Depletion Of NAD Research Articles

Molecular Mechanisms Underlying Cellular Responses to the Loading of Non-thermal Atmospheric Pressure Plasma-activated Solutions

Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues, and has emerged as a novel technology for medical applications, such as wound healing, blood coagulation, and cancer treatment. NTAPP was found to affect cells indirectly through the treatment of cells with previously prepared medium irradiated by NTAPP, termed plasma-activated medium (PAM). The treatment of culture media with NTAPP results in the generation of a large amount of reactive oxygen species and reactive nitrogen species, and their derived species. We found that PAM triggered a spiral apoptotic cascade in the mitochondrial-nuclear network in A549 cancer cells. This process induced the depletion of total cellular NAD+ and elevations in intracellular calcium ion, ultimately leading to cell death. We also detected the production of hydroxyl radical and elevations in intracellular ferrous ions in PAM-treated cells. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. However, difficulties are associated with applying PAM to the clinical phase because culture media cannot be used for medical treatments. The anti-tumor activity of plasma-activated Ringer's solution was significantly stronger than that of PAM. At the end, we herein demonstrated the advantages of the combined application of plasma-activated acetate Ringer's solution and hyperthermia, a heat treatment at 42℃, for A549 cancer cell death and elucidated the underlying mechanisms.

Abstract P319: SARM1 NAD Hydrolase Deficiency Protects Hearts Against Diabetic Cardiomyopathy

NAD depletion is associated with the pathogenesis of diseases such as heart failure. Strategies to replenish cellular NAD levels by activating NAD synthesis pathways have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion are less understood. SARM1 is a novel intracellular NAD hydrolase that degrades NAD and promotes axonal degeneration in neurons. We recently showed that NAD redox imbalance and depletion promote the progression of diabetic cardiomyopathy. Therefore, we hypothesized that SARM1 deficiency might protect hearts against diabetic cardiomyopathy. 16-week diabetic stress initiated by streptozotocin (STZ) injections was applied to wild-type C57BL/6 (WT) and whole-body SARM1-KO mice. Cardiac function was measured after 8-week or 16-week of diabetic stress, and cardiac tissue and plasma samples from these mice were harvested after 16 weeks of diabetes. SARM1 mRNA or protein levels were suppressed in SARM1-KO hearts. STZ induced similar hyperglycemia (~600 mg/dL) in both WT and SARM1-KO male mice after 16 weeks. Chronic diabetic stress led to progressive decline in systolic function (Baseline fractional shortening: 50%; 16-week diabetes: 35%; P<0.05; n=5) in WT male mice, which was ameliorated in SARM1-KO male mice (16-week diabetes: 48%; P<0.05; n=5). Progressive decline in diastolic function induced by chronic diabetes (Baseline WT E’/A’: 1.53; WT 16-week diabetes: 1.12; n=5) was also improved in diabetic SARM1-KO mice (KO E’/A’ ratio at 16-week diabetes: 1.5; P<0.05; n=5). Heart weights were similar in diabetic WT or diabetic SARM1-KO hearts. A similar study is on-going in a female cohort. Despite loss of SARM1 expression, no compensatory changes in expressions of other NAD hydrolases (i.e. Cd38 or Bst1) were observed in diabetic SARM1-KO hearts, while expression levels of genes related to NAD consumption and synthesis pathways were mostly unchanged except Qprt and Haao. Cardiac fibrosis was induced in diabetic WT hearts and were suppressed in diabetic SARM1-KO hearts after 16-week diabetic stress, but these changes were not observed in tissues harvested after 8-week diabetic stress. The results suggest fibrosis is a later event in the progression of diabetic cardiomyopathy. WT and SARM1-KO mice have also been challenged with high fat diet feeding (HFD) for 16 weeks, and increased fasting glucose levels and body weights were similarly observed in HFD-WT and HFD-SARM1-KO mice. Longitudinal cardiac function analyses are on-going. Our data thus far support the protective role of SARM1 deficiency in metabolic stress-induced cardiomyopathy, while pathogenic mechanisms of SARM1 in diabetic hearts remain to be determined.

Measuring NQO1 Bioactivation Using [2H7]Glucose.

Simple SummaryCancer is often characterized by profound changes in metabolism, some of which are appropriate targets for therapeutic intervention. Many cancers overexpress the two-electron reductase NQO1, which can bioactivate the drug β-lapachone, inducing a futile redox cycle that liberates large amounts of reactive oxygen species and results in subsequent cell death. However, β-lapachone has off-target toxicities in red blood cells, which makes minimal dosing for chemotherapeutic response desirable. Here, we show that magnetic resonance-based detection of [2H7]glucose metabolism provides a robust metric of NQO1 activation, as the redox perturbation causes downregulation of glycolytic flux that is detectable in the HDO and lactate signals. Imaging of either metabolic product could provide constraints for a continual reassessment model for controlling therapeutic dosing levels.Treatment of cancers with β-lapachone causes NAD(P)H: quinone oxidoreductase 1 (NQO1) to generate an unstable hydroquinone that regenerates itself in a futile cycle while producing reactive oxygen species (ROS) in the form of superoxide and subsequently hydrogen peroxide. Rapid accumulation of ROS damages DNA, hyperactivates poly-ADP-ribose polymerase-I, causes massive depletion of NAD+/ATP, and hampers glycolysis. Cells overexpressing NQO1 subsequently die rapidly through an NAD+-keresis mechanism. Assessing changes in glycolytic rates caused by NQO1 bioactivation would provide a means of assessing treatment efficacy, potentially lowering the chemotherapeutic dosage, and reducing off-target toxicities. NQO1-mediated changes in glycolytic flux were readily detected in A549 (lung), MiaPaCa2 (pancreatic), and HCT-116 (colon) cancer cell lines by 2H-NMR after administration of [2H7]glucose. The deuterated metabolic products 2H-lactate and HDO were quantified, and linear relationships with glucose consumption for both products were observed. The higher concentration of HDO compared to 2H-lactate allows for more sensitive measurement of the glycolytic flux in cancer. Gas chromatography-mass spectrometry analysis agreed with the NMR results and confirmed downregulated energy metabolism in NQO1+ cells after β-lapachone treatment. The demonstrated method is ideal for measuring glycolytic rates, the effects of chemotherapeutics that target glycolysis, and has the potential for in vivo translation.

NAD+ Homeostasis in Diabetic Kidney Disease.

The redox reaction and energy metabolism status in mitochondria is involved in the pathogenesis of metabolic related disorder in kidney including diabetic kidney disease (DKD). Nicotinamide adenine dinucleotide (NAD+) is a cofactor for redox reactions and energy metabolism in mitochondria. NAD+ can be synthesized from four precursors through three pathways. The accumulation of NAD+ may ameliorate oxidative stress, inflammation and improve mitochondrial biosynthesis via supplementation of precursors and intermediates of NAD+ and activation of sirtuins activity. Conversely, the depletion of NAD+ via NAD+ consuming enzymes including Poly (ADP-ribose) polymerases (PARPs), cADPR synthases may contribute to oxidative stress, inflammation, impaired mitochondrial biosynthesis, which leads to the pathogenesis of DKD. Therefore, homeostasis of NAD+ may be a potential target for the prevention and treatment of kidney diseases including DKD. In this review, we focus on the regulation of the metabolic balance of NAD+ on the pathogenesis of kidney diseases, especially DKD, highlight benefits of the potential interventions targeting NAD+-boosting in the treatment of these diseases.

HGG-33. EXPLOITING METABOLIC DEFECTS WITH NAMPT INHIBITORS IN DIPG

Diffuse intrinsic pontine glioma (DIPG) are universally lethal pediatric brain tumors with limited treatment options. We recently performed synthetic lethal drug screen with a panel of DNA repair and metabolic inhibitors in vitro, in patient-derived DIPG cells and isogenic cell lines engineered to contain key DIPG-associated mutations. Nearly 80% of DIPGs harbor a recurrent H3K27M mutation in H3.3 (H3F3A) or H3.1 (HIST1H3B) histones. This has prompted us to consider H3K27M mutation-induced exploitable defects for a therapeutic gain. This screen identified synthetic lethal interactions between H3K27M mutations and the nicotinamide phosphoribosyl transferase (NAMPT) inhibitor, FK866. The association between H3K27M mutations and NAMPTi sensitivity was validated in follow-up studies using isogenic WT and H3K27M-mutant expressing pairs of human immortalized astrocytes and neural progenitor cells (NPCs). In addition, we tested the effects of FK866 in a panel of unique DIPG patient-derived tumor neurospheres in short-term viability assays. We found that FK866 induced depletion of NAD and exhibited toxicity towards H3K27M mutant DIPG cell lines with an IC50 of ~2.5 nM. These findings were consistent across three structurally unique NAMPT inhibitors. Finally, we found that inducible expression of mutant H3K27M results in a gradual, 50% decrease in cellular NAD levels over the course of five days, suggesting that H3K27M mutations may induce an inherent NAD metabolic defect that is exploited by NAMPTi’s. We now seek to understand the mechanistic basis for the observed metabolic defect and NAMPTi sensitivity associated with mutant H3K27M. In addition, we aim to identify specific NAMPTi and DNA damaging agent combinations which maximally exploit this H3K27M-associated NAD metabolic defects.

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, Histone PARylation Factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here, we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADP-ribose (ADPR), resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

Probable Causes of Alzheimer’s Disease

A three-part mechanism is proposed for the induction of Alzheimer’s disease: (1) decreased blood lactic acid; (2) increased blood ceramide and adipokines; (3) decreased blood folic acid. The age-related nature of these mechanisms comes from age-associated decreased muscle mass, increased visceral fat and changes in diet. This mechanism also explains why many people do not develop Alzheimer’s disease. Simple changes in lifestyle and diet can prevent Alzheimer’s disease. Alzheimer’s disease is caused by a cascade of events that culminates in damage to the blood–brain barrier and damage to neurons. The blood–brain barrier keeps toxic molecules out of the brain and retains essential molecules in the brain. Lactic acid is a nutrient to the brain and is produced by exercise. Damage to endothelial cells and pericytes by inadequate lactic acid leads to blood–brain barrier damage and brain damage. Inadequate folate intake and oxidative stress induced by activation of transient receptor potential cation channels and endothelial nitric oxide synthase damage the blood–brain barrier. NAD depletion due to inadequate intake of nicotinamide and alterations in the kynurenine pathway damages neurons. Changes in microRNA levels may be the terminal events that cause neuronal death leading to Alzheimer’s disease. A new mechanism of Alzheimer’s disease induction is presented involving lactic acid, ceramide, IL-1β, tumor necrosis factor α, folate, nicotinamide, kynurenine metabolites and microRNA.

RETRACTED ARTICLE:The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation

Chloroplast function requires the coordinated action of nuclear- and chloroplast-derived proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) proteins that regulate plastid mRNA metabolism. Despite their large number and importance, regulatory mechanisms controlling PPR expression are poorly understood. Here we show that the Arabidopsis NOT4A ubiquitin-ligase positively regulates the expression of PROTON GRADIENT REGULATION 3 (PGR3), a PPR protein required for translating several thylakoid-localised photosynthetic components and ribosome subunits within chloroplasts. Loss of NOT4A function leads to a strong depletion of cytochrome b6f and NAD(P)H dehydrogenase (NDH) complexes, as well as plastid 30 S ribosomes, which reduces mRNA translation and photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. Quantitative transcriptome and proteome analysis of the not4a mutant reveal it lacks PGR3 expression, and that its molecular defects resemble those of a pgr3 mutant. Furthermore, we show that normal plastid function is restored to not4a through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring robust photosynthetic function during development and stress-response, through promoting PGR3 production and chloroplast translation.

Tissue-Specific 1H-NMR Metabolomic Profiling in Mice with Adenine-Induced Chronic Kidney Disease.

Chronic kidney disease (CKD) results in the impaired filtration of metabolites, which may be toxic or harmful to organs/tissues. The objective of this study was to perform unbiased 1H nuclear magnetic resonance (NMR)-based metabolomics profiling of tissues from mice with CKD. Five-month-old male C57BL6J mice were placed on either a casein control diet or adenine-supplemented diet to induce CKD for 24 weeks. CKD was confirmed by significant increases in blood urea nitrogen (24.1 ± 7.7 vs. 105.3 ± 18.3 mg/dL, p < 0.0001) in adenine-fed mice. Following this chronic adenine diet, the kidney, heart, liver, and quadriceps muscles were rapidly dissected; snap-frozen in liquid nitrogen; and the metabolites were extracted. Metabolomic profiling coupled with multivariate analyses confirm clear separation in both aqueous and organic phases between control and CKD mice. Severe energetic stress and apparent impaired mitochondrial metabolism were observed in CKD kidneys evidenced by the depletion of ATP and NAD+, along with significant alterations in tricarboxylic acid (TCA) cycle intermediates. Altered amino acid metabolism was observed in all tissues, although significant differences in specific amino acids varied across tissue types. Taken together, this study provides a metabolomics fingerprint of multiple tissues from mice with and without severe CKD induced by chronic adenine feeding.

PARP-1 activation after oxidative insult promotes energy stress-dependent phosphorylation of YAP1 and reduces cell viability.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyze the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using WRL68 (HeLa derivate) cells, we previously showed that PARP-1 activation induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promoted cell death. In this work, LC-MS/MS-based phosphoproteomics in WRL68 cells showed that the oxidative damage induced by H2O2 increased the phosphorylation of YAP1, a transcriptional co-activator involved in cell survival, and modified the phosphorylation of other proteins involved in transcription. Genetic or pharmacological inhibition of PARP-1 in H2O2-treated cells reduced YAP1 phosphorylation and degradation and increased cell viability. YAP1 silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells exposed to oxidative damage. Supplementation of NAD+ also reduced YAP1 phosphorylation, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of the metabolic sensor AMPK. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates, we hypothesized that the loss of cellular NAD+ after PARP-1 activation may induce an energy stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in which PARP-1 activation reduced cell viability by promoting the phosphorylation and degradation of YAP1 through a mechanism that involves the depletion of NAD+.

Signaling interplay between PARP1 and ROS regulates stress-induced cell death and developmental changes in Dictyostelium discoideum

Poly (ADP-ribose) polymerase-1 (PARP1) is a DNA damage sensor that gets activated in proportion to the damage, helping cells to determine whether to repair the damage or initiate cell death processes. We have previously shown PARP1's significance in the developmental processes of Dictyostelium discoideum in addition to its role in oxidative stress and UV-C stress induced cell death. In this study, we show the significance of ROS in PARP1 mediated responses of D. discoideum under different stress conditions. Interestingly, our results suggest differential kinetics of PARP1 activation and implications of ROS in starvation and cadmium induced cell death events. Increased accumulation of Poly (ADP-ribose), a product of PARP activation, could be detected within minutes post cadmium stress, whereas PARP1 activation was only a later event with starvation. Starvation induced PARP1 activation was supported by the depletion of ATP and NAD+, while PARP inhibitor confers protective effect during starvation. During starvation, cell death is induced in two phases, a primary ROS driven PARP1 independent early necrotic phase followed by a PARP1 driven ROS dependent paraptotic phase; both of which comprise mitochondrial changes. Cadmium (Cd) exerted a dose-dependent effect on cell death; a low dose of 0.2 mM Cd led to paraptosis and a higher dose of 0.5 mM Cd led to necrosis in D. discoideum cells within 24 h. Interestingly, glutathione (GSH) exposure could rescue cells from Cd stress mediated cell death. Besides unicellular cell death, the developmental arrest induced by cadmium and oxidative stress could be rescued by reinstating the redox equilibrium using GSH. In conclusion, we underscore the significant link between PARP1 and ROS in regulating the process of cell death and development in D. discoideum.

TAMI-20. POLY(ADP-RIBOSE) GLYCOHYDROLASE INHIBITION SEQUESTERS NAD+ TO POTENTIATE THE METABOLIC LETHALITY OF ALKYLATING CHEMOTHERAPY IN IDH MUTANT TUMOR CELLS

Abstract Mutations in IDH1 or IDH2 characterize the majority of diffuse infiltrative gliomas of younger adulthood. DNA alkylator chemotherapy has proven to be an effective treatment for IDH mutant glioma, yet recurrences remain frequent and improved treatments are needed. Upon exposure of cancer cells to DNA alkylating agents, poly(ADP-ribose) polymerases (PARPs) catalyze the transient oligomerization of nicotinamide adenine dinucleotide (NAD+) into poly-ADP-ribose (PAR) chain-assemblies, which stimulate the DNA damage response. Here, we find that, in IDH mutant glioma cells, alkylator-induced cytotoxicity is dramatically augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme poly(ADP-ribose) glycohydrolase (PARG). Mechanistically, we show this augmentation is driven by a metabolic state pairing amplified alkylator-induced DNA damage with catastrophic depletion of NAD+. IDH mutant cancer cells have low basal levels of NAD+, rendering them vulnerable to prolonged NAD+ depletion. Either of two clinically-utilized monofunctional alkylators, procarbazine (PCZ) or temozolomide (TMZ), when exposed to multiple IDH mutant lines, activated PARP conversion of cellular NAD+ monomers into polymerized PAR. Subsequent PAR breakdown and recycling of NAD+ monomers was then halted by concomitant PARG inactivation, amplifying PARylation-mediated DNA damage signaling and critically depleting free NAD+, resulting in profound cytotoxicity. This effect was partially reversed by NAD+ rescue supplementation, confirming the key contribution of metabolic stress. Combined alkylator treatment and PARG inhibition in vivo maximized both NAD+ depletion and cytotoxicity in an endogenous IDH mutant cancer model. Thus, alkyating DNA damaging agents expose a metabolic liability in IDH mutant cancers, and subsequent targeted blockade of PAR breakdown and NAD+ metabolite recovery can exploit this vulnerability to provide genotype-specific therapeutic benefit.

Immunotherapy of COVID-19 with poly (ADP-ribose) polymerase inhibitors: starting with nicotinamide.

COVID-19 induces a proinflammatory environment that is stronger in patients requiring intensive care. The cytokine components of this environment may determine efficacy or otherwise of glucocorticoid therapy. The immunity modulators, the aryl hydrocarbon receptor (AhR) and the nuclear NAD+-consuming enzyme poly (ADP-ribose) polymerase 1 (PARP 1) may play a critical role in COVID-19 pathophysiology. The AhR is overexpressed in coronaviruses, including COVID-19 and, as it regulates PARP gene expression, the latter is likely to be activated in COVID-19. PARP 1 activation leads to cell death mainly by depletion of NAD+ and adenosine triphosphate (ATP), especially when availability of these energy mediators is compromised. PARP expression is enhanced in other lung conditions: the pneumovirus respiratory syncytial virus (RSV) and chronic obstructive pulmonary disease (COPD). I propose that PARP 1 activation is the terminal point in a sequence of events culminating in patient mortality and should be the focus of COVID-19 immunotherapy. Potent PARP 1 inhibitors are undergoing trials in cancer, but a readily available inhibitor, nicotinamide (NAM), which possesses a highly desirable biochemical and activity profile, merits exploration. It conserves NAD+ and prevents ATP depletion by PARP 1 and Sirtuin 1 (silent mating type information regulation 2 homologue 1) inhibition, enhances NAD+ synthesis, and hence that of NADP+ which is a stronger PARP inhibitor, reverses lung injury caused by ischaemia/reperfusion, inhibits proinflammatory cytokines and is effective against HIV infection. These properties qualify NAM for therapeutic use initially in conjunction with standard clinical care or combined with other agents, and subsequently as an adjunct to stronger PARP 1 inhibitors or other drugs.

Protein kinase C activates NAD kinase in human neutrophils

NAD kinase (NADK) is required for the de novo synthesis of NADP+ from NAD+. In neutrophils, NADK plays an essential role by providing sufficient levels of NADPH to support a robust oxidative burst. Activation of NADPH oxidase-2 (NOX-2) in neutrophils by stimulators of protein kinase C (PKC), such as phorbol myristate acetate (PMA), results in the rapid generation of superoxide at the expense of oxidation of NADPH to NADP+. In this study, we measured the levels of pyridine nucleotides following the addition of PMA to neutrophils. PMA elicited a rapid increase in NADP+ in neutrophils, which was not due to oxidation of NADPH, the levels of which also rose. This was mirrored by a rapid reduction in NAD+ levels, suggesting that NADK had been activated. PMA-induced depletion of NAD+ in neutrophils was blocked by PKC inhibitors, but was not dependent on NOX-2, as it was not blocked by the NOX inhibitor, diphenyleneiodonium. PMA also increased NADK activity in neutrophil lysates as well as NADK phosphorylation, as revealed by a monoclonal antibody selective for phospho-NADK. Human recombinant NADK was phosphorylated by PKCδ, resulting in increased immunoreactivity, but unchanged enzyme activity, suggesting that PKC-induced phosphorylation alone is insufficient to increase NADK activity in neutrophils. This leads us to speculate that phosphorylation of NADK promotes the dissociation of an inhibitory molecule from a complex, thereby increasing enzyme activity. Activation of NADK by PKC in phagocytic cells could be critical for the rapid provision of sufficient levels of superoxide for host defence against invading microorganisms.

Targeting Base Excision Repair in Cancer: NQO1-Bioactivatable Drugs Improve Tumor Selectivity and Reduce Treatment Toxicity Through Radiosensitization of Human Cancer.

Ionizing radiation (IR) creates lethal DNA damage that can effectively kill tumor cells. However, the high dose required for a therapeutic outcome also damages healthy tissue. Thus, a therapeutic strategy with predictive biomarkers to enhance the beneficial effects of IR allowing a dose reduction without losing efficacy is highly desirable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in the majority of recalcitrant solid tumors in comparison with normal tissue. Studies have shown that NQO1 can bioactivate certain quinone molecules (e.g., ortho-naphthoquinone and β-lapachone) to induce a futile redox cycle leading to the formation of oxidative DNA damage, hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), and catastrophic depletion of NAD+ and ATP, which culminates in cellular lethality via NAD+-Keresis. However, NQO1-bioactivatable drugs induce methemoglobinemia and hemolytic anemia at high doses. To circumvent this, NQO1-bioactivatable agents have been shown to synergize with PARP1 inhibitors, pyrimidine radiosensitizers, and IR. This therapeutic strategy allows for a reduction in the dose of the combined agents to decrease unwanted side effects by increasing tumor selectivity. In this review, we discuss the mechanisms of radiosensitization between NQO1-bioactivatable drugs and IR with a focus on the involvement of base excision repair (BER). This combination therapeutic strategy presents a unique tumor-selective and minimally toxic approach for targeting solid tumors that overexpress NQO1.

Metabolic alterations caused by the mutation and overexpression of the Tmem135 gene.

Mitochondria are dynamic organelles undergoing fission and fusion. Proper regulation of this process is important for healthy aging process, as aberrant mitochondrial dynamics are associated with several age-related diseases/pathologies. However, it is not well understood how imbalanced mitochondrial dynamics may lead to those diseases and pathologies. Here, we aimed to determine metabolic alterations in tissues and cells from mouse models with over-fused (fusion > fission) and over-fragmented (fusion < fission) mitochondria that display age-related disease pathologies. Our results indicated tissue-dependent sensitivity to these mitochondrial changes, and metabolic pathways likely affected by aberrant mitochondrial dynamics. This study provides new insights into how dysregulated mitochondrial dynamics could lead to functional abnormalities of tissues and cells.

SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration.

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho -/- sarm1 -/- double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho -/- ) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

Lymphocyte senescence in COPD is associated with decreased sirtuin 1 expression in steroid resistant pro-inflammatory lymphocytes

Background:The class III NAD-dependent histone deacetylase (HDAC) sirtuin 1 (SIRT1) is an important regulator of senescence, aging, and inflammation. SIRT1de-acetylates chromatin histones, thereby silencing inflammatory gene transcription. We have reported increased steroid-resistant senescent pro-inflammatory CD28nullCD8+ T cells in patients with chronic obstructive pulmonary disease (COPD). We hypothesized that SIRT1 is reduced in these cells in COPD, and that treatment with SIRT1 activators (resveratrol, curcumin) and agents preventing NAD depletion (theophylline) would upregulate SIRT1 and reduce pro-inflammatory cytokine expression in these steroid-resistant cells.Methods:Blood was collected from n = 10 COPD and n = 10 aged-matched controls. Expression of CD28, SIRT1, and pro-inflammatory cytokines was determined in CD8+ and CD8– T and natural killer T (NKT)-like cells cultured in the presence of ±1 µM prednisolone, ±5 mg/L theophylline, ±1 µM curcumin, ±25 µM resveratrol, using flow cytometry and immunofluorescence.Results:There was an increase in the percentage of CD28nullCD8+ T and NKT-like cells in COPD patients compared with controls. Decreased SIRT1 expression was identified in CD28nullCD8+T and NKT-like cells compared with CD28+ counterparts from both patients and controls (e.g. CD28null 11 ± 3% versus CD28+ 57 ± 9%). Loss of SIRT1 was associated with increased production of IFNγ and TNFα, steroid resistance, and disease severity. SIRT1 expression was upregulated in the presence of all drugs and was associated with a decrease in steroid resistance and IFNγ and TNFα production by CD28nullCD8+T and NKT-like cells. The presence of the SIRT1 inhibitor, EX-527 negated [by 92 ± 12% (median ± SEM)] the effect of the SIRT1 activator SRT720 on the percentage of CD8+ T cells producing IFNγ and TNFα.Conclusions:Steroid resistance in pro-inflammatory CD28nullCD8+ T and NKT-like cells is associated with decreased SIRT1 expression. Treatment with prednisolone, in combination with theophylline, curcumin or resveratrol increases SIRT1 expression, restores steroid sensitivity, and inhibits pro-inflammatory cytokine production from these cells and may reduce systemic inflammation in COPD. The reviews of this paper are available via the supplemental material section.

Targeting NAD+ Synthesis to Potentiate CD38-Based Immunotherapy of Multiple Myeloma

Antibodies targeting CD38, a NAD+-degrading enzyme, have emerged as a promising immunotherapy against multiple myeloma (MM). Currently, the mechanisms by which anti-CD38 antibodies establish their therapeutic effects are poorly understood. Here, we advocate for the depletion of NAD+ to enhance the efficacy of anti-CD38-based immunotherapies in MM.

Nonspecific Binding of RNA to PARP1 and PARP2 Does Not Lead to Catalytic Activation.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2, respectively), upon binding damaged DNA, become activated to add long chains of poly(ADP-ribose) (PAR) to themselves and other nuclear proteins. This activation is an essential part of the DNA damage response. The PAR modifications recruit the DNA repair machinery to sites of DNA damage and result in base excision and single-strand break repair, homologous recombination, nucleotide excision repair, and alternative nonhomologous end joining. More recently, both PARP1 and PARP2 have been shown to bind to or be activated by RNA, a property that could interfere with the function of PARP1 and PARP2 in the response to DNA damage or lead to necrosis by depletion of cellular NAD+. We have quantitatively evaluated the in vitro binding of a variety of RNAs to PARP1 and PARP2 and queried the ability of these RNAs to switch on enzymatic activity. We find that while both proteins bind RNAs without specificity toward sequence or structure, their interaction with RNA does not lead to auto-PARylation. Thus, although PARP1 and PARP2 are promiscuous with respect to activation by DNA, they both demonstrate exquisite selectivity against activation by RNA.