Building in vitro human iPSC-derived motor neuron systems to explore cell autonomous mechanisms and modifiers of TDP-43 perturbation associated neurotoxicity

Abstract

Transactive Response DNA Binding Protein 43 (TDP-43) is a ubiquitously expressed nucleic acid transport and processing factor that is essential for normal cellular development and function. Disruptions to TDP-43 function in neurons, particularly in the form of aggregation in the cytoplasm, have been associated with the development of several neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). Approximately 97% of ALS patients will develop hyper-phosphorylated, hyper-ubiquitinated inclusions of TDP-43 in spinal cord motor neurons. While experimental models of TDP-43 perturbation relevant to ALS have been actively studied by many groups, many of these models are non-human, non-neuronal, inefficient to generate, or a combination of those qualities. These shortcomings limit the efficacy of these cellular systems for use as platforms for future therapeutic development at scale. This dissertation describes the development of genetically engineered human induced pluripotent stem cell (iPSC)-derived motor neuron models of TDP-43 overexpression and mislocalization as well as implementation of these models to elucidate mechanisms of ALS-relevant neurodegenerative processes. To begin, we demonstrate that these engineered motor neuron systems recapitulate key gain-of-function properties of TDP-43 perturbation upon induction, such as visible aggregation of TDP-43, re-localization of TDP-43 to the cytoplasm, and loss of cellular viability. We will then examine the physiologic effects of persistent cytoplasmic localization of TDP-43. Specifically, we will examine how cytoplasmic localization of TDP-43 changes the neuronal response to oxidative stress as well as which transcriptomic pathways are modulated by this disease-associated re-localization. Furthermore, we will use similar assays to examine the effect of genetic knockout of a proposed modifier of TDP-43 neurotoxicity, Ataxin-2 (ATXN2). We will establish that ATXN2 knockout rescues the neurotoxic effect of TDP-43 overexpression as well as positively modulates the stress response of neurons to proteotoxic and oxidative stress. In summary, this dissertation describes an efficient and expandable human iPSC-derived neuronal platform that provides insights to the mechanisms of neurodegeneration. These discoveries have the potential to be starting points for the design of future pharmacological and genetic treatments for ALS and other TDP-43 associated neurologic conditions.