Golgi outpost biogenesis in oligodendrocytes - PROJECT SUMMARY/ABSTRACT Oligodendrocytes are the brain glial cell responsible for the formation and maintenance of the myelin sheath, a lipid-rich insulating material that wraps around neuronal axons. Myelination is crucial for the fast interconnectivity of neurons in vertebrates, as underscored by devastating demyelinating diseases like Multiple Sclerosis (MS). To support this essential function, oligodendrocytes rely on unique mechanisms: in contrast to the classic model in which microtubules are made solely from centrosomes in the cell body, oligodendrocytes also nucleate microtubules from satellite organelles in their branches called Golgi outposts. In oligodendrocytes, these Golgi outposts contain multiple compartments, appearing like “miniature” Golgi apparatuses, and are marked with Tubulin polymerization promoting protein (TPPP), a microtubule nucleating protein. Loss of TPPP in mice results in aberrant aggregation of myelin basic protein (Mbp) mRNA, suggesting a critical role for TPPP+ Golgi outposts in the proper distribution of key myelin components. However, we lack foundational knowledge about TPPP+ Golgi outposts, such as the mechanisms of their biogenesis. The long-term goal of this study is to elucidate cellular mechanisms that support myelination. The objective of this application is to investigate how TPPP+ Golgi outposts, an organelle critical to supporting myelination, is formed. In Specific Aim 1, Lana will use immunofluorescence staining and live-cell imaging of developing oligodendrocytes to elucidate the events leading up to the biogenesis of mature TPPP+ Golgi outposts in primary rat oligodendrocyte cultures. Preliminary fixed and live-cell imaging reveal the emergence of putative Golgi outpost precursors: multi-compartment Golgi tubules which bud, elongate, and fission from the cell body Golgi apparatus and exhibit motility. In Specific Aim 2, Lana seeks to elucidate how Golgi outposts acquire TPPP using imaging modalities to visualize both Tppp mRNA trafficking and translation in primary cells. Preliminary smFISH (single molecule fluorescence in situ hybridization) data demonstrate that Tppp mRNA is found throughout the cell and on Golgi tubules, putative precursors to mature, TPPP+ Golgi outposts. She aims to further elucidate how Tppp mRNA localization, translation, and Golgi outpost biogenesis are coordinated. This work sets the foundation for further studies about the function of the TPPP+ Golgi outpost. With its multiple Golgi compartments and capacity as a microtubule organizing center, the TPPP+ Golgi outpost may serve as a specialized processing and delivery “hub” for myelin cargoes. Further, understanding how TPPP localization to Golgi outposts is regulated may reveal critical insights in understanding the neurodegenerative disease Multiple Systems Atrophy (MSA), in which TPPP is mislocalized in glial cytoplasmic inclusions (GCIs). At the conclusion of Lana’s NRSA-sponsored training, Lana will develop the robust intellectual foundation and technical expertise to emerge as a productive, independent scientist, making her an outstanding candidate for an independent investigator position in the future.
