The role of extracellular vesicles in septic encephalopathy metabolism and immunity - Project Summary/Abstract: Septic encephalopathy (SE) constitutes a common form of encephalopathy with an associated 70% mortality rate, underscoring the need for a highly effective treatment. The pathophysiology of SE is linked to an energetic crisis in the brain combined with immune-mediated neuro-inflammation. Specifically, neural cells shift their energy generation from aerobic oxidative phosphorylation (OXPHOS) to less- efficient glycolysis. Energy deficiency in the brain leads to excess accumulation of reactive oxygen species (ROS) and subsequently to oxidative stress, inflammation, and neuronal cell death. Studies have highlighted a mesenchymal stem cell (MSC)-related improvement in energetics that is partially achieved by the transfer of bioactive molecules such as miRNAs and proteins carried by secreted extracellular vesicles (MSC-EVs). In our preliminary work using a murine model of SE, we showed that MSC-EVs administered intravenously restore cerebellar ATP and improve neuro-inflammation and Purkinje and granule cell viability. However, the mechanisms by which MSC-EV cargo exerts its protective effects on neuronal integrity in SE have not been studied. The goal of this application is to examine the therapeutic potential of MSC-EVs in SE using a mechanistic approach to connect EV cargo with the regulation of metabolic and immune pathways. By including a murine model of SE, we propose to examine whether specific members of the MSC-EV cargo (miRNAs and proteins) alter metabolic and immune pathways, leading to improved neuronal cell viability. We hypothesize that in SE, based on our previous work, MSC-EV cargo directly activates cerebellar OXPHOS-related and other metabolic pathways, improving ATP generation, neuro-inflammation, and neuronal cell death. We will study in vivo the MSC-EV mechanism of action by using inhibitors of MSC-EV miRNA and protein cargo members that are abundant in the utilized EVs. The practical implication of this effort includes the development of EV-based therapeutic protocols to alleviate the metabolic and immune abnormalities that can broadly affect neural cells in encephalopathy and other conditions that cause brain injury. The PI is firmly committed to a career in translational neuroscience research and his goals are strongly supported by his institution and the co-Is. He is currently a tenure-track Assistant Professor of Pediatrics with 75% protected time for research and recently funded by K12/STTR grants, and Children’s National Hospital has been providing him with bench and office space, and computer/software support. The current proposal includes a comprehensive research plan that will advance our knowledge in EV therapeutic properties using techniques such as liquid chromatography and mass spectrometry, ATP measurement using Seahorse© technology, RNA/miRNA sequencing and advanced cerebellar histopathology. This work will lead to an R01-level grant focusing on SE metabolism and EV therapeutics using MSC-EVs and more efficient engineered EVs.
