Approximately 1/1000 people develop multiple sclerosis (MS), but even among people that express the MS- associated MHC II allele HLA-DR2 we cannot predict who will develop disease and only 1/200 HLA-DR2+ people ever will. Surprisingly, even in studies of the murine experimental autoimmune encephalomyelitis (EAE) model of MS, where active or passive disease is induced under well-defined conditions, not all of the genetically-identical mice develop disease with the same trajectory or severity and we cannot accurately predict who will develop disease. In our own preliminary studies we have identified several protein biomarker candidates that correlate with onset, peak, and remission of EAE using a novel quantitative proteomics method that we recently reported (Raphael, I. Electrophoresis, 2012). Furthermore, we have developed a method to induce EAE of predetermined severity using adoptive transfer EAE in combination with pre-transfer cytokine ELISPOT analysis. Importantly, we have preliminary results that predicted candidate protein biomarkers can be detected in serum of mice with EAE by ELISA. By combining these approaches for inducing EAE and correlating protein expression to disease progression we will provide proof-of-principle in a unique system to identifying predictive markers of disease incidence and severity. The objective of this proposal is to provide proof-of-principle of predictive biomarkers using the EAE model. Our central hypothesis is that the clinical onset of EAE symptoms is preceded by release of CNS disease- specific proteins into blood (serum) that can be used to predict which animal will develop disease and how severe. This hypothesis is based on our own preliminary results and supported by published studies. The rationale for the proposed research is that proof-of-principle in the EAE model will provide the basis for developing homologous predictive biomarkers for MS patients, which will revolutionize treatment and drug development for MS. We will test our central hypothesis with the following specific aims: Aim 1. To determine key CNS disease-related protein isoforms with altered expression in CNS tissue prior to the onset of EAE and as a function of disease severity. Aim 2. To determine the CNS disease-related proteins isoforms released into blood (serum) most predictive of EAE incidence and severity. We expect that the proposed studies will provide important information that is currently not available and that will have significant and lasting impact on research and treatment of MS.