BioRxiv. 2022 Jun 2
Liang Xu, Zong-Qin Xiang, Yao-Wei Guo, Yu-Ge Xu, Min-Hui Liu, Wen-Yu Ji, Shu He, Wen-Liang Lei, Wen Li, Zheng Wu and Gong Chen
Products used in the paper Details Operation
AAV vector packaging Recombinant AAV5 and AAV9 Adeno-associated virus were produced by PackGene® Biotech, LLC, purified through iodixanol gradient ultracentrifuge and subsequent concentration. Request Quote

Research Field: CNS

AAV Serotype: AAV5 and AAV9

Dose: Unless otherwise specified, AAV titre used in this study was mostly in the order of 10^11 – 10^12 GC/ml, with the exception of experiments in Fig. 1 to purposely demonstrate high neuronal leakage using high titre of 10^13 GC/ml.

Routes of Administration: Stereotaxic viral injection

Targeted organ: brain

Animal or cell line strain: Brain surgeries were performed on 4-8 weeks old wild-type mice for viral injection.


Regenerating functional new neurons in adult mammalian brains has been proven a difficult task for decades. Recent advancement in direct glia-to-neuron conversion in vivo opens a new field for neural regeneration and repair. However, this emerging new field is facing serious challenges from misuse of viral vectors to misinterpretation of conversion data. Here, we employ a variety of AAV vectors with different promoters and enhancers to demonstrate that astrocytes can be converted into neurons in a NeuroD1 dose-dependent manner in both wildtype (WT) and transgenic mice. Notably, astrocytes in WT mice were relatively easy to convert with higher conversion efficiency, whereas lineage-traced astrocytes in Aldh1l1-CreERT2 mice showed high resistance to reprogramming but were still converted into neurons after enhancing NeuroD1 expression with CMV enhancer. Furthermore, under two-photon microscope, we observed direct astrocyte-to-neuron conversion within 3 weeks of serial live imaging in the mouse cortex. We also demonstrated that high titre AAV reaching 1013 GC/ml caused severe neuronal leakage using a variety of AAV GFAP::GFP vectors, highlighting the necessity to inject low titre AAV into healthy brains to avoid artifactual results. Together, our studies suggest that lineage-traced astrocytes can be converted into neurons but require stronger conversion force such as enhanced NeuroD1 expression. Failure to recognize the difference between WT astrocytes and lineage-traced astrocytes in terms of conversion barrier will lead to misinterpretation of data.

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