Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

Mitochondrial H2O2 release does not directly cause damage to chromosomal DNA.

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging.

Metabolic Alterations in NADSYN1-Deficient Cells.

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662* ; VPS41c.1423-2A>G and VPS41R662* ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

Upregulation of the pathogenic transcription factor SPI1/PU.1 in tuberous sclerosis complex and focal cortical dysplasia by oxidative stress.

Tuberous sclerosis complex (TSC) is a congenital disorder characterized by cortical malformations and concomitant epilepsy caused by loss-of-function mutations in the mTOR suppressors TSC1 or TSC2. While the underlying molecular changes caused by mTOR activation in TSC have previously been investigated, the drivers of these transcriptional change have not been fully elucidated. A better understanding of the perturbed transcriptional regulation could lead to the identification of novel pathways for therapeutic intervention not only in TSC, but other genetic epilepsies in which mTOR activation plays a key role, such as focal cortical dysplasia 2b (FCD). Here, we analyzed RNA sequencing data from cortical tubers and a tsc2-/- zebrafish. We identified differential expression of the transcription factors (TFs) SPI1/PU.1, IRF8, GBX2, and IKZF1 of which SPI1/PU.1 and IRF8 targets were enriched among the differentially expressed genes. Furthermore, for SPI1/PU.1 these findings were conserved in TSC zebrafish model. Next, we confirmed overexpression of SPI1/PU.1 on the RNA and protein level in a separate cohort of surgically resected TSC tubers and FCD tissue, in fetal TSC tissue, and a Tsc1GFAP-/- mouse model of TSC. Subsequently, we validated the expression of SPI1/PU.1 in dysmorphic cells with mTOR activation in TSC tubers. In fetal TSC, we detected SPI1/PU.1 expression prenatally and elevated RNA Spi1 expression in Tsc1GFAP-/- mice before the development of seizures. Finally, in vitro, we identified that in astrocytes and neurons SPI1 transcription was driven by H2 O2 -induced oxidative stress, independent of mTOR. We identified SPI1/PU.1 as a novel TF involved in the pro-inflammatory gene expression of malformed cells in TSC and FCD 2b. This transcriptional program is activated in response to oxidative stress and already present prenatally. Importantly, SPI1/PU.1 protein appears to be strictly limited to malformed cells, as we did not find SPI1/PU.1 protein expression in mice nor in our in vitro models.

Dysregulated RASGRP1 expression through RUNX1 mediated transcription promotes autoimmunity.

RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.

Pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations.

Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo-/-) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo-/- zebrafish develop seizures resulting in only 38% of pnpo-/- zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo-/- zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.

Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6. Mammals cannot synthesize vitamin B6, so they rely on dietary uptake of the different B6 forms, and via the B6 salvage pathway they interconvert them into PLP. Humans possess three enzymes in this pathway: pyridoxal kinase, pyridox(am)ine phosphate oxidase and pyridoxal phosphatase. Besides these, a fourth enzyme has been described in plants and yeast but not in humans: pyridoxal reductase. METHODS: We analysed B6 vitamers in remnant CSF samples of PLP-treated patients and four mammalian cell lines (HepG2, Caco2, HEK293 and Neuro-2a) supplemented with PL as the sole source of vitamin B6. RESULTS: Strong accumulation of pyridoxine (PN) in CSF of PLP-treated patients was observed, suggesting the existence of a PN-forming enzyme. Our in vitro studies show that all cell lines reduce PL to PN in a time- and dose-dependent manner. We compared the amino acid sequences of known PL reductases to human sequences and found high homology for members of the voltage-gated potassium channel beta subunits and the human aldose reductases. Pharmacological inhibition and knockout of these proteins show that none of the candidates is solely responsible for PL reduction to PN. CONCLUSIONS: We show evidence for the presence of PL reductase activity in humans. Further studies are needed to identify the responsible protein. GENERAL SIGNIFICANCE: This study expands the number of enzymes with a role in B6 salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the intracellular amount of free PL and PLP.

A Tuba/Cdc42/Par6A complex is required to ensure singularity in apical domain formation during enterocyte polarization.

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization.

The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size.

PTEN is a tumor suppressor that is frequently lost in epithelial malignancies. A part of the tumor-suppressive properties of PTEN is attributed to its function in cell polarization and consequently its role in maintaining epithelial tissue integrity. However, surprisingly little is known about the function and regulation of PTEN during epithelial cell polarization. We used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene disruption to delete PTEN in intestinal epithelial Ls174T:W4 cells, which upon differentiation form a microvillus-covered apical membrane (brush border) on a part of the cell cortex, independent of cell-cell junctions. We show that loss of PTEN results in the formation of a larger brush border that, in a fraction of the cells, even spans the entire plasma membrane, revealing that PTEN functions in the regulation of apical membrane size. Depletion of the phosphatase PTPL1 resulted in a similar defect. PTPL1 interacts with PTEN, and this interaction is necessary for apical membrane enrichment of PTEN. Importantly, phosphatase activity of PTPL1 is not required, indicating that PTPL1 functions as an anchor protein in this process. Our work thus demonstrates a novel function for PTEN during cell polarization in controlling apical membrane size and identifies PTPL1 as a critical apical membrane anchor for PTEN in this process.

A Two-Tiered Mechanism Enables Localized Cdc42 Signaling during Enterocyte Polarization.

Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.

Prioritization and burden analysis of rare variants in 208 candidate genes suggest they do not play a major role in CAKUT.

The leading cause of end-stage renal disease in children is attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Familial clustering and mouse models support the presence of monogenic causes. Genetic testing is insufficient as it mainly focuses on HNF1B and PAX2 mutations that are thought to explain CAKUT in 5­15% of patients. To identify novel, potentially pathogenic variants in additional genes, we designed a panel of genes identified from studies on familial forms of isolated or syndromic CAKUT and genes suggested by in vitro and in vivo CAKUT models. The coding exons of 208 genes were analyzed in 453 patients with CAKUT using next-generation sequencing. Rare truncating, splice-site variants, and non-synonymous variants, predicted to be deleterious and conserved, were prioritized as the most promising variants to have an effect on CAKUT. Previously reported disease-causing mutations were detected, but only five were fully penetrant causal mutations that improved diagnosis. We prioritized 148 candidate variants in 151 patients, found in 82 genes, for follow-up studies. Using a burden test, no significant excess of rare variants in any of the genes in our cohort compared with controls was found. Thus, in a study representing the largest set of genes analyzed in CAKUT patients to date, the contribution of previously implicated genes to CAKUT risk was significantly smaller than expected, and the disease may be more complex than previously assumed.

ATP8B1-mediated spatial organization of Cdc42 signaling maintains singularity during enterocyte polarization.

During yeast cell polarization localization of the small GTPase, cell division control protein 42 homologue (Cdc42) is clustered to ensure the formation of a single bud. Here we show that the disease-associated flippase ATPase class I type 8b member 1 (ATP8B1) enables Cdc42 clustering during enterocyte polarization. Loss of this regulation results in increased apical membrane size with scattered apical recycling endosomes and permits the formation of more than one apical domain, resembling the singularity defect observed in yeast. Mechanistically, we show that to become apically clustered, Cdc42 requires the interaction between its polybasic region and negatively charged membrane lipids provided by ATP8B1. Disturbing this interaction, either by ATP8B1 depletion or by introduction of a Cdc42 mutant defective in lipid binding, increases Cdc42 mobility and results in apical membrane enlargement. Re-establishing Cdc42 clustering, by tethering it to the apical membrane or lowering its diffusion, restores normal apical membrane size in ATP8B1-depleted cells. We therefore conclude that singularity regulation by Cdc42 is conserved between yeast and human and that this regulation is required to maintain healthy tissue architecture.