The GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 (C9) is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including familial and sporadic forms of the disease, as well as the ALS/FTD overlap syndrome. The C9 HRE is thought to cause disease by a toxic gain of function, mediated by expanded repeat RNAs and/or dipeptide repeat proteins (DPRs), produced by aberrant translation of the HRE. Our laboratory and others recently discovered that the C9 HRE impairs nucleocytoplasmic transport across multiple species and model systems, strongly implicating this fundamental cellular pathway in C9-mediated neurodegeneration. Our more recent, unpublished data suggest that the mechanism of nuclear transport impairment in C9-ALS/FTD involves disruption of a subset of nucleoporin proteins (Nups) with low complexity phenylalanine-glycine domains (FG-Nups). In yeast, FG-Nups line the nuclear pore complex (NPC), playing key roles in transport specificity and permeability, and a subset are functionally essential for nuclear transport and cell survival. Currently, little is known about the biology of FG- Nups in mammalian cells, particularly in the central nervous system (CNS), posing a major barrier for understanding the consequences of FG-Nup disruption in C9-ALS/FTD. In the proposed studies, our goal is to comprehensively evaluate FG-Nup expression and function in ALS/FTD-vulnerable cells of the CNS, to serve as a framework for further investigation of C9 toxicity. We will use the INTACT transgenic mouse (isolation of nuclei tagged in specific cell types) to isolate nuclei from defined neuronal and glial populations, analyze the expression and localization of FG-Nups by mass spectrometry and immuno-EM, and use siRNA knockdown to identify which FG-Nups are essential for nuclear transport and cell survival. Subsequently, we will investigate two potential mechanisms of C9-mediated FG-Nup disruption: (1) altered expression, and (2) cytoplasmic mislocalization and aggregation, which may be triggered by aberrant protein-protein interactions between DPRs and the FG-low complexity domain. Finally, we will test whether manipulating these factors in C9 induced pluripotent stem cell-derived neurons (iPSN) attenuates nuclear transport defects and prevents neurotoxicity. Taken together, these studies will provide the first comprehensive assessment of FG-Nup biology in ALS/FTD-vulnerable cells of the CNS, elucidate mechanisms by which C9 disrupts these essential FG-Nups, and identify novel targets for therapeutic intervention.

Public Health Relevance

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating, fatal neurodegenerative disorders with overlapping clinical and pathologic features. A recently discovered mutation in C9ORF72, now recognized as the most common cause of ALS and FTD, is providing new insights into fundamental cellular pathways that are disrupted in these disorders. This proposal aims to pinpoint the mechanisms of one of these pathways, impaired nuclear transport, and test therapeutic strategies for reversing these defects.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Clinical Investigator Award (CIA) (K08)
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Neurological Sciences Training Initial Review Group (NST)
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Gubitz, Amelie
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Johns Hopkins University
Schools of Medicine
United States
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