ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes.

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens in C. elegans to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting C. elegans ERGU-1 causes excessive H2O2 and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both C. elegans and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, Endoplasmic Reticulum Guardian Aegis of Redox Defense. Human and Drosophila homologs of ERGU-1 can rescue C. elegans mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.

LPD-3 as a megaprotein brake for aging and insulin-mTOR signaling in C. elegans.

Insulin-mechanistic target of rapamycin (mTOR) signaling drives anabolic growth during organismal development; its late-life dysregulation contributes to aging and limits lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here, we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin, INS-7, is drastically overproduced from early life and shortens lifespan in lpd-3 mutants. LPD-3 forms a bridge-like tunnel megaprotein to facilitate non-vesicular cellular lipid trafficking. Lipidomic profiling reveals increased hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1. Reducing the abundance of HYL-1, insulin receptor/DAF-2 or mTOR/LET-363, normalizes INS-7 levels and rescues the lifespan of lpd-3 mutants. LPD-3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age. We propose that LPD-3 acts as a megaprotein brake for organismal aging and that its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Acquired stress resilience through bacteria-to-nematode interdomain horizontal gene transfer.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml-3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L-rhamnose, a cell-wall polysaccharide component. C. elegans rml-3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress-resistant dauer stage. rml-3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L-rhamnose. We propose that interdomain HGT of an ancient bacterial rml-3 homolog has enabled L-rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene.

Acquired stress resilience through bacteria-to-nematode horizontal gene transfer.

Natural selection drives acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisition of functions in immunity, metabolism, and reproduction via interdomain HGT (iHGT) from bacteria. We report that the nematode gene rml-3, which was acquired by iHGT from bacteria, enables exoskeleton resilience and protection against environmental toxins in C. elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most highly similar to bacterial enzymes that biosynthesize L-rhamnose to build cell wall polysaccharides. C. elegans rml-3 is regulated in developing seam cells by heat stress and stress-resistant dauer stage. Importantly, rml-3 deficiency impairs cuticle integrity, barrier functions and organismal stress resilience, phenotypes that are rescued by exogenous L-rhamnose. We propose that iHGT of an ancient bacterial rml-3 homolog enables L-rhamnose biosynthesis in nematodes that facilitates cuticle integrity and organismal resilience in adaptation to environmental stresses during evolution. These findings highlight the remarkable contribution of iHGT on metazoan evolution that is conferred by the domestication of bacterial genes.

Bridge-Like Lipid Transfer Proteins (BLTPs) in C. elegans: From Genetics to Structures and Functions.

In eukaryotic cells, lipid transfer can occur at membrane contact sites (MCS) to facilitate the exchange of various lipids between two adjacent cellular organelle membranes. Lipid transfer proteins (LTPs), including shuttle LTP or bridge-like LTP (BLTP), transport lipids at MCS and are critical for diverse cellular processes, including lipid metabolism, membrane trafficking, and cell signaling. BLTPs (BLTP1-5, including the ATG2 and VPS13 family proteins) contain lipid-accommodating hydrophobic repeating ß-groove (RBG) domains that allow the bulk transfer of lipids through MCS. Compared with vesicular lipid transfer and shuttle LTP, BLTPs have been only recently identified. Their functions and regulatory mechanisms are currently being unraveled in various model organisms and by diverse approaches. In this review, we summarize the genetics, structural features, and biological functions of BLTP in the genetically tractable model organism C. elegans. We discuss our recent studies and findings on C. elegans LPD-3, a prototypical megaprotein ortholog of BLTP1, with identified lipid transfer functions that are evolutionarily conserved in multicellular organisms and in human cells. We also highlight areas for future research of BLTP using C. elegans and complementary model systems and approaches. Given the emerging links of BLTP to several human diseases, including Parkinson's disease and Alkuraya-Kucinskas syndrome, discovering evolutionarily conserved roles of BLTPs and their mechanisms of regulation and action should contribute to new advances in basic cell biology and potential therapeutic development for related human disorders.

LPD-3 as a megaprotein brake for aging and insulin-mTOR signaling in C. elegans.

Insulin-mTOR signaling drives anabolic growth during organismal development, while its late-life dysregulation may detrimentally contribute to aging and limit lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin INS-7 is drastically over-produced in early life and shortens lifespan in lpd-3 mutants, a C. elegans model of human Alkuraya-Kucinskas syndrome. LPD-3 forms a bridge-like tunnel megaprotein to facilitate phospholipid trafficking to plasma membranes. Lipidomic profiling reveals increased abundance of hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1 (Homolog of Yeast Longevity). Reducing HYL-1 activity decreases INS-7 levels and rescues the lifespan of lpd-3 mutants through insulin receptor/DAF-2 and mTOR/LET-363. LPD3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age in wild type animals. We propose that LPD-3 acts as a megaprotein brake for aging and its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Towards the development of phytoextract based healthy ageing cognitive booster formulation, explored through Caenorhabditis elegans model.

The positive effect of herbal supplements on aging and age-related disorders has led to the evolution of natural curatives for remedial neurodegenerative diseases in humans. The advancement in aging is exceedingly linked to oxidative stress. Enhanced oxidative stress interrupts health of humans in various ways, necessitating to find stress alleviating herbal resources. Currently, minimal scientifically validated health and cognitive booster resources are available. Therefore, we explored the impact of plant extracts in different combinations on oxidative stress, life span and cognition using the multicellular transgenic humanized C. elegans, and further validated the same in Mus musculus, besides testing their safety and toxicity. In our investigations, the final product-the HACBF (healthy ageing cognitive booster formulation) thus developed was found to reduce major aging biomarkers like lipofuscin, protein carbonyl, lipid levels and enhanced activity of antioxidant enzymes. Further confirmation was done using transgenic worms and RT-PCR. The cognitive boosting activities analyzed in C. elegans and M. musculus model system were found to be at par with donepezil and L-dopa, the two drugs which are commonly used to treat Parkinson's and Alzheimer's diseases. In the transgenic C. elegans model system, the HACBF exhibited reduced aggregation of misfolded disease proteins α-synuclein and increased the health of nicotinic acetylcholine receptor, levels of Acetylcholine and Dopamine contents respectively, the major neurotransmitters responsible for memory, language, learning behavior and movement. Molecular studies clearly indicate that HACBF upregulated major genes responsible for healthy aging and cognitive booster activities in C. elegans and as well as in M. musculus. As such, the present herbal product thus developed may be quite useful for healthy aging and cognitive boosting activities, and more so during this covid-19 pandemic. Supplementary Information: The online version contains supplementary material available at 10.1007/s13237-022-00407-1.

A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience.

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through conserved homeostatic mechanisms. Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that mediates lipid trafficking to confer cold resilience. We identify lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7. LPD-3 bridges the endoplasmic reticulum (ER) and plasma membranes (PM), forming a structurally predicted hydrophobic tunnel for lipid trafficking. lpd-3 mutants exhibit abnormal phospholipid distribution, diminished FAT-7 abundance, organismic vulnerability to cold, and are rescued by Lecithin comprising unsaturated phospholipids. Deficient lpd-3 homologues in Zebrafish and mammalian cells cause defects similar to those observed in C. elegans. As mutations in BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, LPD-3 family proteins may serve as evolutionarily conserved highway bridges critical for ER-associated non-vesicular lipid trafficking and resilience to cold stress in eukaryotic cells.

Co-opted genes of algal origin protect C. elegans against cyanogenic toxins.

Amygdalin is a cyanogenic glycoside enriched in the tissues of many edible plants, including seeds of stone fruits such as cherry (Prunus avium), peach (Prunus persica), and apple (Malus domestica). These plants biosynthesize amygdalin in defense against herbivore animals, as amygdalin generates poisonous cyanide upon plant tissue destruction.1,2,3,4 Poisonous to many animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals.5 Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1, and cysl-2. A screen of a natural product library for hypoxia-independent regulators of HIF-1 identifies amygdalin as a potent activator of cysl-2, a HIF-1 transcriptional target that encodes a cyanide detoxification enzyme in C. elegans. As a cysl-2 paralog similarly essential for amygdalin resistance, cysl-1 encodes a protein homologous to cysteine biosynthetic enzymes in bacteria and plants but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis indicates that cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Since acquisition, these two genes evolved division of labor in a cellular circuit to detect and detoxify cyanide. Thus, algae-to-nematode HGT and subsequent gene function co-option events may facilitate host survival and adaptation to adverse environmental stresses and biogenic toxins.

Sesquiterpenoids isolated from davana (Artemisia pallens Wall. ex DC) mitigates parkinsonism in Caenorhabditis elegans disease model.

Parkinson's disease (PD) is a multifactorial ailment that severely affects the viability of dopaminergic neurons leading to progressive loss of motor control. The current regimen for PD treatment includes synthetic drugs that lack efficacy and cause serious side effects. Consequently, recent drug development studies are focusing on alternative medicines from plant sources. Artemisia pallens Wall. ex DC, commonly known as davana, is an annual aromatic herb cultivated in southern India. Given the diverse traditional and scientifically documented therapeutic effects of A. pallens, the pharmacological potential of the isolates of the plant, namely bicyclogermacrene (D1), cis-davanone (D3), and cis-hydroxy davanone (D5), was tested for anti-Parkinson's activity in Caenorhabditis elegans model. The tested compounds alleviated α-synuclein (α-syn) aggregation and maximum decline was observed in 25 µM D1 supplemented worms. Additionally, D1 modulated dopamine regulated nonanol-1 repulsion and locomotory behaviour of C. elegans validating its future use as a dopamine-enhancing agent. The genetic regulation mediating the above effects validated through the qPCR study showed that D1 supplementation displayed its anti-Parkinson's effect through upregulation of the antioxidant defence system genes (superoxide dismutase (sod)-1, sod-2, and sod-4) and PD associated pdr-1 gene that maintains the mitochondrial proteostasis. The molecular docking studies of C. elegans PDR-1 with D1 further confirmed its contribution in D1 induced abridgment of Parkinson disease linked pathologies in C. elegans disease model. Hence, this article proposes D1 as an effective regimen for curtailing the Parkinson disease linked pathologies through mechanism of maintaining cellular redox state and proteostasis.

Stress-Induced Phenoptosis: Mechanistic Insights and Evolutionary Implications.

Evolution by natural selection results in biological traits that enable organismic adaptation and survival under various stressful environments. External stresses can be sometimes too severe to overcome, leading to organismic death either because of failure in adapting to such stress, or alternatively, through a regulated form of organismic death (phenoptosis). While regulated cell deaths, including apoptosis, have been extensively studied, little is known about the molecular and cellular mechanisms underlying phenoptosis and its evolutionary significance for multicellular organisms. In this article, we review documented phenomena and mechanistic evidence emerging from studies of stress-induced phenoptosis in the multicellular organism C. elegans and stress-induced deaths at cellular levels in organisms ranging from bacteria to mammals, focusing on abiotic and pathogen stresses. Genes and signaling pathways involved in phenoptosis appear to promote organismic death during severe stress and aging, while conferring fitness and immune defense during mild stress and early life, consistent with their antagonistic pleiotropy actions. As cell apoptosis during development can shape tissues and organs, stress-induced phenoptosis may also contribute to possible benefits at the population level, through mechanisms including kin selection, abortive infection, and soma-to-germline resource allocation. Current models can generate experimentally testable predictions and conceptual frameworks with implications for understanding both stress-induced phenoptosis and natural aging.

Swertiamarin from Enicostemma littorale, counteracts PD associated neurotoxicity via enhancement α-synuclein suppressive genes and SKN-1/NRF-2 activation through MAPK pathway.

The elusive targets and the multifactorial etiology of Parkinson's disease (PD) have hampered the discovery of a potent drug for PD. Furthermore, the presently available medications provide only symptomatic relief and have failed to mitigate the pathogenesis associated with PD. Therefore, the current study was aimed to evaluate the prospective of swertiamarin (SW), a secoiridoid glycoside isolated from a traditional medicinal plant, Enicostemma littorale Blume to ameliorate the characteristic features of PD in Caenorhabditis elegans. SW (25 µM) administration decreased the α-synuclein (α-syn) deposition, inhibited apoptosis and increased dopamine level mediated through upregulating the expression of genes linked to ceramide synthesis, mitochondrial morphology and function regulation, fatty acid desaturase genes along with stress responsive MAPK (mitogen-activated protein kinase) pathway genes. The neuroprotective effect of SW was evident from the robust reduction of 6-hydroxydopamine (6-OHDA) induced dopaminergic neurodegeneration independent of dopamine transporter (dat-1). SW mediated translational regulation of MAPK pathway genes was observed through increase expression of SKN-1 and GST-4. Further, in-silico molecular docking analysis of SW with C. elegans MEK-1 showed a promising binding affinity affirming the in-vivo results. Overall, these novel finding supports that SW is a possible lead for drug development against the multi- factorial PD pathologies.

Wedelolactone Mitigates Parkinsonism Via Alleviating Oxidative Stress and Mitochondrial Dysfunction Through NRF2/SKN-1.

Parkinsonism is an age-associated neurodegenerative disorder characterized by aggregation of α-synuclein (α-syn) protein in the substantia nigra region, degeneration of dopaminergic neurons, and deregulated lipid metabolism. Currently, only symptomatic relief has been provided by FDA-approved therapeutic approaches for Parkinson's disease (PD). The present study aims to evaluate the potential of wedelolactone (WDL), a natural occurring coumestan found in Eclipta alba to mitigate the parkinsonism in Caenorhabditis elegans disease model. In the present studies, supplementation with 37.5 µM WDL exhibited a reduction in the level of α-syn in an age-dependent manner (22% at day 5, p < 0.05; and 16% at day 10, p < 0.001, n = 30), along with improvement in neuronal health through basal movement, and elevated the dopamine levels evident through 1-nonanol repulsion results in wild-type and diseased worms. Moreover, WDL augmented the mitochondrial health in wild-type, PD-diseased, and mev-1 mutant worms that establish the inherent activity of WDL in the alleviation of oxidative stress. Furthermore, WDL supplementation significantly decreases the neutral lipid and triglyceride level and also alleviates protein carbonyl level in PD disease condition. The overall investigation will provide a pioneer to the future insights of PD research related to plant-based drugs. qPCR studies after WDL supplementation revealed alteration of genes involved in the regulation of various stress-responsive (sod-5, gst-4, skn-1), α-syn-suppressing (lrk-1, ymel-1, lagr-1, grk-1), and mitochondrial (pink-1) genes. All together, these findings support that the WDL is a promising candidate to combat age-related multi-factorial PD pathology associated with protein misfolding and accumulation. The results provide sufficient information in the development of therapeutic medicines from natural products for improving the health.

A Bioactive compound Shatavarin IV-mediated longevity as revealed by dietary restriction-induced autophagy in Caenorhabditis elegans.

Plant-based dietary supplements that delay aging are of significant interest now a days because these naturally occurring bioactive molecules effectively provide pharmaceuticals/neutraceuticals to deal with diseases related to the advanced life expectancy. In this paper, we aimed to investigate the effect of Shatavarin IV (SIV), a steroidal saponin isolated from Asparagus racemosus Willd. on dietary restriction (DR) induced longevity in Caenorhabditis elegans. SIV significantly increased the lifespan to 18% which is independent of antimicrobial activity and reduced the aging by-product, lipofuscin along with increased locomotion, and chemotaxis behavior in wild type worms. The longevity effect has been dependent on eat-2, which was further validated via reduced pharyngeal pumping rate that established the effect similar to DR induced longevity. Moreover, like eat-2 mutant worms, SIV reduces the total progeny number of wild type worm along with a significant alleviation of stored fat, which reconfirms the involvement of eat-2 mediated longevity. Further, it was also observed that DR induced longevity mechanism by SIV requires mTOR which works in PHA-4/FOXA dependent manner. In addition to this, the role of autophagy mechanism concerning SIV mediated DR was confirmed via bec-1, unc-51, and lgg-1. The longevity effect achieved by SIV was also dependent on SKN-1/NRF-2 and partially dependent on DAF-16/FOXO. Furthermore, the DR-induced longevity by SIV was found to be independent of hsf-1 exhibiting non-significant alteration in the mRNA expression of downstream target genes hsp-16.2 and hsp-70. Altogether, this study provides first-hand information on the pro-longevity effect of SIV in worms that have been mediated by the DR-regulating gene induced autophagy.

Swertiamarin, a secoiridoid glycoside modulates nAChR and AChE activity.

The ailments related to a malfunction in cholinergic functioning currently employ the use of inhibitors for acetylcholinesterase (AChE) and N-methyl-d-aspartate (NMDA) receptors. The present study was designed to elucidate the potential of swertiamarin (SW), a secoiridoidal glycoside isolated from Enicostemma littorale in curtailing the cholinergic dysfunction. Using Caenorhabditis elegans as a model, SW was found to enhance neurotransmission by modulating AChE and nicotinic acetylcholine receptor (nAChR) activity; being orchestrated through up-regulation of unc-17 and unc-50. SW exhibited AChE inhibition both in vivo and cell-free system. The in silico molecular docking of SW and human AChE (hAChE) displayed good binding energy of -6.02. Interestingly, the increase in aldicarb and levamisole sensitivity post SW treatment was curtailed to a significant level in daf-16 and skn-1 mutants. SW raised the level of the endogenous antioxidant enzymes through up-regulation of sod-3 and gst-4 that act downstream to DAF-16 and SKN-1, imparting protection against neurodegeneration. The outcome of our study displays SW as a potential natural molecule for the amelioration of cholinergic dysfunction. Moreover, the study also indicates that SW elicits antioxidant response via up-modulation of daf-16 possibly through unc-17 upregulation. Further research on SW pertaining to the underlying mechanism and potential is expected to significantly advance the current understanding and design of possible ameliorative or near ameliorative regimens for cholinergic dysfunction.

Anti-ageing and anti-Parkinsonian effects of natural flavonol, tambulin from Zanthoxyllum aramatum promotes longevity in Caenorhabditis elegans.

Ageing is a progressive deterioration in functional and structural well-being of the body, accompanied with age-associated neurological disorders such as Parkinson's disease (PD), Alzheimer's disease and Huntington's disease. PD is marked with motor function decline, progressive neurodegeneration due to aggregation of insoluble α-synuclein in the dopaminergic neuron. Here we investigated the effect of tambulin (3,5-dihydroxy-7,8-dimethoxy-2-(4-methoxyphenyl) chromen-4-one), a hydroxy substituted flavanol isolated from fruits of Zanthoxyllum armatum DC (Family-Rutaceae) for its longevity promoting and neuromodulatory activities using Caenorhabditis elegans model system. Our results show that tambulin treatment significantly enhance lifespan and stress tolerance in worms, along with mitigation of ageing biomarkers like lipofuscin and protein carbonyl. In line with the alleviated ROS levels, tambulin treatment led to upregulated mRNA expression of ROS scavenging genes viz., sod-1, sod-3, and ctl-2. Upregulation in daf-16 gene indicates the involvement of insulin signaling pathway in tambulin mediated longevity. Tambulin treatment exhibited curtailed PD manifestations in terms of reduced α-synuclein levels, lipid accumulation, improved locomotary behavior and dopamine levels. Altogether, our data suggest that tambulin mediated alleviation of PD manifestations possibly involved PD counter protective machinery as evident through upregulated mRNA expression of lagr-1, ymel-1, pdr-1, ubc-12, and lrk-1. Our studies present tambulin as a potential molecule for its properties against ageing and Parkinson's disease. Further studies are speculated to realize the mechanistic and pharmacological aspects of tambulin.