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In vivo imaging of astrocytes in the whole brain with engineered AAVs and diffusion-weighted magnetic resonance imaging

Molecular Psychiatry (2022)Cite this article

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Abstract

Astrocytes constitute a major part of the central nervous system and the delineation of their activity patterns is conducive to a better understanding of brain network dynamics. This study aimed to develop a magnetic resonance imaging (MRI)-based method in order to monitor the brain-wide or region-specific astrocytes in live animals. Adeno-associated virus (AAVs) vectors carrying the human glial fibrillary acidic protein (GFAP) promoter driving the EGFP-AQP1 (Aquaporin-1, an MRI reporter) fusion gene were employed. The following steps were included: constructing recombinant AAV vectors for astrocyte-specific expression, detecting MRI reporters in cell culture, brain regions, or whole brain following cell transduction, stereotactic injection, or tail vein injection. The astrocytes were detected by both fluorescent imaging and Diffusion-weighted MRI. The novel AAV mutation (Site-directed mutagenesis of surface-exposed tyrosine (Y) residues on the AAV5 capsid) significantly increased fluorescence intensity (p < 0.01) compared with the AAV5 wild type. Transduction of the rAAV2/5 carrying AQP1 induced the titer-dependent changes in MRI contrast in cell cultures (p < 0.05) and caudate-putamen (CPu) in the brain (p < 0.05). Furthermore, the MRI revealed a good brain-wide alignment between AQP1 levels and ADC signals, which increased over time in most of the transduced brain regions. In addition, the rAAV2/PHP.eB serotype efficiently introduced AOP1 expression in the whole brain via tail vein injection. This study provides an MRI-based approach to detect dynamic changes in astrocytes in live animals. The novel in vivo tool could help us to understand the complexity of neuronal and glial networks in different pathophysiological conditions.

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Fig. 1: Site-directed AAV5 capsid mutation improved vector-mediated transgene expression in vivo.
Fig. 2: Validation of AQP1 expression for astrocyte targeting rAAV in the cell culture study.
Fig. 3: Validation of the MRI contrast for rAAV2/5-GFAP-AQP1-EGFP in living animals.
Fig. 4: Detection of astrocytes in the whole brain marked by rAAV-PHP.eB-GFAP-AQP1-EGFP.
Fig. 5: Longitudinal detection of astrocytes of the whole brain in living animals with diffusion-weighted MRI.

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Acknowledgements

We thank Mr. Zengpeng Han for drawing the structure of VP1 in AAV Capsid; Ms. Yating Liu (Northwest Minzu University) for drawing the figures, Ms. Qitian Wang, Ms. Dingyu Jin, and Ms. Pingping An for technical support. The current study was supported by the National Natural Science Foundation of China (31970973, 81974170, 31830035, 21921004); the National Key Research and the Development Program of China (2021M693294); the Key-Area Research and Development Program of Guangdong Province (2018B030331001); the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB32030200); the Shenzhen Key Laboratory of Viral Vectors for Biomedicine (ZDSYS20200811142401005), the National Natural Science Foundation (NSF) of Hubei Province (2020CFA059), the Open Project Program of Wuhan National Laboratory for Optoelectronics (2019WNLOKF022).

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Author notes

  1. These authors contributed equally: Mei Li, Zhuang Liu.

Authors and Affiliations

  1. The Brain Cognition and Brain Disease Institute (BCBDI), NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen Key Laboratory of Viral Vectors for Biomedicine Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 518055, China

    Mei Li & Fuqiang Xu

  2. Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, 430071, Wuhan, PR of China

    Zhuang Liu, Yang Wu, Ning Zheng, Aoling Cai, Danhao Zheng, Jinpiao Zhu, Jinfeng Wu, Lingling Xu, Fuqiang Xu & Jie Wang

  3. University of Chinese Academy of Sciences, Beijing, 100049, P.R. China

    Zhuang Liu, Yang Wu, Aoling Cai, Danhao Zheng, Fuqiang Xu & Jie Wang

  4. Department of Anaesthesia and Intensive Care, Peter Hung Pain Research Institute, The Chinese University of Hong Kong, Hong Kong SAR, PR China

    Xiaodong Liu

  5. Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, 430071, China

    Jinpiao Zhu

  6. Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, PR China

    Xihai Li

  7. Department of Pathophysiology, Key Lab of Neurological Disorder of Education Ministry, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Ling-Qiang Zhu

  8. School of Human and Social Sciences, University of West London, Middlesex, TW8 9GA, UK

    Anne Manyande

  9. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, 200031, PR China

    Fuqiang Xu

  10. Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, China

    Fuqiang Xu

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  1. Mei Li
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Contributions

ML, FX, and J Wang designed the experiments and wrote the manuscript. ML and ZL performed the whole experiments; YW performed the virus production; NZ; AC; DZ; JZ and J Wu performed the data analysis; LX performed the fluorescence imaging recordings; XL, XDL, and LQZ performed the discussion of the results and revised the manuscript; AM revised the whole manuscript; FX and J Wang provided funding and designed the project. All authors read and approved the manuscript.

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Correspondence to Fuqiang Xu or Jie Wang.

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Li, M., Liu, Z., Wu, Y. et al. In vivo imaging of astrocytes in the whole brain with engineered AAVs and diffusion-weighted magnetic resonance imaging. Mol Psychiatry (2022). https://doi.org/10.1038/s41380-022-01580-0

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