The neonatal brain is unusually sensitive to sublethal hypoxic injury. As a common accompaniment of extremely premature birth, periods of sublethal hypoxia disrupt synaptic remodeling and maturation in the newborn brain, and may thereby account for the long-term cognitive deficiencies of such children. One mechanism contributing to synaptic organization and function is the neuronal spectrin skeleton. Recent data has revealed that specific isoforms of spectrin subserve distinct roles in axonal transport, receptor trafficking, and receptor organization and receptor turnover. Collectively these activities are crucial to neuronal and synaptic function. Since the spectrin skeleton is regulated by calcium signaling pathways involving calmodulin and calpain proteolysis, as well as by phosphorylation on both ser and thru and by a recently recognized tyrosine phosphorylation, we hypothesize that inappropriate modification of the neuronal spectrin skeleton following mild hypoxic injury contributes to the pathology of premature brain dysfunction. It is important to thus understand the physiologic and pathologic consequences of calcium activated protease-mediated spectrin processing in the developing brain. We will use in vitro analysis to identify the specific calpain cleavage sites in betaI, betaIII, and betaIV spectrin, and determine whether the susceptibility of these spectrins to calpain is allosterically coupled to spectrin cleavage (as is betaII spectrin cleavage). These additional beta-spectrin isoforms have only recently been identified in specialized neuronal compartments, and their susceptibility to proteolysis is unknown, as is the biologic consequences of such cleavage. Using cleavage-specific antibodies, the topographic and temporal in vivo processing of these spectrins and of alphaII spectrin by calpain will be assessed in mice experiencing mild sublethal hypoxia, using both wt mice and animals genetically modified such that exons 28-30 of the spectrin gene have been selectively deleted using the cre-loxp recombinase system. These exons encode the hypersensitive calpain and caspase 3 target sites, two putative sites of tyrosine phosphorylation, and the calmodulin binding domain of alphaII spectrin. This critical central region of spectrin thus serves as a point of convergence between Ca ++ and phosphorylation-mediated signaling pathways in the brain. By generating animals in which each of these actions has been selectively blocked, the contributions of these pathways to neuronal maturation and viability will be determined. Project Core resources will be used to evaluate these phenotypes.
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