G4C2 repeat expansions in C9ORF72 are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Success in developing a treatment for ?c9FTD/ALS? will require a better understanding of disease pathomechanisms, rigorous testing of r(G4C2)exp-binding compounds in pre- clinical models, and the development of pharmacodynamic and clinical biomarkers. Putative pathomechanisms of ?c9FTD/ALS? involve RNA transcribed from the expansion [r(G4C2)exp]. We have shown these transcripts serve as templates for the synthesis of ?c9RAN proteins? through repeat-associated non-ATG (RAN) translation. That neuronal inclusions of c9RAN proteins are a pathological hallmark of c9FTD/ALS implicates RAN translation as a mechanism of disease. Indeed, poly(GR) and poly(PR) c9RAN proteins cause nucleocytoplasmic transport defects in yeast, and our studies reveal nuclear pore protein pathology in poly(GA)-expressing mice. These findings are consistent with those demonstrating that C9ORF72 repeat expansions impair nucleocytoplasmic transport and provoke atypical gene expression in c9FTD/ALS patient- derived cell models. Of note, we found extensive transcriptome changes in the brains of c9ALS patients, including abnormal alternative splicing of genes involved in nucleocytoplasmic transport. Whether this RNA misregulation is caused by c9RAN proteins and/or other events associated with r(G4C2)exp, and whether these transcriptome defects underlie the development of TDP-43 pathology, a hallmark feature of c9FTD/ALS argued to be a primary cause of neurodegeneration, are questions of importance that we will investigate. In addition, we will evaluate the therapeutic benefit of neutralizing r(G4C2)exp; together with leaders of Projects 1 and 3, we have shown that small molecule binders of r(G4C2)exp mitigate c9FTD/ALS-associated defects in patient- derived cell models. Therefore, we will screen r(G4C2)exp probes emerging from Project 1 in c9FTD/ALS patient-derived lymphoblastoid cell lines, and those showing promise will be screened in c9FTD/ALS iPSN by Project 3. Ultimately, we will validate lead compounds in our (G4C2)66 mice, yet another crucial step in moving drugs towards clinical trials. Likewise, because testing potential therapies in clinical trials will demand reliable, efficient means to measure target engagement and patient responses to treatment, and because there is an urgent need for indicators of c9FTD/ALS prognosis and disease stage, we will evaluate whether poly(GP) c9RAN proteins can serve these purposes. Our exciting findings that poly(GP) proteins are detectable in cerebrospinal fluid (CSF) and peripheral blood lymphocytes from c9FTD/ALS patients, coupled with preliminary data suggesting that CSF poly(GP) levels associate with clinical features of disease, provide compelling support for the use of poly(GP) as a biomarker of c9FTD/ALS. Overall, through the pre-clinical validation of lead small molecules and the development of clinical and pharmacodynamic biomarkers, Project 2 aims to increase the likelihood of success in identifying an effective treatment for c9FTD/ALS patients.
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