Biology subjects

Brenner, C.

Publications and source records attributed to Brenner, C..

17 recordsLinked to original sources

bioRxiv2026

Disease-specific differences in particulate matter handling drive pathogenic responses in human derived nasal epithelial cells

BackgroundParticulate matter (PM) exposure is associated with increased risk and exacerbation of chronic rhinosinusitis (CRS), yet underlying mechanisms remain poorly understood. ObjectiveTo define the epithelial mechanisms by which PM exposure promotes and exacerbates CRS, with a focus on epithelial remodeling, inflammation, barrier dysfunction, and cellular uptake of PM. MethodsHuman nasal epithelial cells obtained from ethmoid tissue of CRS (n = 5) and control donors (n = 4) were cultured at an air-liquid interface and exposed to PM. Single-cell RNA sequencing was performed to characterize PM-induced cellular and transcriptional changes. Protein expression, epithelial barrier integrity, cell death, and intracellular PM uptake were evaluated using biochemical, imaging, and ultrastructural approaches. ResultsUnsupervised clustering identified seven epithelial cell populations. Gene set analysis revealed baseline enrichment of inflammatory and keratinization pathways and reduced ciliogenesis in CRS compared with controls. Although PM induced inflammation and squamous differentiation in controls, the pathogenic responses were significantly amplified in CRS, including uniquely enhanced IL-1 signaling. Transcriptional changes were validated by ELISA, transepithelial electrical resistance, and immunofluorescence, demonstrating increased inflammation, epithelial barrier disruption, and cell death following PM exposure. Transmission electron microscopy revealed increased intracellular PM within membrane-bound organelles. Pre-treatment with an endocytosis inhibitor rescued PM-induced epithelial barrier dysfunction and inflammation. ConclusionCRS epithelium exhibits baseline dysfunction that may predispose it to environmental injury. PM exposure both induces CRS-like epithelial changes in controls and exacerbates disease-associated phenotypes. Key MessagesO_LICompared to controls, CRS nasal epithelium exhibits baseline inflammatory, keratinization, and ciliogenesis abnormalities. C_LIO_LIParticulate matter induces inflammation and squamous differentiation, while amplifying epithelial injury that is more robust in CRS epithelium compared to controls. C_LIO_LIInhibition of dynamin-dependent endocytosis rescues PM-induced epithelial barrier leakiness and inflammation, implicating intracellular particulate matter uptake in disease pathogenesis. C_LI Capsule SummaryParticulate matter induces CRS-like epithelial remodeling in controls and exacerbates inflammation and epithelial barrier dysfunction in CRS nasal epithelium, which can be rescued with endocytosis inhibition. This suggests a mechanistic link between baseline CRS vulnerability, intracellular uptake of particulate matter, and disease pathogenesis.

cell biology↗

bioRxiv2026

Combined inhibition of AIF/CHCHD4 interaction and GLS1 to exploit metabolic vulnerabilities in pediatric osteosarcoma

Osteosarcoma is a malignant bone tumor with a high risk of metastatic relapse and poor outcomes due to primary and acquired chemoresistance. This highlights the medical need to develop effective targeted approaches to overcome chemoresistance. Recent studies have revealed the roles of metabolic reprogramming and mitochondria-nucleus crosstalk in osteosarcoma progression, indicating the potential of these cellular processes as therapeutic targets. The complex formed by mitochondrial apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) orchestrates the import and oxidative folding of cysteine-rich, nuclear-encoded proteins, thereby regulating key mitochondrial functions and metabolism. Here, we identified mitoxantrone as an inhibitor of the AIF/CHCHD4 mitochondrial import machinery and revealed a new mitoxantrone-induced metabolic vulnerability in some osteosarcoma cell line models, characterized by intracellular glutamine accumulation and an increase in nucleotide synthesis. As a result, synergy was found between mitoxantrone and the glutaminase inhibitor telaglenastat in both in vitro and in vivo osteosarcoma models. Collectively, our findings position the AIF/CHCHD4 complex as a druggable therapeutic target and provide a combination strategy for mitoxantrone/telaglenastat treatment to overcome metabolic adaptations and chemoresistance in osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/716303v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1229e58org.highwire.dtl.DTLVardef@1c9af45org.highwire.dtl.DTLVardef@120d2borg.highwire.dtl.DTLVardef@11e8216_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

NAPRT expression and epigenetic regulation in pediatric rhabdomyosarcoma as a potential biomarker for NAMPT inhibition

PurposeNew treatments are needed to improve survival in children with rhabdomyosarcoma (RMS). NAD biosynthesis, regulated by the enzymes NAPRT and NAMPT, represents a metabolic vulnerability due to high NAD turnover in cancers. Although NAMPT inhibitors (NAMPTi) show preclinical promise, clinical translation has been limited by toxicity and the lack of predictive biomarkers. Here, we evaluated NAPRT expression in RMS and its potential as an actionable biomarker to guide NAMPTi therapy. Experimental DesignNAPRT promoter methylation, transcript levels, and protein expression were assessed in RMS cells, PDXs, and primary tumors (n=109) from the Childrens Oncology Group. In vitro sensitivity to NAMPTi was tested in molecularly diverse and isogenic RMS cell lines, examining the role of NAPRT expression in mediating cytotoxicity and the ability of nicotinic acid (NA) to rescue viability. In vivo efficacy was assessed using NAPRT-isogenic orthotopic xenograft models. ResultsNAPRT promoter hypermethylation was found in a subset of RMS models and patient samples. Immunohistochemistry showed loss of NAPRT protein in 30-40% of tumors, defined as <1% tumor cell staining. Methylation modestly correlated with protein expression. NAPRT-silenced cells were highly sensitive to NAMPTi, driven by NAD depletion and not reversible with NA. In vivo, NAMPTi induced significant tumor regression, which was not abrogated with NA administration in NAPRT-silenced models. ConclusionsNAPRT loss occurs in a subset of RMS, offering a potential strategy to expand the therapeutic window of NAMPTi. Further research is needed to understand NAPRT regulation and optimize biomarker assay strategies for use in future clinical trials.

cancer biology↗

bioRxiv2025

Astrocytic CREB regulates transcriptional, neuronal, and behavioral responses to cocaine

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREBs influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaines effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

neuroscience↗

bioRxiv2025

Engineered flavonoid disrupts mitochondrial AIF/CHCHD4 complex for targeted cancer therapy

Oncogenic metabolism depends on multifaceted mechanisms, including bidirectional inter-organelle communication between mitochondria and the nucleus, facilitating cellular adaptation at the transcriptomic, proteomic, and metabolomic levels. The mitochondrial protein complex composed of apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is essential for this mitochondrio-nuclear communication. The AIF/CHCHD4 complex mediates the mitochondrial import of cysteine-enriched nuclear gene-encoded proteins, thereby adapting the mitochondrial proteome to cellular energy demands. We report the discovery of M30-E05, an engineered flavonoid that binds to the NADH pocket of AIF, preventing its dimerization and disrupting the AIF/CHCHD4 complex. Molecular docking and gel electrophoresis analysis of mitochondrial AIF/CHCHD4 substrates expression confirm this mechanism. In cancer cells, M30-E05 reduces the expression of nuclear gene-encoded mitochondrial proteins such as AIF, CHCHD4, COX17, and MICU1. In addition, M30-E05 fragments the mitochondrial network and impairs mitochondrial respiration, causing profound alterations, particularly in lipid and aminoacid metabolism, as revealed by kinetic measurements of oxygen consumption and mass spectrometric metabolomics. Importantly, M30-E05 significantly reduces the viability of a human adult and pediatric osteosarcoma cancer cell panel, including those from patient-derived xenografts (PDX) of osteosarcomas, and induces apoptosis. When orally administered for two weeks to immunodeficient NSG mice, M30-E05 inhibited tumor growth in a subcutaneous PDX xenograft model without apparent toxicity. We anticipate that M30-E05, as a first-in-class metabolic inhibitor, could serve as the lead compound for a new class of targeted antineoplastic agents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/642976v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b1add9org.highwire.dtl.DTLVardef@cac7corg.highwire.dtl.DTLVardef@101c9corg.highwire.dtl.DTLVardef@1c6845f_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Glycerol-3-phosphate activates ChREBP, FGF21 transcription and lipogenesis in Citrin Deficiency

Citrin Deficiency (CD) is caused by inactivation of SLC25A13, a mitochondrial membrane protein required to move electrons from cytosolic NADH to the mitochondrial matrix in hepatocytes. People with CD do not like sweets. We discovered that SLC25A13 loss causes accumulation of glycerol-3-phosphate (G3P), which activates carbohydrate response element binding protein (ChREBP) to transcribe FGF21, which acts in the brain to restrain intake of sweets and alcohol, and to transcribe key genes of de novo lipogenesis. Mouse and human data establish G3P-ChREBP as a new mechanistic component of the Randle Cycle that contributes to metabolic dysfunction-associated steatotic liver disease (MASLD) and forms part of a system that communicates metabolic states from liver to brain in a manner that alters food and alcohol choices. The data provide a framework for understanding FGF21 induction in varied conditions, suggest ways to develop FGF21-inducing drugs, and drug candidates for both lean MASLD and support of urea cycle function in CD.

molecular biology↗

bioRxiv2024

Oral supplementation with Nicotinamide Riboside treatment protects RGCs in DBA/2J mouse model

PurposeThe aim of this study was to test whether oral administration of nicotinamide riboside (NR), the nicotinamide adenine dinucleotide (NAD+) precursors, protect retina ganglion cells (RGCs) from neurodegeneration in DBA/2J (D2) mice, which is a widely used mouse model of age-related inherited glaucoma. MethodOral NR or NAM administration (NR low dose: 1150mg/kg; NR high dose: 4200mg/kg; NAM low dose group: 500mg/kg; NAM high dose: 2000mg/kg of body weight per day) essentially started when D2 mice were 4 or 9 months old and continued up to 12 months old. Control cohort identically received food/water without NAM or NR. Intraocular pressure (IOP) was measured every month until experiment completion. Pattern electroretinography (PERG) was recorded. Retinas were harvested for whole mount immunofluorescence staining with RGCs marker Brn3a and imaged by fluorescent confocal microscopy. Optic nerves were harvested for axon staining and quantification. Retinal NAD+ levels were enzymatically assayed. ResultsNR oral supplementary treatment started at 4 months old robustly increased retinal NAD+ levels in D2 mice (NRHigh vs. vehicle: 273.7{+/-}23.59% vs. 108.70{+/-}12.10%, p<0.001). In aged vehicle group (12 months old), there was significantly diminution of the P1 and N2 components of PERG response compare with naive group (naive vs. vehicle: P1: 7.82{+/-} 0.70uV vs 1.63{+/-} 0.17uV, p<0.0001; N2: -13.29{+/-} 0.83uV vs. -3.22{+/-} 0.27uV, p<0.0001; Kruskal-Wallis test with Dunn s multiple comparison test). NR treatment preserved aged D2 visual function when mice were 9 and 12 months old. In addition, long-term NR high dose treatment significantly protected against total RGCs loss and optic nerve atrophy (RGC: NRHigh vs. vehicle: 1412{+/-}62.00vs 475.2{+/-}94.68 cells/field, p<0.00001; axon numbers: NRHigh vs. vehicle: 23990{+/-}1159 vs 8573{+/-}1160, n=41-53, p<0.0001). Furthermore, long-term NR supplementation prevent iris depigmentation and delayed IOP elevation. ConclusionNR oral supplementary treatment significantly preserved RGC and axon numbers, potentially preserves retinal function via elevated retinal NAD+ level in aged D2 mice. Interestingly, NR treatment also prevented iris atrophy, delayed IOP elevation associated with this glaucoma model. NR oral supplementation thus treated several aspects of murine pigment dispersion glaucoma. Given parallels between this model and glaucoma in human, out data indicate that NR is worth exploring as a therapeutic candidate in treatment of glaucoma.

neuroscience↗

bioRxiv2024

Polyamines sustain epithelial regeneration in aged intestines by modulating protein homeostasis

Aging hampers the regenerative potential of intestinal epithelium across species including humans, yet the underlying causes remain elusive. Here, using proteomic and metabolomic profiling of intestinal tissues together with functional assays, we characterized the temporal dynamics of regeneration following injury induced by 5-fluorouracil, a commonly used chemotherapeutic agent. Comparison of regeneration dynamics in mice of different ages revealed the emergence of a proteostasis stress signature and increased levels of polyamines following injury exclusively in old epithelia. Mechanistically, we show that delayed regeneration is an intrinsic feature of aged epithelial cells that display reduced protein synthesis and accumulation of ubiquitylated proteins. Notably, dietary restriction followed by re-feeding prior to injury increases polyamine pathway activation, enhances protein synthesis, and restores the regenerative capacity of aged intestines. Our findings highlight promising epithelial targets for interventions aimed at tackling the decline in tissue repair mechanisms associated with aging.

molecular biology↗

bioRxiv2023

The Interferon-inducible NAMPT acts as a protein phosphoribosylase to restrict viral infection

As obligate intracellular pathogens, viruses often activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the salvage NAD+ synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses with unknown mechanism. Here we report that NAMPT restricts herpes simplex virus 1 (HSV-1) replication via phosphoribosyl-hydrolase activity toward key viral structural proteins, independent of NAD+ synthesis. Deep mining of enriched phosphopeptides of HSV-1-infected cells identified phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins. Indeed, NAMPT de-phosphoribosylates viral proteins in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Moreover, loss of NAMPT renders mice highly susceptible to HSV-1 infection. The work describes a hidden enzyme activity of a metabolic enzyme in viral infection and host defense, offering a system to interrogate roles of phosphoribosylation in metazoans.

molecular biology↗

bioRxiv2022

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome

Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). We report that LS mice also develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular NAD+/ NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+-dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+/ NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+-dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV1.5 current and SERCA2a function measured by Ca2+- transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human iPSC models of LS.

pathology↗

bioRxiv2021

Nicotinamide riboside alleviates Parkinson's disease symptoms but downregulates dopamine metabolism upon lactacystin-induced proteostasis failure

Activation of mitochondrial metabolism and proteostasis with the NAD+ precursor nicotinamide riboside (NR) has emerged as a potential therapeutic approach for neurodegenerative disorders including Parkinsons disease (PD). However, despite recently started clinical trials, studies on NR in animal models of PD are scarce. In this study, we investigated the effect of NR in multiple models of PD. In transgenic C. elegans overexpressing -synuclein, a protein of which aggregation is believed to promote PD, NR rescued PD-like phenotypes including mitochondrial dysfunction and motility defects, decreased oxidative stress, and age-related dopamine (DA) neuron loss. We found that NR eased symptoms of disease by activating the mitochondrial unfolded protein response (UPRmt) via the transcription factor atfs-1. Similarly, in a proteasome inhibitor, lactacystin, -induced mouse model of PD, NR rescued mitochondrial dysfunction and behavioural deficits caused by lactacystin lesion. However, long-term NR supplementation, in conjunction with proteasome inhibition, resulted in decreased DA levels in both the lesioned and unlesioned sides of the substantia nigra with concomitant downregulation of key genes in DA metabolism. Our results suggest specific endpoints that should be monitored in ongoing NR clinical trials.

neuroscience↗

bioRxiv2020

Coronavirus Infection and PARP Expression Dysregulate the NAD Metabolome: A Potentially Actionable Component of Innate Immunity

Poly-ADP-ribose polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using nicotinamide adenine dinucleotide (NAD) as the source of ADPR. While the well-known poly-ADP-ribosylating (PARylating) PARPs primarily function in the DNA damage response, many non-canonical mono-ADP-ribosylating (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust upregulation of several PARPs following infection with Murine Hepatitis Virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly upregulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while downregulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM and NR dramatically decrease the replication of an MHV virus that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD, and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

microbiology↗

bioRxiv2020

The NAD Metabolome is Functionally Depressed in Patients Undergoing Liver Transplantation for Alcohol-related Liver Disease

Nicotinamide adenine dinucleotide (NAD+) and related coenzymes play critical roles in liver function. Though hepatic alcohol metabolism depresses NAD+, current understanding of the NAD+ metabolome in alcohol-related liver disease (ArLD) is based on animal models. We used human liver samples to quantify the NAD+ metabolome in ArLD with samples obtained at the time of liver transplantation or resection at University Hospitals Birmingham NHS Foundation Trust (UHB). The severity of steatohepatitis in liver from patients with ArLD was assessed with standard liver function tests (LFT) and histology. NAD-targeted quantitative metabolomic analysis of liver tissue was performed by liquid chromatography-tandem mass spectrometry (LC-MS). Seventy-two human liver specimens were analyzed including 43 with ArLD. The NAD+ metabolome differed significantly between different types of liver disease (two-way ANOVA p = 0.001). ArLD liver tissue showed markedly depressed concentrations of NAD+ (432 M vs. 616 M in NL) and precursor molecules nicotinic acid and nicotinamide riboside. There was a significant overall difference in the NAD+ metabolome between ArLD samples with and without steatohepatitis (two-way ANOVA p = 0.018). After correcting for multiple comparisons, a significant difference for individual components of the metabolome was observed for the concentration of NAD+ (mean 451 M vs. 381 M, p = 0.045). NAD+ concentration was inversely related to serum bilirubin concentration (r2 -0.127, p = 0.04) and positively correlated with myeloperoxidase activity (r2 0.31, p = 0.003). The concentration of NAD+ and its precursor molecules are significantly reduced in ArLD and are associated with disease activity. Conclusion: Liver samples from people with ArLD show depressed NAD+ and precursor levels as well as depressed myeloperoxidase activity.

pathology↗

bioRxiv2020

IL8 Drives an Elaborate Signal Transduction Process for CD38 to Produce NAADP from NAAD and NADP+ in Endolysosomes to Effect Cell Migration

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an obligate driver of calcium signaling whose formation from other metabolites of nicotinamide adenine dinucleotide (NAD+) has remained elusive. In vitro, CD38-mediated NAADP synthesis requires an acidic pH and a nonphysiological concentration of nicotinic acid (NA). We discovered that the type II membrane form of CD38 catalyzes synthesis of NAADP by exchanging the nicotinamide moiety of nicotinamide adenine dinucleotide phosphate (NADP+) for the NA group of nicotinic acid adenine dinucleotide (NAAD) inside endolysosomes of interleukin 8 (IL8)-treated lymphokine-activated killer cells. Upon IL8 stimulation, cytosolic NADP+ is transported to acidified endolysosomes via connexin 43 via cAMP-EPAC-RAP1-PP2A signaling. Luminal CD38 then performs a base exchange reaction with the donor NA group deriving from NAAD, produced by newly described endolysosomal activities of NA phosphoribosyltransferase and NMN adenyltransferase 3. Thus, the membrane organization of endolysosomal CD38, a signal-mediated transport system for NADP+ and luminal NAD+ biosynthetic enzymes integrate signals from a chemokine and cAMP to specify the spatiotemporal mobilization of calcium to drive cell migration.

cell biology↗

bioRxiv2020

Modulation of the Cardiac Sodium Channel Nav1.5 Peak and Late Currents by NAD+ Precursors

RationaleThe cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown. ObjectiveTo determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,L in vitro and cardiac electrophysiology in vivo. Methods and ResultsThe effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 M: 51 {+/-} 18%, p=0.02, 5 mM: 59 {+/-} 22%, p=0.03) and RNCMs (500 {micro}M: 60 {+/-} 26%, p=0.02, 5 mM: 75 {+/-} 39%, p=0.03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: -45 {+/-} 11%, p=0.04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6J mice (0.35% NR: -4.9 {+/-} 2.0%, p=0.26; 1.0% NR: -9.5 {+/-} 2.8%, p=0.01). ConclusionsNAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

physiology↗

bioRxiv2019

Nicotinamide riboside augments the human skeletal muscle NAD+ metabolome and induces transcriptomic and anti-inflammatory signatures in aged subjects: a placebo-controlled, randomized trial

NAD+ is modulated by conditions of metabolic stress and has been reported to decline with aging, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome, and questioned if tissue NAD+ levels are depressed with aging. We supplemented 12 aged men with NR 1g per day for 21-days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways. NR also depressed levels of circulating inflammatory cytokines. In an additional study, 31P magnetic resonance spectroscopy-based NAD+ measurement in muscle and brain showed no difference between young and aged individuals. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR, while suggesting that NAD+ decline is not associated with chronological aging per se in human muscle or brain.

clinical trials↗

bioRxiv2019

Aging-related inflammation driven by cellular senescence enhances NAD consumption via activation of CD38+ macrophages

Decline in tissue NAD levels during aging is linked to aging and its associated diseases. However, the mechanism for aging-associated NAD decline remains unclear. Here we report that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues including visceral white adipose tissue and the liver during aging. Remarkably, these M1-like macrophages highly express the NAD consuming enzyme CD38 and have enhanced CD38-dependent NADase activity. We also find that senescent cells progressively accumulate in visceral white adipose tissue during aging and that inflammatory cytokines found in the supernatant from senescent cells (Senescence associated secretory proteins, SASP) induce macrophages to proliferate and to express CD38. These results highlight a new causal link between visceral tissue senescence and tissue NAD decline during aging and represent a novel therapeutic opportunity targeting maintenance of NAD levels during aging.

immunology↗