Stress-induced Death Research Articles

Restoring Colistin Sensitivity in Multidrug-Resistant Pathogenic E. coli Using Cinacalcet Hydrochloride

Restoring colistin’s efficacy is crucial in addressing the resistance crisis of colistin. This study utilized a high-throughput screening method to identify 43 compounds from 800 FDA-approved drugs that exhibited significant antibacterial effects when combined with colistin. Among these, cinacalcet hydrochloride (CH) was selected for its potential synergistic effect with colistin against multidrug-resistant (MDR) E. coli strains, including mcr-1-positive strains. A series of experiments revealed that the combination of CH and colistin showed strong synergy, especially in mcr-1-positive strains, restoring colistin sensitivity. The combination significantly inhibited bacterial growth and reduced CFU counts more effectively than either drug alone. Additionally, CH and colistin together significantly inhibited biofilm formation and eradicated existing biofilms, as visualized through confocal microscopy. Mechanistic studies showed that the combination increased bacterial membrane permeability and disrupted membrane integrity. The treatment also elevated extracellular ATP release and ROS production, indicating oxidative stress-induced bacterial death. Safety evaluations showed that the combination did not increase toxicity in host cells. Finally, animal models further validated the combination’s efficacy. Overall, this study showed that the combination of colistin and CH significantly restored colistin sensitivity in mcr-1-positive E. coli, revealing their synergistic antibacterial mechanism involving membrane damage and oxidative stress, with promising clinical applications.

Oocytes maintain low ROS levels to support the dormancy of primordial follicles.

Primordial follicles (PFs) function as the long-term reserve for female reproduction, remaining dormant in the ovaries and becoming progressively depleted with age. Oxidative stress plays an important role in promoting female reproductive senescence during aging, but the underlying mechanisms remain unclear. Here, we find that low levels of reactive oxygen species (ROS) are essential for sustaining PF dormancy. Compared to growing follicles, oocytes within PFs were shown to be more susceptible to ROS, which accumulates and damages PFs to promote reproductive senescence. Mechanistically, oocytes within PFs were shown to express high levels of the intracellular antioxidant enzyme superoxide dismutase 1 (SOD1), counteracting ROS accumulation. Decreased SOD1 expression, as a result of aging or through the experimental deletion of the Sod1 gene in oocytes, resulted in increased oxidative stress and triggered ferroptosis within PFs. In conclusion, this study identified antioxidant defense mechanisms protecting PFs in mouse ovaries and characterized cell death mechanisms of oxidative stress-induced PF death.

Protective Effect of Ergothioneine against Oxidative Stress-Induced Chondrocyte Death.

Reactive oxygen species (ROS) induce oxidative stress in cells and are associated with various diseases, including autoimmune diseases. Ergothioneine (EGT) is a natural amino acid derivative derived from the ergot fungus and has been reported to exhibit an effective antioxidant function in many models of oxidative stress-related diseases. Recently, mutations in OCTN1, a membrane transporter of EGT, have been reported to be associated with rheumatoid arthritis. Therefore, we investigated the chondrocyte-protective function of EGT using a model of oxidative stress-induced injury of chondrocytes by hydrogen peroxide (H2O2). Human chondrocytes were subjected to oxidative stress induced by H2O2 treatment, and cell viability, the activity of lactate dehydrogenase (LDH) released into the medium, dead cell ratio, intracellular ROS production, and mitochondrial morphology were assessed. EGT improved chondrocyte viability and LDH activity in the medium and strongly suppressed the dead cell ratio. EGT also exerted protective effects on intracellular ROS production and mitochondrial morphology. These results provide evidence to support the protective effects of EGT on chondrocytes induced by oxidative stress.

Effectiveness and mechanisms of combined use of antioxidant nutrients in protecting against oxidative stress-induced neuronal loss and related neurological deficits.

Oxidative stress is a well-known pathological factor driving neuronal loss and age-related neurodegenerative diseases. Melatonin, coenzyme Q10 and lecithin are three common nutrients with an antioxidative capacity. Here, we examined the effectiveness of them administrated individually and in combination in protecting against oxidative stress-induced neuronal death invitro, and neurodegenerative conditions such as Alzheimer's disease and associated deficits invivo. Mouse neuroblastoma Neuro-2a (N2a) cells were exposed with H2O2 for 6 h, and subsequently treated with melatonin, coenzyme Q10, and lecithin alone or in combination for further 24 h. Cell viability was assessed using the CCK-8 assay. Eight-week-old male mice were intraperitoneally injected with D-(+)-galactose for 10 weeks and administrated with melatonin, coenzyme Q10, lecithin, or in combination for 5 weeks starting from the sixth week, followed by behavioral tests to assess the effectiveness in mitigating neurological deficits, and biochemical assays to explore the underlying mechanisms. Exposure to H2O2 significantly reduced the viability of N2a cells and increased oxidative stress and tau phosphorylation, all of which were alleviated by treatment with melatonin, coenzyme Q10, lecithin alone, and, most noticeably, by combined treatment. Administration of mice with D-(+)-galactose-induced oxidative stress and tau phosphorylation, brain aging, impairments in learning and memory, anxiety- and depression-like behaviors, and such detrimental effects were mitigated by melatonin, coenzyme Q10, lecithin alone, and, most consistently, by combined treatment. These results suggest that targeting oxidative stress via supplementation of antioxidant nutrients, particularly in combination, is a better strategy to alleviate oxidative stress-mediated neuronal loss and brain dysfunction due to age-related neurodegenerative conditions.

#897 Peritoneal inflammation in PD-related peritonitis induces systemic eryptosis: in vitro and in vivo assessments

Abstract Background and Aims Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis ( = stress-induced RBC death mechanism). Triggers of eryptosis include oxidative stress, inflammation and several uremic toxins. Our preliminary data show a significant increase of eryptosis during peritoneal dialysis (PD) -associated peritonitis in PD patients. The aims of this study were: assessment of incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting. Method We enrolled 34 PD patients in stable condition (without systemic inflammation nor PD-related peritonitis in the last 3 months), 31 PD patients with acute peritonitis, and 17 healthy subjects (CTR). For PD stable patients, blood samples for eryptosis evaluation were drawn during the regular outpatient. For PD patients with peritonitis, blood samples and PDE samples were collected at the time of peritonitis diagnosis. PDE samples were used for peritoneal White Blood Cell count (pWBC), peritoneal Neutrophil Gelatinase-associated Lipocalin (pNGAL) and peritoneal cytokines levels (IL-1β and IL-6) measurements. For in vitro study, healthy RBCs were exposed to plasma of 31 PD patients with peritonitis and plasma of CTR group for 2, 4 and 24 hours. Morphological markers of eryptosis (cell membrane scrambling, cell shrinkage) and eryptosis percentage (based on PS exposure at RBC surface and AnnexinV-binding) were evaluated by flow cytometric analyses in vivo and in vitro. Results Totally, 65 chronic PD patients were included in this study. 57% of patients were treated with continuous ambulatory PD (CAPD) and 43% with automated PD (APD). The median PD vintage was 38 months (IQR 3.6–124.9 months). Table 1 reports clinical data for our PD population (Table 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients than in CTR (2.7%; IQR 1.6–3.9 vs CTR: 0.8; IQR 0.7–1.3 P < .05). Eryptosis levels did not differ significantly between PD pts with and without diabetes, with and without hypertension, with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated by CAPD/APD, and with Kt/Vurea values ≤ 1.7 and > 1.7. Generally, RBCs of all 65 PD patients were dramatically deranged in their morphology. In particular, RBCs from PD patients with peritonitis were characterized by dramatically deranged morphology and increased median cell volume in comparison with PD stable patients (P < .001). The percentages of AnnexinV-binding RBCs were significantly increased in the PD-associated peritonitis group (9.6%; IQR 4.2–16.7 versus 2.7%; IQR 1.6–3.9) (P<.0001) (Fig. 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients with peritonitis than in healthy CTR (P < .001). We confirmed these in vivo data by in vitro results: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase of eryptosis compared to healthy RBCs exposed to plasma from control group at all times (Fig. 2). Furthermore, significant positive strong correlations were observed between eryptosis level and all analysed peritoneal biomarkers of peritonitis (Spearman's rho pWBC = 0.77, P<.001, Spearman's rho pNGAL = 0.64, P = .001, Spearman's rho IL-6 = 0.61, P = .003 and Spearman's rho IL-1β = 0.64, P = .001,) (Fig. 3). Conclusion We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. In particular, we theorized that the eryptosis enhancement is directly connected with peritonitis, and, based on the results, we hypothesized that the peritoneal membrane injury could be related to eryptosis. Upregulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis. In particular, we corroborated this point with our observations about in vitro induction of eryptosis by plasma from PD patients on the first day of peritonitis.

Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Tiliacora triandra (Colebr.) Diels leaf extract alleviates motor deficit and protects against MPTP-induced neurodegeneration in mice

Parkinson’s disease (PD) is a neurological disorder that results from a gradual loss of dopaminergic neurons in the substantia nigra. One of the most important causes of nigrostriatal degeneration in PD is oxidative stress-induced neuronal death. Tiliacora triandra (Colebr) Diels (T. triandra (TT)), known as Ya-nang in Thai, is a plant that is recognized to have antioxidant, anti-inflammatory, and neuroprotective activities. In this study, we explored the neuroprotective effects of TT leaf extract in a PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). TT (100, 200, and 400 mg/kg BW) was orally administered to 9-week-old C57BL/6 mice for 7 days before and 7 days after receiving four injections of 20 mg/kg BW MPTP, with a 2-hour gap between each injection. The results showed that the treatment of TT improved MPTP-induced motor impairment and suppressed the MPTP-induced reductions of tyrosine hydroxylase-positive neurons in the substantia nigra while TT also mitigated MPTP-induced decreasing dopamine (DA) levels in the striatum. TT also alleviated oxidative stress status including decreased malondialdehyde levels and increased the activities of glutathione, catalase, and superoxide dismutase. Our findings suggest that the consumption of TT could offer neuroprotection, potentially delaying or preventing the neurodegenerative process associated with PD in the C57BL/6 mice. However, further research into the safety of the extract should be conducted before proceeding with a study involving human PD.

Characterization of Phytaspase Proteolytic Activity Using Fluorogenic Peptide Substrates.

Within the subtilase family of plant proteolytic enzymes, phytaspases are distinguished by their strict substrate cleavage specificity after an aspartate residue preceded by a characteristic tripeptide amino acid motif. This type of recognition resembles that of animal apoptotic proteases, caspases. Phytaspases attract attention not only because they are critically important for the accomplishment of stress-induced death of plant cells, but also due to their ability to specifically process precursor proteins, thus generating bioactive plant peptide hormones, systemin and phytosulfokine. As the activity of phytaspases appears to be essential for life and death decisions made by the plant cell, elaboration of an approach to characterize and quantitate phytaspase proteolytic activity is of importance. Here we provide a protocol for phytaspase activity determination and characterization using fluorogenic peptide substrates. This approach works well, both with purified phytaspase samples, and with crude extracts from plant tissues. We also discuss advantages of the assay, factors that may influence its sensitivity and specificity, as well as possible pitfalls.

Orally Administered Silicon Hydrogen Nanomaterials as Target Therapy to Treat Intestinal Diseases.

The occurrence and development of inflammatory bowel diseases (IBDs) are inextricably linked to the excessive production of reactive oxygen species (ROS). Thus, there is an urgent need to develop innovative tactics to combat IBDs and scavenge excess ROS from affected areas. Herein, silicon hydrogen nanoparticles (SiH NPs) with ROS-scavenging ability were prepared by etching Si nanowires (NWs) with hydrogen fluoride (HF) to alleviate the symptoms associated with IBD by orally targeting the inflamed colonic sites. The strong reductive Si-H bonds showed excellent stability in the gastric and intestinal fluids, which exhibited efficient ROS-scavenging effects to protect cells from high oxidative stress-induced death. After oral delivery, the negatively charged SiH NPs were specifically adsorbed to the positively charged inflammatory epithelial tissues of the colon for an extended period via electrostatic interactions to prolong the colonic residence time. SiH NPs exhibited significant preventive and therapeutic effects in dextran sodium sulfate-induced prophylactic and therapeutic mouse models by inhibiting colonic shortening, reducing the secretion of pro-inflammatory cytokines, regulating macrophage polarization, and protecting the colonic barrier. As determined using 16S rDNA high-throughput sequencing, the oral administration of SiH NPs treatment led to changes in the abundance of the intestinal microbiome, which improved the bacterial diversity and restored the relative abundance of beneficial bacteria after the inflamed colon. Overall, our findings highlight the broad application of SiH-based anti-inflammatory drugs in the treatment of IBD and other inflammatory diseases.

The Slow Progression of Diabetic Retinopathy Is Associated with Transient Protection of Retinal Vessels from Death.

The purpose of this study was to investigate the reason that diabetic retinopathy (DR) is delayed from the onset of diabetes (DM) in diabetic mice. To this end, we tested the hypothesis that the deleterious effects of DM are initially tolerated because endogenous antioxidative defense is elevated and thereby confers resistance to oxidative stress-induced death. We found that this was indeed the case in both type 1 DM (T1D) and type 2 DM (T2D) mouse models. The retinal expression of antioxidant defense genes was increased soon after the onset of DM. In addition, ischemia/oxidative stress caused less death in the retinal vasculature of DM versus non-DM mice. Further investigation with T1D mice revealed that protection was transient; it waned as the duration of DM was prolonged. Finally, a loss of protection was associated with the manifestation of both neural and vascular abnormalities that are diagnostic of DR in mice. These observations demonstrate that DM can transiently activate protection from oxidative stress, which is a plausible explanation for the delay in the development of DR from the onset of DM.

1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p<0.001) and when they were inhibited (n=6, p<0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

SIRT6 overexpression in the nucleus protects mouse retinal pigment epithelium from oxidative stress.

Retinal pigment epithelium (RPE) is essential for the survival of retinal photoreceptors. To study retinal degeneration, sodium iodate (NaIO3) has been used to cause oxidative stress-induced RPE death followed by photoreceptor degeneration. However, analyses of RPE damage itself are still limited. Here, we characterized NaIO3-induced RPE damage, which was divided into three regions: periphery with normal-shaped RPE, transitional zone with elongated cells, and center with severely damaged or lost RPE. Elongated cells in the transitional zone exhibited molecular characteristics of epithelial-mesenchymal transition. Central RPE was more susceptible to stresses than peripheral RPE. Under stresses, SIRT6, an NAD+-dependent protein deacylase, rapidly translocated from the nucleus to the cytoplasm and colocalized with stress granule factor G3BP1, leading to nuclear SIRT6 depletion. To overcome this SIRT6 depletion, SIRT6 overexpression was induced in the nucleus in transgenic mice, which protected RPE from NaIO3 and partially preserved catalase expression. These results demonstrate topological differences of mouse RPE and warrant further exploring SIRT6 as a potential target for protecting RPE from oxidative stress-induced damage.

Thyroid hormone upregulates LAMP2 expression and lysosome activity

Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.

Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called “phenoptosis” in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

PINK1 deficiency with Ca2+ changes in the hippocampus exacerbates septic encephalopathy in mice

PTEN-induced putative kinase 1 (PINK1) is a mitochondrial kinase that protects against oxidative stress-induced cellular death. PINK1 deletion, on the other hand, disrupts mitochondrial calcium (Ca2+) homeostasis in various brain disorders. This study looked at how PINK1 affects hippocampal intracellular Ca2+ changes in mice with septic encephalopathy. Mice were injected intraperitoneally with lipopolysaccharide (LPS, 5 mg/kg) to induce septic encephalopathy; then, fiber photometry was used to record hippocampal Ca2+ transients during behavioral tests in freely moving mice. Basal cytoplasmic Ca2+ levels were detected under a fluorescent microscope. LPS induced PINK1 expression and neuronal loss in the hippocampus of mice, whereas no difference in neuronal counts was shown between PINK1 knockout LPS mice and WT LPS mice. PINK1 deficiency led to inhibited Ca2+ transients and increased intracellular Ca2+ levels in the hippocampus of mice, thus, significantly aggravating the cognitive dysfunction in septic mice. An analysis of Parkin and PLC-γ1, downstream effectors of PINK1, showed that they are associated with the effects of PINK1. These results demonstrate that PINK1 deficiency disrupts intracellular Ca2+ homeostasis and exacerbates septic encephalopathy. This observation suggests a protective role of PINK1 in septic encephalopathy.

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury.

The complement component C3 is a fundamental plasma protein for host defense, produced largely by the liver. However, recent work has demonstrated the critical importance of tissue-specific C3 expression in cell survival. Here, we analyzed the effects of local versus peripheral sources of C3 expression in a model of acute bacterial pneumonia induced by Pseudomonas aeruginosa. Whereas mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient only in liver-derived C3 remained protected, comparable to wild-type mice. Human lung transcriptome analysis showed that secretory epithelial cells, such as club cells, express high levels of C3 mRNA. Mice with tamoxifen-induced C3 gene ablation from club cells in the lung had worse pulmonary injury compared with similarly treated controls, despite maintaining normal circulating C3 levels. Last, in both the mouse pneumonia model and cultured primary human airway epithelial cells, we showed that stress-induced death associated with C3 deficiency parallels that seen in Factor B deficiency rather than C3a receptor deficiency. Moreover, C3-mediated reduction in epithelial cell death requires alternative pathway component Factor B.Thus, our findings suggest that a pathway reliant on locally derived C3 and Factor B protects the lung mucosal barrier.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic β cells invitro and in models of neurodegeneration and diabetes invivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons invitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine.

High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

HECTD1-Mediated Ubiquitination and Degradation of Rubicon Regulates Autophagy and Osteoarthritis Pathogenesis.

Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery- and aging-induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. HECTD1 was significantly down-regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery- and aging-induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1-mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress-induced chondrocyte death and the subsequent progression of OA. HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation.

The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.