The Centers for Disease Control estimate that approximately 1.7 million people per year sustain a traumatic brain injury (TBI), resulting in an estimated 52,000 deaths. The age groups most likely to sustain a TBI are infants (0-4 years old), adolescents (15-19 years old), and the elderly (65 and older). TBI results in the alteration of many signaling pathways and biological processes. These processes contribute to a variety of deleterious pathophysiological sequelae which significantly affect patient morbidity and mortality, including cerebrovascular dysfunctions such as ischemia and increased susceptibility to secondary ischemia, cerebrovascular edema, and increased blood-brain barrier (BBB) permeability. MicroRNAs (miRNAs) are ubiquitous regulatory RNAs that modulate gene expression at the post-transcriptional level by inhibiting protein translation and/or promoting mRNA degradation. MiRNAs play critical roles in regulating many important biological processes, including vascular integrity. We have shown that miRNA expression levels are altered after a controlled cortical impact (CCI) model of TBI, and these altered miRNAs are predicted to regulate the expression of gene products involved in many pathophysiological processes affected by TBI. MiR-223 expression levels in cerebral microvessels are significantly increased 24-72 hr post-TBI, a time period when peak BBB permeability and cerebral edema is observed. Furthermore, preliminary evidence shows that miR-223 directly targets T-lymphoma invasion and metastasis (Tiam1), a central regulator of actin dynamics that impacts endothelial barrier integrity and adherens and tight junction function. We propose to test the hypothesis that increased miR-223 expression in brain microvascular endothelial cells contributes to BBB dysfunction, and blocking miR-223 activity after TBI will improve BBB integrity. The experiments in this grant proposal employ a combination of molecular biology and biochemical approaches using genetic knockout, in vitro, and in vivo model systems to (i) determine the impact of altered miR-223 expression on tight junction protein expression, localization, and barrier function in primary rat brain microvascular endothelial cells, and (ii) evaluate the impact of miR-223 knockout on BBB compromise after TBI. Examining the functional consequences of altered miRNA expression in complex systems is hampered by a lack of methodologies for acutely manipulating miRNAs in vivo. We propose to utilize the rabies virus glycoprotein (RVG) peptide, which selectively transfect cells that express the nicotinic acetylcholine receptor, including vascular endothelial cells, as a novel targeting strategy to transiently manipulate miR-223 in the brain vasculature in vivo. These studies will contribute to our understanding of BBB regulation after CNS trauma, and help characterize a novel method for in vivo manipulation of miRNA levels. Furthermore, if successful these studies will enable future investigations into the effects of transient changes in miRNA expression on protein regulation in vivo, opening up a previously unexplored area of pathophysiological processes initiated after TBI.
MicroRNAs regulate the expression of 30-60% of mRNAs by inhibiting mRNA translation and/or promoting mRNA degradation, playing a significant role in the wide spread modulation of gene expression. It is currently unclear what role miRNAs play in TBI-associated vascular dysfunctions, such as increased blood-brain permeability, that are initiated after injury. The experiments proposed in this grant application will explore the role of miR-223 in regulating vascular endothelial barrier function, and may lead to the identification of novel targets for intervention.