Determining the pathogenesis of DYT1 dystonia in reprogrammed human neurons

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Title Determining the pathogenesis of dystonia in reprogrammed human neurons PROJECT SUMMARY The overall goal of this project is to determine the pathogenesis of dystonia via reprogramming human neurons from patient fibroblasts. Dystonia is the third most common movement disorder characterized by sustained or intermittent muscle contractions causing abnormal movements, postures, or both. The pathological mechanisms of dystonia remain largely unknown and there is no effective treatment to cure this disease. The early-onset DYT1 dystonia also belongs to neurodevelopmental disorders and represents the most frequent and severe form of dystonia, providing an excellent example to understand the pathogenesis of this disease. However, the limited access to patient neurons and the lack of in vitro human neuron systems greatly impede the progress of dystonia research. Excitingly, using lentiviral delivery of transcription factors, I have successfully generated human neurons from fibroblasts of DYT1 patients and healthy controls via two strategies: 1) direct conversion and 2) induced pluripotent stem cells (iPSCs)-based reprogramming and differentiation. The generation of these disease-relevant human neurons laid a solid foundation for this proposed research. Typically, DYT1 dystonia is caused by a loss-of-function mutation in protein torsin A (ΔE), a membrane- embedded ATPase. The effects of torsin A on neurite extension and synaptic vesicle recycling underscore the critical roles of torsin A in neuronal development and function. Additionally, accumulating evidence now indicates that torsin A also plays critical roles at the nuclear envelope (NE). In flies, torsin is required for mRNA exporting via a nuclear pore complex-independent mechanism (NE-budding). At the cellular level, one pathological hallmark in DYT1 dystonia mice is abnormal neuronal NE morphology, particularly severe in the spinal cord, suggesting that lower motor neurons could be the most severely affected neuron type in DYT1 dystonia. Do disrupted nuclear envelopes occurring in mice also occur in human DYT1 neurons? How do these abnormalities contribute to the human dystonia syndrome? In this project, we will address these pertinent questions directly in disease-relevant human neurons. In our preliminary studies, we found that the nuclear envelope morphology of DYT1 neurons was obviously disrupted at both light and electron microscopy levels and also the neurite outgrowth was significantly slower than that of controls. I hypothesize that the abnormities in DYT1 NE impair nucleocytoplasmic transport. In this project, we will systematically measure the nucleocytoplasmic transport of both mRNA exporting and protein nuclear transport, and to identify dysregulated factors, such as mis-localized mRNAs. Expected results emanating from this study will provide novel insights into dystonia pathology and potentially lead to molecular targets for therapeutic interventions.