In peripheral nerve injury, inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-a), contribute to the progression of Wallerian degeneration and the development of painful neuropathies. Alpha 2-Macroglobulin (a2M) is a broad spectrum protease inhibitor found in the plasma and extracellular spaces, which also regulates the activity of cytokines and growth factors. The latter function reflects the activity of two non-covalent protein interaction domains (PIDs) in the structure of the a2M subunit. A distinct sequence mediates interaction of a2M with the receptor, low density lipoprotein receptor-related protein-1 (LRP-1). Exposure of the PIDs and the LRP-1 recognition site is regulated by a2M conformational change. We developed a method for stabilizing a2M conformational intermediates. The resulting preparation (referred to as MAC) expresses increased binding affinity for TNF-a and interleukin-1beta. MAC also demonstrates potent anti-inflammatory activity in mice. In new unpublished studies, we show that MAC expresses anti-inflammatory activity in injured peripheral nerves and, as a result, may be axonal protective. We demonstrate rapid progress in our work to disrupt various a2M activities by mutation of the recombinant protein. We hypothesize that MAC and other a2M derivatives may be potent experimental therapeutics in peripheral nerve injury, sciatica, and lumbar disc herniation. To test this hypothesis, in Aim 1, the activities of a2M, methylamine-activated a2M, and MAC will be compared in sciatic nerve crush and chronic constriction injury experiments in mice. The ability of these agents to block development of painful neuropathies also will be assessed. To test the activity of naturally occurring a2M, nerve injury experiments will be performed in a2M/murinoglobulin gene knock-out mice. To test the hypothesis that MAC functions by mechanisms in addition to neutralizing TNF-a, nerve injury studies will be performed in TNF-a gene-deleted mice. In Specific Aim 2, full-length human a2M will be engineered to independently neutralize or modify the function of the two PIDs and the LRP-1 recognition site. We will then utilize macrophage and Schwann cell culture model systems to test mechanisms, including cytokine- binding and regulation of LRP-1-dependent cell signaling, by which MAC and other forms of a2M may regulate cell physiology in the injured nerve. In Specific Aim 3, mutated full-length a2M, in which the function of specific domains is disrupted, will be tested in the nerve injury model systems. The goal of these studies is to test the mechanism by which MAC and other a2M derivatives are protective in peripheral nerve injury in vivo. Additional studies are planned in Aim 3 to capitalize on our work elucidating structure-function relation- ships in a2M by designing mutated forms of a2M with enhanced activity in nerve injury. This project will contribute to our understanding of extracellular mediators in peripheral nerve injury and offer the potential for generating novel experimental protein therapeutics.