Poster abstracts

Poster number 50 submitted by Chance Creviston

G4R1/DHX36 knockdown does not impact stress granule assembly in HeLa or C9orf72 ALS neural progenitor cells

Chance S. Creviston (Department of Biology, Ball State University), Nana Akua Koranteng, Magdalene A-Kusi, Kelsey Slattery, Christiana Whittey (Department of Biology, Ball State University), Peter K. Todd (Department of Neurology, University of Michigan), James P. Vaughn (Institute of Innate Immunity and Cancer Elimination), Yuh-Hwa Wang (School of Medicine, University of Virginia), Philip J. Smaldino (Department of Biology, Ball State University)

Abstract:
A repeat expansion (G4C2) mutation to C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The mutation forms extensive G-quadruplexes (G4s) in DNA and RNA, which can lead to C9orf72 haploinsufficiency, inhibit RNA metabolism, and produce toxic dipeptide repeat proteins (DPRs) via non-AUG translation. Nuclear clearance and aggregation of TDP-43 and other resident stress granule (SG) proteins are a pathological hallmark of ALS broadly, including C9 ALS. G4R1 (also known as DHX36) promotes toxic DPR production by unwinding G4s in C9orf72 mRNAs, making it a potential therapeutic target for C9 ALS. However, G4R1 plays a role in stress responses and SG assembly, meaning its reduction could potentially worsen SG protein aggregation or dysregulate normal stress responses. G4R1 knockout in cancer cells increases SG assembly, but it is unknown how G4R1 regulates SG assembly in C9 ALS or in neural cells. Based on previous work in cancer cells, we hypothesized that G4R1 inhibits SG assembly in C9 ALS. To accomplish this, we used HeLa cells and neural progenitor cells (NPCs) derived from ALS patient iPSCs (healthy, C9 ALS, FUS ALS) to assess the effect of shRNA-mediated DHX36 knockdown on SG assembly under oxidative stress. We found that partial G4R1 knockdown did not affect SG assembly in HeLa or neural progenitor cells. Additionally, G4R1 knockdown may impact TDP-43 localization. Future work will aim to answer whether linear loss of G4R1 produces nonlinear effects on stress responses. These data show that reducing G4R1 may have a minimal effect on SG assembly in neural progenitor cells, further characterizing it as a potential therapeutic target for C9 ALS.

Keywords: Stress granules, C9orf72 ALS, G-quadruplexes