Figure 6
Bmal1-/- mediated changes in the SCI transcriptome suggest attenuated disruption of the BSCB and reduced neuroinflammation. RNA from dpi 3 contused spinal cord tissue of WT (M:F = 2:2, including 1 KO littermate) and Bmal1−/− (M:F = 1:2) mice was analyzed by RNASeq (a-g) or qPCR (h). (a) Gene count of Bmal1−/− effects on the transcriptome (see Supplementary Table S1 for details). (b, c) Gene ontology (GO) analysis of the highly affected genes in Bmal1−/− spinal cords. Analysis was run for all genes (b) or separately for upregulated and downregulated genes (c). Note the significant enrichment of GO:Biological Process (BP) terms associated with cell proliferation, inflammation and angiogenesis in both analyses. (d) Cell type-enriched transcripts in Bmal1−/− regulated SCI transcriptome. Note the relatively higher representation of astrocyte (ASTR) and vascular cell transcripts (EC + PC) and the predominantly gene downregulation trend for PC-, EC- and macrophage/microglia (MM)-enriched transcripts. Cell type-enriched gene lists are defined in the “Materials and methods”. (e) Significant enrichment of GO terms in downregulated MM- or PC/EC-enriched genes. Note the presence of neuroinflammation or ECM/cell proliferation-associated terms in MM or PC/EC genes, respectively. Cellular component (CC) and molecular function (MF) GOs are also included. No significant GO enrichment was observed for KO-regulated astrocyte genes. (f) Brain EC genes that are upregulated during BBB/BSCB disruption44 are highly represented among Bmal1−/− downregulated genes. Overlapping genes overrepresent GOs that are associated with BBB/BSCB disruption responses such as synthesis of ECM components and vascular cell proliferation. (g) Heat map of Bmal1−/− downregulated components of the BBB/BSCB disruption module. Note that several of those genes appear to be regulated in a circadian fashion, as shown by the number of experiments detecting their significant circadian oscillations in the CircaDB database (# of CircaDB hits). (h) qPCR analysis of selected vascular cell genes that are downregulated in Bmal1−/− spinal cords. Note the downregulation of genes that likely promote BBB/BSCB failure (Ccl2, Ptgs2, Adamts8) and potentially pro-hemostatic downregulation of Aplnr, Nos3, and Ptgis. Slco2a1 is an arachidonic acid transporter that supports activities of Ptgs2 and Ptgis. Data are means ± SD (*p < .05, u-test).
