Loss Of ATP Research Articles

Ancestral retrovirus envelope protein ERVWE1 upregulates circ_0001810, a potential biomarker for schizophrenia, and induces neuronal mitochondrial dysfunction via activating AK2.

Increasingly studies highlight the crucial role of the ancestral retrovirus envelope protein ERVWE1 in the pathogenic mechanisms of schizophrenia, a severe psychiatric disorder affecting approximately 1% of the global population. Recent studies also underscore the significance of circular RNAs (circRNAs), crucial for neurogenesis and synaptogenesis, in maintaining neuronal functions. However, the precise relationship between ERVWE1 and circRNAs in the etiology of schizophrenia remains elusive. This study observed elevated levels of hsa_circ_0001810 (circ_0001810) in the blood samples of schizophrenia patients, displaying a significant positive correlation with ERVWE1 expression. Interestingly, in vivo studies demonstrated that ERVWE1 upregulated circ_0001810 in neuronal cells. Circ_0001810, acting as a competing endogenous RNA (ceRNA), bound to miR-1197 and facilitated the release of adenylate kinase 2 (AK2). The bioinformatics analysis of the schizophrenia datasets revealed increased levels of AK2 and enrichment of mitochondrial dynamics. Notably, miR-1197 was reduced in schizophrenia patients, while AK2 levels were increased. Additionally, AK2 showed positive correlations with ERVWE1 and circ_0001810. Further studies demonstrated that AK2 led to mitochondrial dysfunction, characterized by loss of intracellular ATP, mitochondrial depolarization, and disruption of mitochondrial dynamics. Our comprehensive investigation suggested that ERVWE1 influenced ATP levels, promoted mitochondrial depolarization, and disrupted mitochondrial dynamics through the circ_0001810/AK2 pathway. Circ_0001810 and AK2 were increased in schizophrenia and positively correlated with ERVWE1. Importantly, ERVWE1 triggered mitochondrial dysfunction through circ_0001810/miR-1197/AK2 pathway. Recent focus on the impact of mitochondrial dynamics on schizophrenia development had led to our discovery of a novel mechanism by which ERVWE1 contributed to the etiology of schizophrenia, particularly through mitochondrial dynamics. Moreover, these findings collectively proposed that circ_0001810 might serve as a potential blood-based biomarker for schizophrenia. Consistent with our previous theories, ERVWE1 is increasingly recognized as a promising therapeutic target for schizophrenia.

A combined analysis of transcriptome and proteome reveals the regulation mechanism of alginate oligosaccharides on alleviating energy deficit in postharvest strawberry

Strawberry exhibits a very short shelf life due to its rapid softening and decay, resulting in postharvest loss. Our previous study showed that AOS, an eco-friendly nontoxic natural product, plays a role in preserving the freshness of strawberries by regulating ABA content and its signaling pathway. Considering the critical role that energy deficit plays in the senescence process of fruits, coupled with the potential influence of plant hormones on energy metabolism. In this study, the molecular mechanism of strawberry postharvest senescence in response to AOS treatment was further explored from the perspective of energy metabolism regulation. This was achieved through a two-layered approach, integrating the direct determination of energy-related substances with a comprehensive joint analysis of transcriptome and proteome data. AOS treatment significantly delayed the loss of ATP, ADP, and AMP contents compared to control fruit, implying that maintaining cell energy is a crucial factor in AOS postponing senescence in strawberry. Subsequently, the mechanisms were further revealed by transcriptome and proteome analysis. Overall, 255 DEGs were screened from the AOS group, mainly related to carbohydrate and energy metabolism, defense, plant hormone, and secondary metabolism. Additionally, A total of 227 proteins were identified that were differentially expressed in the AOS group, and the majority were related to carbohydrate and energy metabolism (85 proteins). Two omics data implied that AOS could effectively regulate the energy metabolism system to maintain the cellular energy level of harvested strawberry during storage, ultimately leading to longer shelf life. Therefore, our findings provide comprehensive information regarding the molecular mechanism underlying the postharvest storage of strawberry treated with AOS. From our results, it can be concluded that AOS postharvest treatment is very useful for keeping fruit quality and extending shelf life by maintaining a higher energy level.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

Sulindac prevents increased mitochondrial VDAC1 expression and cell surface mistargeting induced by pathological conditions in retinal cells

Diabetic retinopathy (DR) continues to be the primary cause of vision loss in poorly controlled diabetic subjects. The molecular mechanisms underlying retinal pigment epithelium (RPE) cell dysfunction in DR still remain elusive. We investigated the role of mitochondrial volt-age-dependent anion channel 1 (VDAC1) in RPE dysfunction under glucotoxic and inflammatory conditions. Our results demonstrate that both glucotoxicity and cytokine treatment reduces cellular viability accompanied by increased VDAC1 and inducible nitric oxide synthase (iNOS) expression, concomitant with decreased expression of mitochondrial VDAC2 and constitutively ex-pressed endothelial NOS (eNOS). Increased VDAC1 expression during these conditions leads to its mistargeting to the cell surface, leading to ATP loss. Additionally, VDAC1 upregulation by glucotoxicity and inflammatory cytokines induces leakage of mitochondrial DNA (mtDNA) into the cytosol. Sulindac, a nonsteroidal anti-inflammatory agent, mitigates the adverse effects associated with increased VDAC1 level under pathophysiological conditions, by suppressing VDAC1 expression. The effect of sulindac on restoring cell viability could be comparably achieved only with VDAC1 inhibitor (VBIT-4) or VDAC1-specific antibody and not with the iNOS inhibitor aminoguanidine.Our findings suggest that sulindac's beneficial effects on ARPE-19 cell function are mediated by prevention of increased VDAC1 expression under pathological conditions, thus preventing mtDNA leakage and ATP loss, which are the key steps in induction of cellular inflammatory responses involved in the development of DR.

Ectonucleotidases in Ischemia Reperfusion Injury: Unravelling the Interplay With Mitochondrial Dysfunction in Liver Transplantation

Ischemia-reperfusion injury (IRI) profoundly impacts organ transplantation, especially in orthotopic liver transplantation (OLT). Disruption of the mitochondrial respiratory chain during ischemia leads to ATP loss and ROS production. Reperfusion exacerbates mitochondrial damage, triggering the release of damage-associated molecular patterns (DAMPs) and inflammatory responses. Mitochondrial dysfunction, a pivotal aspect of IRI, is explored in the context of the regulatory role of ectonucleotidases in purinergic signaling and immune responses. CD39, by hydrolyzing ATP and ADP; and CD73, by converting AMP to adenosine, emerge as key players in mitigating liver IRI, particularly through ischemic preconditioning and adenosine receptor signaling. Despite established roles in vascular health and immunity, the impact of ectonucleotidases on mitochondrial function during hepatic IRI is unclear. This review aims to elucidate the interplay between CD39/73 and mitochondria, emphasizing their potential as therapeutic targets for liver transplantation.This article explores the role of CD39/73 in tissue hypoxia, emphasizing adenosine production during inflammation. CD39 and CD73 upregulation under hypoxic conditions regulate immune responses, demonstrating protective effects in various organ-specific ischemic models. However, prolonged adenosine activation may have dual effects, beneficial in acute settings but detrimental in chronic hypoxia.Herein, we raise questions about ectonucleotidases influencing mitochondrial function during hepatic IRI, drawing parallels with cancer cell responses to chemotherapy.The review underscores the need for comprehensive research into the intricate interplay between ectonucleotidases, mitochondrial dynamics, and their therapeutic implications in hepatic IRI, providing valuable insights for advancing transplantation outcomes.

Consequences of a 2-Deoxyglucose Exposure on the ATP Content and the Cytosolic Glucose Metabolism of Cultured Primary Rat Astrocytes

The glucose analogue 2-deoxyglucose (2DG) has frequently been used as a tool to study cellular glucose uptake and to inhibit glycolysis. Exposure of primary cultured astrocytes to 2DG caused a time- and concentration-dependent cellular accumulation of 2-deoxyglucose-6-phosphate (2DG6P) that was accompanied by a rapid initial decline in cellular ATP content. Inhibitors of mitochondrial respiration as well as inhibitors of mitochondrial uptake of pyruvate and activated fatty acids accelerated the ATP loss, demonstrating that mitochondrial ATP regeneration contributes to the partial maintenance of the ATP content in 2DG-treated astrocytes. After a 30 min exposure to 10 mM 2DG the specific content of cellular 2DG6P had accumulated to around 150 nmol/mg, while cellular ATP was lowered by 50% to around 16 nmol/mg. Following such a 2DG6P-loading of astrocytes, glycolytic lactate production from applied glucose was severely impaired during the initial 60 min of incubation, but was reestablished during longer incubation concomitant with a loss in cellular 2DG6P content. In contrast to glycolysis, the glucose-dependent NADPH regeneration via the pentose phosphate pathway (PPP) was only weakly affected in 2DG6P-loaded astrocytes and in cells that were coincubated with glucose in the presence of an excess of 2DG. Additionally, in the presence of 2DG PPP-dependent WST1 reduction was found to have doubled compared to hexose-free control incubations, indicating that cellular 2DG6P can serve as substrate for NADPH regeneration by the astrocytic PPP. The data presented provide new insights on the metabolic consequences of a 2DG exposure on the energy and glucose metabolism of astrocytes and demonstrate the reversibility of the inhibitory potential of a 2DG-treatment on the glucose metabolism of cultured astrocytes.

Mitochondrial depolarization and ATP loss during high frequency nanosecond and microsecond electroporation

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE − European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca2+ electrochemotherapy in vitro. We provide experimental proof that 7.5–12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca2+ electrochemotherapy, ensuring excellent cytotoxic efficacy.

Effect of cholesterol-loaded cyclodextrin treatment on boar sperm cryopreservation.

This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality. In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing. The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05). The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar.

The inhibitory role of nitric oxide in encephalomyocarditis viral replication in pancreatic β-cells.

Abstract During type 1 diabetes (T1D) onset, insulin-secreting β-cells are selectively destroyed by T-cells. In addition to known genetic factors, viral infection (specifically by members of the picornavirus family) is an environmental factor that has been implicated in T1D onset. Viral infection stimulates macrophage release of IL-1, and in islets, stimulates the production of micromolar levels of nitric oxide (NO) by β-cells. NO impairs β-cell function via inhibition of mitochondrial oxidative metabolism, resulting in depletion of ATP. Since β-cells have a limited capacity for self-renewal, and are essential for organismal survival, we hypothesize that the cytokine-mediated production of NO by β-cells functions in a protective manner during a viral infection. In support of this hypothesis, we’ve shown that NO inhibits encephalomyocarditis viral (EMCV) replication and mediated lysis in a β-cell selective manner. EMCV is an inflammatory, mouse-tropic virus that induces diabetes in several mouse models. In this study, we have identified inositol hexakisphosphate kinase-1 (IP6K1, KmATP=1 mM) as an indirect target of NO (via the loss of ATP) that is responsible for the inhibition of EMCV replication in β-cells. We show that inhibition of IP6K1 attenuates EMCV replication and mediated β-cell lysis in a manner comparable to the actions of NO. Current studies are focused on the mechanism by which inhibition of IP6K activity attenuates EMCV replication and EMCV-mediated lysis in β-cells.

Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

Elevation of the intermediate amino acid metabolite Homocysteine (Hcy) causes Hyperhomocysteinemia (HHcy), a metabolic disorder frequently associated with mutations in the methionine-cysteine metabolic cycle as well as with nutritional deficiency and aging. The previous literature suggests that HHcy is a strong risk factor for cardiovascular diseases. Severe HHcy is well-established to correlate with vascular pathologies primarily via endothelial cell death. Though moderate HHcy is more prevalent and associated with an increased risk of cardiovascular abnormalities in later part of life, its precise role in endothelial physiology is largely unknown. In this study, we report that moderate elevation of Hcy causes endothelial dysfunction through impairment of their migration and proliferation. We established that unlike severe elevation of Hcy, moderate HHcy is not associated with suppression of endothelial VEGF/VEGFR transcripts and ROS induction. We further showed that moderate HHcy induces a sub-lethal ER stress that causes defective endothelial migration through abnormal actin cytoskeletal remodeling. We also found that sub-lethal increase in Hcy causes endothelial proliferation defect by suppressing mitochondrial respiration and concomitantly increases glycolysis to compensate the consequential ATP loss and maintain overall energy homeostasis. Finally, analyzing a previously published microarray dataset, we confirmed that these hallmarks of moderate HHcy are conserved in adult endothelial cells as well. Thus, we identified adaptive UPR and metabolic rewiring as two key mechanistic signatures in moderate HHcy-associated endothelial dysfunction. As HHcy is clinically associated with enhanced vascular inflammation and hypercoagulability, identifying these mechanistic pathways may serve as future targets to regulate endothelial function and health.

Excessive nucleic acid R-loops induce mitochondria-dependent epithelial cell necroptosis and drive spontaneous intestinal inflammation.

Oxidative stress, which can be activated by a variety of environmental risk factors, has been implicated as an important pathogenic factor for inflammatory bowel disease (IBD). However, how oxidative stress drives IBD onset remains elusive. Here, we found that oxidative stress was strongly activated in inflamed tissues from both ulcerative colitis patients and Crohn's disease patients, and it caused nuclear-to-cytosolic TDP-43 transport and a reduction in the TDP-43 protein level. To investigate the function of TDP-43 in IBD, we inducibly deleted exons 2 to 3 of Tardbp (encoding Tdp-43) in mouse intestinal epithelium, which disrupted its nuclear localization and RNA-processing function. The deletion gave rise to spontaneous intestinal inflammation by inducing epithelial cell necroptosis. Suppression of the necroptotic pathway with deletion of Mlkl or the RIP1 inhibitor Nec-1 rescued colitis phenotypes. Mechanistically, disruption of nuclear TDP-43 caused excessive R-loop accumulation, which triggered DNA damage and genome instability and thereby induced PARP1 hyperactivation, leading to subsequent NAD+ depletion and ATP loss, consequently activating mitochondrion-dependent necroptosis in intestinal epithelial cells. Importantly, restoration of cellular NAD+ levels with NAD+ or NMN supplementation, as well as suppression of ALKBH7, an α-ketoglutarate dioxygenase in mitochondria, rescued TDP-43 deficiency-induced cell death and intestinal inflammation. Furthermore, TDP-43 protein levels were significantly inversely correlated with γ-H2A.X and p-MLKL levels in clinical IBD samples, suggesting the clinical relevance of TDP-43 deficiency-induced mitochondrion-dependent necroptosis. Taken together, these findings identify a unique pathogenic mechanism that links oxidative stress to intestinal inflammation and provide a potent and valid strategy for IBD intervention.

Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na+ Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia.

Ischemic conditions cause an increase in the sodium concentration of astrocytes, driving the breakdown of ionic homeostasis and exacerbating cellular damage. Astrocytes express high levels of the electrogenic sodium-bicarbonate cotransporter1 (NBCe1), which couples intracellular Na+ homeostasis to regulation of pH and operates close to its reversal potential under physiological conditions. Here, we analyzed its mode of operation during transient energy deprivation via imaging astrocytic pH, Na+, and ATP in organotypic slice cultures of the mouse neocortex, complemented with patch-clamp and ion-selective microelectrode recordings and computational modeling. We found that a 2 min period of metabolic failure resulted in a transient acidosis accompanied by a Na+ increase in astrocytes. Inhibition of NBCe1 increased the acidosis while decreasing the Na+ load. Similar results were obtained when comparing ion changes in wild-type and Nbce1-deficient mice. Mathematical modeling replicated these findings and further predicted that NBCe1 activation contributes to the loss of cellular ATP under ischemic conditions, a result confirmed experimentally using FRET-based imaging of ATP. Altogether, our data demonstrate that transient energy failure stimulates the inward operation of NBCe1 in astrocytes. This causes a significant amelioration of ischemia-induced astrocytic acidification, albeit at the expense of increased Na+ influx and a decline in cellular ATP.

Alternative oxidase promotes high iron tolerance in Candida albicans.

Although crucial for cellular functioning, iron is toxic when present in excess. Iron overload can create a labile iron pool (LIP) that harms the mitochondrion, resulting in disruption of oxidative respiration. Fungal pathogen C. albicans possesses an unconventional terminal oxidase [alternative oxidase (AOX)] that can disrupt the electron transport chain to remove excess electrons, generated from various stressors, to shield the respiratory apparatus. Here, we identify a novel role for AOX under high iron, whereby it works as "safety valve" to protect the mitochondrion from high iron-induced reactive oxygen species (ROS). C. albicans showed pronounced metabolic flexibility to environmental iron, ranging from 1 to 500 µM. High iron significantly increased intracellular iron, LIP, and levels of cellular ROS. Expression of both AOX1/2 was induced by iron, and cells lacking Aox1/2 showed high levels of mitochondrial ROS as well as significant growth defect under high iron. Thus, Aox1/2 helps mitigate the negative effects of iron. Furthermore, iron-induced expression of AOX1/2 was intermittent, thus preventing ATP loss despite AOX being non-proton motive. Mitochondrial oxygen consumption rate (OCR) was significantly higher (62.8%) under high iron conditions. Contribution of Aox1/2 was greater toward this high OCR, compared to their contribution in low iron conditions. Lastly, enhanced virulence of C. albicans that is observed for wild-type cells under high iron was lost when fungal cells lacking AOX1/2 were used for murine infection. Thus, AOX is crucial for preserving mitochondrial function for survival in a high iron host.IMPORTANCEThe yeast C. albicans exhibits metabolic flexibility for adaptability to host niches with varying availability of nutrients including essential metals like iron. For example, blood is iron deplete, while the oral cavity and the intestinal lumen are considered iron replete. We show here that C. albicans can tolerate very high levels of environmental iron, despite an increase in high iron-induced reactive oxygen species (ROS) that it mitigates with the help of a unique oxidase, known as alternative oxidase (AOX). High iron induces AOX1/2 that limits mitochondrial accumulation of ROS. Genetic elimination of AOX1/2 resulted in diminished virulence during oropharyngeal candidiasis in high iron mice. Since human mitochondria lack AOX protein, it represents a unique target for treatment of fungal infections.

Electrolysis products, reactive oxygen species and ATP loss contribute to cell death following irreversible electroporation with microsecond-long pulsed electric fields

Membrane permeabilization and thermal injury are the major cause of cell death during irreversible electroporation (IRE) performed using high electric field strength (EFS) and small number of pulses. In this study, we explored cell death under conditions of reduced EFS and prolonged pulse application, identifying the contributions of electrolysis, reactive oxygen species (ROS) and ATP loss. We performed ablations with conventional high-voltage low pulse (HV-LP) and low-voltage high pulse (LV-HP) conditions in a 3D tumor mimic, finding equivalent ablation volumes when using 2000 V/cm 90 pulses or 1000 V/cm 900 pulses respectively. These results were confirmed by performing ablations in swine liver. In LV-HP treatment, ablation volume was found to increase proportionally with pulse numbers, without the substantial temperature increase seen with HV-LP parameters. Peri-electrode pH changes, ATP loss and ROS production were seen in both conditions, but LV-HP treatments were more sensitive to blocking of these forms of cell injury. Increases in current drawn during HV-LP was not observed during LV-HP condition where the total ablation volume correlated to the charge delivered into the tissue which was greater than HV-LP treatment. LV-HP treatment provides a new paradigm in using pulsed electric fields for tissue ablation with clinically relevant volumes.

Elaidic acid leads to mitochondrial dysfunction via mitochondria-associated membranes triggers disruption of mitochondrial calcium fluxes

Elaidic acid (EA) stimulation can lead to endoplasmic reticulum stress (ERS), accompanied by a large release of Ca2+, and ultimately the activation of NLRP3 inflammasome in Kupffer cells (KCs). Mitochondrial instability or dysfunction may be the key stimulating factors to activate NLRP3 inflammasome, and sustained Ca2+ transfer can result in mitochondrial dysfunction. We focused on KCs to explore the damage to mitochondria by EA. After EA stimulation, cells produced an oxidative stress (OS) response with a significant increase in ROS release. Immunoprecipitation experiments and the addition of inhibitors revealed that the increase in the level of intracellular Ca2+ led to Ca2+ accumulation in the mitochondrial matrix via mitochondria-associated membranes (MAMs). This was accompanied by a significant release of mROS, loss of MMP and ATP, and a significant increase in mitochondrial permeability transition pore opening, ultimately leading to mitochondrial instability. These findings confirmed the mechanism that EA induced mitochondrial Ca2+ imbalance in KCs via MAM, ultimately leading to mitochondrial dysfunction. Meanwhile, EA induced OS and the decrease of MMP and ATP in rat liver, and significant lesions were found in liver mitochondria. Swelling of the inner mitochondrial cristae and mitochondrial vacuolization occurred, with a marked increase in lipid droplets.

Effect of postharvest nicotinamide treatment on NAD+ metabolism and redox status in strawberry fruit during storage

The increased activity of PARP enzymes is associated with a deficiency of NAD+, as well as with a loss of NADPH and ATP, and consequent deterioration of the redox state in fruits. In this study, we checked whether treatment with nicotinamide (NAM) would affect PARP-1 expression and NAD+ metabolism in strawberry fruit during storage. For this purpose, strawberry fruits were treated with 10 mM NAM and co-treated with NAM and UV-C, and then stored for 5 days at 4 °C. Research showed that nicotinamide contributes to reducing oxidative stress level by reducing PARP-1 mRNA gene expression and the protein level resulting in higher NAD+ availability, as well as improving energy metabolism and NADPH levels in fruits, regardless of whether they are exposed to UV-C. The above effects cause fruits treated with nicotinamide to be characterised by higher anti-radical activity, and a lower level of reactive oxygen species in the tissue.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.03YEMK to analyse the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. We demonstrate that brief chemical ischemia, induced by combined inhibition of glycolysis and oxidative phosphorylation, results in a transient decrease in intracellular ATP. Neurons experienced a larger relative decline and showed less ability to recover from prolonged (>5min) metabolic inhibition than astrocytes. Blocking voltage-gated Na+ channels or NMDA receptors ameliorated the ATP decline in neurons and astrocytes, while blocking glutamate uptake aggravated the overall reduction in neuronal ATP, confirming the central role of excitatory neuronal activity in the cellular energy loss. Unexpectedly, pharmacological inhibition of transient receptor potential vanilloid 4 (TRPV4) channels significantly reduced the ischemia-induced decline in ATP in both cell types. Imaging with Na+ -sensitive indicator dye ING-2 furthermore showed that TRPV4 inhibition also reduced ischemia-induced increases in intracellular Na+ . Altogether, our results demonstrate that neurons exhibit a higher vulnerability to brief metabolic inhibition than astrocytes. Moreover, they reveal an unexpected strong contribution of TRPV4 channels to the loss of cellular ATP and suggest that the demonstrated TRPV4-related ATP consumption is most likely a direct consequence of Na+ influx. Activation of TRPV4 channels thus provides a hitherto unacknowledged contribution to the cellular energy loss during energy failure, generating a significant metabolic cost in ischemic conditions. KEY POINTS: In the ischemic brain, cellular ATP concentrations decline rapidly, which results in the collapse of ion gradients and promotes cellular damage and death. We analysed the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. Our results confirm the central role of excitatory neuronal activity in the cellular energy loss and demonstrate that neurons experience a larger decline in ATP and are more vulnerable to brief metabolic stress than astrocytes. Our study also reveals a new, previously unknown involvement of osmotically activated transient receptor potential vanilloid 4 (TRPV4) channels to the reduction in cellular ATP in both cell types and indicates that this is a consequence of TRPV4-mediated Na+ influx. We conclude that activation of TRPV4 channels provides a considerable contribution to the cellular energy loss, thereby generating a significant metabolic cost in ischemic conditions.

Cyclosporine A (CsA) prevents synaptic impairment caused by truncated tau by caspase-3

During Alzheimer's (AD), tau protein suffers from abnormal post-translational modifications, including cleaving by caspase-3. These tau forms affect synaptic plasticity contributing to the cognitive decline observed in the early stages of AD. In addition, caspase-3 cleaved tau (TauC3) impairs mitochondrial dynamics and organelles transport, which are both relevant processes for synapse. We recently showed that the absence of tau expression reverts age-associated cognitive and mitochondrial failure by blocking the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial complex involved in calcium regulation and apoptosis. Therefore, we studied the effects of TauC3 against the dendritic spine and synaptic vesicle formation and the possible role of mPTP in these alterations. We used mature hippocampal mice neurons to express a reporter protein (GFP, mCherry), coupled to full-length human tau protein (GFP-T4, mCherry-T4), and coupled to human tau protein cleaved at D421 by caspase-3 (GFP-T4C3, mCherry-T4C3) and synaptic elements were evaluated.Treatment with cyclosporine A (CsA), an immunosuppressive drug with inhibitory activity on mPTP, prevented ROS increase and mitochondrial depolarization induced by TauC3 in hippocampal neurons. These results were corroborated with immortalized cortical neurons in which ROS increase and ATP loss induced by this tau form were prevented by CsA. Interestingly, TauC3 expression significantly reduced dendritic spine density (filopodia type) and synaptic vesicle number in hippocampal neurons. Also, neurons transfected with TauC3 showed a significant accumulation of synaptophysin protein in their soma. More importantly, all these synaptic alterations were prevented by CsA, suggesting an mPTP role in these negative changes derived from TauC3 expression.

Low Temperature Delays the Effects of Ischemia in Bergmann Glia and in Cerebellar Tissue Swelling.

Cerebral ischemia results in oxygen and glucose deprivation that most commonly occurs after a reduction or interruption in the blood supply to the brain. The consequences of cerebral ischemia are complex and involve the loss of metabolic ATP, excessive K+ and glutamate accumulation in the extracellular space, electrolyte imbalance, and brain edema formation. So far, several treatments have been proposed to alleviate ischemic damage, yet few are effective. Here, we focused on the neuroprotective role of lowering the temperature in ischemia mimicked by an episode of oxygen and glucose deprivation (OGD) in mouse cerebellar slices. Our results suggest that lowering the temperature of the extracellular 'milieu' delays both the increases in [K+]e and tissue swelling, two dreaded consequences of cerebellar ischemia. Moreover, radial glial cells (Bergmann glia) display morphological changes and membrane depolarizations that are markedly impeded by lowering the temperature. Overall, in this model of cerebellar ischemia, hypothermia reduces the deleterious homeostatic changes regulated by Bergmann glia.

Ablation of GPR56 Causes β-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane

The activation of G Protein-Coupled Receptor 56 (GPR56), also referred to as Adhesion G-Protein-Coupled Ceceptor G1 (ADGRG1), by Collagen Type III (Coll III) prompts cell growth, proliferation, and survival, among other attributes. We investigated the signaling cascades mediating this functional effect in relation to the mitochondrial outer membrane voltage-dependent anion Channel-1 (VDAC1) expression in pancreatic β-cells. GPR56KD attenuated the Coll III-induced suppression of P70S6K, JNK, AKT, NFκB, STAT3, and STAT5 phosphorylation/activity in INS-1 cells cultured at 20 mM glucose (glucotoxicity) for 72 h. GPR56-KD also increased Chrebp, Txnip, and Vdac1 while decreasing Vdac2 mRNA expression. In GPR56-KD islet β-cells, Vdac1 was co-localized with SNAP-25, demonstrating its plasma membrane translocation. This resulted in ATP loss, reduced cAMP production and impaired glucose-stimulated insulin secretion (GSIS) in INS-1 and human EndoC βH1 cells. The latter defects were reversed by an acute inhibition of VDAC1 with an antibody or the VDAC1 inhibitor VBIT-4. We demonstrate that Coll III potentiates GSIS by increasing cAMP and preserving β-cell functionality under glucotoxic conditions in a GPR56-dependent manner by attenuating the inflammatory response. These results emphasize GPR56 and VDAC1 as drug targets in conditions with impaired β-cell function.