ABS 126: Investigating Genetic Modifiers in a Novel C. elegans C9orf72-associated Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Model

Selamawit Asfaw ¹ , Mika Gallati ¹ , Alexander Lin-Moore ¹ , Jeremy Lins ¹ , Ashley Guo ¹ , Anne C. Hart ¹

¹ Brown University

Van Wickle (2025) Volume 1, ABS 126

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder caused by the progressive dysfunction and loss of motor neurons in the brain and spinal cord. A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both ALS and Frontotemporal Dementia (FTD). Healthy individuals typically carry fewer than 11 repeats, while affected individuals may harbor 30 to thousands. These expansions undergo repeat-associated non-AUG (RAN) translation, producing toxic dipeptide repeat proteins (DPRs) that interfere with nucleocytoplasmic transport, contribute to protein aggregation, and induce RNA toxicity. We have developed a Caenorhabditis elegans (C. elegans) model expressing 30 G4C2 repeats within the first intron of C9orf72 (rtIs90), which results in the degeneration of phasmid sensory neurons, providing a quantifiable measure of neurotoxicity. We are currently testing genetic modifiers derived from both published studies and ongoing research within our group, with the aim of understanding G4C2-mediated neurodegeneration. By uncovering conserved genetic pathways that influence DPR toxicity, this work will contribute to a deeper understanding of ALS/FTD pathogenesis and may ultimately guide the development of targeted therapeutic strategies.

Methods: Transgenic Caenorhabditis elegans expressing (G₄C₂)₃₀ and (G₄C₂)₇₅ under distinct neuronal promoters were used to investigate genetic modifiers of DPR-induced neurodegeneration. Loss-of-function mutations were introduced into these transgenic backgrounds, and resulting strains were maintained at 20 °C under standard conditions. For immunohistochemistry, synchronized L4-stage animals were fixed, permeabilized, and stained with anti–poly-GA antibodies. Fluorescence microscopy was used to visualize DPR accumulation in ASI, ASJ, PHA, and PHB neurons. Neurodegeneration was assessed via dye-filling assays: L4 animals were incubated in M9 buffer supplemented with MgSO₄ and 2.5 µL DiI (Invitrogen D3911) for 90 minutes on a shaking platform, then recovered on OP50-seeded plates. Young adults were immobilized using 2,3-butanedione monoxime and imaged under a 63× red fluorescence objective. Neuronal degeneration was defined as loss of dye uptake in PHA and PHB neurons. Scoring was performed blinded to genotype, and statistical significance was determined using one-way ANOVA with multiple comparisons.

Results: Poly-GA accumulation was robustly detected in the (G₄C₂)₃₀ rtIs90 model by anti–poly-GA immunostaining. Loss-of-function mutations in previously reported suppressors of G₄C₂-induced toxicity, including eif-2D and spop-1, did not attenuate neurodegeneration in this model. Similarly, suppressors of SOD-1–mediated toxicity failed to rescue neuronal degeneration in both the rtIs90 and kasIs7 lines. These findings suggest model-specific differences in the mechanisms underlying DPR-induced neurodegeneration and highlight the potential limited generalizability of genetic modifiers across ALS/FTD models. Context-dependent validation will be needed for accurately identifying conserved therapeutic targets in C9orf72-related disease.