Impaired mitochondrial calcium homeostasis in neuronal progenitors from IPSCs of patients with AGC1 deficiency

This thesis aims to investigate the pathogenesis of AGC1 deficiency by generating a model of human neuronal progenitors (NPs) derived from iPSCs of a patient carrying a newly identified mutation in AGC1, the neuromuscular isoform of the mitochondrial aspartate/glutamate carrier. AGC1 catalyses the Ca2+-regulated and unidirectional exchange of mitochondrial aspartate with cytosolic glutamate/H+ and is a key component of the malate-aspartate shuttle MAS. Mutations in SLC25A12 gene encoding AGC1 cause AGC1 deficiency, a severe infantile encephalopathy presenting with brain atrophy, neuromuscular delay, hypotonia, epilepsy and hypomyelination associated with a reduction in cerebral N-acetyl aspartate, the lipid precursor of myelin in the CNS. In the first part of this work, the altered biochemical parameters of NPs of the new identified patient carrying a mutation in the Ca2+ binding domain at position p.L284S of the protein were defined in comparison with those of NPs of patients carrying mutations in the catalytic domain of AGC1, and with those of healthy unrelated individuals. The second part of the thesis focused on the evaluation of calcium homeostasis changes in AGC1 deficiency patient NPs and their effect on cellular bioenergetics. Similarly to NPs of patients with null or strongly reduced AGC1 transport activity, NPs of the patient with mutation that does not affect the catalytic domain exhibited a proliferation deficit associated with increased apoptosis, and elevated lactate levels consistent with increased glycolytic activity and a drastic reduction in mitochondrial oxygen consumption rate (OCR) when measured in the presence of glucose alone or in combination with pyruvate, thus suggesting that changes in Ca2+ binding domain also affect the activity of MAS in this cell models. Furthermore, by examining the activity of the mitochondrial respiratory chain, we observed a significant reduction in the activity of complex I in patient NPs, while the activities of the other complexes were similar to the controls. Importantly, we demonstrated that patient NPs are characterised by a transcriptomic profile with a huge number of downregulated and upregulated genes, as compared with control NPs, such as genes involved in the expression of key enzymes of the TCA cycle or genes involved in glutamate and glutamine metabolism. Since the administration of a ketogenic diet (KD) improves clinical outcomes in AGC1-deficient patients, including recovery of myelination and muscular tone, we assessed OCR in the presence of ketone bodies. We observed the restoration of mitochondrial respiration when ketone bodies were added to NPs in combination with glutamine, which bypasses the impeded import of cytosolic pyruvate in AGC1-deficient NPs and indicating the biochemical basis of the beneficial effect of the ketogenic diet during the diet therapy. Ca2+ homeostasis was assessed in the prepared NP models using the Ca2+-sensitive FURA probes selectively targeted to the cytosol (Fura-2AM) and mitochondria (MtFura-2) in the presence of Ca2+-releasing agonist. Our data showed that after stimulation, cytosolic calcium levels were similar between patient and control cells. By contrast, mitochondrial calcium levels were significantly reduced in patient NPs compared to controls. Whether mitochondrial calcium homeostasis was dysregulated in AGC1-deficient NPs was partly supported by the altered expression of genes encoding the main actors controlling calcium signalling in cells, such as the mitochondrial NCLX Ca2+/Na+ exchanger. However, when basal respiration was measured in stimulated NP cells incubated with glucose, pyruvate and glutamine, patient cell lines showed virtually null increase of basal mitochondrial respiration compared to controls. On the other hand, when the measurement was in the presence of ketone bodies and glutamine, their anaplerotic effect was maintained even in the presence of lower mitochondrial Ca2+ concentrations.