Metabolomics analysis of antiquitin deficiency in cultured human cells and plasma: Relevance to pyridoxine-dependent epilepsy

Abstract

Deficiency of antiquitin (α-aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B₆ -dependent epilepsy (PDE-ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE-ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE-ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin-deficient NPCs and western-blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin-deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE-ALDH7A1 and antiquitin-deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma-glutamyl cycle, suggesting potential oxidative stress-related effects in PN-treated patients with PDE-ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine-dependent epilepsy.

Keywords: NPCs; PDE-ALDH7A1; antiquitin deficiency; lysine catabolism; metabolomics; pyridoxine.

FIGURE 1

Overview of lysine degradation pathway. Dashed lines indicate proposed reactions for which no enzymes have yet been identified in human. The proposed structure of the Knoevenagel condensation product has only been shown in vitro. AASS α‐aminoadipic semialdehyde synthase; ATQ antiquitin (α‐aminoadipic semialdehyde dehydrogenase); CRYM/KR, μ‐crystallin/ketimine reductase; PIPOX pipecolic acid oxidase; P5CR pyrroline‐5‐carboxylase reductase

FIGURE 2

Western blot analysis showing antiquitin and β‐Actin expression in control (untransfected) and CRISPR/Cas transfected colonies of neuronal progenitor cells (NPCs) following single cell expansion

FIGURE 3

Overview of the folate cycle and methionine cycle. Metabolites that are significantly changed in any of the cell culture experiments are indicated by gray boxes. Pathways that require vitamin B₆ as a cofactor are indicated by dashed lines. 5‐methyltetrahydrofolate (5‐MTHF); 5,10‐methylene tetrahydrofolate (5,10 methylene THF); S‐adenosyl methionine (SAM); S‐adenosyl homocysteine (SAH)

FIGURE 4

The gamma‐glutamyl cycle, adapted from Larsson A. (1990) Disorders of the Gamma Glutamyl Cycle. In: Fernandes J., Saudubray JM., Tada K. (eds) Inborn Metabolic Diseases. Springer, Berlin, Heidelberg. Glutathione synthetase (GSS), gamma‐glutamyl transferase (GGT), gamma‐glutamylcyclotransferase (GGCT), 5‐oxoprolinase (OPLAH), glutamate cystein lygase (GCS)