N-acetylcysteine as a potential therapeutic agent for Treacher Collins syndrome: evidence from craniofacial defect suppression in polr1d crispant zebrafish

Treacher Collins syndrome (TCS; OMIM: 154,500) is a rare autosomal dominant craniofacial disorder. POLR1D mutations account for ~ 5% of familial TCS, yet the mechanism underlying POLR1D-mediated cranial neural crest cell (CNCC) dysfunction and targeted therapies for TCS remain elusive. Oxidative stress-induced CNCC loss is a key driver of TCS-related craniofacial malformations, suggesting antioxidants may hold therapeutic potential. We employed polr1d crispant zebrafish, which faithfully recapitulate the core phenotypic features of TCS, to evaluate the therapeutic potential of N-acetylcysteine (NAC), a clinically approved antioxidant agent. Zebrafish embryos were treated with 0.5 mM to 1.0 mM NAC from 6 to 72 h post-fertilization (hpf). PHH3 immunofluorescence staining was performed at 24 hpf to detect the proliferation of CNCCs. TUNEL staining was used to assess CNCC apoptosis at 48 hpf, and Alcian Blue staining was applied at 72 hpf to analyze craniofacial cartilage morphology. Additionally, qRT-PCR was conducted to quantify the expression levels of P53-pathway genes for exploring the underlying mechanism of NAC intervention. NAC treatment reduced CNCC apoptosis by 42% in polr1d crispant zebrafish, with a statistically significant difference relative to untreated controls (p = 0.006). This was accompanied by marked recovery of craniofacial cartilages (Meckel’s, palatoquadrate, ceratohyal), with their length/width parameters approaching wild-type levels (p < 0.001). Notably, mRNA levels of p53 showed no significant differences, suggesting that the p53-mediated apoptotic pathway may not be transcriptionally activated. NAC mitigates CNCCs loss and ameliorates TCS-like craniofacial defects in polr1d crispant zebrafish, independently of transcriptional activation of the p53 pathway. Our findings highlight oxidative stress as a tractable target for POLR1D-related TCS and provide preclinical evidence for antioxidant use in embryonic TCS prevention. Future studies will optimize NAC dosing in mammalian models to advance clinical translation.