Pathological Inflammation Alters Oligodendrocyte Differentiation and Function - PROJECT SUMMARY Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system and stems from autoimmune damage against the myelin sheath, which is necessary for rapid and efficient conduction of electrical impulses along neurons. Oligodendrocyte progenitor cells (OPCs) serve a vital role in regulating the progression of MS because they differentiate into myelinating oligodendrocytes to restore myelin integrity in demyelinated lesions. However, many demyelinated lesions in MS experience remyelination failure, a poorly understood phenomenon that may be triggered by neuroinflammation. Recent evidence has showed that OPCs have notably decreased differentiation to oligodendrocytes and interestingly adopt novel antigen presentation functions in settings that mimic neuroinflammation. Even so, how the potential connection between these two separate phenotypes is biologically meaningful to remyelination failure and what molecular mechanisms drive their occurrence in this disease state remain unclear. To answer these questions, I developed a robust, scalable in vitro culture system to generate pure OPCs from mouse induced pluripotent stem cells that are then treated with interferon-gamma (IFNγ), a proinflammatory cytokine that is greatly increased in MS lesions and can broadly simulate the neuroinflammatory milieu in this disease. Through bulk RNA-seq and high-throughput chemical screen studies, I have found that STAT1 activity controls both IFNγ-induced phenotypes. In this proposal, Aim 1 will assess how IFNγ alters the temporal dynamics of differentiation and immune function activation to gain greater insights into MS pathophysiology. Because I can precisely control IFNγ exposure, I seek to probe potential transcriptional and functional memory in IFNγ-treated OPCs that can be broadly relevant to relapsing-remitting MS, the most common MS subtype. I will also use special nanofiber plates for 3D culturing of oligodendrocytes and interrogation of memory in myelination behavior (i.e. fiber wrapping) by modulating IFNγ exposure over a longer duration. Next, Aim 2 will assess functional targets of STAT1 to determine mechanisms for these two phenotypes. In Ctrl- versus IFNγ-treated OPCs, I will perform CUT&RUN for H3K27ac to assess genome-wide epigenetic changes and for STAT1 to determine its global binding profile and how STAT1 can cause these changes in acetylation and also have context- dependent functions. A rigorous computational pipeline will identify key differentially regulated genes and pathways, and I will validate them in vitro via genetic knockdown studies and in human MS single- cell RNA-seq datasets and patient brain sections. The experiments outlined here will significantly bolster our knowledge of oligodendrocyte functional plasticity in remyelination failure and pave the way for new, exciting therapeutic approaches that go beyond conventional MS disease-modifying therapies aimed at immune cell depletion and instead harness the innate regenerative capacity of oligodendrocytes.
