Transcriptome Analyses Reveal Systematic Molecular Pathology After Optic Nerve Crush

Abstract

The function of glial cells in axonal regeneration after injury has been the subject of controversy in recent years. Thus, deeper insight into glial cells is urgently needed. Many studies on glial cells have elucidated the mechanisms of a certain gene or cell type in axon regeneration. However, studies that manipulate a single variable may overlook other changes. Here, we performed a series of comprehensive transcriptome analyses of the optic nerve head over a period of 90 days after optic nerve crush (ONC), showing systematic molecular changes in the optic nerve head (ONH). Furthermore, using weighted gene coexpression network analysis (WGCNA), we established gene module programs corresponding to various pathological events at different times post-ONC and found hub genes that may be potential therapeutic targets. In addition, we analyzed the changes in different glial cells based on their subtype markers. We revealed that the transition trend of different glial cells depended on the time course, which provides clues for modulating glial function in further research.

Keywords: WGCNA; immune response; microglia; optic nerve crush; transcriptome.

Figure 1

PCA and sample clustering revealed temporal changes following ONC. (A) PCA of 45 ONH samples. The time points following ONC are labeled with different colors. Uninjured control ONHs are labeled with circles. The injured ONHs are labeled with triangles. (B) Heatmap of unbiased hierarchical clustering of pairwise correlations among all 45 samples. The dark red in the heatmap represents Pearson's correlation coefficient of transcriptomes among pairs of samples in all combinations. The traits of all samples are labeled with different colors. The horizontal and vertical frames are symmetrical. All the uninjured control samples were clustered together. ONH, optic nerve head; PCA, principal component analysis; ONC, optic nerve crush.

Figure 2

ONC-induced temporal DEGs and the involved biological processes. (A) Venn plot of DEGs 1/3/7/21 d post-ONC. The temporal DEGs and overlap of the four DEGs are indicated by black arrows. (B) GO enrichment analysis for temporal DEGs showed the top 10 most enriched biological processes. GO, Gene Ontology; DEG, differentially expressed gene; ONC, optic nerve crush.

Figure 3

WGCNA of all 45 ONH samples identified gene modules. (A) With 5,323 time-course-related genes, a hierarchical cluster dendrogram of 45 ONH samples identified coexpression modules. Modules corresponding to the branches are labeled with different colors indicated by the color bands below the tree. Nine gene modules were generated after 0.25 threshold merging. (B) Cell type enrichment analyses for the 9 modules. The two most enriched cell type terms for each module are shown. ONH, optic nerve head.

Figure 4

Gene modules underlying biological and pathological events at different time points post-ONC. (A–I) GO terms associated with the M1–M9 modules are shown. The time-course expression curves of the 9 modules based on the average expression levels of the top 30 genes with the highest MM are shown. The gene symbols of crucial genes in each module are shown. GO, Gene Ontology; ONC, optic nerve crush.

Figure 5

Visualized network of each module. (A–D) Visualized networks based on the top 250 gene connections of the M2 (A), M4 (B), M5 (C), and M6 (D) modules. The top 5 hub genes based on degree in each module are shown.

Figure 6

Expression pattern of ONHs 90 d post-ONC. (A) Volcano plot of DEGs in ONHs 90 d post-ONC compared with uninjured control ONHs. Red points represent DEGs. (B) The changes in the average expression levels of the top 30 genes with the highest MM in ONHs 90 d post-ONC. (C,D). GO enrichment analyses of genes with downregulated (C) and upregulated (D) expression showed the top 10 enriched biological processes. DEG, differentially expressed gene; ONC, optic nerve crush; ONH, optic nerve head.

Figure 7

The changes in expression levels of subpopulations of astrocyte and microglial marker genes after crush injury. Overall, there was an obvious expression pattern difference between the subpopulation of astrocyte and microglial markers. (A,B) Genes expressed by A1- and A2-reactive astrocytes. (C,D) Genes expressed by M1 and M2 microglia, respectively.