Elevated Bile Acid 3ß,5α,6ß-Trihydroxycholanoyl Glycine in a Subset of Adult Ataxias Including Niemann-Pick Type C.

Ataxia is a common neurological feature of Niemann-Pick disease type C (NPC). In this disease, unesterified cholesterol accumulates in lysosomes of the central nervous system and hepatic cells. Oxidation by reactive oxygen species produces oxysterols that can be metabolised to specific bile acids. These bile acids have been suggested as useful biomarkers to detect NPC. Concentrations of 3ß,5α,6ß-trihydroxycholanyl glycine (3ß,5α,6ß-triOH-Gly) and 3ß,7ß-dihydroxy-5-cholenyl glycine (3ß,7ß-diOH-Δ5-Gly) were measured in plasma of 184 adults with idiopathic ataxia. All patients were tested with whole genome sequencing containing hereditary ataxia panels, which include NPC1 and NPC2 mutations and other genetic causes of ataxia. Plasma 3ß,5α,6ß-triOH-Gly above normal (>90 nM) was found in 8 out of 184 patients. One patient was homozygous for the p.(Val1165Met) mutation in the NPC1 gene. The remaining seven included one patient with Friedreich's ataxia and three patients with autoimmune diseases. Oxidative stress is known to be increased in Friedreich's ataxia and in autoimmune diseases. Therefore, this subset of patients possibly shares a common mechanism that determines the increase of this bile acid. In a large cohort of adults with ataxia, plasma 3ß,5α,6ß-triOH-Gly was able to detect the one patient in the cohort with NPC1 disease, but also detected oxidation of cholesterol by ROS in other disorders. Plasma 3ß,7ß-diOH-Δ5-Gly is not a potential biomarker for NPC1.

mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria.

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders.

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell-penetrating autophagy-inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.

Hereditary polyneuropathy with optic atrophy due to PDXK variant leading to impaired Vitamin B6 metabolism.

PDXK encodes for a pyridoxal kinase, which converts inactive B6 vitamers to the active cofactor pyridoxal 5'-phosphate (PLP). Recently, biallelic pathogenic variants in PDXK were shown to cause axonal Charcot-Marie-Tooth disease with optic atrophy that responds to PLP supplementation. We present two affected siblings carrying a novel biallelic missense PDXK variant with a similar phenotype with earlier onset. After detection of a novel PDXK variant using Whole Exome Sequencing, we confirmed pathogenicity through in silico protein structure analysis, determination of pyridoxal kinase activity using liquid chromatography-tandem mass spectrometry, and measurement of plasma PLP concentrations using high performance liquid chromatography. Our in silico analysis shows a potential effect on PDXK dimer stability, as well as a putative effect on posttranslational ubiquitination that is predicted to lead to increased protein degradation. We demonstrate that the variant leads to almost complete loss of PDXK enzymatic activity and low PLP levels. Our patients' early diagnosis and prompt PLP replacement restored the PLP plasma levels, enabling long-term monitoring of clinical outcomes. We recommend that patients presenting with similar phenotype should be screened for PDXK mutations, as this is a rare opportunity for treatment.

Urea Cycle Related Amino Acids Measured in Dried Bloodspots Enable Long-Term In Vivo Monitoring and Therapeutic Adjustment.

BACKGROUND: Dried bloodspots are easy to collect and to transport to assess various metabolites, such as amino acids. Dried bloodspots are routinely used for diagnosis and monitoring of some inherited metabolic diseases. METHODS: Measurement of amino acids from dried blood spots by liquid chromatography-tandem mass spectrometry. RESULTS: We describe a novel rapid method to measure underivatised urea cycle related amino acids. Application of this method enabled accurate monitoring of these amino acids to assess the efficacy of therapies in argininosuccinate lyase deficient mice and monitoring of these metabolites in patients with urea cycle defects. CONCLUSION: Measuring urea cycle related amino acids in urea cycle defects from dried blood spots is a reliable tool in animal research and will be of benefit in the clinic, facilitating optimisation of protein-restricted diet and preventing amino acid deprivation.