Characterization of disease genes in neurodegeneration with brain iron accumulation through the development of cellular models

Neurodegeneration with brain iron accumulation (NBIA) comprises a heterogeneous group of genetically defined disorders clinically characterized by progressive extrapyramidal deterioration and by iron accumulation in the basal ganglia. The NBIA disease genes encode proteins with a variety of functions: some are directly involved in iron homeostasis, other are related to coenzyme A biosynthesis, fatty acid metabolism, autophagy, DNA damage response or lysosomal activity. The clinical and molecular heterogeneity of NBIA disorders causes a large fraction (around 20%) of affected patients to be without a molecular genetics diagnosis. From this scenario, there is clearly a compelling need to use novel tools to both discover new disease genes and develop new in vitro and in vivo models for these disorders in order to understand their pathogenetic mechanisms. So the purpose of the experimental work I carried out during my DIMET course, has been focused on characterization of disease genes in NBIA, in particular of C19orf12 gene, coding for a mitochondrial membrane protein, which mutations are responsible for a form called MPAN (Mitochondrial membrane Protein Associated Neurodegeneration). We showed that wild-type C19orf12 protein is localized not only in the mitochondria but also in the Endoplasmic Reticulum (ER), and MAM (Mitochondria Associated Membrane). Using a GFP-tagged protein, we demonstrated that mutations of C19orf12, cause mis-localization of the protein. Moreover high mitochondrial calcium concentration and inability to respond to oxidative stress were found in MPAN fibroblasts. Then I worked on a project based on identification of new disease genes by exome sequencing on selected patients, and we found that mutations in CoA Synthase (COASY) were responsibible for a form of NBIA, named CoPAN (COASY protein-associated neurodegeneration). COASY is a bifunctional mitochondrial enzyme involved in the two last steps of coenzyme A (CoA) biosynthesis, and together with mutations in PANK2, coding for the first enzyme in CoA biosynthesis, mutations in CoA synthase impinge on the same biosynthetic pathway causing NBIA. For this reason further analysis were performed also on PKAN (Panthothenate kinase 2-associated neurodegeneration), caused by mutations in PANK2 gene, in order to support the concept that dysfunction in CoA synthesis may play a crucial role in the pathogenesis of NBIA. Due to the fact that existing cellular models and PKAN mice don’t recapitulate the neuropathological signs typical of the human disorder, we tried to obtain new cellular models. We generated human induced pluripotent stem cells (hiPSC) reprogramming PKAN fibroblasts. Their derived neurons exhibited functional impairments, such as, premature death, increased ROS production, aberrant mitochondria, reduction of respiratory capacity and major membrane excitability defects. At the end of my PhD project, we generate hiPSC also from MPAN patient, but we haven’t derived neurons yet. In conclusion, as concerning MPAN and considering that the C19orf12 protein has unknown function, we collected evidence on the role of mitochondria-ER connection in the transfer of lipid, calcium metabolism and autophagosome formation. Furthermore we demonstrated that inborn errors of CoA biosynthesis, represent one of the main cause of NBIA disease, although the connection between iron, mitochondria function, lipids and neurodegeneration is still missing. Altogether these data, obtained through the development of cellular models, represent the chance of having human patient’s diseased cells in culture that can provide new insights to research and a helpful tool for therapeutic purposes.