PROJECT SUMMARY The exact mechanism underlying the onset of Multiple Sclerosis (MS), a disease that affects over 2 million people worldwide and ~ 400,000 in the United States, is unknown although most experts in the field agree that MS involves an abnormal immune-mediated response against the body’s central nervous system (CNS). Specifically, in the CNS, components of the immune system attack myelin, the protein-based substance that surrounds nerve fiber. This attack on myelin results in multiple scar lesions (hence, Multiple Sclerosis) that lead to disease symptoms. Mounting evidence suggests that a breakdown in the blood brain barrier (BBB) enables the permeability of toxins and other foreign pathogens into the CNS that attack the myelin. Here, an investigative team with basic and clinical science experience proposes that hypermetabolic red blood cells (RBCs) in the MS circulation are contributing to increased glucose utilization in the brain and subsequent BBB breakdown. Specifically, during our previous project period, the investigative team discovered and recently published several interesting findings involving the MS RBC. First, the MS RBC has a significantly increased amount of the GLUT1 glucose transporter in its membrane. The GLUT1 transporter is the dominant glucose transporter in the RBC (and most circulatory cells) and is not affected by insulin. Secondly, we reported that C-peptide, the 31-amino acid peptide that is co-secreted in equal molecule amounts with insulin from the pancreatic -cells, binds to the MS RBC at a level nearly 50% higher than control RBCs and RBCs obtained from people with other neurological diseases (ONDs, as rigorous controls). Finally, we reported an increase in ATP release from the MS RBC upon stimulation, indicating increased glycolysis. Collectively, our data and other recent data in the literature from other groups, suggest MS RBCs are hypermetabolic. The link to the compromised BBB occurs when considering recent impactful work in the literature that supports decades-old reports of hypermetabolic RBCs in people with MS. The increased metabolism results in a significant increase in lactic acid (lactate) production in the MS circulation, which may compromise BBB integrity through localized changes in pH. During the renewal project period, the investigative team plans to deepen our understanding of MS by further developing novel tools to study the MS RBC at the single cell level, as well as in humans, with novel in vivo imaging strategies. We will also better power our previous studies by increasing the number of MS patients at the different stages of MS. These aims will inform an in vivo PET/MRI clinical study in years 4-5 designed to determine glucose utilization before and after normal dosing of interferon-beta (IFN-, a common MS disease modifying therapy), thereby providing impactful information on the mechanism of MS disease onset.