Dimitriadi M et al. (2013) J Neurosci "The neuroprotective drug riluzole acts via small conductance Ca2+-activated K+ channels to ameliorate defects in spinal muscular atrophy models."

Sahin M, Hart AC, Dimitriadi M, Kalloo G, Kye MJ, Yersak JM

[J Neurosci, 2013]

Spinal muscular atrophy (SMA), a recessive neuromuscular disorder, is caused by diminished function of the Survival Motor Neuron (SMN) protein. To define the cellular processes pertinent to SMA, parallel genetic screens were undertaken in Drosophila and Caenorhabditis elegans SMA models to identify modifiers of the SMN loss of function phenotypes. One class of such genetic modifiers was the small conductance, Ca(2+)-activated K(+) (SK) channels. SK channels allow efflux of potassium ions when intracellular calcium increases and can be activated by the neuroprotective drug riluzole. The latter is the only drug with proven, albeit modest, efficacy in the treatment of amyotrophic lateral sclerosis. It is unclear if riluzole can extend life span or ameliorate symptoms in SMA patients as previous studies were limited and of insufficient power to draw any conclusions. The critical biochemical target of riluzole in motor neuron disease is not known, but the pharmacological targets of riluzole include SK channels. We examine here the impact of riluzole in two different SMA models. In vertebrate neurons, riluzole treatment restored axon outgrowth caused by diminished SMN. Additionally, riluzole ameliorated the neuromuscular defects in a C. elegans SMA model and SK channel function was required for this beneficial effect. We propose that riluzole improves motor neuron function by acting on SK channels and suggest that SK channels may be important therapeutic targets for SMA patients.

Krabbendam IE et al. (2020) Cell Death Dis "SK channel-mediated metabolic escape to glycolysis inhibits ferroptosis and supports stress resistance in C. elegans."

Honrath B, Dolga AM, Branicky RS, Dilberger B, Evers B, Iannetti EF, Hekimi S, Meyer T, Bano D, Bakker BM, Krabbendam IE, Koopman WJH, Beyrath J, Dekker FJ, Eckert GP, Culmsee C, Schmidt M

[Cell Death Dis, 2020]

Metabolic flexibility is an essential characteristic of eukaryotic cells in order to adapt to physiological and environmental changes. Especially in mammalian cells, the metabolic switch from mitochondrial respiration to aerobic glycolysis provides flexibility to sustain cellular energy in pathophysiological conditions. For example, attenuation of mitochondrial respiration and/or metabolic shifts to glycolysis result in a metabolic rewiring that provide beneficial effects in neurodegenerative processes. Ferroptosis, a non-apoptotic form of cell death triggered by an impaired redox balance is gaining attention in the field of neurodegeneration. We showed recently that activation of small-conductance calcium-activated K⁺ (SK) channels modulated mitochondrial respiration and protected neuronal cells from oxidative death. Here, we investigated whether SK channel activation with CyPPA induces a glycolytic shift thereby increasing resilience of neuronal cells against ferroptosis, induced by erastin in vitro and in the nematode C. elegans exposed to mitochondrial poisons in vivo. High-resolution respirometry and extracellular flux analysis revealed that CyPPA, a positive modulator of SK channels, slightly reduced mitochondrial complex I activity, while increasing glycolysis and lactate production. Concomitantly, CyPPA rescued the neuronal cells from ferroptosis, while scavenging mitochondrial ROS and inhibiting glycolysis reduced its protection. Furthermore, SK channel activation increased survival of C. elegans challenged with mitochondrial toxins. Our findings shed light on metabolic mechanisms promoted through SK channel activation through mitohormesis, which enhances neuronal resilience against ferroptosis in vitro and promotes longevity in vivo.

Nam YW et al. (2018) Sci Rep "A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model."

Zhang M, Nam YW, Gazgalis D, Orfali R, Baskoylu SN, Cui M, Hart AC

[Sci Rep, 2018]

Small-conductance Ca²⁺-activated K⁺ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca²⁺-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca²⁺/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca²⁺ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP₂-interacting residue K405, and subsequently enhanced Ca²⁺ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca²⁺ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca²⁺ with increased activity in vivo.

Yu N et al. (2013) Peptides "Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption."

Zabrocki J, Benzi V, Smagghe G, Staljanssens D, Kaczmarek K, Yu N, Nachman RJ, Zotti MJ

[Peptides, 2013]

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.

Kolathur KK et al. (2022) FEBS Lett "The ubiquitin-like protein Hub1/UBL-5 functions in pre-mRNA splicing in Caenorhabditis elegans."

Kolathur KK, Nishitha A, Kadam NY, Babu K, Shahi N, Mishra SK, Sharma P

[FEBS Lett, 2022]

The ubiquitin-like protein Hub1/UBL-5 associates with proteins non-covalently. Hub1 promotes alternative splicing and splicing of precursor mRNAs with weak introns in yeast and mammalian cells; however, its splicing function has remained elusive in multicellular organisms. Here, we demonstrate the splicing function of Hub1/UBL-5 in the free-living nematode Caenorhabditis elegans. Hub1/UBL-5 binds to the HIND-containing splicing factors Snu66/SART-1 and PRP-38 and associates with other spliceosomal proteins. C. elegans hub1/ubl-5 mutants die at the larval L3 stage and show splicing defects for selected targets, similar to the mutants in yeast and mammalian cells. UBL-5 complemented growth and splicing defects in Schizosaccharomyces pombe hub1 mutants, confirming its functional conservation. Thus, UBL-5 is important for C. elegans development and plays a conserved pre-mRNA splicing function.

Misra S et al. (2011) Parasitol Res "Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host."

Srivastava S, Lakshmi V, Misra S, Verma M, Misra-Bhattacharya S, Mishra SK

[Parasitol Res, 2011]

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 g/ml; mf = 13.17 g/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 x 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 g/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 g/ml, CC50 = 212.5 g/ml, SI = 889.1) and photogedunin (IC50 = 0.213 g/ml, CC50 = 262.3 g/ml, SI = 1231.4) at 5 x 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.

Vijayan B et al. (2018) Cell Biol Toxicol "Spermine protects alpha-synuclein expressing dopaminergic neurons from manganese-induced degeneration."

Raj V, Thekkuveettil A, Vijayan B, Nandakumar S, Kishore A

[Cell Biol Toxicol, 2018]

Manganese exposure is among the many environmental risk factors linked to the progression of neurodegenerative diseases, such as manganese-induced parkinsonism. In animal models, chronic exposure to manganese causes loss of cell viability, neurodegeneration, and functional deficits. Polyamines, such as spermine, have been shown to rescue animals from age-induced neurodegeneration in an autophagy-dependent manner; nonetheless, it is not understood whether polyamines can prevent manganese-induced toxicity. In this study, we used two model systems, the Caenorhabditis elegans UA44 strain and SK-MEL-28 cells, both expressing the protein alpha-synuclein (-syn) to determine whether spermine could ameliorate manganese-induced toxicity. Manganese caused a substantial reduction in the viability of SK-MEL-28 cells and hastened neurodegeneration in the UA44 strain. Spermine protected both the SK-MEL-28 cells and the UA44 strain from manganese-induced toxicity. Spermine also reduced the age-associated neurodegeneration observed in the UA44 strain compared with a control strain without -syn expression and led to improved avoidance behavior in a functional assay. Treatment with berenil, an inhibitor of polyamine catabolism, which leads to increased intracellular polyamine levels, also showed similar cellular protection against manganese toxicity. While both translation blocker cycloheximide and autophagy blocker chloroquine caused a reduction in the cytoprotective effect of spermine, transcription blocker actinomycin D had no effect. This study provides new insights on the effect of spermine in preventing manganese-induced toxicity, which is most likely via translational regulation of several candidate genes, including those of autophagy. Thus, our results indicate that polyamines positively influence neuronal health, even when exposed to high levels of manganese and -syn, and supplementing polyamines through diet might delay the onset of diseases involving degeneration of dopaminergic neurons.

Gupta J et al. (2012) Exp Parasitol "Antifilarial activity of marine sponge Haliclona oculata against experimental Brugia malayi infection."

Misra S, Lakshmi V, Srivastava S, Misra-Bhattacharya S, Mishra SK, Gupta J, Srivastava MN

[Exp Parasitol, 2012]

The present study incorporates the findings on in vitro and in vivo antifilarial activity in the marine sponge, Haliclona oculata using an experimental rodent infection of human lymphatic filarial parasite, Brugia malayi. The in vitro antifilarial action was determined on both adult female worms as well as microfilariae using two parameters viz. adverse effect on motility and inhibition in MTT reduction by the treated adult parasite over control worm. The antifilarial activity could be located in the methanol extract and one of its four fractions (chloroform). Bioactivity guided fractionation of chloroform fraction led to localization of in vitro activity in one of its eight chromatographic fractions. Methanol extract, chloroform fraction and one of the chromatographic fractions revealed IC(50) values of 5.00, 1.80, and 1.62g/ml, respectively when adult B. malayi were exposed to these test samples for 72h at 37C. Under similar exposure conditions, the IC(50) values for microfilariae were 1.88, 1.72 and 1.19g/ml, respectively. The active test samples were found to be safe revealing >10 selectivity indices (SI) on the basis of cytotoxicity to Vero cells (monkey kidney cells) and therefore selected for in vivo evaluation against primary (adult B. malayi intraperitoneal transplanted jird) and secondary (subcutaneous infective larvae induced mastomys) screens. In primary jird model, the three test samples at 100mg/kg for five consecutive days by subcutaneous route demonstrated significant macrofilaricidal efficacy to the tune of 51.3%, 64% and 70.7% by methanol extract, chloroform and chromatographic fraction, respectively. The three samples demonstrated 45-50% macrofilaricidal activity with moderate embryostatic effect in secondary model at 5x500, 5x250 and 5x125mg/kg by oral route. Chromatographic fraction possessing highest antifilarial action was primarily found to be a mixture of four alkaloids Mimosamycin, Xestospongin-C, Xestospongin-D and Araguspongin-C in addition to few minor compounds.

Lakshmi V et al. (2010) Acta Trop "Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi."

Dube V, Murthy PK, Joseph SK, Verma SK, Mishra SK, Lakshmi V, Srivastava S, Sahoo MK

[Acta Trop, 2010]

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 g/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 g/ml. The half maximal inhibitory concentration (IC(50)) of naringenin for motility of adult females was 2.5 g/ml. Flavone immobilized female adult worms at 31.2 g/ml (MTT>80%) and microfilariae at 62.5 g/ml. Rutin killed microfilariae at 125 g/ml and inhibited MTT-reduction in female worms for >65% at 500 g/ml. Naringin had adulticidal effects at 125 g/ml while chrysin killed microfilariae at 250 g/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin>flavone=hesperetin>rutin>naringin>chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.

Pandey S et al. (2019) Comp Biochem Physiol C Toxicol Pharmacol "Betula utilis extract prolongs life expectancy, protects against amyloid- toxicity and reduces Alpha Synuclien in Caenorhabditis elegans via DAF-16 and SKN-1."

Bajpai R, Lehri A, Phulara SC, Pandey S, Chauhan PS, Niranjan A, Upreti DK, Mishra SK, Kumar A

[Comp Biochem Physiol C Toxicol Pharmacol, 2019]

Betula utilis (BU), an important medicinal plant that grows in high altitudes of the Himalayan region, has been utilized traditionally due to it's antibacterial, hepatoprotective, and anti-tumor properties. Here, we demonstrated the longevity and amyloid- toxicity attenuating activity of B. utilis ethanolic extract (BUE) in Caenorhabditis elegans. Lifespan of the worms was observed under both the standard laboratory and stress (oxidative and thermal) conditions. Effect of BUE was also observed on the attenuation of age-dependent physiological parameters. Further, gene-specific mutants and green fluorescent protein (GFP)-tagged strains were used to investigate the molecular mechanism underlying the beneficial effects mediated by BUE supplementation. Our results showed that BUE (50g/ml) extended the mean lifespan of C. elegans by 35.99% and increased its survival under stress conditions. The BUE also reduced the levels of intracellular reactive oxygen species (ROS) by 22.47%. A delayed amyloid- induced paralyses was observed in CL4176 transgenic worms. Interestingly, the BUE supplementation was also able to reduce the -synuclein aggregation in NL5901 transgenic strain. Gene-specific mutant studies suggested that the BUE-mediated lifespan extension was dependent on daf-16, hsf-1, and skn-1 but not on sir-2.1 gene. Furthermore, transgenic reporter gene expression assay showed that BUE treatment enhanced the expression of stress-protective genes such as sod-3 and gst-4. Present findings suggested that ROS scavenging activity, together with multiple longevity mechanisms, were involved in BUE-mediated lifespan extension. Thus, BUE might have potential to increase the lifespan and to attenuate neuro-related disease progression.