scAAV2-Mediated Expression of Thioredoxin 2 and C3 Transferase Prevents Retinal Ganglion Cell Death and Lowers Intraocular Pressure in a Mouse Model of Glaucoma.

Elevated intraocular pressure (IOP) in glaucoma causes retinal ganglion cell (RGC) loss and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. In this paper, we aimed to develop a novel gene therapy for glaucoma using an AAV2-based thioredoxin 2 (Trx2)-exoenzyme C3 transferase (C3) fusion protein expression vector (scAAV2-Trx2-C3). We evaluated the therapeutic effects of this vector in vitro and in vivo using dexamethasone (DEX)-induced glaucoma models. We found that scAAV2-Trx2-C3-treated HeLa cells had significantly reduced GTP-bound active RhoA and increased phosphor-cofilin Ser3 protein expression levels. scAAV2-Trx2-C3 was also shown to inhibit oxidative stress, fibronectin expression, and alpha-SMA expression in DEX-treated HeLa cells. NeuN immunostaining and TUNEL assay in mouse retinal tissues was performed to evaluate its neuroprotective effect upon RGCs, whereas changes in mouse IOP were monitored via rebound tonometer. The present study showed that scAAV2-Trx2-C3 can protect RGCs from degeneration and reduce IOP in a DEX-induced mouse model of glaucoma, while immunohistochemistry revealed that the expression of fibronectin and alpha-SMA was decreased after the transduction of scAAV2-Trx2-C3 in murine eye tissues. Our results suggest that AAV2-Trx2-C3 modulates the outflow resistance of the trabecular meshwork, protects retinal and other ocular tissues from oxidative damage, and may lead to the development of a gene therapeutic for glaucoma.

Role of thioredoxin in chronic obstructive pulmonary disease (COPD): a promising future target.

INTRODUCTION: Thioredoxin (Trx) is a secretory protein that acts as an antioxidant, redox regulator, anti-allergic, and anti-inflammatory molecule. It has been used to treat dermatitis and inflammation of the digestive tract. In the lungs, Trx has a significant anti-inflammatory impact. On the other hand, Chronic Obstructive Pulmonary Disease (COPD) is one of the significant causes of death in the developed world, with a tremendous individual and socioeconomic impact. Despite new initiatives and endless treatment trials, COPD incidence and death will likely escalate in the coming decades. AREAS COVERED: COPD is a chronic inflammatory disease impacting the airways, lung parenchyma, and pulmonary vasculature. Oxidative stress and protease-antiprotease imbalances are thought to be involved in the process. The most popular respiratory inflammatory and allergic disorders therapies are corticosteroids and ß-receptor agonists. These medications are helpful but have some drawbacks, such as infection and immunosuppression; thus, addressing Trx signalling treatments may be a viable COPD treatment approach. This review shall cover the pathophysiology of COPD, the pharmacognosy of anti-COPD drugs, including the assets and liabilities of each, and the role and mechanism of Trx in COPD treatment. EXPERT OPINION: Limited research has targeted the thioredoxin system as an anti-COPD drug. Spectating the increase in the mortality rates of COPD, this review article would be an interesting one to research.

Regeneration of cytosolic thiol peroxidases.

Three soluble type two peroxiredoxins (PRXIIB, C, D) and two glutathione peroxidase-like enzymes (GPXL2, 8) reside in the cytosol of Arabidopsis thaliana cells and function both as thiol-dependent antioxidants and redox sensors. Their primary substrate is H2 O2 , but they also accept other peroxides with a distinct preference between PRXII and GPXL. Less known is their regeneration specificity in the light of the large set of thiol reductases, namely eight annotated thioredoxin h isoforms (TRXh1-5, 7-9), a few TRX-like proteins, including CxxS1 (formerly TRXh6) and several glutaredoxins (GRX) associated with the cytosol. This study addressed this open question by in vitro enzyme tests using recombinant protein. GPXL2 and 8 exclusively accepted electrons from the TRX system, namely TRXh1-5 and TDX, while PRXIIB/C/D were efficiently regenerated with GRXC1 and C2 but not the TRX-like protein Picot1. They showed significant but low activity (<3% of GRXC2) with TRXh1-5 and TDX. A similar reduction efficiency with TRX was seen in the insulin assay, only TDX was less active. Finally, the reduction of oxidized cytosolic malate dehydrogenase 1, as measured by regained activity, showed an extremely broad ability to accept electrons from different TRXs and GRXs. The results demonstrate redundancy and specificity in the redox regulatory network of the cytosol.

TXNL1 has dual functions as a redox active thioredoxin-like protein as well as an ATP- and redox-independent chaperone.

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity.

Nuclear translocation of thioredoxin-1 promotes colorectal cancer development via modulation of the IL-6/STAT3 signaling axis through interaction with STAT3.

Background: Thioredoxin 1 (Trx-1) is a small redox protein predominantly localized in the cytoplasm. Its expression is increased in several cancers, including colorectal cancer (CRC). However, the function of Trx-1 translocation to the nucleus in cancer is not clear. In this study, we investigated the role of Trx-1 nuclear translocation in development of CRC. Methods: Expression of Trx-1 and STAT3 was analyzed by Western blot and immunofluorescence. Endogenous interaction of Trx-1, STAT3, and karyopherin α1 in CRC cells was analyzed by co-immunoprecipitation. Trx-1 and pSTAT3 nuclear staining in human CRC tissues was analyzed by immunohistochemistry. A mouse model of AOM/DSS induced colitis-associated cancer (CAC) was utilized to investigate the antitumor effect of PX-12, a Trx-1 inhibitor. A knockin mouse with the Txn1(KK81-82EE) mutation was generated via CRISPR/Cas9, and CAC was induced in knockin and wild-type mice. Results: Nuclear translocation of Trx-1 was induced by IL-6, and inhibition of this translocation reversed IL-6-induced epithelial-to-mesenchymal transition, invasion and metastasis. Karyopherin α1 was found to specifically mediate IL-6-induced translocation of the Trx-1-pSTAT3 complex into the nucleus. Nuclear Trx-1 expression was closely correlated with lymph node metastasis and distant metastasis in human CRC. In addition, nuclear staining of Trx-1 showed significant positive correlation with nuclear staining of pSTAT3 in human CRC tissues. PX-12, an inhibitor of Trx-1, significantly impaired the activation of STAT3 and suppressed the development of AOM/DSS-induced CAC in mice. Moreover, AOM/DSS-induced nuclear Trx-1 expression was suppressed in Txn1(KK81-82EE) mice, which inhibited STAT3 activation and cancer progression. Conclusions: These results provide new insights into the mechanisms of STAT3 activation triggered by IL-6 and identify nuclear translocation of Trx-1 as a potential therapeutic target for the treatment of CRC and CAC.

Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells.

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO or related donor drug delivery systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

x- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light.

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.

The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC coevolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study, we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and reactivates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

Verapamil Prevents Decline of IGF-I in Subjects With Type 1 Diabetes and Promotes ß-Cell IGF-I Signaling.

Verapamil promotes functional ß-cell mass and improves glucose homeostasis in diabetic mice and humans with type 1 diabetes (T1D). Now, our global proteomics analysis of serum from subjects with T1D at baseline and after 1 year of receiving verapamil or placebo revealed IGF-I as a protein with significantly changed abundance over time. IGF-I, which promotes ß-cell survival and insulin secretion, decreased during disease progression, and this decline was blunted by verapamil. In addition, we found that verapamil reduces ß-cell expression of IGF-binding protein 3 (IGFBP3), whereas IGFBP3 was increased in human islets exposed to T1D-associated cytokines and in diabetic NOD mouse islets. IGFBP3 binds IGF-I and blocks its downstream signaling, which has been associated with increased ß-cell apoptosis and impaired glucose homeostasis. Consistent with the downregulation of IGFBP3, we have now discovered that verapamil increases ß-cell IGF-I signaling and phosphorylation/activation of the IGF-I receptor (IGF1R). Moreover, we found that thioredoxin-interacting protein (TXNIP), a proapoptotic factor downregulated by verapamil, promotes IGFBP3 expression and inhibits the phosphorylation/activation of IGF1R. Thus, our results reveal IGF-I signaling as yet another previously unappreciated pathway affected by verapamil and TXNIP that may contribute to the beneficial verapamil effects in the context of T1D. ARTICLE HIGHLIGHTS: Verapamil prevents the decline of IGF-I in subjects with type 1 diabetes (T1D). Verapamil decreases the expression of ß-cell IGF-binding protein 3 (IGFBP3), whereas IGFBP3 is increased in human and mouse islets under T1D conditions. Verapamil promotes ß-cell IGF-I signaling by increasing phosphorylation of IGF-I receptor and its downstream effector AKT. Thioredoxin-interacting protein (TXNIP) increases IGFBP3 expression and inhibits the phosphorylation/activation of IGF1R in ß-cells. Regulation of IGFBP3 and IGF-I signaling by verapamil and TXNIP may contribute to the beneficial verapamil effects in the context of T1D.

Tremella fuciformis polysaccharides alleviates UV-provoked skin cell damage via regulation of thioredoxin interacting protein and thioredoxin reductase 2.

INTRODUCTION: Skin is exposed to a wide range of environmental risk factors including ultraviolet (UV) and all kinds of pollutants. Excessive UV exposure contributes to many disorders, such as photoaging, skin inflammation, and carcinogenesis. Previous studies have shown that Tremella fuciformis polysaccharides (TFPS) have protective effects on oxidative stress in cells, but the specific protective mechanism has not been clarified. METHODS: To determine the effects of TFPS on UV-irritated human skin, we conducted a variety of studies, including Cell Counting Kit-8 (CCK-8), trypan blue, Western blot, apoptosis assays, reactive oxygen species (ROS) detection in primary skin keratinocytes, and chronic UV-irradiated mouse model. RESULTS: We first determined that TFPS protects human skin keratinocytes against UV radiation-induced apoptosis and ROS production. Moreover, TFPS regulates thioredoxin interacting protein (TXNIP) and thioredoxin reductase 2 (TXNRD2) levels in primary skin keratinocytes for photoprotection. Last, we found that topical TFPS treatment could alleviate the UV-induced skin damage in chronic UV-irradiated mouse model. CONCLUSION: Collectively, our work indicates the beneficial role of TFPS in UV-induced skin cell damage and provides a novel therapeutic reagent to prevent or alleviate the progress of photoaging and other UV-provoked skin diseases.

Identification and characterization of archaeal and bacterial F420 -dependent thioredoxin reductases.

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F420 H2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F420 H2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR.

Atypical network topologies enhance the reductive capacity of pathogen thiol antioxidant defense networks.

Infectious diseases are a significant health burden for developing countries, particularly with the rise of multidrug resistance. There is an urgent need to elucidate the factors underlying the persistence of pathogens such as Mycobacterium tuberculosis, Plasmodium falciparum and Trypanosoma brucei. In contrast to host cells, these pathogens traverse multiple and varied redox environments during their infectious cycles, including exposure to high levels of host-derived reactive oxygen species. Pathogen antioxidant defenses such as the peroxiredoxin and thioredoxin systems play critical roles in the redox stress tolerance of these cells. However, many of the kinetic rate constants obtained for the pathogen peroxiredoxins are broadly similar to their mammalian homologs and therefore, their contributions to the redox tolerances within these cells are enigmatic. Using graph theoretical analysis, we show that compared to a canonical Escherichia coli redoxin network, pathogen redoxin networks contain unique network connections (motifs) between their thioredoxins and peroxiredoxins. Analysis of these motifs reveals that they increase the hydroperoxide reduction capacity of these networks and, in response to an oxidative insult, can distribute fluxes into specific thioredoxin-dependent pathways. Our results emphasize that the high oxidative stress tolerance of these pathogens depends on both the kinetic parameters for hydroperoxide reduction and the connectivity within their thioredoxin/peroxiredoxin systems.

Characterization of thioredoxin gene TaTrxh9 associated with heading-time regulation in wheat.

Thioredoxins (Trxs) are thiol-disulfide oxidoreductase proteins that play important roles in a spectrum of processes linking redox regulation and signaling in plants. However, little is known about Trxs and their biological functions in wheat, one of the most important food crops worldwide. This study reports the identification and functional characterization of an h-type Trx gene, TaTrxh9, in wheat. Three homoeologs of TaTrxh9 were identified and the sequences in the coding region were highly consistent among the homoeologs. Protein characterization showed that a conserved Trx_family domain, as well as a typical active site with a dithiol signature (WCGPC), was included in TaTrxh9. Structural modeling demonstrated that TaTrxh9 could fold into a canonical thioredoxin structure consisting of five-stranded antiparallel beta sheets sandwiched between four alpha helices. The insulin disulfide reduction assay demonstrated that TaTrxh9 was catalytically active in vitro. TaTrxh9 overexpression in the Arabidopsis mutant trxh9 complemented the abnormal growth phenotypes of the mutant, suggesting is functionality in vivo. The transcription level of TaTrxh9 was higher in leaf tissues and it was differentially expressed during the development of wheat plants. Interestingly, barley stripe mosaic virus-mediated suppression of TaTrxh9 shortened the seedling-heading period of wheat. Furthermore, CRISPR-Cas9 mediated gene knockout confirmed that the TaTrxh9 mutation resulted in early heading of wheat. To our knowledge, this study is the first to report that Trxh is associated with heading-time regulation, which lays a foundation for further exploring the biological function of TaTrxh9 and provides new ideas for molecular breeding focusing on early heading in wheat.

Effect of folic acid-linked chitosan-coated PLGA-based curcumin nanoparticles on the redox system of glioblastoma cancer cells.

OBJECTIVES: Oxidative stress is one of the carcinogenic mechanisms underlying the development of glioblastoma multiforme (GBM), a highly aggressive brain tumor type associated with poor prognosis. Curcumin is known to be an efficient antioxidant, anti-inflammatory, and anticancer compound. However, its poor solubility in water, inappropriate pharmacokinetics, and low bioavailability limit its use as an antitumor drug. We prepared PLGA-based curcumin nanoparticles changed with folic acid and chitosan (curcumin-PLGA-CS-FA) and evaluated its effects on GBM tumor cells' redox status. METHODS: The nanoprecipitation method was used to synthesize CU nanoparticles (CU-NPs). The size, morphology, and stability were characterized by DLS, SEM, and zeta potential analysis, respectively. The CU-NPs' toxic properties were studied by MTT assay and measuring the intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) concentrations. The study was completed by measuring the gene expression levels and activity of superoxide dismutase, catalase, glutaredoxin, and thioredoxin antioxidant enzymes. RESULTS: The size, polydispersity index, and zeta potential of CU-NPs were 77.27 nm, 0.29, and -22.45 mV, respectively. The encapsulation efficiency was approximately 98%. Intracellular ROS and MDA levels decreased after CU-NP treatment. Meanwhile, the CU-NPs increased gene expression and activity of superoxide dismutase, catalase, glutaredoxin, and thioredoxin antioxidant enzymes. CONCLUSION: CU-NPs might be effective in the prevention and treatment of glioblastoma cancer by modulating the antioxidant-oxidant balance.

Redox regulation, thioredoxins, and glutaredoxins in retrograde signalling and gene transcription.

Integration of reactive oxygen species (ROS)-mediated signal transduction pathways via redox sensors and the thiol-dependent signalling network is of increasing interest in cell biology for their implications in plant growth and productivity. Redox regulation is an important point of control in protein structure, interactions, cellular location, and function, with thioredoxins (TRXs) and glutaredoxins (GRXs) being key players in the maintenance of cellular redox homeostasis. The crosstalk between second messengers, ROS, thiol redox signalling, and redox homeostasis-related genes controls almost every aspect of plant development and stress response. We review the emerging roles of TRXs and GRXs in redox-regulated processes interacting with other cell signalling systems such as organellar retrograde communication and gene expression, especially in plants during their development and under stressful environments. This approach will cast light on the specific role of these proteins as redox signalling components, and their importance in different developmental processes during abiotic stress.

GKLF, a transcriptional activator of Txnip, drives microglia activation in kainic acid-induced murine models of epileptic seizures.

Neuroinflammation is a major component of epilepsy. Gut-enriched Kruppel-like factor (GKLF), a transcription factor of Kruppel-like factor family, has been reported to promote microglia activation and mediate neuroinflammation. However, the role of GKLF in epilepsy remains poorly characterized. This study focused on the function of GKLF in neuron loss and neuroinflammation in epilepsy and the molecular mechanism underlying microglia activation induced by GKLF upon lipopolysaccharides (LPS) treatment. An experimental epileptic model was induced by an intraperitoneal injection of 25 mg/kg kainic acid (KA). Lentivirus vectors (Lv) carrying Gklf CDS or short hairpin RNA targeting Gklf (shGKLF) was injected into the hippocampus, resulting in Gklf overexpression or knockdown in the hippocampus. BV-2 cells were co-infected with Lv-shGKLF or/and Lv carrying thioredoxin interacting protein (Txnip) CDS for 48 h and treated with 1 µg/mL LPS for 24 h. Results showed that GKLF enhanced KA-induced neuronal loss, pro-inflammatory cytokine secretion, activation of NOD-like receptor protein-3 (NLRP3) inflammasomes and microglia, and TXNIP expression in the hippocampus. GKLF inhibition showed negative effects on LPS-induced microglia activation, as evidenced by reduced pro-inflammatory cytokine secretion and activation of NLRP3 inflammasomes. GKLF bound to Txnip promoter and increased TXNIP expression in LPS-activated microglia. Interestingly, Txnip overexpression reversed the inhibitory effect of Gklf knockdown on microglia activation. These findings indicated that GKLF was involved in microglia activation via TXNIP. This study demonstrates the underlying mechanism of GKLF in the pathogenesis of epilepsy and uncovers that GKLF inhibition may be a therapeutic strategy for epilepsy treatment.

Expression of thioredoxin-1 in the ASJ neuron corresponds with and enhances intrinsic regenerative capacity under lesion conditioning in C. elegans.

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. We trigger conditioned regeneration in the Caenorhabditis elegans ASJ neuron by laser surgery or genetic disruption of sensory pathways. Conditioning upregulates thioredoxin-1 (trx-1) expression, as indicated by trx-1 promoter-driven expression of green fluorescent protein and fluorescence in situ hybridization (FISH), suggesting trx-1 levels and associated fluorescence indicate regenerative capacity. The redox activity of trx-1 functionally enhances conditioned regeneration, but both redox-dependent and -independent activity inhibit non-conditioned regeneration. Six strains isolated in a forward genetic screen for reduced fluorescence, which suggests diminished regenerative potential, also show reduced axon outgrowth. We demonstrate an association between trx-1 expression and the conditioned state that we leverage to rapidly assess regenerative capacity.

Effectiveness of Albumin-Fused Thioredoxin against 6-Hydroxydopamine-Induced Neurotoxicity In Vitro.

Parkinson's disease (PD) is a neurodegenerative disorder caused by oxidative stress-dependent loss of dopaminergic neurons in the substantia nigra and elevated microglial inflammatory responses. Recent studies show that cell loss also occurs in the hypothalamus in PD. However, effective treatments for the disorder are lacking. Thioredoxin is the major protein disulfide reductase in vivo. We previously synthesized an albumin-thioredoxin fusion protein (Alb-Trx), which has a longer plasma half-life than thioredoxin, and reported its effectiveness in the treatment of respiratory and renal diseases. Moreover, we reported that the fusion protein inhibits trace metal-dependent cell death in cerebrovascular dementia. Here, we investigated the effectiveness of Alb-Trx against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Alb-Trx significantly inhibited 6-OHDA-induced neuronal cell death and the integrated stress response. Alb-Trx also markedly inhibited 6-OHDA-induced reactive oxygen species (ROS) production, at a concentration similar to that inhibiting cell death. Exposure to 6-OHDA perturbed the mitogen-activated protein kinase pathway, with increased phosphorylated Jun N-terminal kinase and decreased phosphorylated extracellular signal-regulated kinase levels. Alb-Trx pretreatment ameliorated these changes. Furthermore, Alb-Trx suppressed 6-OHDA-induced neuroinflammatory responses by inhibiting NF-κB activation. These findings suggest that Alb-Trx reduces neuronal cell death and neuroinflammatory responses by ameliorating ROS-mediated disruptions in intracellular signaling pathways. Thus, Alb-Trx may have potential as a novel therapeutic agent for PD.