Author ORCID Identifier
Date Available
3-18-2021
Year of Publication
2020
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Toxicology and Cancer Biology
First Advisor
Dr Haining Zhu
Abstract
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in Sarcoma (FUS) is a ubiquitously expressed RNA binding protein implicated in familial ALS and frontotemporal dementia (FTD). FUS is ubiquitously expressed in cells and has a variety of functions in the nucleus and cytoplasm. FUS mutations in the nuclear localization sequence (NLS) causes mislocalization of FUS in the cytoplasm, where it can undergo liquid-liquid phase separation and become stress granules or protein inclusions. Although FUS inclusion bodies can be found in post-mortem tissues from ALS patients, the pathological mechanism of the disease remains to be fully elucidated.
This dissertation includes two independent studies about FUS regulation and function in the cell. In the first study, we discovered a novel post-translational modification, i.e. lysine acetylation on FUS and aimed to understand whether lysine acetylation plays a role in regulating FUS function. We identified three acetylation sites in FUS: K315/K316 in the RRM domain and K510 in the NLS. We found that K510 disrupted the interaction with Transportin-1 and increased cytoplasmic protein aggregates that co-localized with stress granule markers. Interestingly, acetylation in K315/K316 reduced RNA binding to FUS and decreased the cytoplasmic inclusion formation. Similarly, treatment with deacetylase inhibitors also decreased protein aggregation in cells expressing ALS mutation P525L. Furthermore, ALS patient derived fibroblasts showed higher levels of acetylation at K510, compared to healthy controls. Finally, we demonstrated that CBP/p300 acetylates FUS, while both HDACs and Sirtuins contribute to FUS deacetylation. We concluded that FUS acetylation regulates RNA binding, subcellular localization, and inclusion formation, linking acetylation of FUS to a potential molecular mechanism for ALS/FTD.
The second study aimed to determine the function of FUS in the autophagy pathway. We found that mutant FUS did not affect autophagy flux in N2A cells. However, FUS knockout (KO) N2A cells showed a significant decrease in the basal autophagy flux compared to wild-type (WT) cells. Bafilomycin A1 treatment showed a decrease in autophagy flux in FUS KO cells, and induction of autophagy by rapamycin was not as efficient in FUS KO cells compared to WT cells. These results suggest that FUS KO affects early stages of the autophagy pathway. We found that FIP200, ATG16L1, and ATG12 gene and protein expression levels were significantly lower in FUS KO cells. FIP200 is involved in autophagy initiation and Atg16 and Atg12 form a complex to induce phagophore elongation. Overexpressing WT FUS in FUS KO cells was able to rescue gene and protein expression levels of FIP200 and ATG16L1. These findings demonstrate a novel function of FUS in regulating transcription of genes involved in early stages of the autophagy pathway. Failure to maintain protein homeostasis by protein degradation has been demonstrated in neurodegenerative diseases and finding new protein targets can be useful to discover new therapeutic options for ALS.
The two studies of this dissertation produce novel findings of FUS protein modification and function. Each study provides new insights into the role of FUS in neurodegenerative diseases including ALS and FTD, as well as novel therapeutic targets. Future studies that determine the molecular mechanisms connecting these two findings will be necessary to evaluate how FUS acetylation and inclusion formation will impact the autophagy pathway.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2020.404
Funding Information
This research was supported by the National Institutes of Health grant T325T32ES007266 training grant (2017-2020), the Lyman T. Johnson Fellowship (2014-2017), National Institute of Neurological Disorder and Stroke grant, Department of Veteran Affairs Merit Review Award, and National Institute of Neurological Disorders and Stroke grant R21
Recommended Citation
Arenas, Alexandra, "NOVEL POST-TRANSLATIONAL MODIFICATION AND FUNCTION OF FUS: THE RELEVANCE TO AMYOTROPHIC LATERAL SCLEROSIS" (2020). Theses and Dissertations--Toxicology and Cancer Biology. 33.
https://uknowledge.uky.edu/toxicology_etds/33
Included in
Biochemistry Commons, Molecular and Cellular Neuroscience Commons, Molecular Biology Commons, Toxicology Commons
