Deng Tingting, Wu Xingxing, Wang Yujie, Fan Xiaoqin, Hu Bing
Shenzhen Key Laboratory of Nanozymes and Translational Cancer Research, Department of Otolaryngology, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, 518035, China.
Heliyon. 2024 Oct 18;10(20):e39535. doi: 10.1016/j.heliyon.2024.e39535. eCollection 2024 Oct 30.
Toe1 (target of EGR1, member 1) is a 3'- exonuclease in the deadenylase family, essential for the maturation of small nuclear RNAs. Mutations in Toe1 are linked to pontocerebellar hypoplasia 7 (PCH7), a severe neurodegenerative syndrome affecting infants, characterized by progressive neurodegeneration, developmental delay, and genital abnormalities. The pathogenic mechanisms of PCH7 are unclear but are thought to involve abnormal neural stem cell (NSC) development during embryogenesis. This study investigates Toe1's role in NSC development using the C17.2 NSC line. Colony formation, EdU incorporation, and CFSE staining assays showed that Toe1 knockout inhibited C17.2 cell proliferation. Upon inducing differentiation, Toe1 knockout significantly reduced cell dendrites. Immunofluorescence, qPCR, and Western blot analyses indicated that Toe1 knockout suppressed the expression of neuronal marker βIII-tubulin and glial cell marker Gfap, thereby inhibiting C17.2 cell differentiation. Additionally, Toe1 knockout reduced the expression of Dll1 and Jag1, suggesting an inhibition of Notch signaling. High-throughput transcriptome sequencing revealed that Toe1 influenced calcium ion binding, ECM, and amino acid catabolism in undifferentiated C17.2 cells, and peptidase activity, chemotactic factors, ECM, and TNF signaling in differentiated cells. These findings underscore Toe1's critical regulatory role in NSC proliferation and differentiation, with significant implications for developing therapeutic targets for neurodegenerative diseases such as PCH7.
Toe1（EGR1的靶标，成员1）是去腺苷酸化酶家族中的一种3'外切核酸酶，对小核RNA的成熟至关重要。Toe1突变与脑桥小脑发育不全7型（PCH7）相关，PCH7是一种影响婴儿的严重神经退行性综合征，其特征为进行性神经退行性变、发育迟缓及生殖器异常。PCH7的致病机制尚不清楚，但被认为涉及胚胎发育过程中神经干细胞（NSC）的异常发育。本研究使用C17.2 NSC细胞系研究Toe1在NSC发育中的作用。集落形成、EdU掺入及CFSE染色试验表明，Toe1基因敲除抑制了C17.2细胞增殖。诱导分化后，Toe1基因敲除显著减少了细胞树突。免疫荧光、qPCR及蛋白质印迹分析表明，Toe1基因敲除抑制了神经元标志物βIII-微管蛋白和神经胶质细胞标志物Gfap的表达，从而抑制了C17.2细胞分化。此外，Toe1基因敲除降低了Dll1和Jag1的表达，提示Notch信号通路受到抑制。高通量转录组测序显示，Toe1影响未分化C17.2细胞中的钙离子结合、细胞外基质（ECM）和氨基酸分解代谢，以及分化细胞中的肽酶活性、趋化因子、ECM和肿瘤坏死因子（TNF）信号通路。这些发现强调了Toe1在NSC增殖和分化中的关键调节作用，对开发针对PCH7等神经退行性疾病的治疗靶点具有重要意义。
