TITLE Determining the pathogenesis of DYT1 dystonia in reprogrammed human neurons ABSTRACT 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. There is no effective treatment to cure this disease due to largely unknown the pathological mechanisms. The childhood onset DYT1 dystonia represents the most frequent and severe form of dystonia, providing an excellent example to understand the pathogenesis of dystonia disease. The typical DYT1 dystonia is caused by a heterozygous GAG deletion in TOR1A gene. Very interestingly, mice with the identical torsin A mutation as a heterozygote failed to show any pathological phenotypes, suggesting that the substantial differences of pathophysiological mechanisms exist between DYT1 mouse models and human patients. In torsin A knockout mice, one pathological hallmark is the abnormal nuclear envelope (NE) morphology in multiple areas of central nervous system (CNS). The large ventral horn neurons in the spinal cord were particularly severe, suggesting that lower motor neurons (MNs) could be the most severely affected neuron type in DYT1 dystonia. Whether the abnormalities occurred in DYT1 mice also occur in human patients? What are the effects of such abnormalities on neuron functions and what are underlying molecular mechanisms? These pertinent questions remained unanswered because the limited access to patient neurons and the lack of in vitro human neuron systems that greatly impede the progress of dystonia research. Excitingly, using lentiviral delivery of transcription factors, we 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. Using these patient-specific neurons, we will perform two specific aims: (1) to systematically examine the NE morphology and the nucleocytoplasmic transport (NCT) of mRNA and protein cargos, and (2) to identify dysregulated factors in human DYT1 neurons, including mis-localized mRNAs. The contributions of these identified factors to the disease will be focused in our follow-up studies. Expected results emanating from this study will provide novel insights into dystonia pathology and potentially lead to molecular targets for therapeutic interventions.
The limited access to patient neurons greatly impedes the progress of research in neurological diseases. Reprogramming of human neurons from adult fibroblasts provides an unprecedented approach to deciphering the molecular pathogenesis underlying disease condition in vitro. This project aims to determine the pathogenesis of DYT1 dystonia in reprogrammed patient-specific neurons.