*Article* **Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons**

**Teresa Galera-Monge 1,2,3,4, Francisco Zurita-Díaz 1,2,3,4, Isaac Canals 5, Marita Grønning Hansen 5, Laura Rufián-Vázquez 3,4,6, Johannes K. Ehinger 7, Eskil Elmér 7, Miguel A. Martin 3,4,6, Rafael Garesse 1,2,3, Henrik Ahlenius 5,\*,**† **andM.EstherGallardo 1,2,3,4,8,\*,**†


Received: 11 April 2020; Accepted: 28 April 2020; Published: 30 April 2020

**Abstract:** Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgen<sup>t</sup> need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.

**Keywords:** Leigh syndrome; mitochondrial disorder; iPSC; NSC; neuron; disease modeling; mtDNA
