Stephanie A. Redmond, PhD - US grants

DESCRIPTION (provided by applicant): Myelination of correct axons at the right time is critical for overall neural function. During development, all central nervous system (CNS) axons are unmyelinated until oligodendrocytes (OLs) differentiate from their precursor cells (OPCs) and begin to deposit concentric wraps of insulating membrane around most axons to facilitate saltatory conduction of action potentials. Few molecular regulators of OL axon selection are known, but myelination of only appropriate axons may be accomplished by (1) attraction to positive axonal cues, (2) repulsion from inappropriate fibers, or a combination of induction and inhibition. Unexpectedly, our preliminary data suggest a third possibility; that OLs sense and use axon diameter in myelination independent of molecular signaling. OLs cocultured with axon-sized polystyrene nanofibers select only fibers above a threshold diameter to myelinate. Interestingly, the dendrite is geometrically similar to the axon, yet is never myelinated. Thus, we hypothesize that dendrites inhibit their myelination by oligodendrocytes in a contact- dependent manner. To test our hypothesis I will use a novel reductionist coculture system to (1) determine whether dendrites inhibit OL myelination, as well as (2) identify molecular signals localized to dendrite membranes that modulate OL myelination. It is critical to determine how oligodendrocytes interact with, and avoid dendrites to understand normal myelination kinetics and gain insight into dismyelinating diseases.

Ependymal cells (E cells) are essential for cerebrospinal fluid (CSF) flow and prevention of hydrocephalus. CSF flow is thought regulate intracranial pressure and promote waste removal, but it?s likely doing much more. Several recent studies suggest that the CSF contains essential signaling molecules for neuroendocrine signaling, neurogenesis, migration and brain activity. The CSF is constantly being produced and moved by E cells through the ventricular system, turning over three to four times per day in humans. While the ependymal lining was thought to be composed of a homogeneous layer of multiciliated (E1) cells, recent data suggest that E cells are heterogeneous. The Alvarez-Buylla (A.-B.) lab has identified a novel subtype of E cell (E2 cell) that has a unique apical domain with only two motile-type (9+2 microtubule structure) cilia, and complex basal bodies that are 30-100 times larger than those of E1 cells. Furthermore, recent data now in press has revealed that E2 cells have long basal processes that project into the underlying brain parenchyma, including the dorsal raphe nucleus (DRN). The A.-B. lab has previously shown that the DRN modulates B1 cell adult neurogenesis. Most neuronal and glial cell bodies in the brain parenchyma are separated from direct CSF contact. I suspect that E2 cells could be serving as a bridge between the ventricular and parenchymal brain compartments; their apical compartment with large basal bodies and long motile cilia could serve for the detection of CSF components; their long basal process could transmit this information to underlying neurons. I hypothesize that E2 cells are spatially and structurally primed to `bridge' the gap between signaling molecules in the CSF and neurons of the DRN, and that DRN-dependent adult neurogenesis is modulated by E2 cell signaling. In Aim 1 I will test the hypothesis that E2 cell basal processes reach morphological maturity during postnatal development and make contacts among DRN neurons. In Aim 2 I will test the hypothesis that E2 cells modulate DRN circuit dynamics and adult neurogenesis. I predict that rates of neurogenesis will be altered when DRN-contacting E2 cells are selectively ablated.

Project Summary/Abstract: The brain is made up of highly specialized neurons and glia, organized spatially into distinct functional units, interconnected to perform some of the most complex computations in nature. To date, cutting-edge molecular and genetic tools have been applied overwhelmingly to the study of neural structure and function. Less well understood are glial cells, despite the fact that radial glia generate all neurons in the central nervous system during embryonic development. Adult neural stem cells (aNSCs) share many features of radial glia and astrocytes, and in mice give rise to new neurons throughout life. aNSCs are present in the lateral walls of the lateral ventricles, and sit in complex cellular niches that regulate many features of their activity. We and others have previously found that the neurogenic potential of aNSCs depends on the aNSC?s position along the two- dimensional surface of the ventricular wall. Dorsal aNSCs generate superficial-layer granular cells for the olfactory bulb, while ventral aNSCs produce deep-layer granular cells. Adult NSC neurogenic potential is cell- intrinsic, as heterotopically transplanted cells produce neurons consistent with their original position. However, instructive niche cell signaling may also be regionally defined, as recent work demonstrates spatially selective aNSC activation directly or indirectly by projection neurons in response to feeding behavior. Given the widespread interest in stem cell therapies for brain repair, a critical gap in knowledge is the lack of mechanistic insight into molecular determinants of aNSC neurogenic potential and of neurogenic niche regionalization. I hypothesize that regionally-restricted transcriptional signatures define aNSC neurogenic potential, and complementary signatures in niche glia underlie region-specific extrinsic control of adult neurogenesis. In this proposal, I put forward three orthogonal Aims that span the K99 and R00 phases of the award. In the first two Aims completed largely in the K99 phase, I use single cell sequencing to identify dorsal and ventral clusters of aNSCs and niche glia, and mouse genetics to assess signature-driving gene contributions to regional identity. The R00 phase is mainly accomplished in Aim 3, where I build on my existing preliminary single cell RNA- sequencing analyses to gain mechanistic insight into functional heterogeneity among ependymal cells throughout the ventricular system. Together, these data will provide a foundational understanding of neurogenic niche signaling dynamics that together drive neurogenesis, and creates a new avenue of exploration to understand the diverse roles of ependymal cells at the brain/cerebral spinal fluid interface.