PROJECT 2 Recent studies have documented defects in nuclear transport and stress granule/liquid phase transitions as potentially early events in C9orf72 repeat expansion model systems. However, little is actually known about how these initial discoveries relate to ALS vs. FTD, two diseases caused by the same mutation. The hexanucleotide repeat expansion leads to both aberrant RNA as well as dipeptide repeat (DPR) proteins made via non-ATG repeat associated translation (RAN translation). A consensus of studies suggests the mutation disrupts nucleocytoplasmic transport but the mechanism and cell type specificity of this defect remain unclear. This leads to the important question: how does the same mutation differentially involve cortical neurons versus spinal motor neurons in clinically distinct diseases? Emerging data also links stress granule-based biology to nucleocytoplasmic transport in non-neuronal cells. However, whether this is relevant to CNS neurons or glia is unclear. We will employ a model system based on human induced pluripotent stem cell (iPSC)-derived cortical neurons and motor neurons from well-defined patients with either C9orf72 FTD, C9orf72 ALS or C9orf72 ALS and FTD to explore the underlying biological mechanism that may serve to explain the involvement of selective neuronal and/or glial cell types in these widely differing diseases. Specifically, in Aim 1 we will comprehensively assess and compare alterations in the nuclear pore complex (NPC) and nucleocytoplasmic transport in C9orf72 ALS/FTD iPSC-derived motor and cortical neurons. Understanding the differences in motor and cortical neuron nuclear pore complexes will define fundamental neuron-specific biology and may aid in dissecting the disease-specific pathogenesis in C9orf72 ALS and FTD.
In Aim 2, we propose to elucidate molecular pathways altered by C9orf72 repeat expansions in iPSC-derived motor and cortical neurons. Interrogating human cortical versus spinal C9orf72 neurons using ?omics? analytics will provide insight into cell- specific defects and opportunities for mitigating neuronal injury. Finally, in Aim 3, we will investigate the relationship between cellular stress and alterations in the nuclear pore complex in C9orf72 ALS/FTD. Determining the contribution of stress granules to dysfunctional nucleocytoplasmic transport will uncover novel approaches to therapeutic interventions for repairing the nuclear pore complex. In summary, understanding the connection between disease pathomechanisms and the neuronal subtype-specific effects of the C9orf72 repeat expansion on the nuclear pore complex, nucleocytoplasmic transport, and gene expression are essential to our understanding of C9orf72 ALS/FTD pathogenesis.
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