WWOX contributes to DNA damage, but not somatic instability in Huntington's disease

Huntington's disease (HD), caused by a CAG repeat expansion in the huntingtin ( HTT ) gene, is characterized by progressive neurodegeneration and accumulation of DNA damage with multiple disease-modifier genes involved in DNA repair pathways. Previous studies have implicated ataxia telangiectasia mutated (ATM) signaling in the regulation of genomic stability and DNA damage repair (DDR) pathways in HD. ATM has also been linked to the WW domain-containing oxidoreductase (WWOX), a protein involved in DNA repair and maintenance of genomic stability, through the E3 ubiquitin ligase ITCH. However, whether this signaling pathway contributes to HD pathogenesis remains unknown. Here, we investigated the role of ATM-ITCH-WWOX signaling in HD. Our results revealed no significant alterations in total ATM, phosphorylated ATM (pATM-S1981), or ITCH in HD post-mortem prefrontal cortex (PFC) compared to controls. Although treatment of human neuroblastoma SH-SY5Y cells with HD PFC lysates did not alter pATM-S1981 levels, it increased histone H2AX phosphorylation at S139 (γ-H2AX), a marker of DNA double-strand breaks. This finding suggested the presence of persistent DNA damage signaling independent of canonical ATM activation. Conversely, WWOX levels were increased in both HD PFC and HD embryonic stem cell-derived cortical neurons. Additionally, treatment of SH-SY5Y cells with recombinant human WWOX protein or WWOX overexpression increased γ-H2AX levels, supporting a role for WWOX in promoting DNA damage. To determine whether WWOX contributed to DNA damage in HD, SH-SY5Y cells were treated with HD PFC lysates that were depleted of WWOX. Immuno-depletion of WWOX reduced the ability of HD PFC lysates to increase γ-H2AX, suggesting that WWOX contributes to DNA damage in HD. Finally, overexpression of WWOX in RPE1-AAVS1-CAG115 cells did not affect somatic CAG repeat instability, despite persistent increases in γ-H2AX levels. Collectively, our findings identify WWOX as a contributor to DNA damage in HD, acting independently of the ATM pathway.