In the past decade, restoring the intrinsic axon growth ability of mature neurons has received promising results in promoting axon regeneration in the central nervous system (CNS). However, to date, axon regeneration that leads to successful functional recovery in the CNS is still practically impossible, primarily due to the inadequate distance of regeneration and the low number of regenerating axons. Previous studies and my preliminary data have shown that many genes mediating the intrinsic axon growth ability are differentially expressed at different developmental stages in neurons, indicating the altered gene expression level during neuronal maturation is an important factor underlying the diminished intrinsic axon growth capacity. However, how the altered gene expression program is regulated remains largely unknown. Transcription factors (TFs) play important roles during neuronal development, shaping the spatiotemporal gene expression landscape to control cellular activities including axon elongation. Thus, understanding the intricate transcriptional regulatory network orchestrating axon growth during development is critical for solving the challenge of mammalian CNS axon regeneration. In this proposed study, I will perform parallel RNA-seq and ATAC-seq of purified retinal ganglion cells (RGCs) at multiple developmental time points, and use advanced integrative bioinformatics analysis to obtain a comprehensive view of the transcriptional regulatory network controlling the axon elongation function during RGC development, and identify key TFs that function as core regulators of axon growth. The identified TFs will be functionally tested in mouse optic nerve regeneration model to verify if they play important roles in RGC axon regeneration and cell survival. RGCs are comprised of more than forty molecular distinct subtypes. Different RGC subtypes vary in vulnerability to axonal injury and have distinct responses toward gene modulations. I will conduct single-cell RNA-seq (scRNA-seq) in RGCs 2 weeks after optic nerve crush from control and TF- manipulated groups to acquire the frequency of each RGC subtype in the final population, and determine what specific RGC subtypes are protected by the manipulation of a specific TF by comparing the frequencies of RGC subtypes between control and TF-manipulated groups. TFs whose manipulations are found to improve survival in distinct RGC subtypes will be combined in the next step to determine if simultaneously manipulating these TFs could protect a wide variety of RGC subtypes from injury-induced cell death and induce synergistic promoting effect on RGC axon regeneration. In addition, I will also combine the manipulations of these TFs with non-muscle myosin IIA/B deletion in RGCs, which produces axon regeneration by modifying cytoskeletal dynamics in the growth cone of injured axons, to find out if this combinatory approach could lead to unprecedented long-distance axon regeneration.
Understanding the complex gene regulatory network controlling axon growth is a critical step to meet the challenge of poor axon regeneration in the central nervous system. The proposed study will combine RNA-seq and ATAC-seq in purified retinal ganglion cells at various developmental stages and use advanced integrative multi-omics analysis to identify core transcription factors regulating axon growth during retinal ganglion cell development, and functionally test the identified transcription factors in optic nerve regeneration model. The study will not only provide a comprehensive view of the transcriptional regulatory network that governs neuronal axon growth, but also reveal novel therapeutic targets for glaucoma and other neural injuries and neurodegenerative diseases.
