中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (7): 1526-1534.doi: 10.4103/1673-5374.325747

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

BrdU抑制星形胶质细胞向神经元转分化:一项意外发现

  

  • 出版日期:2022-07-15 发布日期:2022-01-17
  • 基金资助:
    广东省自然科学基金项目(2021A1515011237, 2020A1515010854);国家自然科学基金项目(U1801681, 31701291);广东省科技计划项目(2018B030332001)

Unexpected BrdU inhibition on astrocyte-to-neuron conversion

Tao Wang1, #, Jian-Cheng Liao2, #, Xu Wang1, Qing-Song Wang1, Kai-Ying Wan1, Yi-Yi Yang1, Qing He1, Jia-Xuan Zhang1, Gong Chen1, *, Wen Li1, *   

  1. 1Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China; 2Department of Neurosurgery, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China
  • Online:2022-07-15 Published:2022-01-17
  • Contact: Wen Li, PhD, liwenhlb@163.com; Gong Chen, PhD, gongchen@jnu.edu.cn.
  • Supported by:
    This study was supported by the Natural Science Foundation of Guangdong Province of China, Nos. 2021A1515011237 (to WL), 2020A1515010854 (to QSW); the National Natural Science Foundation of China, Nos. U1801681 (to GC), 31701291 (to WL); and the Guangdong Province Science and Technology Planning Project of China, No. 2018B030332001 (to GC).

摘要:

BrdU是一种胸腺嘧啶核苷的类似物,可在细胞周期的S期插入复制的DNA分子中,可在胚胎和成年神经发生的命运映射的研究中识别新生神经元,但有报道显示其对神经干细胞及其后代有副作用。体内星形胶质细胞转分化为神经元是一种直接将内源性星形胶质细胞转化为神经元来产生新生神经元的新方法。BrdU标记已被用于追踪星形胶质细胞转化的神经元,但其是否对星形胶质细胞向神经元转分化有不良影响目前尚不清楚。在此使用BrdU标记缺血性损伤后分裂的反应性星形胶质细胞,并以NeuroD1诱导星形胶质细胞向神经元转分化,结果发现在小鼠皮质中NeuroD1诱导的星形胶质细胞向神经元转分化过程中,BrdU标记的星形胶质细胞逐渐减少。尽管大多数NeuroD1诱导的星形胶质细胞可被转分化为神经元,但令人惊讶的是,BrdU标记的神经元却很少。为排除由缺血性损伤引起的这种BrdU抑制的可能性,在存在或不存在BrdU的情况下培养的皮质星形胶质细胞中过表达NeuroD1,以进一步进行体外星形胶质细胞向神经元转分化的研究。令人惊讶的是,与无BrdU组相比,BrdU组的转分化率明显降低,且转分化的神经元数量更少。这些结果说明BrdU对星形胶质细胞向神经元转分化存在抑制作用,因此未来的转分化研究需谨慎使用这种传统的BrdU标记法,以免错误解读实验结果。实验于2018年3月30日经暨南大学IACUC批准(体内实验批准号为IACUC-20180330-06,体外实验批准号为IACUC-20180319-05)。

https://orcid.org/0000-0002-4632-5754 (Wen Li); https://orcid.org/0000-0002-1857-3670 (Gong Chen)

关键词:

BrdU, NeuroD1, 星形胶质细胞向神经元转分化, 重编程, 神经再生, 反应性星形胶质细胞, 神经元, 谱系追踪, 命运映射, 神经干细胞

Abstract: 5-Bromo-2′-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.

Key words: 5-bromo-2′-deoxyuridine, NeuroD1, astrocyte-to-neuron conversion, reprogramming, neural regeneration, reactive astrocytes, neurons, lineage tracing, fate mapping, neural stem cell