Neural Regeneration Research ›› 2024, Vol. 19 ›› Issue (8): 1781-1788.doi: 10.4103/1673-5374.386401

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Two-photon live imaging of direct glia-to-neuron conversion in the mouse cortex

Zongqin Xiang1, 2, 3, #, Shu He1, #, Rongjie Chen1, #, Shanggong Liu1, Minhui Liu4, Liang Xu1, Jiajun Zheng2, Zhouquan Jiang1, Long Ma1, Ying Sun1, Yongpeng Qin1, Yi Chen1, Wen Li1, *, Xiangyu Wang2, *, Gong Chen1, *, Wenliang Lei1, *   

  1. 1Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China; 2Department of Neurosurgery, The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong Province, China; 3Laboratory for Neuroimmunology in Health and Diseases, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong Province, China; 4VIB-KU Leuven Center for Brain & Disease Research, Leuven, Flemish Region, Belgium
  • Online:2024-08-15 Published:2024-01-03
  • Contact: Wenliang Lei, PhD, leiwenliang@jnu.edu.cn; Gong Chen, PhD, gongchen@jnu.edu.cn; Xiangyu Wang, MD, wang_xy123@126.com; Wen Li, PhD, liwenhlb@163.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 31970906 (to WLei); the Natural Science Foundation of Guangdong Province, No. 2020A1515011079 (to WLei); Key Technologies R&D Program of Guangdong Province, No. 2018B030332001 (to GC); Science and Technology Projects of Guangzhou, No. 202206060002 (to GC); the Youth Science Program of the National Natural Science Foundation of China, No. 32100793 (to ZX); the Pearl River Innovation and Entrepreneurship Team, No. 2021ZT09Y552; and Yi-Liang Liu Endowment Fund from Jinan University Education Development Foundation.

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Abstract: Over the past decade, a growing number of studies have reported transcription factor-based in situ reprogramming that can directly convert endogenous glial cells into functional neurons as an alternative approach for neuroregeneration in the adult mammalian central nervous system. However, many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry. In addition, concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tracing mice. In this study, we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ectopic expression of the neural transcription factor NeuroD1 in both proliferating reactive astrocytes and lineage-traced astrocytes in the mouse cortex. Time-lapse imaging over several weeks revealed the step-by-step transition from a typical astrocyte with numerous short, tapered branches to a typical neuron with a few long neurites and dynamic growth cones that actively explored the local environment. In addition, these lineage-converting cells were able to migrate radially or tangentially to relocate to suitable positions. Furthermore, two-photon Ca2+ imaging and patch-clamp recordings confirmed that the newly generated neurons exhibited synchronous calcium signals, repetitive action potentials, and spontaneous synaptic responses, suggesting that they had made functional synaptic connections within local neural circuits. In conclusion, we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuroregeneration and neural circuit reconstruction.

Key words: astrocyte-to-neuron conversion, Ca2+ imaging, direct lineage conversion, glia, astrocyte, in vivo reprogramming, lineage-tracing mice, NeuroD1, neuron, two-photon imaging