CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1.

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.

MCART1/SLC25A51 is required for mitochondrial NAD transport.

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as coessential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

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.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

A Novel Mutation in MARS in a Patient with Charcot-Marie-Tooth Disease, Axonal, Type 2U with Congenital Onset.

Charcot-Marie-Tooth disease is a phenotypically and genetically heterogeneous group of disorders affecting both motor and sensory neurons. Exome sequencing has driven discovery of genes responsible for Charcot-Marie-Tooth disease with more than 70 genes now associated with this neuromuscular disease. The MARS gene was recently reported as the cause of Charcot-Marie-Tooth 2U, a slowly progressive axonal sensorimotor polyneuropathy with adult-onset reported in six patients. We report here a patient with a progressive, early childhood-onset, motor-predominant form of Charcot-Marie-Tooth disease. Exome sequencing identified a novel MARS variant (c.1189G>A; p.Ala397Thr) that was not present in her unaffected mother; her unaffected father was unavailable. Further studies using structural modeling and a yeast humanization assay support pathogenicity of the variant. Our study expands the phenotype of Charcot-Marie-Tooth 2U, while highlighting the utility of functional assays to evaluate variant pathogenicity.

Glycine decarboxylase deficiency-induced motor dysfunction in zebrafish is rescued by counterbalancing glycine synaptic level.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

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.

Current knowledge for pyridoxine-dependent epilepsy: a 2016 update.

Pyridoxine-dependent epilepsy (PDE) is a rare genetic condition characterized by intractable and recurrent neonatal seizures that are uniquely alleviated by high doses of pyridoxine (vitamin B6). This recessive disease is caused by mutations in ALDH7A1, a gene encoding Antiquitin, an enzyme central to lysine degradation. This results in the pathogenic accumulation of the lysine intermediates Aminoadipate Semialdehyde (AASA) and its cyclic equilibrium form Piperideine-6-carboxylate (P6C) in body fluids; P6C reacts with pyridoxal-5'-phosphate (PLP, the active form of vitamin B6) causing its inactivation and leading to pyridoxine-dependent seizures. While PDE is responsive to pharmacological dosages of pyridoxine, despite lifelong supplementation, neurodevelopment delays are observed in >75% of PDE cases. Thus, adjunct treatment strategies are emerging to both improve seizure control and moderate the delays in cognition. These adjunctive therapies, lysine restriction and arginine supplementation, separately or in combination (with pyridoxine thus termed 'triple therapy'), have shown promising results and are recommended in all PDE patients. Other new therapeutic strategies currently in preclinical phase of study include antisense therapy and substrate reduction therapy. We present here a comprehensive review of current treatment options as well as PDE phenotype, differential diagnosis, current management and views upon the future of PDE research.

Mouse lysine catabolism to aminoadipate occurs primarily through the saccharopine pathway; implications for pyridoxine dependent epilepsy (PDE).

Lysine is catabolized in mammals through the saccharopine and pipecolate pathways - the former is mainly hepatic and renal, and the latter is believed to play a role in the cerebral lysine oxidation. Both pathways lead to the formation of aminoadipic semialdehyde (AASA) that is then oxidized to aminoadipate (AAA) by antiquitin (ALDH7A1). Mutations in the ALDH7A1 gene result in the accumulation of AASA and its cyclic form, piperideine-6-carboxylate (P6C), which causes pyridoxine-dependent epilepsy (PDE). P6C reacts with pyridoxal 5'-phosphate (PLP) causing its inactivation. Here, we used liquid chromatography-mass spectrometry to investigate lysine catabolism in mice injected with lysine labelled at either its nitrogen epsilon (ε-15N) or nitrogen alpha (α-15N). Analysis of ε-15N and α-15N lysine catabolites in plasma, liver and brain suggested the saccharopine as the main pathway for AAA biosynthesis. Although there was evidence for upstream cerebral pipecolate pathway activity, the resulting pipecolate does not appear to be further oxidized into AASA/P6C/AAA. By far the bulk of lysine degradation and therefore, the primary source of lysine catabolites are hepatic and renal. The results indicate that the saccharopine pathway is primarily responsible for body's production of AASA/P6C. The centrality of the saccharopine pathway in whole body lysine catabolism opens new possibilities of therapeutic targets for PDE. We suggest that inhibition of this pathway upstream of AASA/P6C synthesis may be used to prevent its accumulation benefiting PDE patients. Inhibition of the enzyme aminoadipic semialdehyde synthase, for example, could constitute a new strategy to treat PDE and other inherited diseases of lysine catabolism.

Simultaneous detection of lysine metabolites by a single LC-MS/MS method: monitoring lysine degradation in mouse plasma.

Detection and quantification of lysine degradation metabolites in plasma is necessary for the diagnosis and follow-up of diseases such as pyridoxine-dependent epilepsy. The principal metabolites involved in the disease are related to the first steps of lysine oxidation, either through the saccharopine or the pipecolate pathways. Currently, there are three different analytical methods used to assess the content of these metabolites in urine and plasma, but they require different sample preparations and analytical equipment. Here, we describe a protocol that calls for a simple sample preparation and uses liquid chromatography tandem mass spectrometry (LC-MS/MS) that allows simultaneous detection and quantification of underivatized l-saccharopine, l-aminoadipic acid, l-pipecolic acid, piperideine-6-carboxylate, l-glutamic acid, and pyridoxal-5-phosphate in plasma samples. To validate the method we analyzed the time course degradation after intraperitoneal injection of l-lysine in C57BL/6/J mice. We observed that the degradation of lysine through the saccharopine pathway reached a maximum within the first 2 h. At this time point there was an increase in the levels of the metabolites saccharopine, aminoadipic acid, and pipecolic acid by 3-, 24- and 3.4-fold, respectively, compared to time zero levels. These metabolites returned to basal levels after 4-6 h. In conclusion, we have developed a LC-MS/MS approach, which allows simultaneous analysis of lysine degradation metabolites without the need for derivatization.

Overexpression of mitochondrial uncoupling protein 1 (UCP1) induces a hypoxic response in Nicotiana tabacum leaves.

Mitochondrial uncoupling protein 1 (UCP1) decreases reactive oxygen species production under stress conditions by uncoupling the electrochemical gradient from ATP synthesis. This study combined transcriptome profiling with experimentally induced hypoxia to mechanistically dissect the impact of Arabidopsis thaliana UCP1 (AtUCP1) overexpression in tobacco. Transcriptomic analysis of AtUCP1-overexpressing (P07) and wild-type (WT) plants was carried out using RNA sequencing. Metabolite and carbohydrate profiling of hypoxia-treated plants was performed using (1)H-nuclear magnetic resonance spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection. The transcriptome of P07 plants revealed a broad induction of stress-responsive genes that were not strictly related to the mitochondrial antioxidant machinery, suggesting that overexpression of AtUCP1 imposes a strong stress response within the cell. In addition, transcripts that mapped into carbon fixation and energy expenditure pathways were broadly altered. It was found that metabolite markers of hypoxic adaptation, such as alanine and tricarboxylic acid intermediates, accumulated in P07 plants under control conditions at similar rates to WT plants under hypoxia. These findings indicate that constitutive overexpression of AtUCP1 induces a hypoxic response. The metabolites that accumulated in P07 plants are believed to be important in signalling for an improvement in carbon assimilation and induction of a hypoxic response. Under these conditions, mitochondrial ATP production is less necessary and fermentative glycolysis becomes critical to meet cell energy demands. In this scenario, the more flexible energy metabolism along with an intrinsically activated hypoxic response make these plants better adapted to face several biotic and abiotic stresses.

The saccharopine pathway in seed development and stress response of maize.

Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into α-aminoadipic semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into aminoadipic acid (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). Here, we show that LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing endosperm. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD(+) in the micromolar range. In the developing endosperm, the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles, the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in the stressed tissues, the LKR/SDH protein is not produced. In the developing seeds, the saccharopine pathway is used for pipecolic acid synthesis although proline may play a major role in abiotic stress response. The results indicate that the saccharopine pathway in maize seed development and stress responses significantly differ from that observed for dicot plants.