The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide (G4C2)n repeat expansion (HRE) in the first intron of the C9orf72 (C9) gene. RNA and dipeptide repeats (DPRs) that are transcribed and translated from the C9-HRE respectively, have been shown to be neurotoxic. In a series of genetic screens in the fly and yeast, several groups recently showed that both RNA repeats and DPRs impair nucleocytoplasmic transport. However, the identities of the RNA and protein substrates affected by this defect in mutant C9 motor neurons (MNs), the specific downstream effects of these changes, and their contribution towards neurotoxicity remain unknown. What also remains elusive is the broader relevance of this mechanism for sporadic ALS, although cytoplasmic accumulation of nuclear proteins such as TDP43 is a neuropathological hallmark in almost all ALS and FTD patients. In our own preliminary work we have conducted large-scale sub-cellular proteomic analysis in a C9-HRE cellular model and have identified and validated a number of mislocalized candidate proteins including PRMT1. In the present study we will use patient-derived neurons, patient CNS tissue, and in vivo Drosophila models to test the hypothesis that ALS/FTD-related neurotoxicity is caused by a disruption the nucleus/cytoplasmic (N/C) distribution of specific classes of mRNAs and proteins.
In Aim 1, we will use patient-specific iPSC-derived MNs and employ molecular and precise biochemical subcellular fractionation coupled to RNA-Seq and MS-based quantitative proteomics. We will use multiple C9 and control iPSCs, as well as an isogenic control iPSC line, in which we have corrected the HRE though CRISPR/Cas9 gene editing. Identifying the mRNAs and proteins that are miss- compartmentalized in patient MNs is an essential first step towards elucidating the link between defective nucleocytoplasmic transport and neurotoxicity.
In Aim 2, we will use cellular models, patient tissue and in vivo Drosophila models of C9-HRE toxicity to systematically validate these molecular perturbations and assess their contribution towards ALS/FTD-related neurodegeneration.
In Aim 3, we will determine how cytoplasmic accumulation of PRMT1, an essential arginine methyltransferase, impacts MN function and survival. Taken together, our proposed aims will shed light into the cellular mechanisms that are compromised by abnormal nucleocytoplasmic mRNA/protein distribution in patients and will likely uncover therapeutic targets for C9 and potentially sporadic ALS/FTD.

Public Health Relevance

The largest genetic cause of ALS and FTD is a mutation in a gene called C9orf72, which results in the aberrant production of a rather long and irregular shaped RNA and protein dipeptides. The exact mechanisms by which these mutant products become neurotoxic remain elusive. The main goal of this proposal is to determine how C9orf72 mutations compromise the transport of RNA molecules and proteins in and out of the nucleus, what is the contribution of this defect to disease, and what proteins mediate the associated toxicity. These efforts will highlight novel neurotoxic mechanisms and identify points of therapeutic intervention that may be broadly relevant for ALS and FTD patients.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Molecular Neurogenetics Study Section (MNG)
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Gubitz, Amelie
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Northwestern University at Chicago
Schools of Medicine
United States
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Simkin, Dina; Kiskinis, Evangelos (2018) Modeling Pediatric Epilepsy Through iPSC-Based Technologies. Epilepsy Curr 18:240-245
Edelbrock, Alexandra N; Àlvarez, Zaida; Simkin, Dina et al. (2018) Supramolecular Nanostructure Activates TrkB Receptor Signaling of Neuronal Cells by Mimicking Brain-Derived Neurotrophic Factor. Nano Lett 18:6237-6247