Mitochondrial stress in GABAergic neurons non-cell autonomously regulates organismal health and aging.

Mitochondrial stress within the nervous system can trigger non-cell autonomous responses in peripheral tissues. However, the specific neurons involved and their impact on organismal aging and health have remained incompletely understood. Here, we demonstrate that mitochondrial stress in γ-aminobutyric acid-producing (GABAergic) neurons in Caenorhabditis elegans ( C. elegans ) is sufficient to significantly alter organismal lifespan, stress tolerance, and reproductive capabilities. This mitochondrial stress also leads to significant changes in mitochondrial mass, energy production, and levels of reactive oxygen species (ROS). DAF-16/FoxO activity is enhanced by GABAergic neuronal mitochondrial stress and mediates the induction of these non-cell-autonomous effects. Moreover, our findings indicate that GABA signaling operates within the same pathway as mitochondrial stress in GABAergic neurons, resulting in non-cell-autonomous alterations in organismal stress tolerance and longevity. In summary, these data suggest the crucial role of GABAergic neurons in detecting mitochondrial stress and orchestrating non-cell-autonomous changes throughout the organism.

Genetic and Chemical Controls of Sperm Fate and Spermatocyte Dedifferentiation via PUF-8 and MPK-1 in Caenorhabditis elegans.

Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) repress sperm fate at 20 °C and the dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") at 25 °C. Thus, double mutants lacking both PUF-8 and LIP-1 produce excess sperm at 20 °C, and their spermatocytes return to mitotically dividing cells via dedifferentiation at 25 °C, resulting in germline tumors. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes of three mutant strains that produce excess sperm-fem-3(q20gf), puf-8(q725); fem-3(q20gf), and puf-8(q725); lip-1(zh15). Spermatocyte dedifferentiation was not observed in fem-3(q20gf) mutants, but it was more severe in puf-8(q725); lip-1(zh15) than in puf-8(q725); fem-3(q20gf) mutants. These results suggest that MPK-1 (the C. elegans ERK1/2 MAPK ortholog) activation in the absence of PUF-8 is required to promote spermatocyte dedifferentiation. This idea was confirmed using Resveratrol (RSV), a potential activator of MPK-1 and ERK1/2 in C. elegans and human cells, respectively. Notably, spermatocyte dedifferentiation was significantly enhanced by RSV treatment in the absence of PUF-8, and its effect was blocked by mpk-1 RNAi. We, therefore, conclude that PUF-8 and MPK-1 are essential regulators for spermatocyte dedifferentiation and tumorigenesis. Since these regulators are broadly conserved, we suggest that similar regulatory circuitry may control cellular dedifferentiation and tumorigenesis in other organisms, including humans.

Non-invasive cancer detection in canine urine through Caenorhabditis elegans chemotaxis.

Cancer is the leading cause of death in companion animals, and successful early treatment has been a challenge in the veterinary field. We have developed the Non-Invasive Cancer Screening (N.C.S.) Study to perform cancer detection through the analysis of canine urine samples. The test makes use of the strong olfactory system of the nematode Caenorhabditis elegans, which was previously shown to positively respond to urine samples from human cancer patients. We performed a proof-of-concept study to optimize the detection capability in urine samples obtained from dogs with naturally occurring cancers. In this study, we established a scale for identifying the cancer risk based on the magnitude of the chemotaxis index of C. elegans toward a canine urine sample. Through validation, the N.C.S. Study achieved a sensitivity of 85%, showing that it is highly sensitive to indicate the presence of cancer across multiple types of common canine cancers. The test also showed a 90% specificity to cancer samples, indicating a low rate of over-identifying cancer risk. From these results, we have demonstrated the ability to perform low-cost, non-invasive cancer detection in companion animals-a method that can increase the ability to perform cancer diagnosis and treatment.

Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death.

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.

Triclosan induces apoptosis in Burkitt lymphoma-derived BJAB cells through caspase and JNK/MAPK pathways.

Burkitt's lymphoma (BL) is the fastest growing human tumor. Current treatment consists of a multiagent regimen of cytotoxic drugs with serious side effjects including tumor lysis, cardiotoxicity, hepatic impairment, neuropathy, myelosuppression, increased susceptibility to malignancy, and death. Furthermore, therapeutic interventions in areas of BL prevalence are not as feasible as in high-income countries. Therefore, there exists an urgent need to identify new therapies with a safer profile and improved accessibility. Triclosan (TCS), an antimicrobial used in personal care products and surgical scrubs, has gained considerable interest as an antitumor agent due to its interference with fatty acid synthesis. Here, we investigate the antitumor properties and associated molecular mechanisms of TCS in BL-derived BJAB cells. Dose-dependent cell death was observed following treatment with 10-100 µM TCS for 24 h, which was associated with membrane phospholipid scrambling, compromised permeability, and cell shrinkage. TCS-induced cell death was accompanied by elevated intracellular calcium, perturbed redox balance, chromatin condensation, and DNA fragmentation. TCS upregulated Bad expression and downregulated that of Bcl2. Moreover, caspase and JNK MAPK signaling were required for the full apoptotic activity of TCS. In conclusion, this report identifies TCS as an antitumor agent and provides new insights into the molecular mechanisms governing TCS-induced apoptosis in BL cells.

The soma-germline communication: implications for somatic and reproductive aging.

Aging is characterized by a functional decline in most physiological processes, including alterations in cellular metabolism and defense mechanisms. Increasing evidence suggests that caloric restriction extends longevity and retards age-related diseases at least in part by reducing metabolic rate and oxidative stress in a variety of species, including yeast, worms, flies, and mice. Moreover, recent studies in invertebrates - worms and flies, highlight the intricate interrelation between reproductive longevity and somatic aging (known as disposable soma theory of aging), which appears to be conserved in vertebrates. This review is specifically focused on how the reproductive system modulates somatic aging and vice versa in genetic model systems. Since many signaling pathways governing the aging process are evolutionarily conserved, similar mechanisms may be involved in controlling soma and reproductive aging in vertebrates. [BMB Reports 2021; 54(5): 253-259].

Nucleotide Excision Repair, XPA-1, and the Translesion Synthesis Complex, POLZ-1 and REV-1, Are Critical for Interstrand Cross-Link Repair in Caenorhabditis elegans Germ Cells.

Interstrand cross-links (ICLs) are adducts of covalently linked nucleotides in opposing DNA strands that obstruct replication and prime cells for malignant transformation or premature cell death. ICLs may be caused by alkylating agents or ultraviolet (UV) irradiation. These toxic lesions are removed by diverse repair mechanisms such as the Fanconi anemia (FA) pathway, nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR). In mammals, the xeroderma pigmentosum group F (XP-F) protein participates in both the FA pathway and NER, while DNA polymerase ζ (POLZ-1) and REV-1 mediate TLS. Nevertheless, little is known regarding the genetic determinants of these pathways in ICL repair and damage tolerance in germ cells. In this study, we examined the sensitivity of Caenorhabditis elegans germ cells to ICLs generated by trimethylpsoralen/ultraviolet A (TMP/UV-A) combination, and embryonic mortality was employed as a surrogate for DNA damage in germ cells. Our results show that XPA-1, POLZ-1, and REV-1 were more critical than FA pathway mediators in preserving genomic stability in C. elegans germ cells. Notably, mutant worms lacking both XPA-1 and POLZ-1 (or REV-1) were more sensitive to ICLs compared to either single mutant alone. Moreover, knockdown of XPA-1 and REV-1 leads to the retarded disappearance of RPA-1 and RAD-51 foci upon ICL damage. Since DNA repair mechanisms are broadly conserved, our findings may have ramifications for prospective therapeutic interventions in humans.

Distinct roles of two eIF4E isoforms in the germline of Caenorhabditis elegans.

Germ cells use both positive and negative mRNA translational control to regulate gene expression that drives their differentiation into gametes. mRNA translational control is mediated by RNA-binding proteins, miRNAs and translation initiation factors. We have uncovered the discrete roles of two translation initiation factor eIF4E isoforms (IFE-1, IFE-3) that bind 7-methylguanosine (m7G) mRNA caps during Caenorhabditiselegans germline development. IFE-3 plays important roles in germline sex determination (GSD), where it promotes oocyte cell fate and is dispensable for spermatogenesis. IFE-3 is expressed throughout the germline and localizes to germ granules, but is distinct from IFE-1 and PGL-1, and facilitates oocyte growth and viability. This contrasts with the robust expression in spermatocytes of IFE-1, the isoform that resides within P granules in spermatocytes and oocytes, and promotes late spermatogenesis. Each eIF4E is localized by its cognate eIF4E-binding protein (IFE-1:PGL-1 and IFE-3:IFET-1). IFE-3 and IFET-1 regulate translation of several GSD mRNAs, but not those under control of IFE-1. Distinct mutant phenotypes, in vivo localization and differential mRNA translation suggest independent dormant and active periods for each eIF4E isoform in the germline.

Dose-Dependent Effects of GLD-2 and GLD-1 on Germline Differentiation and Dedifferentiation in the Absence of PUF-8.

PUMILIO/FBF (PUF) proteins have a conserved function in stem cell regulation. Caenorhabditis elegans PUF-8 protein inhibits the translation of target mRNAs by interacting with PUF binding element (PBE) in the 3' untranslated region (3' UTR). In this work, an in silico analysis has identified gld-2 [a poly(A) polymerase] as a putative PUF-8 target. Biochemical and reporter analyses showed that PUF-8 specifically binds to a PBE in gld-2 3' UTR and represses a GFP reporter gene carrying gld-2 3' UTR in the C. elegans mitotic germ cells. GLD-2 enhances meiotic entry at least in part by activating GLD-1 (a KH motif-containing RNA-binding protein). Our genetic analyses also demonstrated that heterozygous gld-2(+/-) gld-1(+/-) genes in the absence of PUF-8 are competent for meiotic entry (early differentiation), but haplo-insufficient for the meiotic division (terminal differentiation) of spermatocytes. Indeed, the arrested spermatocytes return to mitotic cells via dedifferentiation, which results in germline tumors. Since these regulators are broadly conserved, we thus suggest that similar molecular mechanisms may control differentiation, dedifferentiation, and tumorigenesis in other organisms, including humans.

RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs.

The differentiation of mesenchymal stem cells (MSCs) into unwanted lineages can generate potential problems in clinical trials. Thus, understanding the molecular mechanisms, involved in this process, would help prevent unexpected complications. Regulation of gene expression, at the posttranscriptional level, is a new approach in cell therapies. PUMILIO is a conserved posttranscriptional regulator. However, the underlying mechanisms of PUMILIO, in vertebrate stem cells, remain elusive. Here, we show that depletion of PUMILIO2 (PUM2) blocks MSC adipogenesis and enhances osteogenesis. We also demonstrate that PUM2 works as a negative regulator on the 3'-untranslated regions of JAK2 and RUNX2 via direct binding. CRISPR/Cas9-mediated gene silencing of Pum2 inhibited lipid accumulation and induced excessive bone formation in zebrafish larvae. Our findings reveal novel roles of PUM2 in MSCs and provide potential therapeutic targets for related diseases.

Hypersensitivity to DNA double-strand breaks associated with PARG deficiency is suppressed by exo-1 and polq-1 mutations in Caenorhabditis elegans.

Deficiency of either of the two homologs of poly(ADP-ribose) glycohydrolase (PARG), PARG-1 and PARG-2, in Caenorhabditis elegans leads to hypersensitivity to ionizing radiation (IR). In the germ cells of parg-2 mutant worms, the dissipation of recombinase RAD-51 foci was slower than in wild-type (WT) cells, suggesting defects in DNA double-strand break (DSB) repair via homologous recombination (HR). Nevertheless, RPA-1, the large subunit of replication protein A, accumulated faster in parg-2 worms and disappeared earlier than in WT worms. This accelerated RPA-1 accumulation may result from the enhanced expression of exonuclease-1 (EXO-1) after IR treatment. Accordingly, an exo-1 mutation reduced IR sensitivity and accumulation of RPA-1 in parg-2 worms. A mutation of polq-1, encoding for a key factor in the alternative end-joining (Alt-EJ) pathway, suppressed the IR hypersensitivity phenotype of parg-2 worms and normalized the kinetics of RAD-51 dissipation. This indicates that error-prone Alt-EJ may mediate DSB repair in parg-2 worms, causing hypersensitivity to IR. In summary, PARG-2 deficiency in C. elegans causes hyperactive DSB end resection likely through EXO-1 overproduction. DSBs with long single-stranded DNA ends in parg-2 worms are thought to be repaired by Alt-EJ instead of HR, causing genomic instability.

Enhancement of Mesenchymal Stem Cell-Driven Bone Regeneration by Resveratrol-Mediated SOX2 Regulation.

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine. However, MSCs age rapidly during long-term ex vivo culture and lose their therapeutic potential before they reach effective cell doses (ECD) for cell therapy. Thus, a prerequisite for effective MSC therapy is the development of cell culture methods to preserve the therapeutic potential during long-term ex vivo cultivation. Resveratrol (RSV) has been highlighted as a therapeutic candidate for bone disease. Although RSV treatment has beneficial effects on bone-forming cells, in vivo studies are lacking. The current study showed that long-term (6 weeks from primary culture date)-cultured MSCs with RSV induction retained their proliferative and differentiation potential despite reaching ECD. The mechanism of RSV action depends entirely on the SIRT1-SOX2 axis in MSC culture. In a rat calvarial defect model, RSV induction significantly improved bone regeneration after MSC transplantation. This study demonstrated an example of efficient MSC therapy for treating bone defects by providing a new strategy using the plant polyphenol RSV.

Triclosan: An Update on Biochemical and Molecular Mechanisms.

Triclosan (TCS) is a synthetic, chlorinated phenolic antimicrobial agent commonly used in commercial and healthcare products. Items made with TCS include soaps, deodorants, shampoos, cosmetics, textiles, plastics, surgical sutures, and prosthetics. A wealth of information obtained from in vitro and in vivo studies has demonstrated the therapeutic effects of TCS, particularly against inflammatory skin conditions. Nevertheless, extensive investigations on the molecular aspects of TCS action have identified numerous adversaries associated with the disinfectant including oxidative injury and influence of physiological lifespan and longevity. This review presents a summary of the biochemical alterations pertaining to TCS exposure, with special emphasis on the diverse molecular pathways responsive to TCS that have been elucidated during the present decade.

Disruption of erythrocyte membrane asymmetry by triclosan is preceded by calcium dysregulation and p38 MAPK and RIP1 stimulation.

Triclosan (TCS) is a broad-spectrum antimicrobial used in personal care products, household items, and medical devices. Owing to its apoptotic potential against tumor cells, TCS has been proposed for the treatment of malignancy. A major complication of chemotherapy is anemia, which may result from direct erythrocyte hemolysis or premature cell death known as eryptosis. Similar to nucleated cells, eryptotic cells lose membrane asymmetry and Ca2+ regulation, and undergo oxidative stress, shrinkage, and activation of a host of kinases. In this report, we sought to examine the hemolytic and eryptotic potential of TCS and dissect the underlying mechanistic scenarios involved there in. Hemolysis was spectrophotometrically evaluated by the degree of hemoglobin release into the medium. Flow cytometry was utilized to detect phosphatidylserine (PS) exposure by annexin-V binding, intracellular Ca2+ by Fluo-3/AM fluorescence, and oxidative stress by 2-,7-dichlorodihydrofluorescin diacetate (DCFH2-DA). Incubation of cells with 10-100 µM TCS for 1-4 h induced time- and dose-dependent hemolysis. Moreover, TCS significantly increased the percentage of eryptotic cells as evident by PS exposure (significantly enhanced annexin-V binding). Interestingly, TCS-induced eryptosis was preceded by elevated intracellular Ca2+ levels but was not associated with oxidative stress. Cotreatment of erythrocytes with 50 µM TCS and 50 µM SB203580 (p38 MAPK inhibitor), or 300 µM necrostatin-1 (receptor-interacting protein 1 (RIP1) inhibitor) significantly ameliorated TCS-induced PS externalization. We conclude that TCS is cytotoxic to erythrocytes by inducing hemolysis and stimulating premature death at least in part through Ca2+ mobilization, and p38 MAPK and RIP1 activation.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET).

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5 mM (0.02%-0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

Single-strand annealing mediates the conservative repair of double-strand DNA breaks in homologous recombination-defective germ cells of Caenorhabditis elegans.

A missense mutation in C. elegans RAD-54, a homolog of RAD54 that operates in the homologous recombination (HR) pathway, was found to decrease ATPase activity in vitro. The hypomorphic mutation caused hypersensitivity of C. elegans germ cells to double-strand DNA breaks (DSBs). Although the formation of RAD-51 foci at DSBs was normal in both the mutant and knockdown worms, their subsequent dissipation was slow. The rad-54-deficient phenotypes were greatly aggravated when combined with an xpf-1 mutation, suggesting a conservative role of single-strand annealing (SSA) for DSB repair in HR-defective worms. The phenotypes of doubly-deficient rad-54;xpf-1 worms were partially suppressed by a mutation of lig-4, a nonhomologous end-joining (NHEJ) factor. In summary, RAD-54 is required for the dissociation of RAD-51 from DSB sites in C. elegans germ cells. Also, NHEJ and SSA exert negative and positive effects, respectively, on genome stability when HR is defective.