Inborn errors of the malate aspartate shuttle - Update on patients and cellular models.

The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted.

Induced pluripotent stem cell-derived hepatocytes reveal TCA cycle disruption and the potential basis for triheptanoin treatment for malate dehydrogenase 2 deficiency.

Mitochondrial malate dehydrogenase 2 (MDH2) is crucial to cellular energy generation through direct participation in the tricarboxylic acid (TCA) cycle and the malate aspartate shuttle (MAS). Inherited MDH2 deficiency is an ultra-rare metabolic disease caused by bi-allelic pathogenic variants in the MDH2 gene, resulting in early-onset encephalopathy, psychomotor delay, muscular hypotonia and frequent seizures. Currently, there is no cure for this devastating disease. We recently reported symptomatic improvement of a three-year-old girl with MDH2 deficiency following treatment with the triglyceride triheptanoin. Here, we aimed to better characterize this disease and improve our understanding of the potential utility of triheptanoin treatment. Using fibroblasts derived from this patient, we generated induced pluripotent stem cells (hiPSCs) and differentiated them into hepatocytes (hiPSC-Heps). Characterization of patient-derived hiPSCs and hiPSC-Heps revealed significantly reduced MDH2 protein expression. Untargeted proteotyping of hiPSC-Heps revealed global dysregulation of mitochondrial proteins, including upregulation of TCA cycle and fatty acid oxidation enzymes. Metabolomic profiling confirmed TCA cycle and MAS dysregulation, and demonstrated normalization of malate, fumarate and aspartate following treatment with the triheptanoin components glycerol and heptanoate. Taken together, our results provide the first patient-derived hiPSC-Hep-based model of MDH2 deficiency, confirm altered TCA cycle function, and provide further evidence for the implementation of triheptanoin therapy for this ultra-rare disease. Synopsis: This study reveals altered expression of mitochondrial pathways including the tricarboxylic acid cycle and changes in metabolite profiles in malate dehydrogenase 2 deficiency and provides the molecular basis for triheptanoin treatment in this ultra-rare disease.