Isolation of corpus callosum from juvenile and young adult mice and the assessmentThe diagram in Fig. 1a summarizes the experimental approach in this study, i.e., expression levels of mRNA in the corpus callosum of juvenile (2 week-old) and young adult (12 week-old) mice were compared with RNA-seq experiments (Fig. 1a). PCA analysis revealed that these samples were clustered into two groups (Fig. 1b). To validate the successful isolation of the corpus callosum, we assessed the gene expression levels of specific markers for oligodendrocytes (Mbp and Mobp) and cortical neurons (Reln, Rasfrf2, Pou3f2, and Foxp2) and confirmed that the sample was white matter tissue based on the high expression of oligodendrocyte markers (Fig. 1c). Additionally, we examined the expression of markers for astrocytes (Gfap and Aldh1l1), microglia (Aif1, Cd68 and Itgam), and OPCs (Pdgfra, Cspg4, and Sox10) to confirm the presence of various cell types in our samples (Supplementary Fig. S1).Fig. 1Corpus callosum sampling and RNA-seq analysis from juvenile to adult: (a) Schematic representation illustrating the collection of the corpus callosum (CC) in 2 week-old and 12 week-old mice. The isolated RNA was analyzed using RNA-seq. (b) Principal Component Analysis (PCA) plot of the samples. (c) Violin plots representing the expression of oligodendrocyte markers (Mbp and Mobp) and cortical neuron markers (Reln for layer I, Rasgrf2 for layer II/III, Pou3f2 for layer II-V, and Foxp2 for layer IV).Transcriptome profiling of corpus callosum at different agesWe next explored the differentially expressed genes (DEGs), visualized in volcano plots (2 week vs. 12 week), and found that 439 genes were upregulated and 620 genes were downregulated in 12 week-old mice (a total of 1059 genes; Fig. 2a). The top 10 up-regulated genes by padj were Serpinb1a, Gm21984, C030029H02Rik, Adamtsl4, Il33, Neat1, Ndrg1, Cbx7, Efhd1, and Qdpr, while the top 10 down-regulated genes by padj were Gng4, Col4a1, Dnmt3a, Bdh1, Apcdd1, Cd93, Nrep, Nid1, Emid1, and Marcksl1 (Fig. 2b,c, Supplementary Tables S1, S2).Fig. 2Transcriptome profiling of the corpus callosum from juvenile to adult: (a) Volcano plot illustrating DEGs (a total of 1564 genes) between 2-week-old (n = 4) and 12 week-old (n = 3) mice. The DEGs cutoff was set at padj < 0.01 and | Fold Change (FC) |> 2 (|log2FC|> 1). (b) Bar plot of the top 10 genes upregulated and downregulated in 12 week-old WT mice compared to 2 week-old WT mice (by padj). (c) Violin plot of the top 10 genes upregulated and downregulated in 12 week-old WT mice compared to 2-week-old WT mice (by padj). (d) Classification of the top 10 gene lists (Gm21984, C030029H02Rik, and Apccd1 were not registered in this database) using public single-cell RNA-seq data21. For abbreviations in this figure, see Supplementary Figure S4. (e) Top 10 GO terms enriched among all DEGs in 2-week-old vs. 12-week-old. Upregulated genes (Up); red, Downregulated genes (Down); blue.To further investigate the cell types of these highly up- or down-regulated genes, we used publicly available single-cell RNA-seq data from adult mouse brains (GSE129788)21. Among the upregulated genes at 12 weeks of age, we verified that Serpinb1a, Adamtsl4, Il3, Efhd1, and Qdpr are predominantly expressed in oligodendrocyte cells, and Ndrg1 and Neat1 are in oligodendrocyte and vascular cells (ex. vascular smooth muscle cells [VSMC], endothelial cells [EC], and pericytes [PC]) (Fig. 2d). On the other hand, among the downregulated genes, Emid1 is predominantly expressed in OPC, Nid1, Cbx7, Cd93, and Col4a1 are in vascular cells (ex. VSMC, EC, and PC), Nrep is in immature neurons, and Gng4 are in mature neurons (Fig. 2d).Gene ontology (GO) analysis of the DEGs further revealed significant enrichment in various biological processes and pathways. Specifically, upregulated genes were significantly enriched in processes such as actin filament-based process, actin cytoskeleton organization, reversible hydration of carbon dioxide, actin filament organization, nitrogen metabolism in Mus musculus (house mouse), supramolecular fiber organization, regulation of interleukin-4 production, positive regulation of transcription in response to endoplasmic reticulum stress, protein refolding, and positive regulation of inflammatory response (Fig. 2e, Supplementary Table S3). Downregulated genes were predominantly involved in extracellular matrix organization, external encapsulating structure organization, extracellular structure organization, extracellular matrix organization, ECM-receptor interaction in mus musculus (house mouse), mitotic cell cycle, mitotic cell cycle process, collagen formation, focal adhesion in mus musculus, and supramolecular fiber organization (Fig. 2e, Supplementary Table S3).When analyzing genes with notable expression in central nervous system cells (neuron, astrocyte, microglia, OPCs, newly formed oligodendrocytes, and myelinating oligodendrocytes) based on public RNA-seq datasets22,23, the majority of upregulated DEGs were derived from myelinating oligodendrocytes, while the majority of downregulated DEGs were derived from OPCs and newly formed oligodendrocytes in 2 week vs. 12 week (Fig. 3a). Our analysis showed that among the genes with increased expression, 95 genes were abundantly expressed in myelinating oligodendrocytes (Fig. 3a, Supplementary Table S4). Additionally, among the genes with decreased expression, 56 genes were most abundantly expressed in OPCs, and 68 genes in newly formed oligodendrocytes (Supplementary Table S4). To elucidate the biological processes enriched in these specific cell populations from juvenile to adult stages, these subsets of genes were extracted and subjected to further GO analysis. The GO analysis revealed that for myelinating oligodendrocytes, enriched biological processes included linoleic acid metabolic process, small molecule biosynthetic process, aspartate family amino acid metabolic process, carboxylic acid biosynthetic process, organic acid biosynthetic process, positive regulation of nervous system process, actomyosin structure organization, actin cytoskeleton organization, unsaturated fatty acid metabolic process, and regulation of canonical NF-kappaB signal transduction (Fig. 3b, Supplementary Table S5). Among the genes downregulated in OPCs, processes such as cell division, mitotic cell cycle process, mitotic cell cycle, mitotic nuclear division, collagen degradation, mitotic sister chromatid segregation, sister chromatid segregation, nuclear division, nuclear chromosome segregation, collagen chain trimerization were prominent (Fig. 3c, Supplementary Table S6). In newly formed oligodendrocytes, neuron projection morphogenesis, myelination, plasma membrane bounded cell projection morphogenesis, ensheathment of neurons, axon ensheathment, cell projection morphogenesis, ECM-receptor interaction—mus musculus (house mouse), semaphorin-plexin signaling pathway, axon guidance, and neuron projection guidance were among the top enriched processes (Fig. 3d, Supplementary Table S7).Fig. 3Gene ontology analysis of myelinating oligodendrocyte-, OPC-, newly formed oligodendrocyte-enriched genes in the corpus callosum from juvenile to adult: (a) Classification of DEGs in 2 week vs. 12 week using cellular markers from previously published RNA-seq data22,23. (b) Top GO terms enriched among the some upregulated DEGs in 2 week-old vs. 12 week-old, which predominantly expressed in myelinating oligodendrocytes. (c) Top GO terms enriched among the some downregulated DEGs in 2 week-old vs. 12 week-old, which predominantly expressed in OPCs. (d) Top GO terms enriched among the some downregulated DEGs in 2 week-old vs. 12 week-old, which predominantly expressed in newly formed oligodendrocytes. Cell type-specific gene lists were based on enriched genes from purified cell populations22,23.In addition, we performed GO analysis on the downregulated genes that were abundantly expressed in neurons and astrocytes. Among the downregulated genes, 39 were highly expressed in neurons and another 39 in astrocytes (Fig. 3a). The GO analysis of these neuron-associated genes revealed enrichment in the following processes: regulation of synaptic transmission (glutamatergic), neuron projection development, positive regulation of synaptic transmission (glutamatergic), positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction, glutamatergic synapse, axonogenesis, regulation of neuron projection development, axon guidance, regulation of nervous system development, and neuron projection guidance (Supplementary Figure S2a). For the astrocyte-associated genes, the top enriched GO terms included: extracellular matrix organization, regulation of neural precursor cell proliferation, ECM-receptor interaction, regulation of cell-substrate adhesion, positive regulation of cell development, ECM proteoglycans, negative regulation of cell-substrate adhesion, regulation of neuroblast proliferation, positive regulation of neurogenesis, and positive regulation of neural precursor cell proliferation (Supplementary Figure S2b).When we performed Gene Set Enrichment Analysis (GSEA) using a gene list ranked by fold change, we found that many functions related to the vascular system, such as circulatory system process, endothelial cell proliferation, vascular transport, endothelial cell migration, positive regulation of vasculature development, negative regulation of vasculature development, sprouting angiogenesis, regulation of vasculature development, vascular process in circulatory system, aorta development, artery development, and artery morphogenesis, were enriched among the genes that decreased from 2 to 12 weeks of age (Supplementary Table S8, Supplementary Fig. S3).Comparative analysis of corpus callosum DEGs from juvenile to adult and adult to aged miceWe also compared these DEGs (i.e., 2-between 2 week-old vs. 12 week-old) with our previous data sets of 20-week-old and 96-week-old mice (PRJNA1011381). The Venn diagram revealed that 13 genes are shared between these two comparisons (Fig. 4a–d, Supplementary Fig. S4). Among the common genes, 10 genes (Spp1, Etnppl, C4b, Lyz2, Mki67, Tnc, Chst3, Gpr17, Kif19a, and Marcksl1) showed consistent trends of either up or down-regulation from 2- to 12 week-old and from 20-to 96 week-old (Fig. 4a–d). Furthermore, 1046 genes are unique in the 2 week vs. 12 week comparison and 46 genes are unique in 20 week and 96 week comparison (Fig. 4a). For example, among the top 10 DEGs of 2 week vs. 12 week mice (10 upregulated and 10 downregulated in 12-wek mice, a total of 20 DEGs) (Fig. 2b,c), 19 genes (Serpinb1a, Gm21984, C030029H02Rik, Adamtsl4, Il33, Neat1, Ndrg1, Cbx7, Efhd1, Qdpr, Gng4, Col4a1, Dnmt3a, Bdh1, Apcdd1, Cd93, Nrep, Nid1, and Emid1), excluding Marcksl1, were identified as DEGs unique to the 2- and 12 week comparison (Supplementary Fig. S5a). Conversely, genes such as Gfap, Col11a1, Cd68, B2m, Fcgr3, Podhb14, Cd84, and Mpeg1 were identified as DEGs unique to the 20-week vs. 96-week comparison (Supplementary Fig. S5b). Furthermore, to validate our RNA-seq findings, we performed reverse transcription quantitative PCR (RT-qPCR) on selected genes potentially involved in the development, aging processes, and pathology, which also show moderate to high expression in oligodendrocyte lineage cells. We focused on Marcksl124, Chst325, and C4b15,16, which were identified as DEGs common to both the 2 week vs 12 week and 20 week vs 96 week comparisons, and the RT-qPCR results for these genes showed consistent expression patterns with our RNA-seq data (Fig. 5a). Additionally, we examined Neat126, Ndrg127, and Emid128,29, which were identified as DEGs specific to the 2 week vs 12 week comparison, and the RT-qPCR results for these genes also demonstrated consistency with our RNA-seq data (Fig. 5b). These validation experiments support the reliability of our RNA-seq data and the biological significance of the identified DEGs.Fig. 4Comparative analysis of corpus callosum DEGs from juvenile to adult and adult to aged mice: (a) Venn diagram showing the overlapping DEGs between the comparisons of 2 week-old vs. 12 week-old and 20 week-old vs. 96 week-old. (b) Heatmap displaying the expression profiles of genes common between the DEGs of 2 week-old vs. 12 week-old and 20 week-old vs. 96 week-old, using data from 2 week-old and 12 week-old mice. (c) Heatmap displaying the expression profiles of genes common between the DEGs of 2 week-old vs. 12 week-old and 20 week-old vs. 96 week-old, using data from 20 week-old and 96 week-old mice. (d) Bar plot (with dot plot) of the common genes, using data from 2 week-old (2wk), 12 week-old (12wk), 20 week-old (20wk), and 96 week-old (96wk) mice. Error bars indicate SEM.Fig. 5In vivo validation of RNA-seq findings by RT-qPCR: (a) Expression levels of Marcksl1, Chst3, and C4b in 2 week-old (2wk) and 12 week-old (12wk) mouse corpus callosum. (b) Expression levels of Neat1, Ndrg1, and Emid1 in 2 week-old (2wk) and 12 week-old (12wk) mouse corpus callosum. Gene expression was analyzed by RT-qPCR and normalized to Hprt as an internal control. Statistical analysis was performed using two-tailed Student’s t-test.
