Engineering Tools to Study and Control Signaling in Neural Stem Cells

Adult neurogenesis, the process by which new neurons develop in the adult mammalian central nervous system, was thought to be nonexistent by accepted scientific dogma until the discovery of adult neural stem cells (NSCs) in the 1990s. NSCs have been found in two regions of the adult brain: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, and have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Importantly, hippocampal NSCs play key roles in learning and memory, and have been implicated in a number of pathologies, including Alzheimer’s disease. NSCs reside in complex niches that provide the physical, chemical, and biological signals regulating stem cell maintenance and differentiation. A thorough understanding of NSC biology and niche signals can provide both insight into the mechanisms of adult neurogenesis, and inform stem cell based therapeutics to treat neurological injury and disease. Since the discovery of NSCs, a body of work has emerged characterizing the wide array of signals and intracellular pathways that mediate NSC behavior. Some of these findings, however, point to complex signaling mechanisms, the further study of which requires techniques outside the standard biological “toolbox”. The goal of this dissertation, therefore, was to engineer novel tools to enable the study and control of complex signaling systems in NSCs, and their application towards novel biological discoveries./nThe work presented here investigates two aspects of NSC biology: heterogeneity and cell-cell signaling. Stem cells are inherently heterogeneous; NSCs give rise to diverse progeny, including neurons and astrocytes, and in vitro, NSCs can differentially respond to the same set of cues. To probe this heterogeneity, a single cell Western blotting (scWestern) platform was developed. scWesterns enabled the interrogation of the proteome of thousands of single cells in about four hours, and multiplexing allowed for up to eleven targets to be detected from a single cell. We utilized the scWestern to probe heterogeneity in NSC signaling upon mitogen stimulation and differentiation. These studies provided unprecedented insight into differential single NSC responses to homogenous presentation of proliferation and differentiation factors./nCell-cell signaling in the NSC niche is comprised of paracrine and juxtacrine signals presented by supportive niche cells. Ephrin-B2 on hippocampal astrocytes was recently discovered to induce NSC neuronal differentiation through the receptor EphB4. Ephs and ephrins are both cell-surface bound and require oligomerization for downstream signal activation, so further investigation of ephrin-B2-mediated neurogenesis can benefit from novel tool development. This system was the focus of three studies, comprising the remainder of the dissertation. First, to better recapitulate the physical interactions between membrane-bound receptors and ligands, a supported lipid bilayer system was developed to present laterally mobile, monomeric ephrin-B2 to NSCs. We observed EphB4/ephrin-B2 co-clustering, and for the first time showed membrane-bound monomeric ephrin-B2 activation of EphB4 signaling and NSC differentiation in a synthetic system. By employing spatial mutation strategies to control ephrin-B2 diffusion and receptor-ligand complex size, we demonstrated that EphB4 signaling and NSC differentiation are sensitive to spatial properties of apposing cell membranes. This finding reveals novel regulatory mechanisms of both EphB4 signaling and NSC niche dynamics./nEstablished Eph:ephrin signaling targets do not overlap with known neurogenic factors, so we next investigated ephrin-B2-induced downstream signaling in NSCs. Utilizing multivalent ephrin-B2 conjugates to enhance stimulation and mass spectrometry to identify signaling effectors, a number of novel kinases were identified, including activated Cdc42 kinase 1 (Ack1), Fyn proto-oncogene (Fyn), and Src proto-oncogene (Src). CRISPR/Cas9 genome engineering was then employed to knock down these proteins, which prevented ephrin-B2-induced NSC neurogenesis, demonstrating a role for these kinases in signaling downstream of EphB4 stimulation. Ack1 is a novel effector of Eph:ephrin signaling, and Ack1 and Fyn are novel regulators of NSC differentiation; therefore, this work reveals a number of previously unknown biological mechanisms./nFinally, to examine dynamics of EphB4 signaling in NSCs, optogenetic methods for ephrin-independent Eph clustering and activation in response to blue light were created. We first developed generalized tools to apply a recently published method of optically targeting, clustering, and stimulating transmembrane receptors towards any signaling system of interest. We then harnessed these tools to study EphB4 signaling in NSCs by testing a number of EphB4-targeting optogenetic constructs. While none of the tested vectors were able to cluster and activate EphB4, a number of interesting observations were made that point to future areas for optimization. Moreover, we were able to perform a proof-of-concept study of the application of the generalized optogenetic tools developed./nIn summary, this dissertation presents work on both the development of novel tools for probing stem cell complexities, and the implementation of these tools towards the discovery of a number of novel biological mechanisms.

Advisor: Schaffer, David V