Transport of TNFalpha across the BBB

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DESCRIPTION (provided by applicant): The blood-brain and blood-spinal cord barriers (BBB/BSCB), lying between the CNS (brain and spinal cord) and its supplying capillary blood vessels, provide an immense interface for interaction and exchange of information between the CNS and the rest of the body. We have shown that the BSCB is not static or physically passive, but undergoes dynamic changes, to regulate the availability of the cytokine tumor necrosis factor a (TNFa) from blood to the CNS. In the proposed studies, we will test the hypothesis that TNFa, after being transported across the BBB/BSCB, can facilitate functional recovery after spinal cord injury (SCI). We will also characterize the mechanisms by which this specific transport can be regulated. To determine the functional implications of TNFe transport, we will first show that spinal cord uptake of radioactively labeled TNFe will increase specifically in a modified mouse model of thoracic hemisection, and that this increase will not coincide with passive disruption of the barrier (measured by increased paracellular permeability of albumin). We will then determine the effects of TNFa treatment by evaluating locomotor behavior, intraspinal conduction of evoked potentials, and histological evidence of long tract regeneration. We expect that small doses of TNFa will facilitate recovery whereas large doses will worsen the deficits resulting from SCI. To identify the roles of p55- and p75-receptors, we will study TNFa transport in the presence of receptor antibodies and in the receptor knockout mice. We predict that increased spinal cord uptake of TNFe after SCI will be dependent upon upregulation of the receptors, as determined by Western blot and quantitative PCR analysis. Since transcytosis of a cytokine across the BBB/BSCB is a complicated process involving not only the receptors but also other regulatory proteins, we will identify novel transport regulatory proteins by co-immunoprecipitation, comparative proteomics, transfection and transport assays. We predict that the transport process will be regulated by the receptors, vesicular and cytoskeletal proteins, and chaperons in different phosphorylation states. By completing the proposed study, we will better understand the dual roles of TNFa in SCI and in its transport process involving the regulatory proteins. These transporters could be novel drug targets and therefore provide promising therapeutic potential.