TRANSCRIPTOME ANALYSIS OF DIRECT ASTROCYTE-TO-NEURON CONVERSION
Open Access
- Author:
- Ma, Ningxin
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 13, 2019
- Committee Members:
- Gong Chen, Dissertation Advisor/Co-Advisor
Gong Chen, Committee Chair/Co-Chair
Yingwei Mao, Committee Member
Shaun Mahony, Committee Member
Qunhua Li, Outside Member - Keywords:
- reprogramming
astrocyte
neuron
RNA sequencing - Abstract:
- Reprogramming of astrocytes into neurons represents a promising approach to regenerate new neurons for brain repair, but the underlying mechanisms driving this trans-differentiation process are not well understood. In previous work, we have demonstrated that astrocytes can be effectively reprogrammed into functional neurons in two ways. The ectopic expression of a single transcription factor NeuroD1 converted reactive astrocytes into neurons both in vitro and in vivo. Moreover, we recently identified four small molecules - CHIR99021, DAPT, LDN193189 and SB431542 - that could reach highly efficient neuronal conversion in cultured human fetal astrocytes. Here we employ the next generation of RNA-sequencing technology to investigate the transcriptome changes during the astrocyte-to-neuron (AtN) conversion process. The four small molecules together can rapidly activate the hedgehog signaling pathway while downregulating many glial genes such as FN1 and MYL9 within 24 hours of treatment. Chemical reprogramming is mediated by several waves of differential gene expression, including upregulation of hedgehog, Wnt/β- catenin, and Notch signaling pathways, together with downregulation of TGF-β and JAK/STAT signaling pathways. Co-expression gene network analyses identify functional gene modules that may directly respond to chemical treatment. In addition, we reveal many well-connected hub genes such as RGMA, neuronatin (NNAT), neurogenin 2 (NEUROG2), NPTX2, MOXD1, JAG1, and GAP43, which may coordinate the chemical reprogramming process. In comparison to chemical administration, use of virus triggers strong inflammation- related gene expression and impedes cell cycle. With a more than 100-fold increase by virus, NeuroD1 also directly upregulate its target genes, including neurotransmitter receptors, MAPK and cAMP signaling pathways. Moreover, network analyses suggest significant NeuroD1-correlated genes such as CABP7 and LRRTM2, and also indicate the extensive interactions between neurogenic genes (e.g. SOX13, NEUROD6) and glial genes (e.g. HHEX, PRDM1). Together, these findings provide critical insights into the molecular cascades triggered by either a combination of small molecules or a proneural transcription factor, and depict a trajectory that gradually reprogram astrocytes into neurons. Understanding the molecular mechanisms of cell fate determination during chemical or transcription factor-mediated reprogramming will be instrumental for further development of an efficient clinical application in the future.