Research on ALS has recently been the subject of major advances. Two independent groups have identified an expansion of GGGGCC repeats in the non-coding region of the first intron of the C9ORF72 gene as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and FTD (frontotemporal dementia) identified to date. Accumulation of RNA transcripts containing GGGGCC (sense) or CCCCGG (antisense) repeats were found to aggregate in nuclear foci in frontal cortex and spinal cord in C9ORF72 ALS/FTLD patients. Recently, toxicity of dipeptide repeat products (DPRs) generated via repeat associated non-ATG (RAN) translation of GGGGCC or CCCCGG repeat expansions has been proposed as potential pathogenic mechanism in C9- ALS/FTD, and DPR aggregates have been detected in affected and non-affected regions in ALS and ALS/FTD patients. RAN translation from sense and anti-sense directions has been reported in several nucleotide repeat disorders, including C9-ALS/FTD, making RAN translation an established occurrence in these expansion disorders, and also implicating a pathogenic role for RAN translated proteins. We established cellular models of C9-ALS/FTD using primary motor and cortical neurons, which are known to degenerate in ALS/FTD. By expressing fluorescently tagged homopolymeric C9RAN proteins (or DPRs) we were able to decipher their respective impact on neuronal viability using longitudinal time-lapse live-cell imaging, transgenic Drosophila models and found that Proline-Arginine dipeptides (PR) are robustly neurotoxic when expressed in vitro and in vivo. As next step in our investigation, we propose here to generate transgenic mice that express PR dipeptide repeats. Our hypothesis is that expression of PR aggregates in disease-relevant neuronal populations in mice will result in development of key phenotypes and pathologies that resemble those in ALS/FTD patients. This hypothesis is formulated based on extensive evidence of gained toxicity of PR aggregates in neuronal cell culture models as well as eye degeneration and lethality phenotypes when PR proteins are expressed in Drosophila eye and motor neurons. The rationale of the proposed research is that, once the model is fully characterized, it will have the potential to become a vital tool to unravel the basic mechanisms behind neurodegenerative processes in C9-ALS/FTD and, ultimately, for development of therapeutic interventions.
The discovery of an expanded RNA repeat in intron 1 of the C9ORF72 gene is of great importance for the field of ALS research because it accounts for a larger proportion of familial and sporadic ALS cases than SOD1. This discovery therefore represents an opportunity to develop different therapeutic approach from which a vast proportion of ALS patients could potentially benefit. However, we lack knowledge of the toxic species that originates from the genetic anomaly in the C9ORF72 gene. In this study we are proposing to address this gap in knowledge and develop a mouse model in which to study in vivo mechanisms of toxicity related to protein products that are generated in ALS patients carrying the C9ORF72 genetic anomaly. This mouse model will lead to subsequent investigation and therapeutic testing to prevent the progression and alleviate the symptoms of ALS.