This is a Research Initiation Grant to Broaden Participation in the Biological Sciences (Program Solicitation NSF 05-581).
Intellectual Merit: Neuronal myelination is necessary for establishing the mature axonal diameters that, along with saltatory conduction, are essential for rapid impulse transmission. Axonal diameter is regulated through accumulation and modification of the neuronal specific intermediate filaments (neurofilaments). A series of nerve grafting and genetic experiments has led to the proposal of a myelin derived "outside-in" signal that results in stoichiometrically phosphorylated neurofilaments. Loss of neurofilaments or mature myelin results in axons that fail to achieve mature diameters and display reduced conduction velocities. Axons respond to "outside-in" signals via neurofilament carboxy terminal phosphorylation, resulting in increased axonal diameter through phosphorylation dependent formation of carboxy-terminal crossbridges. Recent work has established that an essential target for myelin-derived signals exists within the carboxy terminal 426 amino acids of so-called "neurofilament medium" (NF-M), one of three neurofilament component polypeptides that combine as heteropolymers to form neurofilaments (the other two are "neurofilament light" and "neurofilament heavy"). The precise identity of the essential amino acids within the NF-M tail domain that serve as this target have yet to be elucidated. Identification of the putative signaling cascade and intra-axonal targets will establish the basic mechanism of cell-cell communication that is required to establish and maintain cellular volume during nervous system development. The general hypothesis of this project is that phosphorylation of serine residues within lysine-serine-proline (KSP) motifs in the NF-M tail domain is the mechanism of myelin-dependent regulation of axonal diameter in both the central and peripheral nervous systems. Toward that end, Dr. Garcia plans to use gene replacement in mice to: mutate all known phosphorylation sites within NF-M's tail to alanine, thereby preventing phosphorylation, to determine which of these sites are the essential target within NF-M's tail domain for the outside-in signaling cascade; to mutate the phosphorylation sites to glutamate, to mimic the charge associated with phosphorylation to determine the consequences to axonal growth and organization associated with chronic phosphorylation; and to expand the number of KSP motifs of the NF-M tail domain by generating a chimeric protein consisting of mouse and bovine NF-M to determine if the number of available phosphorylation sites within NF-M's tail domain regulates axonal caliber. He will also use existing neurofilament modified mice to determine if phosphorylation of NF-M tail domain KSP motifs is essential for radial growth of CNS axons. These experiments will establish the mechanism of axonal response to myelination by identifying the amino acids within the NF-M tail domain and the method of post-translational modification that together with myelination facilitate radial axonal growth in both central and peripheral nervous systems. Moreover, this constitutes the first necessary step in elucidating the signaling cascade that derives from myelinating cells resulting in radial axonal growth through neurofilament modification. Elucidation of the signaling cascade and intra-axonal targets are crucial to understanding the mechanism of cell-cell communication required to establish mature axonal diameters during nervous system development.
Broader Impacts: Dr. Garcia will host undergraduate students in his laboratory through his participation in established undergraduate research programs at the University of Missouri, including the EXPRESS (Exposure to Research for Science Students) program, that provide opportunities for undergraduates who are members of underrepresented groups to work in faculty research laboratories during the academic years. In addition, because he himself is a member of an underrepresented minority group, he serves as a role model for minority students who aspire to careers in science.
