Date of Award
Doctor of Philosophy (PhD)
David M Feliciano
Extracellular vesicles (EVs) are nanometer sized particles released from all cells. EVs are found in all biological fluids, including cerebrospinal fluid (CSF) and blood. EVs modulate intercellular communication through the transfer of nucleic acids and proteins from donor to recipient cells. During early postnatal neurogenesis, subventricular zone (SVZ) neural stem cells (NSCs) asymmetrically divide to give rise to neuroblasts that migrate along the rostral migratory stream (RMS) and populate the olfactory bulb (OB). Other, non-neuronal cells populate the SVZ, such as microglia and endothelial cells. Microglia have been shown to regulate SVZ NSCs, but it remains unclear if this communication is bidirectional. The purpose of this study was to determine if SVZ NSCs release EVs and what physiological impact this has on postnatal SVZ development. First, we generated a protocol in which SVZ NSCs were placed in culture and EVs were isolated from NSC conditioned media. To study EVs in vivo, EVs were labeled with DiI and transplanted into the SVZ of neonatal mice. Immunocytochemistry, immunohistochemistry, and electron microscopy were used to confirm the isolation and transplantation of NSC EVs. Using this methodology, SVZ NSC EVs were transplanted into the lateral ventricles of neonatal mice. We found a majority of DiI particles coalesced with Iba1-positive microglia in the SVZ. Furthermore, Iba1-positive microglia underwent a morphological shift from a stellate to rounded phenotype. RNA sequencing and analysis of EV treated microglia revealed that immune system processes and inflammatory responses were the most highly enriched and represented terms. Small RNA sequencing of NSC EVs uncovered families of miRNA, such as Let-7, that have been shown to regulate microglia physiology and morphology. The upregulation of inflammatory response transcripts included interleukin 1α (IL-1α), IL-1β, and IL-6. In agreement with RNA sequencing data, Luminex assays revealed cytokines, such as IL-1α, IL-1β, and IL-6, were significantly upregulated in treated microglia. EV-depleted microglia media was transplanted into the lateral ventricles of neonatal mice, and media collected from EV-treated microglia reduced SVZ NSC proliferation. To further investigate if SVZ NSCs release EVs in vivo, we generated a transgenic model system in which EV marker protein tetraspanin CD9 was fused to Turbo-GFP (CD9-GFP), which is derived from the copepod Pontellina plumata. CD9-GFP was inserted downstream of a STOP codon flanked by loxP sites. CD9-GFP was found in Nestin-positive cells in the SVZ of transgenic mice electroporated with Cre-recombinase. Taken together, our data supports the release of EVs from SVZ NSCs both in vivo and in vitro. Furthermore, EVs released from SVZ NSCs regulate microglia during early postnatal development.
Morton, Mary C., "Subventricular Zone Neural Stem Cells Release Extracellular Vesicles During Early Postnatal Neurogenesis" (2018). All Dissertations. 2561.