Transcriptional regulatory network during axonal regeneration of dorsal root ganglion neurons: laser-capture microdissection and deep sequencing

Li-Li Zhao; Tao Zhang; Wei-Xiao Huang; Ting-Ting Guo; Xiao-Song Gu

doi:10.4103/1673-5374.366494

Transcriptional regulatory network during axonal regeneration of dorsal root ganglion neurons: laser-capture microdissection and deep sequencing

Neural Regen Res. 2023 Sep;18(9):2056-2066. doi: 10.4103/1673-5374.366494.

Authors

Li-Li Zhao¹, Tao Zhang², Wei-Xiao Huang², Ting-Ting Guo³, Xiao-Song Gu¹

Affiliations

¹ Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing; Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China.
² Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong; School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, China.
³ Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China.

Abstract

The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified. In high-throughput sequencing, various factors influence the final sequencing results, including the number and size of cells, the depth of sequencing, and the method of cell separation. There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon. In this study, we performed laser-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0, 3, 6, and 12 hours and 1, 3, and 7 days after sciatic nerve crush in rats. We identified three stages after dorsal root ganglion injury: early (3-12 hours), pre-regeneration (1 day), and regeneration (3-7 days). Gene expression patterns and related function enrichment results showed that one module of genes was highly related to axonal regeneration. We verified the up-regulation of activating transcription factor 3 (Atf3), Kruppel like factor 6 (Klf6), AT-rich interaction domain 5A (Arid5a), CAMP responsive element modulator (Crem), and FOS like 1, AP-1 transcription factor Subunit (Fosl1) in dorsal root ganglion neurons after injury. Suppressing these transcription factors (Crem, Arid5a, Fosl1 and Klf6) reduced axonal regrowth in vitro. As the hub transcription factor, Atf3 showed higher expression and activity at the pre-regeneration and regeneration stages. G protein-coupled estrogen receptor 1 (Gper1), interleukin 12a (Il12a), estrogen receptor 1 (ESR1), and interleukin 6 (IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage. Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury. These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.

Keywords: Arid5a; Atf3; Crem; Fosl1; Klf6; dorsal root ganglion; gene expression profile; laser-capture microdissection; neuron; smart-seq2; transcription factor.