Publication Date




Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Biochemistry and Molecular Biology (Medicine)

Date of Defense


First Committee Member

Richard S. Myers

Second Committee Member

Claes Wahlestedt

Third Committee Member

Zane Zeier

Fourth Committee Member

Antonio Barrientos

Fifth Committee Member

Mario A. Saporta


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 15 new cases per day in the United States alone. To date there is no cure, only a modestly effective treatment to extend life by only a few months. A repeat expansion mutation within the C9ORF72 (chromosome 9 open reading frame 72) gene is the most common known cause of both sporadic and familial cases of ALS and frontotemporal dementia, a related neurodegenerative disease. Despite the identification of the C9ORF72 expansion mutation, the mechanism by which it confers neurotoxicity remains unknown. C9ORF72 expansion mutation produces toxic dipeptide repeats by noncanonical translation. Of the five dipeptide repeats produced, proline-arginine (PR) appears to be the most toxic; In the nucleolus PR co-localizes with nucleophosmin (NPM1), a multifunctional nucleolar protein that binds ribosomal RNA, facilitates DNA damage repair, and has been implicated in C9ORF72 ALS. Here we sought to unveil the mechanism by which PR localizes to the nucleolus and causes neurotoxicity by generating NPM1 mutants tagged with GFP to study its interaction with PR. A role for PR and NPM1 in DNA damage and repair was also studied. Several in vitro model systems were used including induced motor neurons a relevant cell type for ALS. To identify potential therapies, a highthroughput cell-based screen of an epigenetic small molecule library in PR expressing cells was performed. This study shows that: 1- PR binds directly to the acidic loop of NPM1 and its ectopic expression increases DNA damage markers, an effect that is enhanced when NPM1 is depleted (p<0.01, n=6). 2- PR significantly impedes single-strand annealing DNA repair (p<0.0001) in replicating cell lines (U2OS). 3- Increased levels of RAD52, a marker of single-strand annealing, in C9ALS neurons and human post-mortem brain tissue. Lastly, a library of >25 biosensors was developed to address nucleocytoplasmic disruption, another facet of C9ALS pathology. These were generated by fusing a fluorescent protein to different combinations of nuclear localization and export signals. These represent a versatile tool for unraveling the role of different dipeptide repeats in nucleocytoplasmic disruption in C9ALS. Additionally, the application of these biosensors is not limited to C9ALS and could be used for several screening campaigns for indications such as cancer, viral infections, muscular dystrophy, drug addiction, and others.


DNA damage; nucleophosmin; C9ALS

Available for download on Wednesday, August 05, 2020