The C9orf72 nucleotide repeat expansion (C9-NRE) mutation has been identified as the most common genetic mutation link to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Although there has been significant work investigating the primary pathogenic mechanisms to explain the pathological features found in patient post-mortem disease tissues, there is still a gap in our assessment of what repeat length leads to disease pathogenesis. Of these pathogenic mechanisms, the neuro-specific contribution for loss-of-function (LOF) of the C9orf72 protein is not well described, while two C9-NRE gain-of-function (GOF) mechanisms, RNA toxicity from the bidirectional transcription of the NRE and/or dipeptide repeat (DPR) toxicity from the non-AUG-dependent translation of the C9-NRE transcripts, have been shown to contribute to neurotoxic mechanisms. However, epigenetic modifications to the C9-NRE region can modulate gain-of-function toxicity by reducing the pathogenic allele transcript levels, which has been proposed to be protective in patients. It is unknown if the LOF or GOF disease mechanisms and these epigenetic modifications emerge in a C9-NRE track-length-dependent manner. Therefore, further investigation is crucial to understand the requirements for these pathological and epigenetic observations identified in patients carrying the C9-NRE and to reconcile the relationship between C9-NRE-linked disease pathogenesis and C9-NRE track-length in neurons. To this end, we propose (Aim 1) to develop a series of C9-NRE isogenic iPSC lines with increasing repeat track-lengths that extend from the known non-pathogenic to the disease pathogenic range. Moreover, these isogenic iPSC lines are derived from a well-characterized patient with ALS/FTD that carries a C9-NRE mutation, which was corrected to generate the isogenic control. Additionally, these lines have been engineered to allow direct conversion to cortical or motor neurons using inducible neuronal-specific transcription factors, called I3Neruons. Utilizing this series of C9-NRE I3Neurons, we will then (Aim 2) perform a rigorous comprehensive biochemical and cellular assessment to quantitatively characterize pathological and phenotypic features as well as C9-NRE-specific epigenetic changes, transcript usage, and repeat instability as a function of C9-NRE track-length in cortical and motor neurons. The development of this robust series of C9-NRE track-length I3Neuronal models and quantitative characterization will produce vital disease-modeling resources to be shared with the scientific community; will enhance our understanding of the C9-NRE disease pathogenesis; and will serve as a prototypical system that can be used to improve translation of potential therapeutic interventions and diagnostic tools to the clinic.
A C9orf72 nucleotide repeat expansion is the most common genetic mutation associated with amyotrophic lateral sclerosis and frontotemporal dementia, yet it is still unclear whether or not repeat size matters. In this proposal we will develop new patient-derived neuronal models to examine the relationship between repeat size and pathology/phenotypes associated with disease. Accomplishing this proposal will determine a repeat threshold that leads to disease pathology/phenotypes identified in patients with the potential to translate these findings to identify at-risk patients that carry this repeat expansion mutation.