RNA splicing analysis deciphers developmental hierarchies and reveals therapeutic targets in adult glioma.

Widespread alterations in RNA alternative splicing (AS) have been identified in adult gliomas. However, their regulatory mechanism, biological significance, and therapeutic potential remain largely elusive. Here, using a computational approach with both bulk and single cell RNA-sequencing, we uncover a prognostic AS signature linked with neural developmental hierarchies. Using advanced iPSC glioma models driven by glioma driver mutations, we show that this AS signature could be enhanced by EGFRvIII and inhibited by in situ IDH1 mutation. Functional validation of two isoform switching events in CERS5 and MPZL1 shows regulations of sphingolipid metabolism and SHP2 signaling, respectively. Analysis of upstream RNA binding proteins reveals PTBP1 as a key regulator of the AS signature where targeting of PTBP1 suppresses tumor growth and promotes the expression of a neuron marker TUJ1 in glioma stem-like cells. Overall, our data highlights the role of AS in impacting glioma malignance and heterogeneity and its potential as a therapeutic vulnerability for treating adult gliomas.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Protein biomarkers GDF15 and FGF21 to differentiate mitochondrial hepatopathies from other pediatric liver diseases.

BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.

Simple Preoperative Imaging Measurements Predict Postoperative Pancreatic Fistula After Pancreatoduodenectomy.

OBJECTIVE: Postoperative pancreatic fistula is a potentially devastating complication after pancreatoduodenectomy (PD). The purpose of this study was to identify features on preoperative computed tomography (CT) imaging that correlate with an increased risk of postoperative pancreatic fistula (POPF). METHODS: Patients who underwent PD at our high-volume pancreatic surgery center from 2019 to 2021 were included if CT imaging was available within 8 weeks of surgical intervention. Pancreatic neck thickness (PNT), abdominal wall thickness (AWT), and intra-abdominal distance from pancreas to peritoneum (PTP) were measured by two board-certified radiologists who were blinded to the clinical outcomes. Radiographic measurements, as well as preoperative patient characteristics and intraoperative data, were assessed with univariate and multivariable analysis (MVA) to determine risk for clinically relevant POPF (CR-POPF, grades B and C). RESULTS: A total of 204 patients met inclusion criteria. Median PTP was 5.8 cm, AWT 1.9 cm, and PNT 1.3 cm. CR-POPF occurred in 33 of 204 (16.2%) patients. MVA revealed PTP > 5.8 cm (odds ratio [OR] 2.86, p = 0.023), PNT > 1.3 cm (OR 2.43, p = 0.047), soft pancreas consistency (OR 3.47, p = 0.012), and pancreatic duct size ≤ 3.0 mm (OR 4.55, p = 0.01) as independent risk factors for CR-POPF after PD. AWT and obesity were not associated with increased risk of CR-POPF. Patients with PTP > 5.8 cm or PNT > 1.3 cm were significantly more likely to suffer a major complication after PD (39.6% vs. 22.3% and 40% vs. 22.1%, p < 0.008). CONCLUSIONS: Patients with a thick pancreatic neck and increased intra-abdominal girth have a heightened risk of CR-POPF after pancreatoduodenectomy, and they experience more serious postoperative complications. We defined a simple CT scan-based measurement tool to identify patients at increased risk of CR-POPF.

ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

Trans-vaccenic acid reprograms CD8+ T cells and anti-tumour immunity.

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6-8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

PARK7 Catalyzes Stereospecific Detoxification of Methylglyoxal Consistent with Glyoxalase and Not Deglycase Function.

The protein PARK7 (also known as DJ-1) has been implicated in several diseases, with the most notable being Parkinson's disease. While several molecular and cellular roles have been ascribed to DJ-1, there is no real consensus on what its true cellular functions are and how the loss of DJ-1 function may contribute to the pathogenesis of Parkinson's disease. Recent reports have implicated DJ-1 in the detoxification of several reactive metabolites that are produced during glycolytic metabolism, with the most notable being the α-oxoaldehyde species methylglyoxal. While it is generally agreed that DJ-1 is able to metabolize methylglyoxal to lactate, the mechanism by which it does so is hotly debated with potential implications for cellular function. In this work, we provide definitive evidence that recombinant DJ-1 produced in human cells prevents the stable glycation of other proteins through the conversion of methylglyoxal or a related alkynyl dicarbonyl probe to their corresponding α-hydroxy carboxylic acid products. This protective action of DJ-1 does not require a physical interaction with a target protein, providing direct evidence for a glutathione-free glyoxalase and not a deglycase mechanism of methylglyoxal detoxification. Stereospecific liquid chromatography-mass spectrometry (LC-MS) measurements further uncovered the existence of nonenzymatic production of racemic lactate from MGO under physiological buffer conditions, whereas incubation with DJ-1 predominantly produces l-lactate. Collectively, these studies provide direct support for the stereospecific conversion of MGO to l-lactate by DJ-1 in solution with negligible or no contribution of direct protein deglycation.

Aberrant Vascular Anatomy Associated With Artifactual Focal Avidity in the Liver on PSMA PET.

ABSTRACT: 68 Ga-prostate-specific membrane antigen (PSMA) PET/CT is a valuable tool for staging and restaging of prostate cancer. Prostate-specific membrane antigen expression is not specific to prostate cancer, as it is expressed in normal tissues as well as in neoplastic and nonneoplastic processes. Awareness of the broad possibility of lesions with PSMA avidity is necessary to recognize normal variants and avoid potential pitfalls in image interpretation. We present a series of cases showing physiologic focal PSMA avidity in hepatic segment IVb. We correlate this uptake with aberrant hepatic vasculature. The awareness of this variant is important for accurate image interpretation to prevent additional invasive procedures, undue treatment escalation, and denial of curative treatment to patients.

Pancreatic tumors exhibit myeloid-driven amino acid stress and upregulate arginine biosynthesis.

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs for survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors. Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling us to study PDAC metabolism ex vivo under physiological nutrient conditions. We show that PDAC cells cultured in TIFM adopt a cellular state closer to that of PDAC cells present in tumors compared to standard culture models. Further, using the TIFM model, we found arginine biosynthesis is active in PDAC and allows PDAC cells to maintain levels of this amino acid despite microenvironmental arginine depletion. We also show that myeloid derived arginase activity is largely responsible for the low levels of arginine in PDAC tumors. Altogether, these data indicate that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity to in vivo systems and enable the discovery of novel cancer metabolic phenotypes.

Congenital Hypermetabolism and Uncoupled Oxidative Phosphorylation.

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the ß subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.

Desidustat in Anemia due to Non-Dialysis-Dependent Chronic Kidney Disease: A Phase 3 Study (DREAM-ND).

BACKGROUND: Desidustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, is being developed to treat anemia in patients with chronic kidney disease (CKD) without dialysis dependency. METHODS: In total, 588 patients with a clinical diagnosis of anemia due to CKD without dialysis need and with baseline hemoglobin of 7.0-10.0 g/dL (inclusive) were randomized in a 1:1 ratio to receive either desidustat 100 mg oral tablets thrice a week for 24 weeks or biosimilar darbepoetin subcutaneous injection 0.75 µg/kg once in 2 weeks for 24 weeks. The primary outcome was the change from baseline in hemoglobin to evaluation period of Weeks 16-24. Key secondary outcomes included the number of patients with hemoglobin response, changes in the hepcidin levels, changes in the vascular endothelial growth factor (VEGF) levels, and changes in the lipid and lipoprotein profiles. RESULTS: Hemoglobin change from baseline to Weeks 16-24 was 1.95 g/dL in the desidustat group and 1.83 g/dL in the darbepoetin group (difference: 0.11 g/dL; 95% CI: -0.12, 0.34), which met prespecified non-inferiority margin (-0.75 g/dL). The hemoglobin responders were significantly higher (p = 0.0181) in the desidustat group (196 [77.78%]) compared to the darbepoetin group (176 [68.48%]). The difference of change in hepcidin from baseline to Week 12 and Week 24 (p = 0.0032 at Week 12, p = 0.0016 at Week 24) and the difference of change in low-density lipoprotein from baseline to Week 24 (p value = 0.0269) between the two groups was statistically significant. The difference of change from baseline in VEGF to Weeks 12 and 24 between the two groups was not statistically significant. CONCLUSION: Desidustat is non-inferior to darbepoetin in the treatment of anemia due to non-dialysis dependent CKD and it is well-tolerated.

Mucin-producing Cystic Hepatobiliary Neoplasms: Updated Nomenclature and Clinical, Pathologic, and Imaging Features.

Cystic hepatobiliary neoplasms with mucin-producing epithelium-mucinous cystic neoplasm of the liver (MCN) and intraductal papillary neoplasm of the bile duct (IPNB)-are rare and distinct entities that have unique clinical, pathologic, and imaging features. They are differentiated pathologically by the presence of subepithelial ovarian-like hypercellular stroma (OLS), which is the defining histopathologic feature of MCN. MCN is commonly a benign, large, solitary, symptomatic, multiloculated cystic mass without biliary communication that occurs in middle-aged women. On the other hand, IPNBs are a heterogeneous spectrum of tumors, which are commonly associated with invasive carcinoma, occur in older patients, and can be differentiated from MCN by communication with the biliary tree, intraductal masses, associated biliary ductal dilatation, and absent OLS. Understanding of these rare neoplasms has grown and evolved over time and continues to today, but uncertainty and controversy persist, related to the rarity of these tumors, relatively recent designation as separate entities, inherent clinicopathologic heterogeneity, overlapping imaging features, and the fact that many prior studies likely included MCN and cystic IPNB together as a single entity. Confusion regarding these neoplasms is evident by historical inconsistencies and nonstandardized nomenclature through the years. Awareness of these entities is important for the interpreting radiologist to suggest a particular diagnosis or generate a meaningful differential diagnosis in the appropriate setting, and is of particular significance as MCN and cystic IPNB have overlapping imaging features with other more common hepatobiliary cystic masses but have different management and prognosis. Online supplemental material is available for this article. Work of the U.S. Government published under an exclusive license with the RSNA.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and pathology of multiple organs in individuals under 21 years of age in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although an autoimmune pathogenesis has been proposed, the genes, pathways and cell types causal to this new disease remain unknown. Here we perform RNA sequencing of blood from patients with MIS-C and controls to find disease-associated genes clustered in a co-expression module annotated to CD56dimCD57+ natural killer (NK) cells and exhausted CD8+ T cells. A similar transcriptome signature is replicated in an independent cohort of Kawasaki disease (KD), the related condition after which MIS-C was initially named. Probing a probabilistic causal network previously constructed from over 1,000 blood transcriptomes both validates the structure of this module and reveals nine key regulators, including TBX21, a central coordinator of exhausted CD8+ T cell differentiation. Together, this unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.