Date of Award

Summer 8-26-2016

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

Advisor

Roger Sher

Second Committee Member

Robert Gundersen

Third Committee Member

Melissa Maginnis

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that causes selective death of upper and lower motor neurons resulting in paralysis and death. There is no cure for ALS and the average duration of survival upon diagnosis is only 3-5 years1, making research designed towards better understanding mechanisms leading to ALS an urgent need. ALS can either be sporadic or genetically inheritable in origin, and much of what we know about the cellular mechanisms that underlie ALS has come from the discovery and study of hundreds of mutations in 27 different genes that have been shown to cause the disease2. A common pathological phenotype in genetic forms of ALS is the subcellular mislocalization and aggregation of mutant proteins. This phenomenon may be explained by recent findings demonstrating that defects in nucleocytoplasmic transport (NCT) are a direct result of the most common ALS mutations3-5. NCT is a highly regulated process that controls the entry and exit of proteins and RNA to and from the nucleus through large, multi-protein structures that span the nuclear envelope called Nuclear Pore Complexes (NPC). NCT of large molecules through the NPC requires energy, which is provided by the small GTPase Ran, which exists as either RanGTP or RanGDP. NCT also requires the action of transport receptors which, recognize and bind specific cargo molecules. Import receptors require RanGTP binding in the nucleus to release their cargo, while export receptors require RanGTP to bind their cargo in the nucleus. A concentration gradient of high to low nuclear to cytoplasmic RanGTP is essential for NCT. This gradient is maintained by Ran effector proteins RCC1 and RanGAP1, which help exchange GDP for GTP on Ran in the nucleus and help catalyze RanGTP to RanGDP in the cytoplasm, respectively. Our group has identified a novel protein linked to both ALS and NCT. Nuclear Export Mediator Factor (NEMF) has been suggested to play a role in nuclear export6, and we have found that Nemf mutations causes neurodegeneration in mice. In order to determine effect of Nemf mutation on NCT, we used our NemfR86S mouse model. We performed immunofluorescence labeling to determine subcellular protein localization and nuclear transport assays to determine effects of the NemfR86S mutation upon Importin-β and Transportin-1 mediated nuclear import, Crm1 mediated nuclear export, and RNA export. Real-time quantitative PCR (RT-qPCR) was performed to investigate effects of the NemfR86S mutation upon endogenous gene expression. We found that the NemfR86S mutation causes mislocalization of Nemf and other ALS-related proteins, as well as inhibition of protein import and export. The NemfR86S mutation also disrupts the Ran gradient, which may be a result of the observed mislocalization of Ran effector molecule, RanGAP1. We observed no effect on RNA transport in our model. RT-qPCR demonstrated upregulation of genes involved in NCT. Overall this research demonstrated that Nemf plays a critical role in proper nucleocytoplasmic transport and that a mutation in Nemf causes NCT defects and neurodegeneration in a mammalian model, providing further evidence that NCT may be a common cellular mechanism affected in ALS.

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