Srivastava, S. et al. (2017) International Worm Meeting "2-Isopropyl-5-methylphenol ameliorates alpha-synuclein proteotoxicity and intracellular ROS accumulation in Caenorhabditis elegans."

Srivastava, S., Pandey, R.

[International Worm Meeting, 2017]

Parkinson's disease (PD) is a progressive neuronal disorder, leading to cognitive disorder and bradykinesia in patients. PD is characterized by aggregated a-synuclein and dopaminergic neurodegeneration, related with elevated ROS level and other biological pathways. As hitherto, a paucity of any potent medication against PD suggesting everlasting efficacy has stimulated advances in terms of research and quest for newer drugs. However, some novel therapies involving plant molecules are being exploited for adjunctive therapy and synthetic medicaments. Caenorhabditis elegans serves as a suitable in-vivo model with a healthier approximation of the real environment in the human body than in-vitro tests. In this venture, the present study was fabricated for the first time to decipher anti-Parkinson's effects of 2-Isopropyl-5-methylphenol (IPMP) employing C. elegans. IPMP is derived from Trachyspermum ammi, which contains anti-inflammatory and anti-apoptotic activity but no reports have yet described its effect on PD. Our study showed that IPMP significantly reduces a- synuclein aggregation within the worm's body. IPMP administration resulted in enhanced repulsive behavior towards chemorepellant, 1-nonanol indicating increased dopamine levels, which was further quantified by LC-MS/MS. Additionally, upon treatment with 6-OHDA, IPMP attenuates 6-OHDA-induced degeneration of DA neuron in worms. IPMP curtails ROS level and oxidative stress using H2-DCFDA and 5-Hydroxy-1,4-naphthoquinone, respectively. In the lead, staining with acridine orange, IPMP exhibited a significant decline in apoptosis. Altered lipid levels, another manifestation of PD were also found to normalize significantly in both wild type worms and NL5901 worms (Punc-54::a-synuclein::YFP+unc-119). Moreover, we furthered our studies towards deploying a qPCR technique for enumerating transcript expression of selected genes screened as a-synuclein modifiers from a genome-wide screen in C. elegans model of Parkinson's disease. In wild-type worms, we perceived a significant up-regulation of pink-1, ubc-12, spp-9, grk-2, lrk-1 and lagr-1 whereas pink-1, ubc-12, spp-9, grk-2 and pdr-1, mRNA expression levels were augmented in NL5901 worms. In concise, we conclude imminent areas of investigation, about the emerging nature of IPMP that can be exploited to provide mechanistic perceptions toward therapeutic intervention for PD. We hypothesize that the observed beneficial effect establishes the potential of IPMP as a possible candidate for ameliorative or supportive roles in the management of Parkinsonism.

Srivastava S et al. (2016) Environ Toxicol Pharmacol "Curcumin and -caryophellene attenuate cadmium quantum dots induced oxidative stress and lethality in Caenorhabditis elegans model system."

Trivedi S, Pandey R, Pant A, Srivastava S

[Environ Toxicol Pharmacol, 2016]

Curcumin (CUR) and -caryophellene (BCP) are well known bioactive phytomolecules which are known to reduce oxidative stress in living organisms. Therefore, the present study was envisaged to explore the possible effects of CUR and BCP in suppression of cadmium quantum dots (CdTe QDs) induced toxicity in Caenorhabditis elegans. CdTe QD are luminescent nanoparticles extensively exploited for in vivo imaging, but long term bioaccumulation confer deleterious effects on living organisms. The 24-h LC50 and LC100 of CdTe QD were found to be 18.40g/ml and 100g/ml respectively. The CdTe QD exposure elevated HSP-16.2 expression mediating induction of the stress response. The CdTe QD lethality was due to increment in ROS and decline in SOD and GST expression. The present study demonstrates improved survival in BCP (50M) and CUR (20M) treated worms by over 60% (P<0.01) and 50% (P<0.029) in CdTe QD (100g/ml) exposed worms. Furthermore, BCP and CUR attenuate oxidative stress triggered by QD. The present study for the first time demonstrates CdTe QD toxicity remediation via BCP and CUR. The future investigations can unravel underlying protective effects of phytomolceules for remediating cyotoxicolgical effects of QDs.

Srivastava S et al. (2005) Mol Cell Biol "The phosphatidylinositol 3-phosphate phosphatase myotubularin- related protein 6 (MTMR6) is a negative regulator of the Ca2+-activated K+ channel KCa3.1."

Liu G, Li Z, Ko K, Skolnik EY, Coetzee WA, Srivastava S, Lin L

[Mol Cell Biol, 2005]

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3' position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P(2). MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca(2+)-activated K channel, K(Ca)3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K(Ca)3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K(Ca)3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K(Ca)3.1, indicating that MTM CC domains are sufficient to confer target specificity. K(Ca)3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K(Ca)3.1 by MTMR6 overexpression. These data, when taken together, indicate that K(Ca)3.1 is regulated by PI(3)P and that MTMR6 inhibits K(Ca)3.1 by dephosphorylating the 3' position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K(Ca)3.1. K(Ca)3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K(Ca)3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Deforce D, Coenye T, Van Nieuwerburgh F, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Rathor L et al. (2015) Age (Dordr) "Folic acid supplementation at lower doses increases oxidative stress resistance and longevity in Caenorhabditis elegans."

Pandey S, Rathor L, Srivastava S, Pandey R, Akhoon BA

[Age (Dordr), 2015]

Folic acid (FA) is an essential nutrient that the human body needs but cannot be synthesized on its own. Fortified foods and plant food sources such as green leafy vegetables, beans, fruits, and juices are good sources of FA to meet the daily requirements of the body. The aim was to evaluate the effect of dietary FA levels on the longevity of well-known experimental aging model Caenorhabditis elegans. Here, we show for first time that FA extends organism life span and causes a delay in aging. We observed that FA inhibits mechanistic target of rapamycin (mTOR) and insulin/insulin growth factor 1 (IGF-1) signaling pathways to control both oxidative stress levels and life span. The expression levels of stress- and life span-relevant gerontogenes, viz. daf-16, skn-1, and sir. 2.1, and oxidative enzymes, such as glutathione S-transferase 4 (GST-4) and superoxide dismutase 3 (SOD-3), were also found to be highly enhanced to attenuate the intracellular reactive oxygen species (ROS) damage and to delay the aging process. Our study promotes the use of FA to mitigate abiotic stresses and other aging-related ailments.

Pokharel DR et al. (2006) Vaccine "Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in jirds."

Nandakumar Kodumudi K, Rai R, Pokharel DR, Rathaur S, Reddy MV

[Vaccine, 2006]

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Vandecandelaere I et al. (2014) Pathog Dis "Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms."

Coenye T, Vandecandelaere I, Depuydt P, Nelis HJ

[Pathog Dis, 2014]

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Kamp F et al. (2010) EMBO J "Inhibition of mitochondrial fusion by -synuclein is rescued by PINK1, Parkin and DJ-1."

Haass C, Hegermann J, Giese A, Eimer S, Kamp F, Lutz AK, Nuscher B, Wender N, Brunner B, Winklhofer KF, Exner N, Beyer K, Bartels T

[EMBO J, 2010]

Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.