Brain development is regulated by a variety of molecules that are used both to build the brain and to help it function normally after it is built. This project focuses on Reelin, a large molecule that critically controls the embryonic formation of brain structures that contain different cell types organized into layers. Reelin also affects brain development after birth by promoting the maturation of cells, and the formation of connections that nerve cells make with each other (called synapses). Finally, in the adult and in old-aged brains, Reelin modulates the transmission of information at synapses, influencing cognitive brain function. The molecular mechanisms that underlie these diverse biological functions of Reelin are not well understood. The purpose of the present project is to address this knowledge gap by advancing the scientific understanding of the mechanisms through which Reelin affects mammalian brain development and function. The knowledge and materials generated by this study, including novel purified protein reagents and structural models, will be made available to the broad research community, accelerating the rate of discovery and promoting new scientific advances. This work will also benefit society by providing research training for undergraduate and graduate students, including women and other groups of people who are currently under-represented in science, fostering the development of a more diverse and globally competitive S.T.E.M. workforce.Reelin is a large, modular secreted glycoprotein that controls many aspects of brain development and function, from neuronal migration at embryonic ages, to dendrite development, synapse formation, plasticity, and learning at postnatal ages. The mechanisms that mediate these biological functions are not well understood. A major obstacle that hindered progress in the field is the lack of availability of purified Reelin. This stems not only from the difficulty of expressing such a large glycoprotein, but also from the challenges associated with proteolytic cleavage events, which generate multiple protein isoforms. Through collaborative efforts and the establishment of novel protocols, the present research team was able to overcome this limitation, and has obtained large amounts of highly-purified, bioactive Reelin protein. The goals of the project are to: 1) characterize the three-dimensional structure of Reelin proteins by Cryo- Electron Tomography (Cryo-ET), protein crystallography and small angle X-ray scattering; 2) ; identify and characterize novel receptor(s) by a combination of biochemical and proteomic technologies such as ELISA and mass spectrometry; 3) and identify signal transduction mechanisms that are elicited by these new receptors in distinct neuronal populations, including excitatory neurons, inhibitory neurons and astroglia. Together, these experiments will either validate or rule out the main hypothesis guiding the work, that full-length Reelin and its physiological proteolytic fragments are bioactive ligands that affect brain development and function by engaging a variety of cell-surface receptors expressed by different cell types, and activating multiple signal transduction pathways. The approach followed here with Reelin may also provide a paradigm for gaining a deeper understand of other molecules that play a variety of roles in the brain from early development through adulthood and senescence.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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