**1. Introduction**

Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) with a prevalence of 1 in 40,000 births [**???** ]. Forty-five percent of LS patients die before reaching 20 years of age, most of them by respiratory failure. However, there are several complications that may further increase morbidity and mortality, such as refractory seizures and cardiovascular deterioration [**?** ]. The common feature of these patients is the presence of bilateral symmetric necrotic areas in the basal ganglia or the brain stem, which correspond with regions of demyelination, neuronal death and astrogliosis [**? ?** ]. However, LS is characterized by a prominent clinical and genetic variability. More than 75 genes have been associated to LS [**?** ], all of them involved in mitochondrial energy production [**?** ].

Mitochondria are cellular organelles considered to be the powerhouse of the cell because of their participation in cellular energy production through a process known as oxidative phosphorylation (OXPHOS) [**?** ]. The OXPHOS process is carried out by five multiheteromeric complexes located in the inner mitochondrial membrane and collectively termed the respiratory chain (RC) [**?** ]. Moreover, mitochondria are also involved in other pivotal processes such as reactive oxygen species (ROS) production, apoptosis and calcium homeostasis, whose role in pathology is being increasingly recognized. Given the crucial role of mitochondria for functionality of neuronal cells, it is not surprising that diseases affecting mitochondria result in neurological conditions such as LS [**?** ].

Mitochondria possess their own DNA [**?** ] and the human mitochondrial DNA (mtDNA) is a double-stranded circular molecule of 16.5 kb that encodes 13 subunits of the OXPHOS complexes as well as two rRNAs and 22 tRNAs [**?** ]. Hundreds to thousands of copies of mtDNA are present per cell [**? ?** ], allowing the possibility of coexistence of healthy and pathogenic mtDNA molecules, a phenomenon called heteroplasmy. Genetically, LS is highly heterogeneous; to date, a broad variety of causative mutations have been described in nuclear- and mitochondrial-encoded genes involved in energy metabolism. mtDNA mutations are responsible for 10–20% of LS cases and, more specifically, mutations in genes affecting complex I of the respiratory chain have been a well-recognized cause of LS. Among them, the m.13513G>A mutation located in the *MT-ND5* gene is a frequent cause of LS [**?** ]. In spite of the advances in the molecular diagnosis of LS, the molecular pathogenesis of this disease remains poorly understood due, in part, to the lack of suitable disease models.

For that reason, generation of induced pluripotent stem cells (iPSCs) and differentiation into the affected tissue could be an interesting approach for modeling LS [**?** ]. Several studies have used iPSC technology to generate in vitro models of LS harboring the mutation m.8993T>G in the *MT-ATP6* gene [**? ?** ]. These models recapitulate the mitochondrial dysfunction in muscle [**?** ] or in neurons, the principally affected cell type [**?** ]. Moreover, this model recapitulated the typical neurodegeneration in brains of LS patients [**?** ]. The m.13513G>A mutation in the *MT-ND5* gene is responsible for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) and LS. Until now, one report has described the behavior of heteroplasmy during reprogramming and extended culture of iPSCs harboring this mutation in association with MELAS syndrome, but neuronal characterization is missing. Here, we generate iPSC-derived neurons from a described patient suffering LS caused by m.13513G>A mutation in heteroplasmy [**?** ] and explore the mechanisms by which this mutation could cause the disease. Although LS iPSC-derived neurons were electrophysiologically normal, they manifested a decreased respiration and a diminished calcium buffering capacity. The slower removal of cytoplasmic calcium could lead to an overload and the consequent neuronal death observed in patients.
