594 Tracking newly regenerated oligodendrocytes in a preclinical mouse model of multiple sclerosis

Objectives/Goals: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system that affects 2 million people worldwide causing severe disability. This study uses the Gamt-GFP transgenic mouse line as a novel approach to track oligodendrocyte lineage cell regeneration in inflammatory demyelination for identifying potential therapies. Methods/Study Population: We previously showed that Gamt, an enzyme required for creatine synthesis, is essential for oligodendrocyte (OL) maturation and survival using the Gamt-Green Fluorescent Protein (Gamt-GFP) reporter line. In this study, we capitalize on this finding and track OL lineage cells in an experimental autoimmune encephalomyelitis (EAE) mouse model by inducing immune-mediated demyelination in the Gamt-GFP reporter line. At 7 days postimmunization (dpi), both control and EAE mice receive 4 mg/kg tamoxifen for 4 consecutive days to induce GFP expression. GFP+ cells and those also expressing OL lineage markers [Olig2 (pan-OL lineage cell marker), NG2 (OL precursor cells; OPCs), and CC1 (mature OL)] are quantitated by immunofluorescent staining of spinal cord sections collected at 28 dpi. Results/Anticipated Results: Preliminary data using immunofluorescent staining demonstrated GFP was expressed in Olig2+ cells in the inflammatory ventral white matter lesions of mice with EAE, whereas no GFP labeling was present in the control mice. Moreover, GFP+ cells also expressed NG2+. In contrast, few CC1+ cells were detected in the inflammatory lesions. The low number of dual labeled GFP+CC1+ cells in these lesions suggests OPCs under the EAE environment are unable to efficiently differentiate into mature OL. Therefore, the Gamt-GFP reporter mouse can be used to identify and track activated OL lineage cell populations (i.e., GFP+CC1) in inflammatory lesions in the EAE mouse model. Discussion/Significance of Impact: The Gamt-GFP reporter line identifies activated OL lineage cells responding to inflammatory demyelination, making it a valuable tool for testing potential therapeutics aimed at enhancing remyelination. This model helps bridge the gap between preclinical and clinical research to guide MS therapy development.