Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative diseases with significant clinical and neuropathological overlap. A recent groundbreaking study now highlights a genetic link - hexanucleotide (GGGGCC) repeat expansion in C9ORF72 - as the most common cause of ALS and FTLD-TDP. This discovery raises an important question: how do the expanded repeats in a non-coding region of C9ORF72 contribute to disease pathogenesis? We hypothesize that RNA-mediated toxicity is a likely mechanism given that transcripts containing the expanded GGGGCC repeat accumulate as nuclear RNA foci in the frontal cortex and spinal cord of C9ORF72 mutation carriers. RNA foci formation, and the subsequent sequestration and altered activity of RNA-binding proteins by the foci, are emerging as a common pathogenic mechanism in many neurodegenerative diseases caused by non-coding repeat expansions. To determine whether RNA foci formation is a primary neurotoxic mechanism in c9FTD/ALS, novel in vitro and in vivo models of C9ORF72 hexanucleotide repeat expansions are required. To this end, we created two constitutive expression constructs to drive expression of RNA containing 18 GGGGCC repeats (18R) or 51 GGGGCC repeats (51R), the latter to mimic hexanucleotide repeat expansion. Notably, expression of the 51R transcript, but not the 18R transcript, leads to the formation of distinct, intranuclear RNA foci in HeLa and COS1 cell lines reminiscent of the foci observed in C9FTD/ALS cases. We also found foci formation is associated with enhanced cytotoxicity, as well as the sequestration of the RNA-binding proteins SAM68 and hnRNP-K. These findings not only illustrate that we can model the RNA foci formation observed in C9FTD/ALS, but also show that we have generated a valuable tool to study the mechanisms by which they exert toxicity. On the heels of these exciting findings, the goals of this project are: 1) to generate and characterize transgenic mice overexpressing non-pathogenic (non-foci-forming) and pathogenic (foci-forming) C9ORF72 repeat expansions;2) to evaluate whether RNA foci formation results in behavioral deficits and neurodegeneration in our foci-forming (GGGGCC)51 mouse model;3) to identify RNA-binding proteins sequestered by the foci;4) and to evaluate whether RNA targets of the sequestered proteins are altered in our novel cell and animal models, as well as in brain tissue and fibroblasts of C9FTD/ALS cases. Overall, we believe the proposed studies examining RNA foci formation consequences will lead to a better understanding of C9FTD/ALS-related mechanisms and help to uncover promising therapeutic targets.

Public Health Relevance

We seek to determine how a recently discovered abnormality in C9ORF72, specifically a (GGGGCC) expanded repeat in a non-coding region of the gene, leads to the formation of toxic RNA foci, or clumps, that are present in the brain and spinal cord of patients suffering from frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We are developing novel cell and transgenic mouse models that mimic disease features, such as foci-forming GGGGCC repeat expansions, to identify which RNA-binding proteins the foci sequester and prevent from carrying out their normal functions within cells. In so doing, we aim to identify new targets for therapy that could combat the neuronal death, and related symptoms such as language and motor impairments, associated with expansions in C9ORF72, the most common cause of FTD and ALS for which there is currently no cure.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-MDCN-N (03))
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Sutherland, Margaret L
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Mayo Clinic Jacksonville
United States
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Gendron, Tania F; Chew, Jeannie; Stankowski, Jeannette N et al. (2017) Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci Transl Med 9:
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