Modeling Friedreich Ataxia with Patient iPSC-derived Neurons

Friedreich Ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by an elongated intronic GAA repeat in the gene encoding the mitochondrial protein frataxin. Sensory neurons from the dorsal root ganglia are the most susceptible cells for FRDA pathophysiology. Animal models of FRDA reproduced GAA repeat expansion, frataxin deficiency, mitochondrial alterations and neurodegeneration observed for the human disease. However, central questions concerning FRDA pathophysiology remained elusive: why are specific neuronal populations particularly susceptible in FRDA and when during ontogeny does the pathology manifest in susceptible neurons? We aim to address these questions by in vitro and in vivo experiments with induced pluripotent stem cell (iPSC)- derived neurons from FRDA patients. We have previously generated iPSC lines from FRDA patients, which carry pathological GAA repeat expansions and exhibit frataxin deficiency, and from control individuals. We were also successful in differentiating them to a neuronal population with peripheral sensory phenotype (Eigentler et al 2013). In order to determine why specific neuronal types differ in their susceptibility to frataxin deficiency, we will investigate molecular and cellular FRDA disease hallmarks in different types of neurons derived from the same patient. FRDA peripheral sensory neurons will be compared to telencephalic neurons, which will be generated by a differentiation protocol that we have already established for human embryonic stem cells (Nat et al 2012). In order to test the hypothesis that a developmental defect contributes to the disease process, functional characteristics will be compared during in vitro maturation of control and FRDA iPSC-derived sensory neurons. Furthermore, we will analyze whether the frataxin deficit affects the in vivo development of peripheral sensory neurons and their circuitries. To this end we will transplant human FRDA and control sensory precursors in chicken embryos and investigate their maturation and integration into spinal monosynaptic circuits.

Patient-specific induced pluripotent stem cells (iPSCs) are powerful models for studying human diseases, as they offer direct human relevance and the opportunity to generate cell types not accessible from living patients, such as neurons. Different neuronal types of both peripheral and central nervous systems are affected in genetic mitochondrial diseases, although mutations are present in all cells. This is the case in Friedreich Ataxia (FRDA), an autosomal recessive disease caused by a mutation in the gene encoding the mitochondrial protein frataxin. In order to elucidate the mechanisms underlying mitochondrial dysfunction and neuronal susceptibility in FRDA, we generated peripheral and central neurons from FRDA patients and matched control iPSCs, following their developmental stages: stem cells, progenitor cells and post-mitotic neurons. Phenotypic milestones for each developmental stage allowed an efficient and homogeneous differentiation and a proper comparison of cells carrying or not the FRDA mutation. In order to detect mitochondrial defects in FRDA cells we addressed mitochondrial morphology, respiration, ATP and reactive oxygen species (ROS) generation, and apoptosis. Mitochondrial morphology, ROS generation and apoptosis changed during differentiation but did not differ between stage-related FRDA and control cells. A shift from glycolysis in stem cells to oxidative phosphorylation in progenitors and neurons, with increasing ATP production, paralleled the neural differentiation. ATP production and maximal respiratory capacity were lower in all FRDA cell stages, the difference being the most pronounced in peripheral sensory neurons. This model also allowed to investigating the dynamic and disease-related expression of frataxin and other mitochondrial and cellular stress and inflammation markers, including iron, lipids and calcium biochemical pathways. Their differential expression explained some of the neuronal-specific phenotypes in FRDA but also raised new questions and hypotheses. Furthermore, we addressed whether the frataxin deficit affects human peripheral sensory progenitors and neurons transplanted in chicken embryos. While the neurons successfully integrated in chicken ganglia, their functional evaluation needs further investigations. These in vitro and in vivo findings helped us to understand the events that mirror the otherwise inaccessible human early neurodevelopment. Further elucidation of mitochondrial networking and pathophysiology is critical for the identification of novel and effective therapies in FRDA and other mitochondrial neurological diseases.