mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1. We demonstrate that in human cells, the release of mTORC1 from lysosomes depends on its kinase activity. In accordance with a negative feedback mechanism, activated mTOR mutants display low lysosome occupancy, causing hypo-phosphorylation and nuclear localization of the lysosomal substrate TFE3. Surprisingly, mTORC1 activated by Rheb does not increase the cytoplasmic/lysosomal ratio of mTORC1, indicating the existence of mTORC1 pools with distinct substrate specificity. Dysregulation of either pool results in aberrant TFE3 activity and may explain nuclear accumulation of TFE3 in epileptogenic malformations in focal cortical dysplasia type II (FCD II) and tuberous sclerosis (TSC).

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Pyridox (am) ine 5'-phosphate oxidase deficiency induces seizures in Drosophila melanogaster.

Pyridox (am) ine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5'-phosphate (PLP), the biologically active form of VB6 and involved in the synthesis of neurotransmitters including γ-aminobutyric acid (GABA), dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously, we identified a c.95 C>A missense mutation in sugarlethal (sgll)-the Drosophila homolog of human PNPO (hPNPO)-and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here, we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with the GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

The malate-aspartate shuttle is important for de novo serine biosynthesis.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD+. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-13C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD+/NADH ratio), consistent with the NAD+ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD+-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders.

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.

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

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.

Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare disease characterized by mutations in the lysine degradation gene ALDH7A1 leading to recurrent neonatal seizures, which are uniquely alleviated by high doses of pyridoxine or pyridoxal 5'-phosphate (vitamin B6 vitamers). Despite treatment, neurodevelopmental disabilities are still observed in most PDE patients underlining the need for adjunct therapies. Over 60 years after the initial description of PDE, we report the first animal model for this disease: an aldh7a1-null zebrafish (Danio rerio) displaying deficient lysine metabolism and spontaneous and recurrent seizures in the larval stage (10 days postfertilization). Epileptiform electrographic activity was observed uniquely in mutants as a series of population bursts in tectal recordings. Remarkably, as is the case in human PDE, the seizures show an almost immediate sensitivity to pyridoxine and pyridoxal 5'-phosphate, with a resulting extension of the life span. Lysine supplementation aggravates the phenotype, inducing earlier seizure onset and death. By using mass spectrometry techniques, we further explored the metabolic effect of aldh7a1 knockout. Impaired lysine degradation with accumulation of PDE biomarkers, B6 deficiency, and low γ-aminobutyric acid levels were observed in the aldh7a1-/- larvae, which may play a significant role in the seizure phenotype and PDE pathogenesis. This novel model provides valuable insights into PDE pathophysiology; further research may offer new opportunities for drug discovery to control seizure activity and improve neurodevelopmental outcomes for PDE.

Vitamin B6 in plasma and cerebrospinal fluid of children.

BACKGROUND: Over the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce. MATERIALS AND METHODS: B6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated. RESULTS: The B6 vitamer composition of plasma (pyridoxal phosphate (PLP) > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF. CONCLUSION: We provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6.

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.

Vitamin B-6 vitamers in human plasma and cerebrospinal fluid.

BACKGROUND: Vitamin B-6 comprises a group of 6 interrelated vitamers and is essential for numerous physiologic processes, including brain functioning. Genetic disorders disrupting vitamin B-6 metabolism have severe clinical consequences. OBJECTIVE: To adequately diagnose known and novel disorders in vitamin B-6 metabolism, a reference set is required containing information on all vitamin B-6 vitamers in plasma and cerebrospinal fluid (CSF). DESIGN: Concentrations of vitamin B-6 vitamers in the plasma and CSF of 533 adult subjects were measured by ultra high-performance liquid chromatography-tandem mass spectrometry. RESULTS: The relative vitamin B-6 vitamer composition of plasma [pyridoxal phosphate (PLP) > pyridoxic acid (PA) > pyridoxal] differed from that of CSF (pyridoxal > PLP > PA > pyridoxamine). Sex influenced vitamin B-6 vitamer concentrations in plasma and CSF and should therefore be taken into account when interpreting vitamin B-6 vitamer concentrations. The strict ratios and strong correlations between vitamin B-6 vitamers point to a tight regulation of vitamin B-6 vitamer concentrations in blood and CSF. Given the unique design of this study, with simultaneously withdrawn blood and CSF from a large number of subjects, reliable CSF:plasma ratios and correlations of vitamin B-6 vitamers could be established. CONCLUSIONS: We provide an extensive reference set of vitamin B-6 vitamer concentrations in plasma and CSF. In addition to providing insight on the regulation of individual vitamers and their intercompartmental distribution, we anticipate that these data will prove to be a valuable reference set for the diagnosis and treatment of conditions associated with altered vitamin B-6 metabolism.

The intestine plays a substantial role in human vitamin B6 metabolism: a Caco-2 cell model.

BACKGROUND: Vitamin B6 is present in various forms (vitamers) in the diet that need to be metabolized to pyridoxal phosphate (PLP), the active cofactor form of vitamin B6. In literature, the liver has been reported to be the major site for this conversion, whereas the exact role of the intestine remains to be elucidated. OBJECTIVE: To gain insight into the role of the intestine in human vitamin B6 metabolism. MATERIALS AND METHODS: Expression of the enzymes pyridoxal kinase (PK), pyridox(am)ine phosphate oxidase (PNPO) and PLP-phosphatase was determined in Caco-2 cells and in lysates of human intestine. Vitamin B6 uptake, conversion and excretion were studied in polarized Caco-2 cell monolayers. B6 vitamer concentrations (pyridoxine (PN), pyridoxal (PL), PLP, pyridoxamine (PM), pyridoxamine phosphate (PMP)) and pyridoxic acid (PA) were quantified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using stable isotope-labeled internal standards. RESULTS: The enzymatic system involved in vitamin B6 metabolism (PK, PNPO and PLP-phosphatase) is fully expressed in Caco-2 cells as well as in human intestine. We show uptake of PN, PM and PL by Caco-2 cells, conversion of PN and PM into PL and excretion of all three unphosphorylated B6 vitamers. CONCLUSION: We demonstrate, in a Caco-2 cell model, that the intestine plays a substantial role in human vitamin B6 metabolism.

Vitamin B6 vitamer concentrations in cerebrospinal fluid differ between preterm and term newborn infants.

BACKGROUND AND OBJECTIVE: Vitamin B(6) plays a pivotal role in brain development and functioning. Differences in vitamin B(6) homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B(6) vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. METHODS: B(6) vitamer concentrations were determined in 69 CSF samples of 36 newborn infants (26 born preterm and 10 born term) by ultra performance liquid chromatography-tandem mass spectrometry. CSF samples, taken from a subcutaneous intraventricular reservoir, were bedside frozen and protected from light. RESULTS: Concentrations of pyridoxal (PL), pyridoxal phosphate (PLP), pyridoxic acid (PA), and pyridoxamine (PM) in preterm newborns (postmenstrual age 30-37 weeks) were at least twice as high as in older newborns (postmenstrual age ≥ 42 weeks). Pyridoxine and pyridoxamine phosphate concentrations were below limits of quantification in all newborns. In CSF of 2 very preterm newborns (postmenstrual age <30 weeks), significant amounts of pyridoxine were present besides high concentrations of PL, PA, and PM, whereas PLP concentrations were relatively low. B(6) vitamers in CSF were positively correlated, especially PA, PLP, and PL. CONCLUSIONS: In CSF of newborn infants, PL, PLP, PA, and PM are present, and concentrations are strongly dependent on postmenstrual age. Our results indicate that vitamin B(6) homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B(6) deficiency in newborn infants.