CRISPR Screen for Protein Inclusion Formation Uncovers SRRD as a Novel Regulator of Aggresome Formation and Cellular Proteostasis / Katelyn M Sweeney.

Format:

Book

Thesis/Dissertation

Author/Creator:

Sweeney, Katelyn M., author.

Contributor:

University of Pennsylvania. Cell and Molecular Biology, degree granting institution.

Language:

English

Subjects (All):

Cellular biology.

Molecular biology.

Genetics.

Cell and Molecular Biology--Penn dissertations.

Penn dissertations--Cell and Molecular Biology.

Local Subjects:

Cellular biology.

Molecular biology.

Genetics.

Cell and Molecular Biology--Penn dissertations.

Penn dissertations--Cell and Molecular Biology.

Physical Description:

1 online resource (210 pages)

Distribution:

Ann Arbor : ProQuest Dissertations & Theses, 2023

Contained In:

Dissertations Abstracts International 85-08B.

Place of Publication:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2022.

Language Note:

English

Summary:

A defining feature of human neurodegenerative diseases (NDs) is pathological protein aggregation. In the related NDs, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein (RBP) TDP-43 is the primary aggregated protein in up to 97% of ALS cases and 45% of FTD, despite disease causing mutations in TDP-43 explaining fewer than 1% of disease incidence. A related RBP, FUS, is the primary pathology in about a third of ALS and 45% of FTD cases that lack TDP-43 pathology, while mutations in FUS are incredibly rare. It is unclear why TDP-43 and FUS aggregation occurs, but the high incidence provides an opportunity to identify the underlying mechanisms and suggest therapeutics that may be applicable to many patients regardless of genetic etiology. To this end, we developed fluorescent aggregation reporters for TDP-43 and FUS that, when coupled to Pulse Shape Analysis (PulSA), a FACS based methodology, enabled detection of protein inclusions at the single cell level. These reporters were leveraged against genome-wide CRISPR-Cas9 screens and protein disaggregase systems to find novel modifiers of protein aggregation. The TDP-43 aggregation screen and follow up APEX proximity labeling and immunofluorescence assays found SRRD, a previously uncharacterized protein, to be a regulator of intermediate filament levels and organization, as well as a regulator of aggresome assembly. The TDP-43 reporter coupled to PulSA also enabled us to find HSP104 homologs that can specifically reduce TDP-43 aggregation without inducing cellular toxicity. Finally, we identified RUFY3, a regulator of neuron polarity and axon growth, as a modifier of FUS aggregation. Validation studies in yeast and Drosophila models show that RUFY3 expression exacerbates FUS toxicity. This work highlights the feasibility of and provides a framework for screening for modifiers of complex protein phenotypes like aggregation, where we uncovered novel regulators of proteostasis.

Notes:

Source: Dissertations Abstracts International, Volume: 85-08, Section: B.

Advisors: Shalem, Ophir; Committee members: Lee, Edward B.; Shorter, James; Maday, Sandra; Rhoades, Elizabeth.

Department: Cell and Molecular Biology.

Ph.D. University of Pennsylvania 2023.

Local Notes:

School code: 0175

ISBN:

9798381509878

Access Restriction:

Restricted for use by site license.