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New Insights Into Human Lysine Degradation Pathways with Relevance to Pyridoxine‐Dependent Epilepsy Due to Antiquitin Deficiency

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New Insights Into Human Lysine Degradation Pathways with Relevance to Pyridoxine‐Dependent Epilepsy Due to Antiquitin Deficiency Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 New insights into human lysine degradation pathways with relevance to pyridoxine‐dependent epilepsy due to antiquitin deficiency Crowther, Lisa M ; Mathis, Déborah ; Poms, Martin ; Plecko, Barbara Abstract: Deficiency of antiquitin (ATQ), an enzyme involved in lysine degradation, is the major causeof vitamin B6 -dependent epilepsy. Accumulation of the potentially neurotoxic ฀-aminoadipic semialdehyde (AASA) may contribute to frequently associated developmental delay. AASA is formed by ฀-aminoadipic semialdehyde synthase (AASS) via the saccharopine pathway of lysine degradation, or, as has been postulated, by the pipecolic acid (PA) pathway, and then converted to ฀-aminoadipic acid by ATQ. The PA pathway has been considered to be the predominant pathway of lysine degradation in mammalian brain; however, this was refuted by recent studies in mouse. Consequently, inhibition of AASS was proposed as a potential new treatment option for ATQ deficiency. It is therefore of utmost importance to determine whether the saccharopine pathway is also predominant in human brain cells. The route of lysine degradation was analyzed by isotopic tracing studies in cultured human astrocytes, ReNcell CX human neuronal progenitor cells and human fibroblasts, and expression of enzymes of the two lysine degradation pathways was determined by Western blot. Lysine degradation was only detected through the saccharopine pathway in all cell types studied. The enrichment of 15 N-glutamate as a side product of AASA formation through AASS furthermore demonstrated activity of the saccharopine pathway. We provide first evidence that the saccharopine pathway is the major route of lysine degradation in cultured human brain cells. These results support inhibition of the saccharopine pathway as a new treatment option for ATQ deficiency. DOI: https://doi.org/10.1002/jimd.12076 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-181856 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Crowther, Lisa M; Mathis, Déborah; Poms, Martin; Plecko, Barbara (2019). New insights into human lysine degradation pathways with relevance to pyridoxine‐dependent epilepsy due to antiquitin deficiency. Journal of Inherited Metabolic Disease, 42(4):620-628. DOI: https://doi.org/10.1002/jimd.12076 Received: 6 July 2018 Accepted: 12 February 2019 DOI: 10.1002/jimd.12076 ORIGINAL ARTICLE New insights into human lysine degradation pathways with relevance to pyridoxine-dependent epilepsy due to antiquitin deficiency Lisa M. Crowther1,2,3 | Déborah Mathis2,3,4 | Martin Poms1,2,3 | Barbara Plecko1,2,3,5 1Division of Child Neurology, University Children's Hospital Zurich, Zurich, Abstract Switzerland Deficiency of antiquitin (ATQ), an enzyme involved in lysine degradation, is the 2 CRC Clinical Research Center, University major cause of vitamin B6-dependent epilepsy. Accumulation of the potentially Children's Hospital Zurich, Zurich, neurotoxic α-aminoadipic semialdehyde (AASA) may contribute to frequently Switzerland associated developmental delay. AASA is formed by α-aminoadipic semialdehyde 3Radiz – Rare Disease Intiative Zurich, Clinical Research Priority Program for Rare synthase (AASS) via the saccharopine pathway of lysine degradation, or, as has Diseases, University of Zurich, Zurich, been postulated, by the pipecolic acid (PA) pathway, and then converted to Switzerland α-aminoadipic acid by ATQ. The PA pathway has been considered to be the pre- 4Department of Clinical Chemistry and Biochemistry, University Children's Hospital dominant pathway of lysine degradation in mammalian brain; however, this was Zurich, Zurich, Switzerland refuted by recent studies in mouse. Consequently, inhibition of AASS was pro- 5Department of Pediatrics and Adolescent posed as a potential new treatment option for ATQ deficiency. It is therefore of Medicine, Division of General Pediatrics, utmost importance to determine whether the saccharopine pathway is also predomi- Medical University of Graz, Graz, Austria nant in human brain cells. The route of lysine degradation was analyzed by isotopic Correspondence tracing studies in cultured human astrocytes, ReNcell CX human neuronal progeni- Barbara Plecko, Department of Pediatrics and Adolescent Medicine, Division of tor cells and human fibroblasts, and expression of enzymes of the two lysine degra- General Pediatrics, University Childrens' dation pathways was determined by Western blot. Lysine degradation was only Hospital Graz, Medical University of Graz, detected through the saccharopine pathway in all cell types studied. The enrich- Auenbruggerplatz 34/2, 8036 Graz, Austria. 15 Email: [email protected] ment of N-glutamate as a side product of AASA formation through AASS fur- thermore demonstrated activity of the saccharopine pathway. We provide first Communicating Editor: Sander M. Houten evidence that the saccharopine pathway is the major route of lysine degradation in Funding information cultured human brain cells. These results support inhibition of the saccharopine Hartmann Müller-Stiftung für Medizinische pathway as a new treatment option for ATQ deficiency. Forschung, Grant/Award Number: 1802; Schweizerischer Nationalfonds zur Förderung KEYWORDS der Wissenschaftlichen Forschung, Grant/ α-Aminoadipic semialdehyde, antiquitin, lysine catabolism, pipecolic acid, saccharopine Award Number: 320030_163302; Universität Zürich, Grant/Award Number: Clinical Abbreviations: AAA, α-aminoadipic acid; AADAT/KAT2, kynurenine/alpha-aminoadipate aminotransferase; AASA, α-aminoadipic semialdehyde; AASS, α-aminoadipic semialdehyde synthase; ATQ, antiquitin; CRYM/KR, μ-crystallin/ketimine reductase; LC-MS/MS, liquid chromatography-mass spectrometry; MPE, molar percent enrichment; NPC, neural progenitor cells; P5CR, pyrroline-5-carboxylate reductase; P6C, Δ1-piperideine-6-carboxylate; PA, pipecolic acid; PIPOX, pipecolic acid oxidase; PLP, pyridoxal 50-phosphate. Lisa M. Crowther and Déborah Mathis contributed equally to this work. Barbara Plecko is the guarantor. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM 620 wileyonlinelibrary.com/journal/jimd J Inherit Metab Dis. 2019;42:620–628. CROWTHER ET AL. 621 research priority program Radiz; Hartmann- Müller Stiftung, Grant/Award Number: 1802; University of Zurich; Clinical Research Priority Program Radiz (Rare Disease Initiative Zurich); SNF, Grant/Award Number: 320030_163302 1 | INTRODUCTION In this study, we used isotopic tracing studies to examine whether in human brain cells, AAA is formed by the PA path- Lysine degradation has been considered to have two distinct way as has been deduced from previous studies, or by the sac- pathways, through saccharopine formation by ε-deamination, charopine pathway as was recently demonstrated in mouse or through pipecolic acid (PA) formation by α-deamination brain. It has been shown that ATQ is expressed in human or transamination (Figure 1), although the first enzyme of the astrocytes but not in neurons20; therefore, we examined the PA pathway has not yet been identified.1 Both proposed path- formation of AAA in astrocytes as well as in a human neural ways lead to formation of Δ1-piperideine-6-carboxylate progenitor cell (NPC) line derived from the cortical region of (P6C) and its open form α-aminoadipic semialdehyde human fetal brain (ReNcell CX NPCs), which we demonstrate (AASA), which is then converted to α-aminoadipic acid express AASS and ATQ. In addition, we examined whether (AAA) by the AASA dehydrogenase antiquitin (ATQ). activity through the PA pathway might increase when the sac- Based on rat and mouse studies, it has long been believed that charopine pathway is impaired, by comparing AASS-deficient the PA pathway is predominant in the brain, while in other and ATQ-deficient human fibroblasts to control fibroblasts. tissues the saccharopine pathway is more active.1–5 In ATQ deficiency, the most frequent cause of vitamin 2 | METHODS B6/pyridoxine-dependent epilepsy, AASA and P6C accumu- 0 late, leading to inactivation of pyridoxal 5 -phosphate (PLP), 2.1 | Cell culture the active vitamer of vitamin B6. This occurs due to a con- densation reaction between PLP and accumulated P6C.6 Human fibroblasts (Life Technologies, Carlsbad, California, Despite seizure control and reduction of AASA with high- US), human astrocytes derived from human cortex (Sciencell dose vitamin B6 (pyridoxine) treatment, around 70% of Research Laboratories, Carlsbad, California, US), and ReNcell ATQ-deficient patients still suffer from developmental CX human NPCs (Millipore, Billerica, Massachusetts) were cul- delay.6 This could be due to AASA, which is highly reactive tured according to the manufacturers' directions. Astrocytes and possibly neurotoxic and which levels do not were cultured for a maximum of 10 population doublings (four normalize.7–11 passages). ATQ-deficient fibroblasts were provided by In the search for new treatment modalities, the lysine deg- B. Plecko and P. Mills (Institute of Child Health, University
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