# Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2022 · $34,179

## Abstract

Project Summary
The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic
Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological
aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing
mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation
or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the
aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape
Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting,
quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR-
Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known
interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several
novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD,
revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP-
43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation.
 The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43
aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro
studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four
genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized
proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better
understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the
role of TDP-43 aggregation in disease pathogenesis and progression.
 By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to
therapeutically modulate the pathways that contribute to disease.

## Key facts

- **NIH application ID:** 10404620
- **Project number:** 5F31NS116999-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Katelyn Sweeney
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $34,179
- **Award type:** 5
- **Project period:** 2020-06-01 → 2023-05-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10404620

## Citation

> US National Institutes of Health, RePORTER application 10404620, Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen (5F31NS116999-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10404620. Licensed CC0.

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