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The role of RNA in antagonizing aberrant phase transitions of RNA-binding proteins in ALS/FTD

Mann, Jacob (2020) The role of RNA in antagonizing aberrant phase transitions of RNA-binding proteins in ALS/FTD. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Aberrant aggregation of RNA-binding proteins (RBPs) is a common pathological hallmark of neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In these diseases, RBPs like TDP-43 and FUS are observed to be both depleted from the nuclear compartment, where they are normally localized, and found within cytoplasmic inclusions in degenerating regions of patient postmortem tissue. The mechanisms responsible for the aggregation of these proteins has remained elusive, largely due to technological limitations, but current hypotheses have proposed that liquid-liquid phase separation (LLPS) might serve as a critical nucleation step in the formation of pathological inclusions. This process of phase separation also seems to underlie the formation of a number of membraneless organelles (MLOs) throughout the cell, some of which have been shown to contain TDP-43, FUS, and other disease-linked RBPs. While various in vitro assays have been the predominant method to investigate protein phase behavior, here we describe the development of novel optogenetic-based tools to investigate the endogenous forces regulating phase separation of these proteins in the native intracellular environment. We also demonstrate the application of these tools to selectively induce pathologically-relevant aggregation of these proteins under the spatiotemporal control of light, which has allowed for the first direct testing of neurotoxicity resulting from TDP-43/FUS inclusions. Along with established in vitro assays, the use of these models has also led to the discovery of intracellular RNA as a strong modulator of TDP-43 and FUS LLPS and aggregation. Furthermore, we show here that short, specific RNA oligonucleotides mimicking this endogenous buffer system are capable of preventing and reversing aberrant TDP-43/FUS phase transitions, both in vitro and in cells, resulting in a rescue of cellular toxicity associated with pathological aggregation. While the exact mechanisms underlying RNA-mediated antagonization of TDP-43/FUS aggregation are still unclear, in-depth analysis of various RNA inhibitors outlined in this dissertation has begun to identify specific molecular properties, such as length, sequence, and secondary structure, that may mediate these effects. Together, this work may represent the discovery of a new potential therapeutic opportunity to target aberrant RBP aggregation observed in neurodegenerative disease.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Mann, Jacobjrm218@pitt.edujrm218
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairThathiah, Amanthaamantha@pitt.eduamantha
Thesis AdvisorDonnelly, Christopherchrisdonnelly@pitt.educhrisdonnelly
Committee MemberAizenman, Eliasredox@pitt.eduredox
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.edujbrodsky
Committee MemberPandey, Udaiudai@pitt.eduudai
Date: 30 August 2020
Date Type: Publication
Defense Date: 13 May 2020
Approval Date: 30 August 2020
Submission Date: 4 August 2020
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 265
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Protein aggregation; RNA-binding proteins; ALS; FTD
Date Deposited: 30 Aug 2020 12:38
Last Modified: 30 Aug 2020 12:38
URI: http://d-scholarship.pitt.edu/id/eprint/38765

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