Project Summary/Abstract Traumatic brain injury (TBI) is a major cause of death and disability worldwide. There are no effective therapies available for TBI patients. Thus, there is a compelling need to develop novel therapeutics in order to improve neurological recovery after TBI. Mesenchymal stem cells (MSCs) are adult multipotent cells that give rise to various mesodermal cell types. The use of MSCs for tissue repair is of great interest because of their ability to home to damaged and inflammatory tissues. However, previous studies from us and others show that only a small proportion of transplanted MSCs actually survive and few MSCs differentiate into neural cells in injured brain tissues. The predominant mechanisms by which MSCs participate in brain remodeling and functional recovery are related to their secretion-based paracrine effect rather than a cell replacement effect. Our recent data suggest that posttraumatic treatment with cell-free exosomes isolated from rat and human MSCs improves functional recovery in male rats after TBI. Exosomes play an important role in intercellular communication. Exosomes transfer not only proteins and lipids but also genetic materials including mRNAs and microRNAs (miRNAs) to recipient cells, thereby mediating a variety of biological responses. Our preliminary data further demonstrate that the labeled exosomes administered intravenously after TBI reach the brain and are incorporated into brain cells as well as in macrophages in peripheral organs. Our encouraging findings indicate that MSC-derived exosomes have equivalent restorative effects as their cellular counterparts on brain remodeling and functional recovery after TBI. Thus, MSC-generated exosomes are novel candidates as a cell- free therapy that can overcome the obstacles and risks associated with the use of naive or engineered stem cells or MSCs. While our results are promising, the precise therapeutic mechanisms underlying exosome therapy for TBI recovery warrant further elucidation. In this proposal, we will first determine therapeutic efficacy of nave MSC-exosomes for improvement in functional recovery in male and female rats after TBI. We will then evaluate the effect of MSC-exosomes on brain neuroplasticity, and growth factor expression as well as on the brain and peripheral immune response, effects that likely underlie and contribute to functional recovery (Aim 1). We will then evaluate the role of the miRNA content of the MSC-derived exosomes on brain angiogenesis, neurogenesis, synaptogenesis, cell death, growth factors and immune responses underlying functional recovery (Aim 2). Finally, we propose to enhance the therapeutic effects of exosome treatment of TBI by generating and employing tailored MSC-derived exosomes enriched with the miR-17-92 cluster as a treatment for TBI. In addition, we will investigate the molecular mechanisms underlying cellular exosome uptake (Aim 3). This proposal is innovative, and highly translational. This study will provide novel insights into mechanisms underlying the MSC-derived exosome-promotion of functional recovery after TBI, develop a means to amplify the therapeutic effects of exosome therapy for TBI, and form the foundation for clinical translation of exosome therapy for TBI.