/mL, respectively as compared to that of standard control V C (5.139 ± 0.098 μg/ mL). It is worth mentioning that ligands and starting precursor were demonstrated lower DPPH radical scavenging activity as compared to complexes and conventional standard. Considering the antioxidant potency, we then assayed the in vivo antioxidant efficiency using a nematode model C. elegans. In the in vivo assay system, we first assessed the safety property of Ru(ƞ 6 -p-cymene) complexes on wild type C. elegans. When assessing the safety property, we found that exposure with 0-18 µM concentration of the complexes 1-4 did not significantly alter the survival, development and reproduction of nematodes ( Supplementary Fig. S16). In addition, ligands and precursor were found to be less toxic and affects the reproduction and development. In C. elegans, D-type GABAergic motor neurons regulate the locomotion behaviour 60 . It was found that complexes 1-4 did not obliviously alter the development/morphology of D-type GABAergic motor neurons. On the contrary, ligands and precursor exhibited some toxicity on the morphology of D-type GABAergic motor neurons ( Supplementary Fig. S17). Neurons  and reproductive organs are the two important secondary targets of any toxins and drugs in C. elegans. From these observations, it was apparent that new complexes are relatively safe in C. elegans. Hence, the ligands and precursor were excluded for the following experiments.
We have shown that pretreatment with complexes 1-4 at different pharmacological doses (2, 6, 10, 14 and 18 μM) significantly inhibited the induction of ROS generation and increased the survival rate of C. elegans under juglone intoxicated conditions (Fig. 6). We also performed lifespan assay using mitochondrial mutant strain TK22 [mev-1 (kn-1)]. This strain has a mutation in succinate dehydrogenase cytochrome b, an integral membrane protein that is a subunit in complex 2 of mitochondrial respiratory chain. MEV-1 is required for oxidative phosphorylation, loss of function in mev-1 resulted in the overproduction of free radicals and lead to a shortened lifespan 61 . We found that treatment with complexes 1-4 at 10 µM appeared to prolong the mean lifespan to 15.82%, 21.19%, 27.38% and 28.72% respectively ( Fig. 7; Supplementary Table S3). The results indicated that the optimal dose (10 µM) of complexes 1-4 involved in the endogenous detoxification pathway thereby prolongs the lifespan of mev-1 worms. Apart from metabolic control and gene expression pattern, the reduced level of ROS has been concomitantly associated with lengthening of organismal lifespan and healthspan 62,63 . In general, the progression of aging has been linked with declined redox regulation which makes the organism vulnerable to lethal diseases 64 . With such understanding from previous literatures, we examined the lifespan of wild type worms raised on the NGM plates in the presence or absence of complexes 1-4 (6, 10 and 14 μM). Of the three doses, animals raised on 10 μM of complexes 1-4 had increased the mean lifespan of N2 animals (p < 0.0001) to 12.60%, 16.64%, 22.32% and 22.95% respectively (Supplementary Fig. S18 and Supplementary Table S3). In all these experiments, we have noticed that complexes 1-4 displayed a hormetic-like concentration-dependent biphasic effects on C. elegans (Figs 6 and 7 and Supplementary Fig. S18). Several previous studies showed that stress hormesis mechanism extend the lifespan of C. elegans and confers neuroprotection in Danio rerio [65][66][67] . The reduction in the ROS level can be anticipated to be the major reason behind oxidative stress resistance and prolongevity in C. elegans. Taken collectively, these findings confirmed that complexes 1-4 reduced the intracellular ROS accumulation, conferred the resistance to stress and extended the lifespan of mev-1 mutant as well as wild type worms by its potent antioxidative properties. Herein it is interesting to note that all the complexes exhibited better antioxidative than the standard vitamin C. Among the complexes, the activity varied based on their N-terminal substitution. Complex 3, with more electron donating ethyl exhibited better activity than the all other complexes and they follow the  Table 2. In vitro free radical scavenging activities of ligands, starting precursor and complexes. All complexes showed far similar radical scavenging activity. Ru(ƞ 6 -p-cymene) complexes regulates the JNK-1/DAF-16 signalling axis in C. elegans to resist against stress. To elucidate the molecular mechanism of stress resistance and life promoting effects of complexes 1-4, we perused the lifespan assay using daf-16 and mek-1 mutant alleles. The DAF-16/FOXO is an evolutionary conserved transcription factor plays an indispensable role in the regulation of signal transduction pathways associated with stress modulation and longevity phenotype in C. elegans 72 . Prior genetic study has been probed that JNK-1, a member of mitogen-activated protein kinase (MAPK) family, promotes the lifespan of C. elegans in response to stress via DAF-16 73 . JNK-1 is positively regulated by the upstream MAP kinase kinase (MAPKK) super family protein MEK-1, which is encoded by mek-1 gene. Our mechanistic study showed that, complexes 1-4 treatment fails to considerably increase the lifespan of daf-16 and mek-1 null mutants ( Fig. 8; Supplementary Table S3). Moreover, all complexes failed to increase the survival rate of daf-16 and mek-1 mutant worms under juglone exposure, in contrast to results obtained with N2 C. elegans ( Supplementary Fig. S19). The hormetically-induced life extension was further blocked by the mutation in daf-16 66,74 . It represents that DAF-16 is an essential transcription factor driving the hormesis-induced beneficial effects in C. elegans and it was consistent with previous results (i.e. lifespan and oxidative stress resistance of wild-type worms). Therefore, stress modulatory effects of complexes 1-4 is probably mediated through JNK-1/DAF-16 pathway 73 . To further confirm these results, we investigated whether complexes 1-4 treatment activates DAF-16 subcellular localization under normal condition. The complexes 1 and 2 treatments resulted in the partial/intermediate translocation of DAF-16, whereas a greater percentage of worms treated with 3 and 4 exhibited a nuclear relocation pattern of DAF-16 compared with that of untreated control group (Fig. 9). We then treated the transgenic worms, which contains sod-3::GFP, hsp-16.2::GFP gst-4::GFP and ctl-1,2,3::GFP reporter transgene with complexes 1-4. As a result, complex 3 and 4 treated groups showed a significant increase of fluorescence intensity in CF1553, CL2070, CL2166 and GA800 C. elegans respectively. However, no corresponding increase in expressions were observed in the groups treated with complexes 1 and 2 ( Fig. 10; Supplementary Fig. S20). All these genes offered a conserved protection against stress and its expressions can be modulated by antioxidants. Moreover, the master regulator of stress resistance and longevity, DAF-16 transcription factor, can also regulates the expression of these antioxidant gene (i.e. sod-3, hsp-16.2 and ctl-1,2,3) 62,75-78 . Thus, we concluded that, Ru(ƞ 6 -p-cymene) complexes, especially 3 and 4, offers stress resistance not only by its redoubtable antioxidative potential but additionally by modulating the expression of stress-responsive genes in C. elegans. Ru(ƞ6-p-cymene) complexes alleviates PolyQ-mediated toxicity. We also investigated the possibility of whether complexes 1-4 could reduce the Huntington's disease (HD) associated pathologies in C. elegans. Results showed that complex 3 and 4 significantly reduced the polyQ (polyQ40::YFP) aggregate score, whereas complex 1 and 2 had no effects on polyQ aggregation in AM141 C. elegans (Fig. 11A). We next examined the protective effect of complexes against polyQ mediated neuronal death in HA759 C. elegans expressing polyQ tract (Htn-Q150::GFP) exclusively in ASH neurons. As shown in Fig. 11B, only 37.78 ± 3.02% ASH neurons were survived in control group worms which indicated that aggregation of polyQ induces the neuronal death. We did not observed any significant changes in the protection upon treatment with complex 1 and 2, while complex 3 and 4 were proficient of increasing the neuronal survival at 10 μM to 66.67 ± 3.82% and 60.56 ± 4.37% respectively. At this molarity, complex 3 and 4 treatments found to be more effective in improving the chemosensory index to ~0.83 and ~0.77 respectively indicating the healthy status of ASH chemosensory neurons when compared to untreated (~0.41) and complex 1 (~0.40) -2 (~0.39) treated groups (Fig. 11C). Therefore, this result suggests that complex 3 and 4 improved the intracellular protein homeostasis (proteostasis) via inhibition of unwanted proteomic changes in C. elegans. The impaired proteostasis network and accumulation of non-native protein aggregates in various tissues are common features of aging and neurodegenerative diseases including HD 79 . Emerging scientific evidences suggest that delaying the aging process and clearing the affected protein or preventing the aggregate formation might be an effective strategy to normalize the protein misfolding diseases [80][81][82] . These polyQ protein aggregations were greatly delayed or even halted by the influences of JNK-1 51 and DAF-16 83 . A genetic study revealed that DAF-16 interrupt the formation of polyQ aggregation through the regulation of four small heat-shock factors (sHsp) genes viz., hsp-16.1, hsp-16.49, hsp-12. 6 and sip-1, since these sHSP has had the DNA binding site for DAF-16 47 . In addition polysaccharide from Astragalus membranaceus offers protection against polyQ proteotoxicity through regulating DAF-16/FOXO pathway 84 . In the present study, we confirmed that Ru(ƞ 6 -p-cymene) complexes mediate stress resistance in C. elegans probably via JNK-1/DAF-16 pathway. Taken together, these results suggest that complexes exert anti-aggregative and neuroprotective activity through regulating stress-response pathways.  Hence, further studies on higher models are required to elucidate the detailed mechanism of action of Ru(ƞ 6 -p-cymene) complexes before making into a drug candidate.
Measurements. All the reagents used were analar grade, were purified and dried according to the standard procedure 85 . 3-methoxysalicylaldehyde, thiosemicarbazide, 4(N)-substituted thiosemicarbazides were obtained from Himedia. Melting points were measured in a Lab India apparatus. Infrared spectra were measured as KBr pellets on a Jasco FT-IR 400-4100 cm −1 range. Elemental analyses of carbon, hydrogen, nitrogen and sulphur were determined by using Vario EL III CHNS at the Department of Chemistry, Bharathiar University, Coimbatore, India. Electronic absorption spectra of the compounds were recorded in dichloromethane using JASCO 600 spectrophotometer. Molar conductance of the complexes was determined in dichloromethane at room temperature by using a Jenway model 4070 conductivity meter. 1 H and 13 C spectra were recorded in CDCl 3 and DMSO at  A similar method was followed to synthesize other complexes.
Assessment of oxidative stress resistance. To assess the effect of complexes 1-4 on oxidative stress resistance in wild-type C. elegans, an intracellular redox cycler 5-hydroxy-1,4-naphthoquinone (Juglone, Sigma) was used. The age-sorted L1 stage populations of N2 worms (25~30 individuals/replicate) were exposed to complexes 1-4 and 0.1% DMSO (solvent control) at 20 °C. At late L4 stage (on 6 th day of adulthood), they were then transferred to fresh NGM plates containing 240 μM juglone. The viability of worms was scored after 5 h of the continuous exposure. Three independent trails were performed with appropriate replicates.
C. elegans longevity analysis. For longevity analysis, age synchronized L1 stage worms (25~30 individuals/treatment) were raised on the NGM plates with and without complexes 1-4. A total of 50 μM 5-fluoro-2′-deoxyuridine (FUdR) was added to each plate to prevent the progeny development. During the course of experiment, worms being tested were periodically transferred to new treatment plates to prevent contaminations and to avoid starvation. The live/dead worms were scored at regular interval of every 2-3 days until the end of life. N2, mev-1, daf-16 and mek-1 mutant worms were employed in the lifespan evaluation.
Neuronal survival assay. The transgenic strain HA759 expressing PolyQ tract in ASH neurons was used to detect the neuroprotective effects of Ru(ƞ6-p-cymene) complexes. Synchronized L1 stage worms of HA759 strain was grown on the NGM plates in the presence/absence of complexes 1-4 at 20 °C for 72 h. After that, control and treated C. elegans were placed on a microscopic slide and imaged under fluorescence microscope. About 20~30 randomly selected individuals/treatment were scored for GFP positive/negative in ASH neurons. For chemosensory behaviour assay, the experimental method was taken from Yang 94 .
Assay for PolyQ aggregation. PolyQ aggregation assay was performed using AM141 C. elegans expressing polyQ40::YFP fusion protein in muscle cells, as described earlier 84 . Briefly, age sorted L1 worms were continuously exposed to complex 1-4 at 20 °C for indicated period of time. Thereafter, GFP images were taken using fluorescence microscope and the polyQ40::YFP aggregates in muscle cells of AM141 worms were quantified.
Statistical analysis. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corporation, Armonk, NY, USA) and Excel, Microsoft office 2010 (MicrosoftCorporation, Redmond, WA, USA). For the lifespan assessment, survival of C. elegans was estimated using Kaplan-Meier survival curves and analysed by the log-rank test using MedCalc software, Version 14. Means were compared with untreated/DMSO treated control group using one way analysis of variance (ANOVA) followed by Bonferroni test (posthoc), values of P < 0.05 were considered as statically significant.