Expansion of a GGGGCC (G4C2) sequence in the first intron of the C9orf72 gene is the most common genetic cause of Frontotemporal Dementia (FTD) and Amyotrophic lateral sclerosis (ALS), two clinically linked neurodegenerative diseases. The mechanisms by which expansion of the G4C2 sequence lead to neurodegeneration of specific neurons are unknown. G4C2 RNA is transcribed in both sense and antisense directions and both RNA strands can undergo an unusual type of translation called Repeat Associated non-AUG dependent translation (RANT). RANT of the sense and antisense G4C2 RNA produces five distinct dipeptide repeat proteins (DPRs), two of which (PR and GR) confer strong toxicity in multiple model systems. The mechanisms by which these DPRs cause cellular toxicity are poorly understood. To understand the pathogenesis of C9orf72-mediated FTD/ALS, we generated C. elegans models expressing pure dipeptide repeat proteins. Both PR and GR were toxic in worms and caused neurodegeneration. To define genes and pathways causing toxicity, we performed an unbiased genetic suppressor and discovered several highly conserved genes that blocked PR50 toxicity. A subset of these suppressors affected protein ubiquitination, including a nuclear E3 ligase (
ufd-2/UBE4B) and a nuclear E3 ligase adaptor (
bath-43/SPOP). PR toxicity requires nuclear localization. CRISPR GFP/RFP-tagged endogenous genes show that both
ufd-2 and
bath-43 localize to the nucleus. Loss of
ufd-2 causes dramatic re-localization of toxic PR50, but not PR5, to the cytoplasm. PR50 localization is unaffected in
bath-43 mutants. SPOP is widely studied in cancer but has never been linked to a neurodegenerative disease until now. We acquired an SPOP inhibitor that blocks SPOP-dependent tumor growth and discovered that this small molecule also blocks DPR toxicity in mammalian primary neurons. Furthermore, miRNA-mediated SPOP knockdown protects primary neurons from DPR toxicity. Initial analysis of C9 patient brain and spinal cord tissue suggests that SPOP expression and localization is altered in disease tissue. Therefore, the SPOP/UBE4B pathway may be a conserved and pathologically relevant pathway for C9 diseases. The discovery of this novel ubiquitination system could lead to new therapeutic insights for these incurable neurodegenerative diseases.