Profiling microglia-specific transcriptional changes after ischemic stroke Kendra J. Lechtenberg1, Todd C. Peterson1, Li Zhu1 and Marion S. Buckwalter1 1Stanford University, Department of Neurology, Woodside, CA
Ischemic stroke is a leading cause of death and long term disability worldwide. Neuroinflammation after stroke has significant influence on stroke outcomes, because it is necessary for injury resolution and repair, but can also exacerbate cell death. Microglia, the brain's resident immune cells, are the major producers of inflammatory molecules in the hours following stroke but also have neuroprotective functions such as clearance of harmful debris and release of anti-inflammatory factors. However, the specific molecular mechanisms that regulate the progression of the microglial response to stroke remain poorly understood. This is in large part due to the challenge of dissecting the microglial response from that of other inflammatory cell types infiltrating the brain after stroke. To address this, we used the RiboTag technique to obtain transcripts specifically from microglia 3 days after stroke, which is the peak of the neuroinflammatory response. By crossing the RiboTag and Cx3cr1-CreER mouse models, we expressed a hemagglutinin tag on ribosomes specifically in microglia, allowing for immunoprecipitation of ribosomes followed by isolation of actively translating microglial mRNA. We verified by immunohistochemistry that this tag is expressed by 99% of microglia following recombination. Targeted gene expression analysis showed that transcripts obtained using this technique were highly enriched in microglia-specific genes such as Tmem119 and de-enriched in genes characteristic of other brain cell types, such as GFAP and Tubb3. We performed RNA-sequencing on microglia-specific transcripts obtained from male and female mice 3 days after distal middle cerebral artery occlusion or sham surgery, and found 583 genes were upregulated and 186 genes were downregulated between stroke and sham animals. Of note, many of the genes upregulated at this time point are involved in monocyte recruitment, suggesting microglia are particularly involved in attracting peripheral immune cells to the injury site at this time point. Our data indicates that the RiboTag technique can effectively profile the microglial translatome after stroke, and our ongoing research will comprehensively determine the microglia-specific molecular response at multiple time points after stroke.
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