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Activity-Dependent Differential Regulation of Auts2 Isoforms In Vitro and In Vivo

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Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder of unknown cause, although one hypothesis suggests a potential imbalance between excitation and inhibition that leads to changes in neuronal activity and a disturbance in the brain network. However, the mechanisms through which neuronal activity contributes to the development of ASD remain largely unexplained. In this study, we described that neuronal activity at the transcriptional and translational levels regulated the expression of Auts2 isoforms. The prolonged stimulation of cultured cortical neurons significantly reduced the auts2 transcripts, accompanied by the decrease of FL-Auts2 protein, as well as one of the short isoforms (S-Auts2 var.1). Blocking neuronal activity increased the number of auts2 transcripts but not protein levels. Furthermore, blocking the NMDA receptors during stimulation could partially restore the FL-Auts2 and S-Auts2 var.1 at protein level, but not at mRNA level. Finally, Auts2 expression in the hippocampus was reduced in mice exposed to an enriched environment, a behavior paradigm designed to increase the brain activity through abundant sensory and social stimulations. Thus, our study revealed a novel regulatory effect of neuronal activity on the transcription and translation of ASD-risk gene auts2.

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Availability of Data and Materials

The datasets used during current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Feng Shi and Dr. Wen Shilei for kind suggestions to our manuscript and experimental techniques.

Funding

This study is supported by the National Natural Science Foundation of China (31960170) and the Hainan Province Science and Technology Special Fund (ZDYF2020216) to L.X., the Hainan Province Science and Technology Special Fund (ZDYF2021SHFZ088) and Hainan Major Science and Technology Projects (ZDKJ2019010) to W.X., the Natural Science Foundation of Hainan Province (821QN1001) to W.B.P., and the Hainan Graduate Students Innovation Projects (Qhys2021-357) to M.J.W. and (Qhys2021-355) to Q.S.B.. The study also received financial support from the Hainan Province Clinical Medical Center Grant (QWYH202175) and the Excellent Talent Team of Hainan Province (QRCBT202121).

Author information

Authors and Affiliations

  1. Hainan Women and Children’s Medical Center, Hainan Medical University, Haikou, China

    Wenbin Pang, Hongai Li, Wei Xiang & Le Xiao

  2. School of Pediatrics, Hainan Medical University, Haikou, China

    Wenbin Pang, Hongai Li, Qionglin Zhou, Xiaoshan Ye, Wei Xiang & Le Xiao

  3. School of Basic Medicine and Life Science, Hainan Medical University, Haikou, China

    Meijuan Wang & Qingshang Bi

  4. National Health Commission (NHC) Key Laboratory of Control of Tropical Diseases, Hainan Medical University, Haikou, China

    Wei Xiang

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  1. Wenbin Pang
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Contributions

L.X. and W.X. designed the study. L.X., W.X., and W.B.P. wrote the paper. W.B.P. and M.J.W. performed most of the experiments, including neuronal culture, qPCR, Wes Assay, and enriched environmental paradigm. Q.S.B., H.A.L., and Q.L.Z. participated in the neuronal culture experiments. X.S.Y. helped with the qPCR experiments. All authors contributed to the article and approved the submitted version.

Corresponding authors

Correspondence to Wei Xiang or Le Xiao.

Ethics declarations

Ethics Approval

All animal experiments were performed according to the Ethics Committee of Hainan Medical University (HYLL-2022–259) and conformed to the ethical principles of welfare of laboratory animals.

Consent to Participate

This study did not involve human subjects.

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This study did not involve patients.

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The authors declare no competing interests.

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Wenbin Pang and Meijuan Wang are the co-first author.

Supplementary Information

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Supplementary Figure 1.

Representative amplification curves and agarose gel image of auts2 qPCR products. A. Representative amplification curves from qPCR experiments of auts2 transcripts. RFU, Relative fluorescence units. B. A representative agarose gel image of auts2 qPCR products. Predicted band sizes: FL-auts2: 165 bp; Total auts2: 107 bp; GAPDH: 183 bp. (PNG 57 kb)

High resolution image (TIF 2078 kb)

Supplementary Figure 2.

The expression of c-fos after various durations of KCl stimulation. A. Level of c-fos transcripts in cultured neurons, with or without KCl incubation of 2 h, 6 h and 12 h. Each symbol represents one biological replicate (n=7). ***p<0.001, *p<0.05. The p-values were determined by two-way ANOVA and Sidak’s multiple comparisons test. B. The c-fos mRNA level with or without KCl incubation for the indicated durations. C-E. Level of c-fos proteins in cultured neurons, with or without KCl incubation for 2 h (C), 6 h (D) and 12 h (E), detected by Wes Assay. Upper panels: Example images of Wes Assay. Lower panels: Quantitative analysis. F. Level of c-fos proteins with or without KCl incubation for the indicated durations. Data are represented as mean ± SD. Each symbol represents one biological replicate (n=5). ***p<0.001, *p<0.05. The p-values were determined by unpaired student’s t test. (PNG 161 kb)

High resolution image (TIF 2778 kb)

Supplementary Figure 3.

The level of Auts2 transcripts in the hippocampus and cortex after stimulation in vivo. A-C. Level of c-fos (A), FL-auts2 (B) and total auts2 (C) transcripts from the hippocampi (HP) of EE mice and SH mice. Each symbol represents one biological replicate (n=4 or 3). **p<0.01. The p-values were determined by unpaired student’s t test. D-F. Level of c-fos (D), FL-auts2 (E) and total auts2 (F) transcripts from the cortices (Cx) of EE mice and SH mice. One of the SH points was normalized to 1. Data are represented as mean ± SD. Each symbol represents one biological replicate (n=4 or 3). *p<0.05. The p-values were determined by unpaired student’s t test. (PNG 57.8 kb)

High resolution image (TIF 2874 kb)

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Pang, W., Wang, M., Bi, Q. et al. Activity-Dependent Differential Regulation of Auts2 Isoforms In Vitro and In Vivo. Mol Neurobiol 60, 2973–2985 (2023). https://doi.org/10.1007/s12035-023-03241-x

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