Leigh syndrome (LS) is a progressive neurodegenerative disorder characterized by the presence of bilateral symmetrical lesions predominantly in basal ganglia, brainstem, and cerebellum. LS affects 1 in 40,000 live births and typically manifests in infancy or early childhood, although adult onset has also been described. Clinical presentations vary between individuals but usually include failure to thrive, lactic acidosis, hypotonia, seizures, ataxia, encephalopathy and premature death. Mutations in more than 75 genes in both the nuclear and the mitochondrial genome have been described to cause LS. Consequences of the mutations arise commonly due to defects of mitochondrial functionality and bioenergetic metabolism. To date, there is no effective treatment or cure for this inherited disease, and the phenotypic and genetic heterogeneity of LS complicates disease modelling and the development of new treatment options. Animal models often fail to recapitulate the pathology of human LS and particularly for mtDNA-related diseases there is a lack of relevant model systems, mainly due to the difficulties of engineering mtDNA. Existing cell models do not exhibit functional and metabolic features of neuronal cells, which are predominantly affected by the disease-related mutations, and do not provide the patient-specific match of mitochondrial and nuclear genomes. The development of more suitable model systems is therefore of crucial importance. In my main project, I used patient-derived induced pluripotent stem cells (iPSCs) to generate neural progenitor cells (NPCs) and neurons for the investigation of LS disease mechanisms and for the establishment of a phenotype-based drug discovery platform. To this goal, I generated iPSC-derived NPC lines of three patients carrying the homoplasmic mtDNA mutation MT-ATP6 (m.9185T>C), which is associated with maternally inherited LS. Characterization of NPCs showed that they retained the patient-specific nuclear and mitochondrial matched genotypes and exhibited a metabolic switch from glycolysis toward oxidative phosphorylation. Furthermore, I examined mitochondrial bioenergetics and calcium handling in patient-specific neural cells, and observed mitochondrial defects, which are associated with the clinical features of the diseases. MT-ATP6 (m.9185T>C) NPCs showed defective ATP production, abnormally high mitochondrial membrane potential (MMP), and altered calcium homeostasis, which could represent a possible cause of neural impairment. The MMP phenotype was then used to carry out the first phenotypic compound screen in patient-derived NPCs using imaging-based high-content analysis (HCA). Among the FDA-approved compounds screened, the PDE5 inhibitor avanafil was discovered to reduce the mitochondrial hyperpolarization. Next, I performed experiments to assess the influence of avanafil on calcium homeostasis in MT-ATP6 (m.9185T>C) NPCs and differentiated neurons and showed that avanafil was able to partially rescue the calcium defect in both. Overall, the results of this project demonstrated that patient-specific iPSC-derived NPCs represent an effective model system for mtDNA disorders affecting the nervous system and for establishing compound screening approaches. For the second project, I generated human iPSC lines from dermal fibroblasts from four patients carrying homoplasmic mutations in the mitochondrial MT-ATP6 gene (m.8993T>G or m.8993T>C) associated with LS and neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP). In addition, I have contributed to the cell line characterization and pluripotency tests, and confirmed the cell identity and quality of the newly generated iPSC lines according to widely used validation criteria. With this project, I contributed to the generation of a cohort of patient-specific cells and made them available to the research community in order to support the development of disease modeling and drug discovery platforms for the rare disease LS. In the third project, the first adult patients with a 3-hydroxyisobutyryl-CoA hydrolase (HIBCH)-associated movement disorder were reported. HIBCH enzyme deficiency is a rare metabolic disease and typically causes a neurodegenerative phenotype resembling Leigh syndrome, including the presence of bilateral lesions of the basal ganglia. The five patients described carry a new homozygous missense mutation (c.913A>G; p.T305A) in the nuclear HIBCH gene and, compared to the literature, showed a mild phenotype that allows survival into adulthood. I contributed to the study of bioenergetic defects in patient-derived fibroblasts and observed reduced oxygen consumption rates suggesting that HIBCH deficiency may cause a general alteration of electron flux along the respiratory chain. This study contributes to the description of the phenotypic spectrum of the disease and may help to establish a genotype-phenotype correlation in HIBCH-related Leigh-like disorders. The studies presented in this cumulative dissertation have helped to advance the understanding of the pathogenic pathways of Leigh syndrome and Leigh-like disease HIBCH deficiency and may pave the way for the development of new therapeutic options.
