Significant phenotypic differences exist between the vascular endothelium of different organs, including cell-cell junctions, paracellular fluid transport, shape, and mural cell coverage. These organ-specific morphological features ultimately manifest as different functional capacities, as demonstrated by the dramatic differences in capillary permeability between the leaky vessels of the liver compared to the almost impermeable vasculature found in the brain. While these morphological and functional differences have been long appreciated, the molecular basis of endothelial organ specialization remains unclear. To determine the epigenetic and transcriptional mechanisms driving this functional heterogeneity, we profiled accessible chromatin, as well as gene expression, in six different organs, across three distinct time points, during murine development and in adulthood. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we then focused our studies on the endothelium of the central nervous system. Using single cell RNA-seq, we identified key gene regulatory networks governing brain blood vessel maturation, including TCF/LEF and FOX transcription factors. Critically, these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. Collectively, this work provides a valuable resource for identifying the transcriptional regulators controlling organ-specific endothelial specialization and provides novel insight into the gene regulatory networks governing the maturation and maintenance of the cerebrovasculature.
Microglia are key mediators of inflammatory responses within the brain, as they regulate pro-inflammatory responses while also limiting neuroinflammation via reparative phagocytosis. Thus, identifying genes that modulate microglial function may reveal novel therapeutic interventions for promoting better outcomes in diseases featuring extensive inflammation, such as stroke. To facilitate identification of potential mediators of inflammation, we performed single-cell RNA sequencing of aged mouse brains following stroke and found that Ifi27l2a was significantly up-regulated, particularly in microglia. The increased Ifi27l2a expression was further validated in microglial culture, stroke models with microglial depletion, and human autopsy samples. Ifi27l2a is known to be induced by interferons for viral host defense, however the role of Ifi27l2a in neurodegeneration is unknown. In vitro studies in cultured microglia demonstrated that Ifi27l2a overexpression causes neuroinflammation via reactive oxygen species. Interestingly, hemizygous deletion of Ifi27l2a significantly reduced gliosis in the thalamus following stroke, while also reducing neuroinflammation, indicating Ifi27l2a gene dosage is a critical mediator of neuroinflammation in ischemic stroke. Collectively, this study demonstrates that a novel gene, Ifi27l2a, regulates microglial function and neuroinflammation in the aged brain and following stroke. These findings suggest that Ifi27l2a may be a novel target for conferring cerebral protection post-stroke.
Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. However, how the transcriptional signature of these cells is altered in aging and stroke remains poorly defined. Here, we compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq). Our unbiased approach identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG with aging or ischemic stroke. Further analyses suggested that this gene may be a novel regulator of MG inflammation in aging and stroke. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke and sham controls. We used qRT-PCR to validate our scRNAseq findings and compare regional transcriptional changes. Human microglial cells (HMC3; subjected to OGD) and human autopsy brain sections (stroke versus non-stroke) were used to examine IFI27l2 protein expression. Results: Using comprehensive scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes. In contrast, a negative correlation was shown with genes involved in promoting the phagocytic MG phenotype (e.g. Spp1, Cst7, Lpl, and Itgax). Our qRT-PCR results validated the central scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in thalamus of aged brains (p = 0.0412) and trending in cortex (p = 0.23) (n=4 young/aged). Following stroke, Ifi27l2a and other markers of MG activation were significantly increased in both brain regions (n= 4-6). In human tissues, we observed an increase in IFI27l2 protein in HMC3 cells with OGD and in brain of stroke patients. Conclusions: We identified Ifi272la as a highly up-regulated gene in MG in aging and following stroke. Our findings suggest that Ifi27l2a may be a novel regulator of neuroinflammation in MG, and thus provide the basis for a new therapeutic target to reduce inflammation in aging and stroke.
Interferon signaling drives neuroinflammation in AD and stroke. Microglia (MG) play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. We compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq) to identify novel key players in inflammation. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice (3 & 20 months). scRNAseq was performed to define the transcriptional signature in brains at 14-days after stroke versus sham surgery. Primary MG culture was used to compare the mRNA levels of Ifi27l2a and Ifitm3 after cytokine treatment. We used qRT-PCR to validate scRNAseq findings and compare specific MG genes. Results: Using comprehensive scRNAseq analysis, our unbiased approach identified Ifi27l2a and Ifitm3 as the most highly up-regulated genes in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and Ifitm3 in MG ( R =0.4, p<0.001) as well as in all cells. Our qRT-PCR results validated scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in the thalamus of aged brains (p=0.04; n=4 young/aged). Expression was increased in cortex, but below significance level (p=0.23). We also found upregulation of Ifitm3 in brain at PSD 3 ( p =0.01), and trending at PSD 14 ( p =0.08) (n=5-6 stroke vs. sham). In contrast, Ifitm1 and Ifitm2 were not changed. Interestingly, Ifitm3 was recently proposed as an activator to γ-secretase. In vitro exposure of MG to inflammatory cytokines (TNFα and IFNγ, 6 hrs) caused significant increase in Ifi27l2a ( p =0.03, n=5-6) and Ifitm3 mRNA (p=0.02, n=5-6) compared to untreated MG. The in vitro co-expression of these genes was also well correlated ( R =0.5). Conclusions: We identified Ifi27l2a and Ifitm3 as highly up-regulated and correlated genes in MG in aging and following stroke. Our findings suggest that co-expression of Ifi27l2a and Ifitm3 may contribute to neuroinflammation. Further studies will be required to identify the specific functions of these genes and to determine the therapeutic potential of targeting these proteins to regulate neuroinflammation in aging and stroke.
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