The goal of this proposal is to investigate the pathogenic mechanism of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) induced by hexanucleotide repeat expansion in the C9ORF72 gene. The expansion of GGGGCC repeats in the first intron of C9ORF72 is the most common genetic cause of both ALS and FTD. The disease has been predominantly attributed to the RNA repeats induced toxicity, including RNA granules that sequester essential RNA binding proteins (RBP), and the toxic poly-dipeptides produced through repeat-associated non-AUG (RAN) translation. Previous biochemical and genetic studies have revealed cellular pathways impaired by the repeat expansion. However, the biophysical properties of RNA granules and DPR biogenesis are still poorly understood at the molecular level. Here we combine cutting edge in vivo single molecule imaging approach with biochemical analysis and high-throughput screening to study the repeat RNA metabolisms at every RNA processing step, including transcription and splicing, turnover and aggregation properties, RAN translation, RNA localization and trafficking in reporter cell lines, patient derived cells and mouse models. We will fluorescently label both sense and antisense RNA with different colors and study the biophysical property of RNA granules and their interactions in live cells. We will measure the translation efficiency, initiation and elongation dynamics, and possible frameshift in live cells for each reading frame of both sense and antisense repeats. We will label C9ORF72 intron and exon with different colors and examine the splicing and repeat-mediated intron export. We will test candidate genetic modifiers of both RNA granules and RAN translation, as well as screen for candidates involved in the nuclear export of repeat-containing intron. All these pathways will be examined at single molecule level in live cells for the dynamic properties. We will also determine whether the repeat RNA metabolisms have cell type-specific features and how stress stimuli and neuronal activities could influence these properties. The approach proposed here allows us to address the mechanistic problems intractable by any other techniques. The molecular insights resulting from this study will help understand the etiology of the disease and develop novel therapeutic strategy.

Public Health Relevance

We propose to investigate the pathogenic mechanism of amyotrophic lateral sclerosis (ALS) / frontotemporal degeneration (FTD) caused by repeat expansion in the C9ORF72 gene by using in vivo single molecule fluorescence imaging, biochemical analysis and high-throughput screening. We will fluorescently label C9ORF72 RNA and newly synthesized protein to study the repeat RNA metabolism at every RNA processing step, including transcription, splicing, aggregation, turnover, trafficking and translation. The study will address disease mechanism of repeat RNA in single molecule resolution, and help understand the etiology of the disease and develop therapeutic strategy.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
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Gubitz, Amelie
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Johns Hopkins University
Schools of Medicine
United States
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