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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with genetic and phenotypic heterogeneity. Pathogenic genetic variants remain the only validated cause of disease, the majority of which were discovered in familial ALS patients. While causal gene variants are a lesser contributor to sporadic ALS, an increasing number of risk alleles (low penetrance genetic variants associated with a small increase in disease risk) and variants of uncertain significance have been reported. To examine the pathogenic potential of genetic variation in ALS, we sought to characterise variant- and gene-level attributes of previously reported ALS-implicated variants. A list of 1,087 genetic variants reported in ALS to March 2021 was compiled through comprehensive literature review. Individual variants were annotated using in silicotools and databases across variant features including pathogenicity scores, localisation to protein domains, evolutionary conservation, and minor allele frequencies. Gene level attributes of genic tolerance, gene expression in ALS-relevant tissues and gene ontology terms were assessed for 33 ALS genes. Statistical analysis was performed for each characteristic, and we compared the most penetrant variants found in familial cases with risk alleles exclusive to sporadic cases, to explore genetic variant features that associate with disease penetrance. We provide spreadsheet (hg19 and GRCh38) and variant call format (GRCh38) resources for all 1,087 reported ALS-implicated variants, including detailed summaries for each attribute. We demonstrate that the characteristics of variants found exclusively in sporadic ALS cases are less severe than those observed in familial ALS. We provide a comprehensive, literature-derived catalogue of genetic variation in ALS thus far and reveal crucial attributes that contribute to ALS pathogenicity. Our variant- and gene-level observations highlight the complexity of genetic variation in ALS, and we discuss important implications and considerations for novel variant interpretation.

Friedreich’s ataxia (FRDA) is caused primarily by expanded GAA repeats in intron 1 of both alleles of the FXNgene, which causes transcriptional silencing and reduced expression of frataxin mRNA and protein. FRDA is characterized by slowly progressive ataxia and cardiomyopathy. Symptoms generally appear during adolescence, and patients slowly progress to wheelchair dependency usually in the late teens or early twenties with death on average in the 4th decade. There are two known mature proteoforms of frataxin. Mitochondrial frataxin (frataxin-M) is a 130-amino acid protein with a molecular weight of 14,268 Da, and there is an alternatively spliced N-terminally acetylated 135-amino acid form (frataxin-E) with a molecular weight of 14,953 Da found in erythrocytes. There is reduced expression of frataxin in the heart and brain, but frataxin is not secreted into the systemic circulation, so it cannot be analyzed in serum or plasma. Blood is a readily accessible biofluid that contains numerous different cell types that express frataxin. We have found that pig blood can serve as an excellent surrogate matrix to validate an assay for frataxin proteoforms because pig frataxin is lost during the immunoprecipitation step used to isolate human frataxin. Frataxin-M is expressed in blood cells that contain mitochondria, whereas extra-mitochondrial frataxin-E is found in erythrocytes. This means that the analysis of frataxin in whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types. In the current study, we observed that the distributions of frataxin levels for a sample of 25 healthy controls and 50 FRDA patients were completely separated from each other, suggesting 100% specificity and 100% sensitivity for distinguishing healthy controls from FRDA cases, a very unusual finding for a biomarker assay. Additionally, frataxin levels were significantly correlated with the GAA repeat length and age of onset with higher correlations for extra-mitochondrial frataxin-E than those for mitochondrial frataxin-M. These findings auger well for using frataxin levels measured by the validated stable isotope dilution ultrahigh-performance liquid chromatography–multiple reaction monitoring/mass spectrometry assay to monitor therapeutic interventions and the natural history of FRDA. Our study also illustrates the utility of using whole blood for protein disease biomarker discovery and validation.

As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become “addicted” may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically “starve” oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.

IMPORTANCE: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. OBJECTIVE: To identify the genetic variants associated with juvenile ALS. DESIGN, SETTING, AND PARTICIPANTS: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. MAIN OUTCOMES AND MEASURES: De novo variants present only in the index case and not in unaffected family members. RESULTS: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. CONCLUSIONS AND RELEVANCE: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

The high mobility group box 1 (HMGB1), which is released during acute acetaminophen (APAP) overdose, is thought to mediate a subsequent immune response, particularly hepatic infiltration of macrophages. The redox behavior of HMGB1 and the proteoforms of HMGB1 present in oxidative environments has been the subject of a number of confusing and contradictory studies. Therefore, a stable isotope dilution two-dimensional nanoultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry method was developed in order to characterize and quantify oxidative modifications to the cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1. Disulfide linkages were determined using carbamidoethyl derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample to sample differences in immunoaffinity precipitation, derivatization, and electrospray ionization. Four discrete HMGB1 proteoforms were found to be released from a hepatocarcinoma cell model of APAP overdose after 24 h. Fully reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond, and 12% was present as the terminally oxidized cysteic acid. Surprisingly, there was no evidence for the formation of HMGB1 disulfides with GSH or other low molecular weight thiols. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury.

Amyotrophic Lateral Sclerosis (ALS) is recognised to be a complex neurodegenerative disease involving both genetic and non-genetic risk factors. The underlying causes and risk factors for the majority of cases remain unknown; however, ever-larger genetic data studies and methodologies promise an enhanced understanding. Recent analyses using published summary statistics from the largest ALS genome-wide association study (GWAS) (20,806 ALS cases and 59,804 healthy controls) identified that schizophrenia (SCZ), cognitive performance (CP) and educational attainment (EA) related traits were genetically correlated with ALS. To provide additional evidence for these correlations, we built single and multi-trait genetic predictors using GWAS summary statistics for ALS and these traits, (SCZ, CP, EA) in an independent Australian cohort (846 ALS cases and 665 healthy controls). We compared methods for generating the risk predictors and found that the combination of traits improved the prediction (Nagelkerke-R2) of the case–control logistic regression. The combination of ALS, SCZ, CP, and EA, using the SBayesR predictor method gave the highest prediction (Nagelkerke-R2) of 0.027 (Pvalue = 4.6 × 10−8), with the odds-ratio for estimated disease risk between the highest and lowest deciles of individuals being 3.15 (95% CI 1.96–5.05). These results support the genetic correlation between ALS, SCZ, CP and EA providing a better understanding of the complexity of ALS.

Multi-omics approaches are increasingly being adopted to understand the complex networks underlying disease. The coisolation of high-quality nucleotides from affected tissues is paramount for the parallel analysis of transcriptomic, genomic, and epigenomic data sets. Although nucleotides extracted from postmortem central nervous system (CNS) tissue are widely used in the study of neurodegenerative disease, assessment of methods for the simultaneous isolation of DNA and RNA is limited. Herein, we describe a strategy for the isolation of high-quality DNA and RNA from postmortem human tissue from 7 CNS regions. Motor cortex, frontal cortex, hippocampus, occipital cortex, anterior cingulate cortex, cerebellum, and spinal cord tissues were obtained from 22 individuals diagnosed with motor neuron disease (MND) and 13 neurologically normal controls (n = 245 tissues). We demonstrated that the Qiagen AllPrep DNA/RNA kit consistently isolated DNA and RNA of high yield and quality from all 6 brain regions. Importantly, phenol-chloroform-based extraction was required to isolate high-yield RNA from spinal cord. RNA sequencing using RNA extracted from 6 CNS regions (n = 60) generated high-quality transcriptomes. Hierarchical clustering of data from motor cortex, using an MND susceptibility gene panel and marker genes of disease-associated microglia, demonstrated that MND-specific gene expression signatures could be detected in the transcriptome data.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with phenotypic and genetic heterogeneity. Approximately 10% of cases are familial, while remaining cases are classified as sporadic. To date, >30 genes and several hundred genetic variants have been implicated in ALS.MethodsSeven hundred and fifty-seven sporadic ALS cases were recruited from Australian neurology clinics. Detailed clinical data and whole genome sequencing (WGS) data were available from 567 and 616 cases, respectively, of which 426 cases had both datasets available. As part of a comprehensive genetic analysis, 853 genetic variants previously reported as ALS-linked mutations or disease-associated alleles were interrogated in sporadic ALS WGS data. Statistical analyses were performed to identify correlation between clinical variables, and between phenotype and the number of ALS-implicated variants carried by an individual. Relatedness between individuals carrying identical variants was assessed using identity-by-descent analysis.ResultsForty-three ALS-implicated variants from 18 genes, including C9orf72, ATXN2, TARDBP, SOD1, SQSTM1and SETX,were identified in Australian sporadic ALS cases. One-third of cases carried at least one variant and 6.82% carried two or more variants, implicating a potential oligogenic or polygenic basis of ALS. Relatedness was detected between two sporadic ALS cases carrying a SOD1p.I114T mutation, and among three cases carrying a SQSTM1p.K238E mutation. Oligogenic/polygenic sporadic ALS cases showed earlier age of onset than those with no reported variant.ConclusionWe confirm phenotypic associations among ALS cases, and highlight the contribution of genetic variation to all forms of ALS.

The crosstalk between deregulated hepatocyte metabolism and cells within the tumour microenvironment, as well as the consequent effects on liver tumorigenesis, are not completely understood. We show here that hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression. FBP1is universally silenced in both human and murine liver tumours. Hepatocyte-specific Fbp1deletion results in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype. Depleting senescent HSCs by ‘senolytic’ treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, the subsequent development of the senescence-associated secretory phenotype and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a critical crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth.

ObjectiveSince the first report of CHCHD10gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia.MethodsWhole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology.ResultsNo causal, novel or disease-associated variants in CHCHD10were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease.ConclusionsGenetic variation in CHCHD10is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for their effects on inflammatory gene expression. Although NSAIDs are known to impact multiple cellular signaling mechanisms, a recent finding is that the NSAID salicylate can disrupt histone acetylation, in part through direct inhibition of the lysine acetyltransferase (KAT) p300/CBP. While salicylate is a relatively weak KAT inhibitor, its CoA-linked metabolite is more potent; however, the ability of NSAID metabolites to inhibit KAT enzymes biochemically and in cells remains relatively unexplored. Here we define the role of metabolic and nonmetabolic mechanisms in inhibition of KAT activity by NSAID chemotypes. First, we screen a small panel of NSAIDs for biochemical inhibition of the prototypical KAT p300, leading to the finding that many carboxylate-containing NSAIDs, including ibuprofen, are able to function as weak inhibitors. Assessing the inhibition of p300 by ibuprofen-CoA, a known NSAID metabolite, reveals that linkage of ibuprofen to CoA increases its biochemical potency toward p300 and other KAT enzymes. In cellular studies, we find that carboxylate-containing NSAIDs inhibit histone acetylation. Finally, we exploit the stereoselective metabolism of ibuprofen to assess the role of its acyl-CoA metabolite in regulation of histone acetylation. This unique strategy reveals that formation of ibuprofen-CoA and histone acetylation are poorly correlated, suggesting metabolism may not be required for ibuprofen to inhibit histone acetylation. Overall, these studies provide new insights into the ability of NSAIDs to alter histone acetylation, and illustrate how selective metabolism may be leveraged as a tool to explore the influence of metabolic acyl-CoAs on cellular enzyme activity.

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. Alkylated phenanthrenes are the most abundant PPAHs present in the crude oil and could contaminate the food chain. We describe the metabolism of a C1-phenanthrene regioisomer 1-methylphenanthrene (1-MP) and a C2-phenanthrene regioisomer 9-ethylphenanthrene (9-EP) in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. Side chain hydroxylation of 1-MP and 9-EP was observed as the major metabolic pathway. The formation of 1-(hydroxymethyl)-phenanthrene was confirmed by reference to an authentic synthetic standard. However, formation of the bioactivated sulfate was not detected. Tetraols were also identified as signature metabolites of 1-MP and 9-EP, indicating that metabolic activation occurred via the diol-epoxide pathway. O-Monosulfonated-catechols were discovered as signature metabolites of the o-quinone pathway of metabolic activation of 1-MP and 9-EP, respectively. The identification of O-monosulfonated-catechols supports the metabolic activation of 1-MP and 9-EP by P450 and AKR isozymes followed by metabolic detoxification of the o-quinone through interception of redox cycling by phase II isozymes. The signature metabolites identified could be used as biomarkers of human exposure to 1-MP and 9-EP resulting from oil spills.

Metabolism of bardoxolone methyl (BARD-Me), an oleanolic acid derivative, and its epoxide metabolite was studied in different in vitro systems. BARD-Me also undergoes glutathione (GSH)-adduct formation via direct nucleophilic attack at the β-carbon of the α,β-unsaturated ketone substituent on the A-ring. The presence of an electron-withdrawing nitrile residue on the α-carbon increases the α,β-unsaturated ketone’s susceptibility to nucleophilic attack by thiols. This allows BARD-Me to generate reversible adducts with the thiol groups of cysteine residues in target proteins without the potential toxic liabilities of irreversible covalent adduct formation. However, BARD-Me epoxide can also react with thiols irreversibly. Therefore, the epoxide was synthesized and its metabolic fate studied in vitro. BARD-Me epoxide was found to undergo two novel metabolic biotransformations: epoxide reduction and oxidative elimination of nitrile moiety. Both metabolic pathways proceed via nucleophilic attack of the thiol group of GSH at each of the two carbon atoms of the epoxide as evidenced by the formation of two β-hydroxy sulfide regioisomers. Oxidative elimination of nitrile moiety proceeds via nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is β to the cyano group to give a cyanohydrin metabolite, which spontaneously decomposes to release cyanide and the corresponding ketone. Nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is α to the cyano group results in the formation of the GSH monoadduct that undergoes intermolecular reduction with another GSH molecule, followed by elimination of oxidized GSH (GS-SG) and the formation of an enolate intermediate. Upon protonation, the enolate intermediate gives rise to hydroxylated BARD-Me, which is readily converted back to BARD-Me through the elimination of water. The chemical reactivity of the epoxide metabolite and the liberation of cyanide are of significant toxicological interest for the potential utility of BARD-Me as a therapeutic agent.

AbstractAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p< 0.002) and traveled a significantly shorter distance during behavioral testing (p< 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2= 0.357, p< 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been previously implicated as the major nitroreductase responsible for 3-NBA activation, but it has recently been reported that human aldo-keto reductase 1C3 (AKR1C3) displays nitroreductase activity toward the chemotherapeutic agent PR-104A. We sought to determine whether AKR1C isoforms could display nitroreductase activity toward other nitrated compounds and bioactivate 3-NBA. Using discontinuous enzymatic assays monitored by UV-HPLC, we determined that AKR1C1–1C3 catalyze three successive two-electron nitroreductions toward 3-NBA to form the reduced product 3-aminobenzanthrone (3-ABA). Evidence of the nitroso- and hydroxylamino- intermediates were obtained by UPLC-HRMS. Km, kcat, and kcat/Kmvalues were determined for recombinant AKR1C and NQO1 and compared. We found that AKR1C1, AKR1C3, and NQO1 have very similar apparent catalytic efficiencies (8 vs 7 min–1mM–1) despite the higher kcatof NQO1 (0.058 vs 0.012 min–1). AKR1C1–1C3 possess a Kmmuch lower than that of NQO1, which suggests that they may be more important than NQO1 at the low concentrations of 3-NBA to which humans are exposed. Given that inhalation represents the primary source of 3-NBA exposure, we chose to evaluate the relative importance of AKR1C1–1C3 and NQO1 in human lung epithelial cell lines. Our data suggest that the combined activities of AKR1C1–1C3 and NQO1 contribute equally to the reduction of 3-NBA in A549 and HBEC3-KT cell lines and together represent approximately 50% of the intracellular nitroreductase activity toward 3-NBA. These findings have significant implications for the metabolism of nitrated polycyclic aromatic hydrocarbons and suggest that the hitherto unrecognized nitroreductase activity of AKR1C enzymes should be further investigated.

T cells compete with malignant cells for limited nutrients within the solid tumor microenvironment. We found that effector memory CD4 T cells respond distinctly from other T cell subsets to limiting glucose and can maintain high levels of interferon-γ (IFN-γ) production in a nutrient-poor environment. Unlike naive (TN) or central memory T (TCM) cells, effector memory T (TEM) cells fail to upregulate fatty acid synthesis, oxidative phosphorylation, and reductive glutaminolysis in limiting glucose. Interference of fatty acid synthesis in naive T cells dramatically upregulates IFN-γ, while increasing exogenous lipids in media inhibits production of IFN-γ by all subsets, suggesting that relative ratio of fatty acid metabolism to glycolysis is a direct predictor of T cell effector activity. Together, these data suggest that effector memory T cells are programmed to have limited ability to synthesize and metabolize fatty acids, which allows them to maintain T cell function in nutrient-depleted microenvironments.

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80%of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

Background:The L25 mouse line was generated by random genomic insertion of a lens-specific transgene. Inbreeding of L25 hemizygotes revealed an unanticipated spastic phenotype in the hind limbs. Objective:The goals were to characterize the motor phenotype in the L25 mice and to map the transgene insert site within the mouse genome. Methods:Six pairs of L25+/–mice were repeatedly mated. Beginning at weaning, all progeny were inspected for body weight and motor signs twice weekly until they displayed predefined ethical criteria for termination. The transgene insert site was determined by whole genome sequencing. Western blotting was used to compare the expression levels of beta-IV spectrin protein in the brain. Results:Matings of hemizygous L25+/–× L25+/–mice yielded 20% (29/148) affected weanlings, identified by an abnormal retraction of the hind limbs when lifted by the tail, and a fine tremor. Affected mice were less mobile and grew more slowly than wild-type littermates. All affected mice required termination due to >15% loss of body weight (50% survival age 92 days). At the endpoint, mice showed varying degrees of spastic paresis or spastic paralysis localised to the hind limbs. Motor endplates remained fully innervated. Genome sequencing confirmed that the transgene was inserted in the locus of βIV spectrin of L25 mice. Western blotting indicated that this random insertion had greatly reduced the expression of βIV spectrin protein in the affected L25 mice. Conclusions:The results confirm the importance of βIV spectrin for maintaining central motor pathway control of the hind limbs, and provide a developmental time course for the phenotype.

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1–10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology.

Context:Estrogen levels and their metabolites are higher in obese vs lean postmenopausal women, and obesity increases breast cancer risk. Quinone derivatives of 4-hydroxylated estrogen metabolites, independently of the estrogen receptor, cause depurination and impaired DNA repair (genotoxic). 16α-Hydroxy (16α-OH)-estrone (E1), eg, promotes tumor proliferation and 2-methoxy-estradiol (E2) may be chemoprotective. Childhood obesity increases breast cancer death risk in women, but levels of estrogen derivatives had not been previously studied in young children.Objective:The objective of the study was to investigate whether total and genotoxic estrogens are increased in prepubertal obese girls compared with lean controls.Design:Stored sera from 12 lean and 23 obese prepubertal girls (Tanner stage I breast and pubic hair) studied previously were assayed for E1, E2, and their multiple metabolites (12 steroids total) using highly sensitive liquid chromatography and tandem mass spectrometry.Results:E2concentrations were significantly higher in obese [3.45 (0.5, 4.65) pg/ml (median [quartile 1, quartile 3])] vs lean girls [0.5 (0.5, 2.37), P= .04], 57% of values upper quartile or greater (quartile 3) of controls. Concentrations of 16α-OH-E1were higher in obese [7.17 (0.5, 9.64) pg/mL] vs lean girls [0.5 (0.5, 1.72, P= .007)], 65% of values quartile 3 or greater of controls. 2-Methoxy-E2concentrations were lower in the obese group (P= .012). 16α-OH-E1concentrations were positively correlated with body mass index, percentage fat mass, and IL-6 concentrations (P< .001).Conclusions:E2and genotoxic metabolites were higher in obese vs lean prepubertal girls. These data suggest that obesity is associated with an increased extraglandular estrogen production and metabolism before the onset of puberty in girls. Long-term epidemiological studies are needed to assess any potential increase in breast cancer risk.

Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM) plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP) levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD) activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence.

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [13C1]propionate and [13C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[2H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.